Wafik S. El‐Deiry

The ability of p53 to activate transcription from specific sequences suggests that genes induced by p53 may mediate its biological role as a tumor suppressor. Using a subtractive hybridization approach, we identified a gene, named WAF1, whose induction was associated with wild-type but not mutant p53 gene expression in a human brain tumor cell line. The WAF1 gene was localized to chromosome 6p21.2, and its sequence, structure, and activation by p53 was conserved in rodents. Introduction of WAF1 cDNA suppressed the growth of human brain, lung, and colon tumor cells in culture. Using a yeast enhancer trap, a p53-binding site was identified 2.4 kb upstream of WAF1 coding sequences. The WAF1 promoter, including this p53-binding site, conferred p53-dependent inducibility upon a heterologous reporter gene. These studies define a gene whose expression is directly induced by p53 and that could be an important mediator of p53-dependent tumor growth suppression.

Over the past decade, the Nomenclature Committee on Cell Death (NCCD) has formulated guidelines for the definition and interpretation of cell death from morphological, biochemical, and functional perspectives. Since the field continues to expand and novel mechanisms that orchestrate multiple cell death pathways are unveiled, we propose an updated classification of cell death subroutines focusing on mechanistic and essential (as opposed to correlative and dispensable) aspects of the process. As we provide molecularly oriented definitions of terms including intrinsic apoptosis, extrinsic apoptosis, mitochondrial permeability transition (MPT)-driven necrosis, necroptosis, ferroptosis, pyroptosis, parthanatos, entotic cell death, NETotic cell death, lysosome-dependent cell death, autophagy-dependent cell death, immunogenic cell death, cellular senescence, and mitotic catastrophe, we discuss the utility of neologisms that refer to highly specialized instances of these processes. The mission of the NCCD is to provide a widely accepted nomenclature on cell death in support of the continued development of the field.

Cell cycle checkpoints can enhance cell survival and limit mutagenic events following DNA damage. Primary murine fibroblasts became deficient in a G1 checkpoint activated by ionizing radiation (IR) when both wild-type p53 alleles were disrupted. In addition, cells from patients with the radiosensitive, cancer-prone disease ataxia-telangiectasia (AT) lacked the IR-induced increase in p53 protein levels seen in normal cells. Finally, IR induction of the human GADD45 gene, an induction that is also defective in AT cells, was dependent on wild-type p53 function. Wild-type but not mutant p53 bound strongly to a conserved element in the GADD45 gene, and a p53-containing nuclear factor, which bound this element, was detected in extracts from irradiated cells. Thus, we identified three participants (AT gene(s), p53, and GADD45) in a signal transduction pathway that controls cell cycle arrest following DNA damage; abnormalities in this pathway probably contribute to tumor development.

Different types of cell death are often defined by morphological criteria, without a clear reference to precise biochemical mechanisms. The Nomenclature Committee on Cell Death (NCCD) proposes unified criteria for the definition of cell death and of its different morphologies, while formulating several caveats against the misuse of words and concepts that slow down progress in the area of cell death research. Authors, reviewers and editors of scientific periodicals are invited to abandon expressions like 'percentage apoptosis' and to replace them with more accurate descriptions of the biochemical and cellular parameters that are actually measured. Moreover, at the present stage, it should be accepted that caspase-independent mechanisms can cooperate with (or substitute for) caspases in the execution of lethal signaling pathways and that 'autophagic cell death' is a type of cell death occurring together with (but not necessarily by) autophagic vacuolization. This study details the 2009 recommendations of the NCCD on the use of cell death-related terminology including 'entosis', 'mitotic catastrophe', 'necrosis', 'necroptosis' and 'pyroptosis'.

In 2009, the Nomenclature Committee on Cell Death (NCCD) proposed a set of recommendations for the definition of distinct cell death morphologies and for the appropriate use of cell death-related terminology, including ‘apoptosis’, ‘necrosis’ and ‘mitotic catastrophe’. In view of the substantial progress in the biochemical and genetic exploration of cell death, time has come to switch from morphological to molecular definitions of cell death modalities. Here we propose a functional classification of cell death subroutines that applies to both in vitro and in vivo settings and includes extrinsic apoptosis, caspase-dependent or -independent intrinsic apoptosis, regulated necrosis, autophagic cell death and mitotic catastrophe. Moreover, we discuss the utility of expressions indicating additional cell death modalities. On the basis of the new, revised NCCD classification, cell death subroutines are defined by a series of precise, measurable biochemical features.

Fusions involving one of three tropomyosin receptor kinases (TRK) occur in diverse cancers in children and adults. We evaluated the efficacy and safety of larotrectinib, a highly selective TRK inhibitor, in adults and children who had tumors with these fusions.We enrolled patients with consecutively and prospectively identified TRK fusion-positive cancers, detected by molecular profiling as routinely performed at each site, into one of three protocols: a phase 1 study involving adults, a phase 1-2 study involving children, or a phase 2 study involving adolescents and adults. The primary end point for the combined analysis was the overall response rate according to independent review. Secondary end points included duration of response, progression-free survival, and safety.A total of 55 patients, ranging in age from 4 months to 76 years, were enrolled and treated. Patients had 17 unique TRK fusion-positive tumor types. The overall response rate was 75% (95% confidence interval [CI], 61 to 85) according to independent review and 80% (95% CI, 67 to 90) according to investigator assessment. At 1 year, 71% of the responses were ongoing and 55% of the patients remained progression-free. The median duration of response and progression-free survival had not been reached. At a median follow-up of 9.4 months, 86% of the patients with a response (38 of 44 patients) were continuing treatment or had undergone surgery that was intended to be curative. Adverse events were predominantly of grade 1, and no adverse event of grade 3 or 4 that was considered by the investigators to be related to larotrectinib occurred in more than 5% of patients. No patient discontinued larotrectinib owing to drug-related adverse events.Larotrectinib had marked and durable antitumor activity in patients with TRK fusion-positive cancer, regardless of the age of the patient or of the tumor type. (Funded by Loxo Oncology and others; ClinicalTrials.gov numbers, NCT02122913 , NCT02637687 , and NCT02576431 .).

For over three decades, a mainstay and goal of clinical oncology has been the development of therapies promoting the effective elimination of cancer cells by apoptosis. This programmed cell death process is mediated by several signalling pathways (referred to as intrinsic and extrinsic) triggered by multiple factors, including cellular stress, DNA damage and immune surveillance. The interaction of apoptosis pathways with other signalling mechanisms can also affect cell death. The clinical translation of effective pro-apoptotic agents involves drug discovery studies (addressing the bioavailability, stability, tumour penetration, toxicity profile in non-malignant tissues, drug interactions and off-target effects) as well as an understanding of tumour biology (including heterogeneity and evolution of resistant clones). While tumour cell death can result in response to therapy, the selection, growth and dissemination of resistant cells can ultimately be fatal. In this Review, we present the main apoptosis pathways and other signalling pathways that interact with them, and discuss actionable molecular targets, therapeutic agents in clinical translation and known mechanisms of resistance to these agents. The authors of this Review present the main pathways that regulate apoptosis as well as other signalling pathways that interact with them, highlighting actionable molecular targets for anticancer therapy. They also provide an overview of therapeutic agents exploiting apoptosis currently in clinical translation and known mechanisms of resistance to these agents.

Apoptosis plays an important role in development and maintenance of tissue homeostasis. Intensive efforts have been made to explore the molecular mechanisms of the apoptotic signaling pathways including the initiation, mediation, execution and regulation of apoptosis. Caspases are central effectors of apoptosis. Cells undergo apoptosis through two major pathways, namely the extrinsic pathway (death receptor pathway) or the intrinsic pathway (the mitochondrial pathway). Finally, the contents of dead cells are packaged into apoptotic bodies, which are recognized by neighboring cells or macrophages and cleared by phagocytosis. Cellular apoptosis is tightly controlled by a complex regulatory networks including balancing pro-survival signals. De-regulation of apoptosis may lead to pathological disorders such as developmental defects, autoimmune diseases, neurodegeneration or cancer. Increasing attention is being focused on alternative signaling pathways leading to cell death including necrosis, autophagy, and mitotic catastrophe. Understanding of cell death signaling pathways is relevant to understanding cancer and to developing more effective therapeutics.

FADD (also known as Mort-1) is a signal transducer downstream of cell death receptor CD95 (also called Fas). CD95, tumor necrosis factor receptor type 1 (TNFR-1), and death receptor 3 (DR3) did not induce apoptosis in FADD-deficient embryonic fibroblasts, whereas DR4, oncogenes E1A and c-myc, and chemotherapeutic agent adriamycin did. Mice with a deletion in the FADD gene did not survive beyond day 11.5 of embryogenesis; these mice showed signs of cardiac failure and abdominal hemorrhage. Chimeric embryos showing a high contribution of FADD null mutant cells to the heart reproduce the phenotype of FADD-deficient mutants. Thus, not only death receptors, but also receptors that couple to developmental programs, may use FADD for signaling.

Circulating tumor cells (CTCs) have become an important biomarker for early cancer diagnosis, prognosis, and treatment monitoring. Recently, a replication-competent recombinant adenovirus driven by a human telomerase gene (hTERT) promoter was shown to detect live CTCs in blood samples of cancer patients. Here, we report a new class of adenoviruses containing regulatory elements that repress the hTERT gene in normal cells. Compared to the virus with only the hTERT core promoter, the new viruses showed better selectivity for replication in cancer cells than in normal cells. In particular, Ad5GTSe, containing three extra copies of a repressor element, displayed a superior tropism for cancer cells among leukocytes and was thus selected for CTC detection in blood samples. To further improve the efficiency and specificity of CTC identification, we tested a combinatory strategy of microfiltration enrichment using flexible micro spring arrays (FMSAs) and Ad5GTSe imaging. Our experiments showed that this method efficiently detected both cancer cells spiked into healthy blood and potential CTCs in blood samples of breast and pancreatic cancer patients, demonstrating its potential as a highly sensitive and reliable system for detection and capture of CTCs of different tumor types.

Cells exposed to extreme physicochemical or mechanical stimuli die in an uncontrollable manner, as a result of their immediate structural breakdown. Such an unavoidable variant of cellular demise is generally referred to as 'accidental cell death' (ACD). In most settings, however, cell death is initiated by a genetically encoded apparatus, correlating with the fact that its course can be altered by pharmacologic or genetic interventions. 'Regulated cell death' (RCD) can occur as part of physiologic programs or can be activated once adaptive responses to perturbations of the extracellular or intracellular microenvironment fail. The biochemical phenomena that accompany RCD may be harnessed to classify it into a few subtypes, which often (but not always) exhibit stereotyped morphologic features. Nonetheless, efficiently inhibiting the processes that are commonly thought to cause RCD, such as the activation of executioner caspases in the course of apoptosis, does not exert true cytoprotective effects in the mammalian system, but simply alters the kinetics of cellular demise as it shifts its morphologic and biochemical correlates. Conversely, bona fide cytoprotection can be achieved by inhibiting the transduction of lethal signals in the early phases of the process, when adaptive responses are still operational. Thus, the mechanisms that truly execute RCD may be less understood, less inhibitable and perhaps more homogeneous than previously thought. Here, the Nomenclature Committee on Cell Death formulates a set of recommendations to help scientists and researchers to discriminate between essential and accessory aspects of cell death.

Tumor necrosis factor-related apoptosis-inducing ligand or Apo 2 ligand (TRAIL/Apo2L) is a member of the tumor necrosis factor (TNF) family of ligands capable of initiating apoptosis through engagement of its death receptors. TRAIL selectively induces apoptosis of a variety of tumor cells and transformed cells, but not most normal cells, and therefore has garnered intense interest as a promising agent for cancer therapy. TRAIL is expressed on different cells of the immune system and plays a role in both T-cell- and natural killer cell-mediated tumor surveillance and suppression of suppressing tumor metastasis. Some mismatch-repair-deficient tumors evade TRAIL-induced apoptosis and acquire TRAIL resistance through different mechanisms. Death receptors, members of the TNF receptor family, signal apoptosis independently of the p53 tumor-suppressor gene. TRAIL treatment in combination with chemo- or radiotherapy enhances TRAIL sensitivity or reverses TRAIL resistance by regulating the downstream effectors. Efforts to identify agents that activate death receptors or block specific effectors may improve therapeutic design. In this review, we summarize recent insights into the apoptosis-signaling pathways stimulated by TRAIL, present our current understanding of the physiological role of this ligand and the potential of its application for cancer therapy and prevention.

The p53 tumor suppressor is the most commonly mutated gene in human cancer. p53 protein is stabilized in response to different checkpoints activated by DNA damage, hypoxia, viral infection, or oncogene activation resulting in diverse biological effects, such as cell cycle arrest, apoptosis, senescence, differentiation, and antiangiogenesis. The stable p53 protein is activated by phosphorylation, dephosphorylation and acetylation yielding a potent sequence-specific DNA-binding transcription factor. The wide range of p53's biological effects can in part be explained by its activation of expression of a number of target genes including p21WAFI, GADD45, 14-3-3 sigma, bax, Fas/APO1, KILLER/DR5, PIG3, Tsp1, IGF-BP3 and others. This review will focus on the transcriptional targets of p53, their regulation by p53, and their relative importance in carrying out the biological effects of p53.

Circulating tumor cells (CTCs) are important targets for cancer biology studies. To further elucidate the role of CTCs in cancer metastasis and prognosis, effective methods for isolating extremely rare tumor cells from peripheral blood must be developed. Acoustic-based methods, which are known to preserve the integrity, functionality, and viability of biological cells using label-free and contact-free sorting, have thus far not been successfully developed to isolate rare CTCs using clinical samples from cancer patients owing to technical constraints, insufficient throughput, and lack of long-term device stability. In this work, we demonstrate the development of an acoustic-based microfluidic device that is capable of high-throughput separation of CTCs from peripheral blood samples obtained from cancer patients. Our method uses tilted-angle standing surface acoustic waves. Parametric numerical simulations were performed to design optimum device geometry, tilt angle, and cell throughput that is more than 20 times higher than previously possible for such devices. We first validated the capability of this device by successfully separating low concentrations (∼100 cells/mL) of a variety of cancer cells from cell culture lines from WBCs with a recovery rate better than 83%. We then demonstrated the isolation of CTCs in blood samples obtained from patients with breast cancer. Our acoustic-based separation method thus offers the potential to serve as an invaluable supplemental tool in cancer research, diagnostics, drug efficacy assessment, and therapeutics owing to its excellent biocompatibility, simple design, and label-free automated operation while offering the capability to isolate rare CTCs in a viable state.

Cell death is essential for a plethora of physiological processes, and its deregulation characterizes numerous human diseases. Thus, the in-depth investigation of cell death and its mechanisms constitutes a formidable challenge for fundamental and applied biomedical research, and has tremendous implications for the development of novel therapeutic strategies. It is, therefore, of utmost importance to standardize the experimental procedures that identify dying and dead cells in cell cultures and/or in tissues, from model organisms and/or humans, in healthy and/or pathological scenarios. Thus far, dozens of methods have been proposed to quantify cell death-related parameters. However, no guidelines exist regarding their use and interpretation, and nobody has thoroughly annotated the experimental settings for which each of these techniques is most appropriate. Here, we provide a nonexhaustive comparison of methods to detect cell death with apoptotic or nonapoptotic morphologies, their advantages and pitfalls. These guidelines are intended for investigators who study cell death, as well as for reviewers who need to constructively critique scientific reports that deal with cellular demise. Given the difficulties in determining the exact number of cells that have passed the point-of-no-return of the signaling cascades leading to cell death, we emphasize the importance of performing multiple, methodologically unrelated assays to quantify dying and dead cells.

Mutations of the BRCA1 tumor suppressor gene are the most commonly detected alterations in familial breast and ovarian cancer. Although BRCA1 is required for normal mouse development, the molecular basis for its tumor suppressive function remains poorly understood. We show here that BRCA1 increases p53-dependent transcription from the p21WAF1/CIP1 and bax promoters. We also show that BRCA1 and p53 proteins interact both in vitro and in vivo. The interacting regions map, in vitro, to aa 224-500 of BRCA1 and the C-terminal domain of p53. Tumor-derived transactivation-deficient BRCA1 mutants are defective in co-activation of p53-dependent transcription and a truncation mutant of BRCA1 that retains the p53-interacting region acts as a dominant inhibitor of p53-dependent transcription. BRCA1 and p53 cooperatively induce apoptosis of cancer cells. The results indicate that BRCA1 and p53 may coordinately regulate gene expression in their role as tumor suppressors.

A MESSAGE FROM ASCO’S PRESIDENT I am pleased to present Clinical Cancer Advances 2017, which highlights the most promising advances in patient-oriented cancer research over the past year. The report gives us an opportunity to reflect on what an exciting time it is for cancer research and how swiftly our understanding of cancer has improved. One year ago, the White House announced the national Cancer Moonshot program to accelerate progress against cancer. This shared vision of progress has reinvigorated the research community, identified new areas of scientific collaboration, and raised our ambitions regarding what may be possible beyond the progress we have already made. When I entered the field 35 years ago, I could not have imagined where we would be today. We can now detect cancer earlier, target treatments more effectively, and manage adverse effects more effectively to enable patients to live better, more fulfilling lives. Today, two of three people with cancer live at least 5 years after diagnosis, up from roughly one of two in the 1970s. This progress has resulted from decades of incremental advances that have collectively expanded our understanding of the molecular underpinnings of cancer. There is no better current example of this than ASCO’s 2017 Advance of the Year: Immunotherapy 2.0. Over the last year, there has been a wave of new successes with immunotherapy. Research has proven this approach can be effective against a wide range of hard-to-treat advanced cancers previously considered intractable. Researchers are now working to identify biologic markers that can help increase the effectiveness of treatment and determine who is most likely to benefit from immunotherapy. This knowledge will enable oncologists to make evidence-based decisions so as many patients as possible might benefit from this new type of treatment. Each successive advance builds on the previous hard work of generations of basic, translational, and clinical cancer researchers. Importantly, the advances described in this report would not have been possible without the individuals who volunteered to participate in clinical trials as part of their treatment. To turn the promising vision of a cancer moonshot into meaningful advances, we need sustained, robust federal funding for continued research and innovation. Approximately 30% of the research highlighted in this report was funded, at least in part, through federal dollars appropriated to the National Institutes of Health or the National Cancer Institute. Without this federal investment—unique internationally in scale, duration, and impact for decades—I fear we may lose the forward momentum needed to further the progress we see highlighted in this report. Federal lawmakers can further fuel progress by advancing initiatives that facilitate the use of big data to achieve the common good of high-quality care for all patients. Such programs, like ASCO’s CancerLinQ, will rapidly increase the pace of progress and dramatically expand the reach of treatment advances to the millions of patients who are living with cancer today or who will do so in the future. This investment will yield medical, scientific, economic, and societal benefits for years to come. Much work still lies ahead. Many questions remain about how cancer develops and spreads and how best to treat it. As you read through Clinical Cancer Advances 2017, I hope you are as inspired as I am by the gains the clinical cancer research community has made over the past year and by the promise of a new era of advances just over the horizon. Daniel F Hayes, MD, FASCO, FACP ASCO President, 2016 to 2017

Although several biochemical features of p53 have been described, their relationship to tumor suppression remains uncertain. We have compared the ability of p53-derived proteins to act as sequence-specific transcriptional (SST) activators with their ability to suppress tumor cell growth, using an improved growth-suppression assay. Both naturally occurring and in vitro derived mutations that abrogated the SST activity of p53 lost the ability to suppress tumor cell growth. Additionally, the N- and C-terminal ends of p53 were shown to be functionally replaceable with foreign transactivation and dimerization domains, respectively, with concordant preservation of both SST and tumor-suppressive properties. Only the central region of p53, conferring specific DNA binding, was required to suppress growth by such hybrid proteins. The SST activity of p53 thus appeared to be essential for the protein to function as a tumor suppressor.

A newly cloned gene named wild-type p53-activated fragment 1 (WAF1; also known as p21, Pic-1, Cip-1, or SDI1) is directly regulated by p53 and can itself suppress tumor cell growth in culture. Induction of expression of WAF1 may be an important means by which cells with DNA injury arrest their growth to repair DNA or undergo apoptosis. Based on the hypothesis that mutations of this gene may play a role in carcinogenesis, we have studied 351 DNAs from 14 kinds of malignancies, as well as 36 human transformed cell lines, for alterations of WAF1 gene by single-strand conformation polymorphism analysis of polymerase chain reaction amplification of the DNA coding region of the WAF1 gene. No abnormal band shifts of WAF1 were noted in any of the samples or cell lines, but three major variants in exons 2 and 3 of the gene were found that are consistent with the existence of two different DNA polymorphisms. Sequence analysis of the amplified products producing these three variants in each exon from normal DNAs confirmed the presence of the polymorphisms in the WAF1 gene. Of 290 selected tumor samples previously evaluated for p53 mutations by single-strand conformation polymorphism, 90% had no detectable p53 alterations. In summary, mutations within the coding portion of the WAF1 gene were undetectable in a large series of human tumors, many of which had a normal p53 gene. This suggests that WAF1 alterations are generally caused indirectly, through p53 mutations rather than through intragenic mutation of the WAF1 itself.

Myeloid-derived suppressor cells (MDSCs) dampen the immune response thorough inhibition of T cell activation and proliferation and often are expanded in pathological conditions.Here, we studied the fate of MDSCs in cancer.Unexpectedly, MDSCs had lower viability and a shorter half-life in tumor-bearing mice compared with neutrophils and monocytes.The reduction of MDSC viability was due to increased apoptosis, which was mediated by increased expression of TNF-related apoptosis-induced ligand receptors (TRAIL-Rs) in these cells.Targeting TRAIL-Rs in naive mice did not affect myeloid cell populations, but it dramatically reduced the presence of MDSCs and improved immune responses in tumor-bearing mice.Treatment of myeloid cells with proinflammatory cytokines did not affect TRAIL-R expression; however, induction of ER stress in myeloid cells recapitulated changes in TRAIL-R expression observed in tumor-bearing hosts.The ER stress response was detected in MDSCs isolated from cancer patients and tumor-bearing mice, but not in control neutrophils or monocytes, and blockade of ER stress abrogated tumor-associated changes in TRAIL-Rs.Together, these data indicate that MDSC pathophysiology is linked to ER stress, which shortens the lifespan of these cells in the periphery and promotes expansion in BM.Furthermore, TRAIL-Rs can be considered as potential targets for selectively inhibiting MDSCs.

This review provides an overview of the structure, regulation and physiological functions of p21, the product of the cyclin-dependent kinase inhibitor 1A (CDKN1A) gene, with a focus on its dual role in promoting and repressing biological processes that are hallmarks of tumourigenesis.Recent work has provided a better understanding of the molecular mechanisms of how oncogenic signalling pathways influence p21 expression. In response to cellular stimuli, p21 expression is tightly regulated at transcriptional and post-translational levels through mechanisms involving RNA stabilization, phosphorylation and ubiquitination. As a result, growing evidence reveals that several important tumour suppressor and oncogenic signalling pathways alter p21 expression to elicit their effects on cell cycle progression and survival. Thus, p21 expression can both promote and inhibit tumourigenic processes, depending on the cellular context.Since its discovery, it has become increasingly clear that p21 can function as both a classical tumour suppressor and an oncogene. In order to effectively utilize p21 as a therapeutic target, it will be necessary to design therapeutic strategies that preferentially block the ability of p21 to promote senescence, stem cell renewal and cyclin/CDK activation, while leaving its tumour suppressive functions intact.

Recombinant tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is an antitumor protein that is in clinical trials as a potential anticancer therapy but suffers from drug properties that may limit efficacy such as short serum half-life, stability, cost, and biodistribution, particularly with respect to the brain. To overcome such limitations, we identified TRAIL-inducing compound 10 (TIC10), a potent, orally active, and stable small molecule that transcriptionally induces TRAIL in a p53-independent manner and crosses the blood-brain barrier. TIC10 induces a sustained up-regulation of TRAIL in tumors and normal cells that may contribute to the demonstrable antitumor activity of TIC10. TIC10 inactivates kinases Akt and extracellular signal-regulated kinase (ERK), leading to the translocation of Foxo3a into the nucleus, where it binds to the TRAIL promoter to up-regulate gene transcription. TIC10 is an efficacious antitumor therapeutic agent that acts on tumor cells and their microenvironment to enhance the concentrations of the endogenous tumor suppressor TRAIL.

c-Myc stimulates angiogenesis in tumors through mechanisms that remain incompletely understood. Recent work indicates that c-Myc upregulates the miR-17∼92 microRNA cluster and downregulates the angiogenesis inhibitor thrombospondin-1, along with other members of the thrombospondin type 1 repeat superfamily. Here, we show that downregulation of the thrombospondin type 1 repeat protein clusterin in cells overexpressing c-Myc and miR-17∼92 promotes angiogenesis and tumor growth. However, clusterin downregulation by miR-17∼92 is indirect. It occurs as a result of reduced transforming growth factor-β (TGFβ) signaling caused by targeting of several regulatory components in this signaling pathway. Specifically, miR-17-5p and miR-20 reduce the expression of the type II TGFβ receptor and miR-18 limits the expression of Smad4. Supporting these results, in human cancer cell lines, levels of the miR-17∼92 primary transcript MIR17HG negatively correlate with those of many TGFβ-induced genes that are not direct targets of miR-17∼92 (e.g., clusterin and angiopoietin-like 4). Furthermore, enforced expression of miR-17∼92 in MIR17HG(low) cell lines (e.g., glioblastoma) results in impaired gene activation by TGFβ. Together, our results define a pathway in which c-Myc activation of miR-17∼92 attenuates the TGFβ signaling pathway to shut down clusterin expression, thereby stimulating angiogenesis and tumor cell growth.

TP53 is the most commonly mutated gene in human cancer with over 100,000 literature citations in PubMed. This is a heavily studied pathway in cancer biology and oncology with a history that dates back to 1979 when p53 was discovered. The p53 pathway is a complex cellular stress response network with multiple diverse inputs and downstream outputs relevant to its role as a tumor suppressor pathway. While inroads have been made in understanding the biology and signaling in the p53 pathway, the p53 family, transcriptional readouts, and effects of an array of mutants, the pathway remains challenging in the realm of clinical translation. While the role of mutant p53 as a prognostic factor is recognized, the therapeutic modulation of its wild-type or mutant activities remain a work-in-progress. This review covers current knowledge about the biology, signaling mechanisms in the p53 pathway and summarizes advances in therapeutic development.

p53 is one of the most important tumor suppressor genes that is frequently mutated in human cancers. Generally, p53 functions as a transcription factor that is stabilized and activated by various genotoxic and cellular stress signals, such as DNA damage, hypoxia, oncogene activation and nutrient deprivation, consequently leading to cell cycle arrest, apoptosis, senescence and metabolic adaptation. p53 not only becomes functionally deficient in most cancers, but not infrequently mutant p53 also acquires dominant negative activity and oncogenic properties. p53 has remained an attractive target for cancer therapy. Strategies targeting p53 have been developed including gene therapy to restore p53 function, inhibition of p53-MDM2 interaction, restoration of mutant p53 to wild-type p53, targeting p53 family proteins, eliminating mutant p53, as well as p53-based vaccines. Some of these p53-targeted therapies have entered clinical trials. We discuss the therapeutic potential of p53, with particular focus on the therapeutic strategies to rescue p53 inactivation in human cancers. In addition, we discuss the challenges of p53-targeted therapy and new opportunities for the future.

The world of molecular profiling has undergone revolutionary changes over the last few years as knowledge, technology, and even standard clinical practice have evolved. Broad molecular profiling is now nearly essential for all patients with metastatic solid tumors. New agents have been approved based on molecular testing instead of tumor site of origin. Molecular profiling methodologies have likewise changed such that tests that were performed on patients a few years ago are no longer complete and possibly inaccurate today. As with all rapid change, medical providers can quickly fall behind or struggle to find up-to-date sources to ensure he or she provides optimum care. In this review, the authors provide the current state of the art for molecular profiling/precision medicine, practice standards, and a view into the future ahead.

p21 (WAF1/CIP1; CDKN1a) is a universal cell-cycle inhibitor directly controlled by p53 and p53-independent pathways. Knowledge of the regulation and function of p21 in normal and cancer cells has opened up several areas of investigation and has led to novel therapeutic strategies. The discovery in 1993 and subsequent work on p21 has illuminated basic cellular growth control, stem cell phenotypes, the physiology of differentiation, as well as how cells respond to stress. There remain open questions in the signaling networks, the ultimate role of p21 in the p53-deficiency phenotype in the context of other p53 target defects, and therapeutic strategies continue to be a work in progress. Cancer Res; 76(18); 5189-91. ©2016 AACRSee related article by El-Deiry et al., Cancer Res 1994;54:1169-74Visit the Cancer Research 75(th) Anniversary timeline.

ONC201 (also called TIC10) is a small molecule that inactivates the cell proliferation- and cell survival-promoting kinases Akt and ERK and induces cell death through the proapoptotic protein TRAIL. ONC201 is currently in early-phase clinical testing for various malignancies. We found through gene expression and protein analyses that ONC201 triggered an increase in TRAIL abundance and cell death through an integrated stress response (ISR) involving the transcription factor ATF4, the transactivator CHOP, and the TRAIL receptor DR5. ATF4 was not activated in ONC201-resistant cancer cells, and in ONC201-sensitive cells, knockdown of ATF4 or CHOP partially abrogated ONC201-induced cytotoxicity and diminished the ONC201-stimulated increase in DR5 abundance. The activation of ATF4 in response to ONC201 required the kinases HRI and PKR, which phosphorylate and activate the translation initiation factor eIF2α. ONC201 rapidly triggered cell cycle arrest, which was associated with decreased abundance of cyclin D1, decreased activity of the kinase complex mTORC1, and dephosphorylation of the retinoblastoma (Rb) protein. The abundance of X-linked inhibitor of apoptosis protein (XIAP) negatively correlated with the extent of apoptosis in response to ONC201. These effects of ONC201 were independent of whether cancer cells had normal or mutant p53. Thus, ONC201 induces cell death through the coordinated induction of TRAIL by an ISR pathway.

Tumor sidedness has emerged as an important prognostic and predictive factor in the treatment of colorectal cancer. Recent studies demonstrate that patients with advanced right-sided colon cancers have a worse prognosis than those with left-sided colon or rectal cancers, and these patient subgroups respond differently to biological therapies. Historically, management of patients with metastatic colon and rectal cancers has been similar, and colon and rectal cancer patients have been grouped together in large clinical trials. Clearly, the differences in molecular biology among right-sided colon, left-sided colon, and rectal cancers should be further studied in order to account for disparities in clinical outcomes. We profiled 10,570 colorectal tumors (of which 2,413 were identified as arising from the left colon, right colon, or rectum) using next-generation sequencing, immunohistochemistry, chromogenic in-situ hybridization, and fragment analysis (Caris Life Sciences, Phoenix, AZ). Right-sided colon cancers had higher rates of microsatellite instability, more frequent aberrant activation of the EGFR pathway including higher BRAF and PIK3CA mutation rates, and increased mutational burden compared to left-sided colon and rectal cancers. Rectal cancers had higher rates of TOPO1 expression and Her2/neu amplification compared to both left- and right-sided colon cancers. Molecular variations among right-sided colon, left-sided colon, and rectal tumors may contribute to differences in clinical behavior. The site of tumor origin (left colon, right colon, or rectum) should certainly be considered when selecting treatment regimens and stratifying patients for future clinical trials.

Abstract Pancreatic ductal adenocarcinoma (PDAC) has a poor 5-year survival rate and lacks effective therapeutics. Therefore, it is of paramount importance to identify new targets. Using multiplex data from patient tissue, three-dimensional coculturing in vitro assays, and orthotopic murine models, we identified Netrin G1 (NetG1) as a promoter of PDAC tumorigenesis. We found that NetG1+ cancer-associated fibroblasts (CAF) support PDAC survival, through a NetG1-mediated effect on glutamate/glutamine metabolism. Also, NetG1+ CAFs are intrinsically immunosuppressive and inhibit natural killer cell–mediated killing of tumor cells. These protumor functions are controlled by a signaling circuit downstream of NetG1, which is comprised of AKT/4E-BP1, p38/FRA1, vesicular glutamate transporter 1, and glutamine synthetase. Finally, blocking NetG1 with a neutralizing antibody stunts in vivo tumorigenesis, suggesting NetG1 as potential target in PDAC. Significance: This study demonstrates the feasibility of targeting a fibroblastic protein, NetG1, which can limit PDAC tumorigenesis in vivo by reverting the protumorigenic properties of CAFs. Moreover, inhibition of metabolic proteins in CAFs altered their immunosuppressive capacity, linking metabolism with immunomodulatory function. See related commentary by Sherman, p. 230. This article is highlighted in the In This Issue feature, p. 211

The integrated stress response (ISR) is an evolutionarily conserved intra-cellular signaling network which is activated in response to intrinsic and extrinsic stresses. Various stresses are sensed by four specialized kinases, PKR-like ER kinase (PERK), general control non-derepressible 2 (GCN2), double-stranded RNA-dependent protein kinase (PKR) and heme-regulated eIF2α kinase (HRI) that converge on phosphorylation of serine 51 of eIF2α. eIF2α phosphorylation causes a global reduction of protein synthesis and triggers the translation of specific mRNAs, including activating transcription factor 4 (ATF4). Although the ISR promotes cell survival and homeostasis, when stress is severe or prolonged the ISR signaling will shift to regulate cellular apoptosis. We review the ISR signaling pathway, regulation and importance in cancer therapy.

Apoptosis is a form of regulated cell death (RCD) that involves proteases of the caspase family. Pharmacological and genetic strategies that experimentally inhibit or delay apoptosis in mammalian systems have elucidated the key contribution of this process not only to (post-)embryonic development and adult tissue homeostasis, but also to the etiology of multiple human disorders. Consistent with this notion, while defects in the molecular machinery for apoptotic cell death impair organismal development and promote oncogenesis, the unwarranted activation of apoptosis promotes cell loss and tissue damage in the context of various neurological, cardiovascular, renal, hepatic, infectious, neoplastic and inflammatory conditions. Here, the Nomenclature Committee on Cell Death (NCCD) gathered to critically summarize an abundant pre-clinical literature mechanistically linking the core apoptotic apparatus to organismal homeostasis in the context of disease.

Abstract Purpose: KRAS mutation (MT) is a major oncogenic driver in pancreatic ductal adenocarcinoma (PDAC). A small subset of PDACs harbor KRAS wild-type (WT). We aim to characterize the molecular profiles of KRAS WT PDAC to uncover new pathogenic drivers and offer targeted treatments. Experimental Design: Tumor tissue obtained from surgical or biopsy material was subjected to next-generation DNA/RNA sequencing, microsatellite instability (MSI) and mismatch repair status determination. Results: Of the 2,483 patients (male 53.7%, median age 66 years) studied, 266 tumors (10.7%) were KRAS WT. The most frequently mutated gene in KRAS WT PDAC was TP53 (44.5%), followed by BRAF (13.0%). Multiple mutations within the DNA-damage repair (BRCA2, ATM, BAP1, RAD50, FANCE, PALB2), chromatin remodeling (ARID1A, PBRM1, ARID2, KMT2D, KMT2C, SMARCA4, SETD2), and cell-cycle control pathways (CDKN2A, CCND1, CCNE1) were detected frequently. There was no statistically significant difference in PD-L1 expression between KRAS WT (15.8%) and MT (17%) tumors. However, KRAS WT PDAC were more likely to be MSI-high (4.7% vs. 0.7%; P < 0.05), tumor mutational burden–high (4.5% vs. 1%; P < 0.05), and exhibit increased infiltration of CD8+ T cells, natural killer cells, and myeloid dendritic cells. KRAS WT PDACs exhibited gene fusions of BRAF (6.6%), FGFR2 (5.2%), ALK (2.6%), RET (1.3%), and NRG1 (1.3%), as well as amplification of FGF3 (3%), ERBB2 (2.2%), FGFR3 (1.8%), NTRK (1.8%), and MET (1.3%). Real-world evidence reveals a survival advantage of KRAS WT patients in overall cohorts as well as in patients treated with gemcitabine/nab-paclitaxel or 5-FU/oxaliplatin. Conclusions: KRAS WT PDAC represents 10.7% of PDAC and is enriched with targetable alterations, including immuno-oncologic markers. Identification of KRAS WT patients in clinical practice may expand therapeutic options in a clinically meaningful manner.

The p53-regulated gene product p21WAF1/CIP1 is the prototype of a family of small proteins that negatively regulate the cell cycle. To learn more about p21WAF1/CIP1 regulation in vivo, monoclonal antibodies were developed for immunohistochemistry. These revealed that p21WAF1/CIP1 expression followed radiation-induced DNA damage in human skin in a pattern consistent with its regulation by p53. A detailed comparison of the human, rat, and mouse p21WAF1/CIP1 promoter sequences revealed that this induction was probably mediated by conserved p53-binding sites upstream of the transcription start site. In unirradiated tissues, p21WAF1/CIP1 expression was apparently independent of p53 and was observed in a variety of cell types. Moreover, there was a striking compartmentalization of p21WAF1/CIP1 expression throughout the gastrointestinal tract that correlated with proliferation rather than differentiation. As epithelial cells migrated up the crypts, the Ki67-expressing proliferating compartment near the crypt base ended abruptly, with the coincident appearance of a nonproliferating compartment expressing p21WAF1/CIP1. In colonic neoplasms, this distinct compartmentalization was largely abrogated. Cell cycle inhibitors are thus subject to precise topological control, and escape from this regulation may be a critical feature of neoplastic transformation.

DNA methylation abnormalities occur consistently in human neoplasia including widespread hypomethylation and more recently recognized local increases in DNA methylation that hold potential for gene inactivation events. To study this imbalance further, we have cloned and localized to chromosome 19 a portion of the human DNA methyltransferase gene that codes for the enzyme catalyzing DNA methylation. Expression of this gene is low in normal human cells, significantly increased (30- to 50-fold by PCR analysis) in virally transformed cells, and strikingly elevated in human cancer cells (several hundredfold). In comparison to colon mucosa from patients without neoplasia, median levels of DNA methyltransferase transcripts are 15-fold increased in histologically normal mucosa from patients with cancers or the benign polyps that can precede cancers, 60-fold increased in the premalignant polyps, and greater than 200-fold increased in the cancers. Thus, increases in DNA methyltransferase gene expression precede development of colonic neoplasia and continue during progression of colonic neoplasms. These increases may play a role in the genetic instability of cancer and mark early events in cell transformation.

The extrinsic cell death pathway is initiated upon ligand-receptor interactions at the cell surface including FAS ligand-FAS/APO1, TNF-TNF receptors, and TRAIL-TRAIL receptors. Abnormalities of various components of these pathways have been identified in human cancer including loss of FAS expression, deletion or loss of TRAIL receptor DR4, mutation of TRAIL receptor DR5, overexpression of TRAIL decoy TRID or overexpression of Fas decoy, as well as overexpression of the caspase activation inhibitor, FLIP. Death ligands have been explored as potential therapeutics in cancer therapy with some limitations in the case of FAS and TNF due to toxicities. TRAIL remains promising as a therapeutic and has potential for combination with chemo- or radio-therapy. The death receptor signaling pathways include cross-talk with the mitochondrial pathway and can in some cases be influenced by mitochondrial membrane potential changes or NF-kappaB. FLIP and BCL-XL expression may reduce sensitivity of cancer cells to combination therapies.

The present study assessed the role of the p53 tumor suppressor gene in cell cycle arrest and apoptosis following treatment of Burkitt's lymphoma and lymphoblastoid cell lines with gamma-rays, etoposide, nitrogen mustard, and cisplatin. Cell cycle arrest was measured by flow cytometry; p53 and p21Waf1/Cip1 protein levels were measured by Western blotting; cell survival was measured in 72-96-h growth inhibition assays and by trypan blue staining, and apoptotic DNA fragmentation was assessed by either agarose gel electrophoresis or a modified filter elution method. We found that gamma-rays and etoposide induced a strong G1 arrest in the wild-type p53 lines while nitrogen mustard and cisplatin induced relatively little G1 arrest. All agents failed to induce G1 arrest in cells containing mutant p53 genes. The degree of G1 arrest observed with these agents correlated with the rate of p53 and p21Waf1/Cip1 protein accumulation: gamma-rays and etoposide induced rapid accumulation of both p53 and p21Waf1/Cip1; nitrogen mustard and cisplatin induced slow accumulation of p53 and no major accumulation of the p21Waf1/Cip1 protein. Despite differences in G1 arrest and kinetics of p53 or p21Waf1/Cip1 protein accumulation, all agents tended to decrease survival to a greater extent in the wild-type p53 lines compared to the mutant p53 lines. Cell death in the wild-type p53 lines was associated with intracellular DNA degradation into oligonucleosomal sized DNA fragments, indicative of apoptosis. We also observed an inverse sensitivity relationship between nitrogen mustard/cisplatin and etoposide in the mutant p53 lines and this was found to correlate with topoisomerase II mRNA levels in the cells. Our results suggest that p53 gene status is an important determinant of both radio- and chemosensitivity in lymphoid cell lines and that p53 mutations are often associated with decreased sensitivity to DNA damaging agents.

Cells expressing oncogenic c-Myc are sensitized to TNF superfamily proteins. c-Myc also is an important factor in determining whether a cell is sensitive to TRAIL-induced apoptosis, and it is well established that the mitochondrial pathway is essential for apoptosis induced by c-Myc. We investigated whether c-Myc action on the mitochondria is required for TRAIL sensitivity and found that Myc sensitized cells with defective intrinsic signaling to TRAIL. TRAIL induced expression of antiapoptotic Mcl-1 and cIAP2 through activation of NF-kappaB. Both Myc and the multikinase inhibitor sorafenib block NF-kappaB. Combining sorafenib with TRAIL in vivo showed dramatic efficacy in TRAIL-resistant tumor xenografts. We propose the combination of TRAIL with sorafenib holds promise for further development.

Tumor necrosis factor alpha (TNF-alpha)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF-alpha family of death receptor ligands and holds great therapeutic potential as a tumor cell-specific cytotoxic agent. Using a panel of established tumor cell lines and normal cells, we found a significant difference between the number of TRAIL-sensitive cells expressing high levels of c-myc and TRAIL-resistant cells expressing low levels of c-myc (P < 0.05, n = 19). We also found a direct linear correlation between c-myc levels and TRAIL sensitivity in TRAIL-sensitive cell lines (r = 0.94, n = 6). Overexpression of c-myc or activation of a myc-estrogen receptor (ER) fusion sensitized TRAIL-resistant cells to TRAIL. Conversely, small interfering RNA (siRNA)-mediated knockdown of c-myc significantly reduced both c-myc expression and TRAIL-induced apoptosis. The gene encoding the inhibitor of caspase activation, FLICE inhibitory protein (FLIP), appears to be a direct target of c-myc-mediated transcriptional repression. Overexpression of c-myc or activation of myc-estrogen receptor (ER) decreased FLIP levels both in cell culture and in mouse models of c-myc-induced tumorigenesis, while knocking down c-myc using siRNA increased FLIP expression. Chromatin immunoprecipitation and luciferase reporter analyses showed that c-myc binds and represses the human FLIP promoter. c-myc expression enhanced TRAIL-induced caspase 8 cleavage and FLIP cleavage at the death-inducing signaling complex. Combined siRNA-mediated knockdown of FLIP and c-myc resensitized cells to TRAIL. Therefore, c-myc down-regulation of FLIP expression provides a universal mechanism to explain the ability of c-myc to sensitize cells to death receptor stimuli. In addition, identification of c-myc as a major determinant of TRAIL sensitivity provides a potentially important screening tool for identification of TRAIL-sensitive tumors.

We have cloned a series of overlapping cDNA clones encoding a 5194 bp transcript for human DNA methyltransferase (DNA MTase). This sequence potentially codes for a protein of 1495 amino acids with a predicted molecular weight of 169 kDa. The human DNA MTase cDNA has eighty percent homology at the nucleotide level, and the predicted protein has seventy-four percent identity at the amino acid level, to the DNA MTase cDNA cloned from mouse cells. Like the murine DNA MTase, the amino terminal two-thirds of the human protein contains a cysteine-rich region suggestive of a metal-binding domain. The carboxy terminal one-third of the protein shows considerable similarity to prokaryotic (cytosine-5)-methyltransferases. The arrangement of multiple motifs conserved in the prokaryotic genes is preserved in the human DNA MTase, including the relative position of a proline-cysteine dipeptide thought to be an essential catalytic site in all (cytosine-5)-methyltransferases. A single 5.2 kb transcript was detected in all human tissues tested, with the highest levels of expression observed in RNA from placenta, brain, heart and lung. DNA MTase cDNA clones were used to screen a chromosome 19 genomic cosmid library. The DNA MTase-positive cosmids which are estimated to span a genomic distance of 93 kb have been localized to 19p13.2-p13.3 by fluorescence in situ hybridization. Isolation of the cDNA for human DNA MTase will allow further study of the regulation of DNA MTase expression, and of the role of this enzyme in establishing DNA methylation patterns in both normal and neoplastic cells.

Many tumor cell types are sensitive to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. Incubation of TRAIL-sensitive cells with TRAIL invariably leads to resistant survivors even when high doses of TRAIL are used. Because the emergence of resistance to apoptosis is a major concern in successful treatment of cancer, and TRAIL survivors may contribute to therapeutic failure, we investigated potential resistance mechanisms. We selected TRAIL-resistant SW480 human colon adenocarcinoma cells by repeatedly treating them with high and/or low doses of TRAIL. The resulting TRAIL-resistant clones were not cross-resistant to Fas or paclitaxel. Expression of modulators of apoptosis was not changed in the resistant cells, including TRAIL receptors, cFLIP, Bax, Bid, or IAP proteins. Surprisingly, we found that DISC formation was deficient in multiple selected TRAIL-resistant clones. DR4 was not recruited to the DISC upon TRAIL treatment, and caspase-8 was not activated at the DISC. Although total cellular DR4 mRNA and protein were virtually identical in TRAIL-sensitive parental and TRAIL-resistant clones, DR4 protein expression on the cell surface was essentially undetectable in the TRAIL-resistant clones. Moreover, exogenous DR4 and KILLER/DR5 were not properly transported to the cell surface in the TRAIL-resistant cells. Interestingly, TRAIL-resistant cells were resensitized to TRAIL by tunicamycin pretreatment, which increased cell surface expression of DR4 and KILLER/DR5. Our data suggest that tumor cells may become resistant to TRAIL through regulation of the death receptor cell surface transport and that resistance to TRAIL may be overcome by the glycosylation inhibitor/endoplasmic reticulum stress-inducing agent tunicamycin. Many tumor cell types are sensitive to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. Incubation of TRAIL-sensitive cells with TRAIL invariably leads to resistant survivors even when high doses of TRAIL are used. Because the emergence of resistance to apoptosis is a major concern in successful treatment of cancer, and TRAIL survivors may contribute to therapeutic failure, we investigated potential resistance mechanisms. We selected TRAIL-resistant SW480 human colon adenocarcinoma cells by repeatedly treating them with high and/or low doses of TRAIL. The resulting TRAIL-resistant clones were not cross-resistant to Fas or paclitaxel. Expression of modulators of apoptosis was not changed in the resistant cells, including TRAIL receptors, cFLIP, Bax, Bid, or IAP proteins. Surprisingly, we found that DISC formation was deficient in multiple selected TRAIL-resistant clones. DR4 was not recruited to the DISC upon TRAIL treatment, and caspase-8 was not activated at the DISC. Although total cellular DR4 mRNA and protein were virtually identical in TRAIL-sensitive parental and TRAIL-resistant clones, DR4 protein expression on the cell surface was essentially undetectable in the TRAIL-resistant clones. Moreover, exogenous DR4 and KILLER/DR5 were not properly transported to the cell surface in the TRAIL-resistant cells. Interestingly, TRAIL-resistant cells were resensitized to TRAIL by tunicamycin pretreatment, which increased cell surface expression of DR4 and KILLER/DR5. Our data suggest that tumor cells may become resistant to TRAIL through regulation of the death receptor cell surface transport and that resistance to TRAIL may be overcome by the glycosylation inhibitor/endoplasmic reticulum stress-inducing agent tunicamycin. Tumor necrosis factor (TNF) 1The abbreviations used are: TNF, tumor necrosis factor; TRAIL, TNF-related apoptosis-inducing ligand; FBS, fetal bovine serum; PI, propidium iodide; PBS, phosphate-buffered saline; FITC, fluorescein isothiocyanate; RT, reverse transcriptase; IP, immunoprecipitation; GFP, green fluorescent protein.1The abbreviations used are: TNF, tumor necrosis factor; TRAIL, TNF-related apoptosis-inducing ligand; FBS, fetal bovine serum; PI, propidium iodide; PBS, phosphate-buffered saline; FITC, fluorescein isothiocyanate; RT, reverse transcriptase; IP, immunoprecipitation; GFP, green fluorescent protein.-related apoptosis-inducing ligand (TRAIL), a member of the TNF cytokine family and a type II transmembrane protein, is highly homologous to other TNF-related proteins, such as Fas ligand (1Wiley S.R. Schooley K. Smolak P.J. Din W.S. Huang C.P. Nicholl J.K. Sutherland G.R. Smith T.D. Rauch C. Smith C.A. Goodwin R.G. Immunity. 1995; 3: 673-682Abstract Full Text PDF PubMed Scopus (2638) Google Scholar). TRAIL, unlike Fas ligand, appears to preferentially induce apoptosis in tumor cells over normal cells (2Pitti R.M. Marsters S.A. Ruppert S. Donahue C.J. Moore A. Ashkenazi A. J. Biol. Chem. 1996; 271: 12687-12690Abstract Full Text Full Text PDF PubMed Scopus (1633) Google Scholar). TRAIL binds to five distinct TRAIL receptors including death receptor 4 (TRAIL-R1) (3Pan G. O'Rourke K. Chinnaiyan A.M. Gentz R. Ebner R. Ni J. Dixit V.M. Science. 1997; 276: 111-113Crossref PubMed Scopus (1546) Google Scholar), KILLER/DR5 (TRAIL-R2, TRICK2) (4Pan G. Ni J. Wei Y.F. Yu G. Gentz R. Dixit V.M. Science. 1997; 277: 815-818Crossref PubMed Scopus (1373) Google Scholar, 5Wu G.S. Burns T.F. McDonald III, E.R. Jiang W. Meng R. Krantz I.D. Kao G. Gan D.D. Zhou J.Y. Muschel R. Hamilton S.R. Spinner N.B. Markowitz S. Wu G. El-Deiry W.S. Nat. Genet. 1997; 17: 141-143Crossref PubMed Scopus (937) Google Scholar, 6Walczak H. Degli-Esposti M.A. Johnson R.S. Smolak P.J. Waugh J.Y. Boiani N. Timour M.S. Gerhart M.J. Schooley K.A. Smith C.A. Goodwin R.G. Rauch C.T. EMBO J. 1997; 16: 5386-5397Crossref PubMed Scopus (1013) Google Scholar), DcR1 (TRID, TRAIL-R3) (7Sheridan J.P. Marsters S.A. Pitti R.M. Gurney A. Skubatch M. Baldwin D. Ramakrishnan L. Gray C.L. Baker K. Wood W.I. Goddard A.D. Godowski P. Ashkenazi A. Science. 1997; 277: 818-821Crossref PubMed Scopus (1520) Google Scholar), DcR2 (TRUNDD OR TRAIL-R4) (8Pan G. Ni J. Yu G. Wei Y.F. Dixit V.M. FEBS Lett. 1998; 424: 41-45Crossref PubMed Scopus (279) Google Scholar), and osteoprotegerin (9Emery J.G. McDonnell P. Burke M.B. Deen K.C. Lyn S. Silverman C. Dul E. Appelbaum E.R. Eichman C. DiPrinzio R. Dodds R.A. James I.E. Rosenberg M. Lee J.C. Young P.R. J. Biol. Chem. 1998; 273: 14363-14367Abstract Full Text Full Text PDF PubMed Scopus (1049) Google Scholar). These receptors have been classified into two groups, death-inducing receptors (TRAIL-R1 and -R2) and death-inhibitory receptors (TRAIL-R3, TRAIL-R4, and osteoprotegerin). Both TRAIL-R1 and TRAIL-R2 contain a C-terminal death domain that signals downstream caspase activation to mediate TRAIL-induced apoptotic cell death in a variety of tumor cell types. In contrast to these death-inducing receptors, TRAIL-R3, which shares homology with DR4/KILLER/DR5, is devoid of a cytoplasmic domain and exists as a glycophospholipid-anchored protein on the cell surface. TRAIL-R4 has a cytoplasmic domain containing a truncated death domain that cannot transmit a death signal but can weakly activate NF-κB, which may protect cells from TRAIL-mediated apoptosis (10Degli-Esposti M.A. Dougall W.C. Smolak P.J. Waugh J.Y. Smith C.A. Goodwin R.G. Immunity. 1997; 7: 813-820Abstract Full Text Full Text PDF PubMed Scopus (742) Google Scholar). It has been suggested that these two decoy receptors can protect cells from TRAIL-induced apoptosis by competing with the death-inducing TRAIL-Rs for TRAIL binding. Since TRAIL-R3 mRNA was preferentially found in normal cells but not in transformed cells, it is thought that TRAIL-R3 might be responsible for the cellular resistance of some normal cells to TRAIL-mediated cytotoxicity (11MacFarlane M. Ahmad M. Srinivasula S.M. Fernandes-Alnemri T. Cohen G.M. Alnemri E.S. J. Biol. Chem. 1997; 272: 25417-25420Abstract Full Text Full Text PDF PubMed Scopus (497) Google Scholar, 12Degli-Esposti M.A. Smolak P.J. Walczak H. Waugh J. Huang C.P. DuBose R.F. Goodwin R.G. Smith C.A. J. Exp. Med. 1997; 186: 1165-1170Crossref PubMed Scopus (556) Google Scholar, 13Schneider P. Bodmer J.L. Thome M. Hofmann K. Holler N. Tschopp J. FEBS Lett. 1997; 416: 329-334Crossref PubMed Scopus (247) Google Scholar). These findings suggest a complex regulation of cellular susceptibility to TRAIL-mediated apoptosis at the level of receptor expression.Apoptosis can be initiated by two distinct pathways: one is the “intrinsic pathway” mediated by the mitochondria, and the other is the “extrinsic pathway” mediated by death receptors. Activation of those two pathways ultimately results in cleavage of caspase-3 and induction of apoptosis. Cross-talk between the extrinsic and intrinsic pathways has been observed mainly as an amplification loop at the level of execution of each cascade. Depending on the relative contribution of mitochondria in death receptor-mediated apoptosis, tumor cells can be classified as type I or type II cells (14Igney F.H. Krammer P.H. Nat. Rev. Cancer. 2002; 2: 277-288Crossref PubMed Scopus (1615) Google Scholar). Apoptosis in type I cells can be induced by TRAIL or Fas without early involvement of the mitochondria pathway, and this death is not blocked by Bcl-2, Bcl-XL, or caspase-9 inhibitors (15Ozoren N. El-Deiry W.S. Neoplasia. 2002; 4: 551-557Crossref PubMed Scopus (179) Google Scholar). In type II cells, the amount of DISC-activated caspase-8 is not sufficient, and the mitochondria act as “amplifiers” of the apoptotic signal. Type II cell death is sensitive to inhibition by Bcl-2, Bcl-XL, or caspase-9 inhibitors.TRAIL is a promising anti-cancer agent because it can preferentially kill tumor cells. Sensitivity to TRAIL-induced apoptosis is a key factor influencing the efficacy of TRAIL treatment. However, the basis for the sensitivity and resistance of cells to TRAIL-induced apoptosis is not fully understood. In addition, TRAIL contributes to immune surveillance against tumor metastasis, which implies that tumors may gain more metastatic potential after acquiring resistance to TRAIL-induced apoptosis (16Cretney E. Takeda K. Yagita H. Glaccum M. Peschon J.J. Smyth M.J. J. Immunol. 2002; 168: 1356-1361Crossref PubMed Scopus (519) Google Scholar, 17Takeda K. Hayakawa Y. Smyth M.J. Kayagaki N. Yamaguchi N. Kakuta S. Iwakura Y. Yagita H. Okumura K. Nat. Med. 2001; 7: 94-100Crossref PubMed Scopus (588) Google Scholar).Thus far, most studies of TRAIL resistance have focused on natural resistance, such as resistance of normal cells to TRAIL-induced apoptosis (18Zhang X.D. Nguyen T. Thomas W.D. Sanders J.E. Hersey P. FEBS Lett. 2000; 482: 193-199Crossref PubMed Scopus (199) Google Scholar). Mechanisms of natural or intrinsic resistance may be different from those of acquired or adapted resistance. In the latter, tumor cells may survive after pro-longed treatment with TRAIL in cancer therapy or escape from TRAIL-mediated immune surveillance in vivo. Tumor cells can acquire resistance to apoptosis through interference with either intrinsic or extrinsic apoptotic signaling pathways. Mutation of the Bcl-2 family member Bax can confer resistance to TRAIL induced apoptosis in HCT116 cells (19LeBlanc H. Lawrence D. Varfolomeev E. Totpal K. Morlan J. Schow P. Fong S. Schwall R. Sinicropi D. Ashkenazi A. Nat. Med. 2002; 8: 274-281Crossref PubMed Scopus (482) Google Scholar). It also has been reported that Jurkat cells acquire resistance to Fas-mediated apoptosis because their mitochondria lose the ability to release intermembrane proteins in response to Bid or Bax (20Wang G.Q. Gastman B.R. Wieckowski E. Goldstein L.A. Rabinovitz A. Yin X.M. Rabinowich H. J. Biol. Chem. 2001; 276: 3610-3619Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Both HCT116 and Jurkat cells behave as Type II cells in which the mitochondrial pathway appears to play a major role in death receptor-mediated apoptosis. Type II cells may develop resistance by modulation of the intrinsic pathway instead of the extrinsic pathway. Resistance in these cells may be overcome in some cases by combined application of chemotherapeutic drugs (19LeBlanc H. Lawrence D. Varfolomeev E. Totpal K. Morlan J. Schow P. Fong S. Schwall R. Sinicropi D. Ashkenazi A. Nat. Med. 2002; 8: 274-281Crossref PubMed Scopus (482) Google Scholar), which might bypass the mitochondrial pathway (21Wang S. El-Deiry W.S. Proc. Natl. Acad. Sci. U. S. A. 2004; 100: 15095-15100Crossref Scopus (130) Google Scholar). Elucidation of acquired TRAIL resistance may lead to new therapeutic approaches to overcome resistance in cancer therapy with TRAIL. In addition, it may improve our understanding of mechanisms underlying how tumor cells escape from surveillance by the immune system and thereby provide novel strategies to prevent tumor development.In the present study, we investigated how tumor cells acquire resistance to TRAIL-induced apoptosis. TRAIL-resistant tumor cells were selected by eliminating apoptotic cells resulting from treatment with TRAIL. TRAIL-resistant clones were found not to be cross-resistant to Fas or paclitaxel-induced apoptosis. DISC formation was abrogated in TRAIL-resistant clones, apparently due to a defect in localization of DR4 in the plasma membrane. Strikingly, we found that pretreatment with the glycosylation inhibitor tunicamycin specifically and efficiently restored the susceptibility of resistant clones to TRAIL-induced apoptosis. Our studies provide a new mechanism through which tumor cells may acquire resistance to TRAIL-induced apoptosis. This mechanism suggests that pharmacological manipulation of cell surface expression of death receptors may be crucial to overcome resistance to TRAIL-induced apoptosis or maintain tumor cell sensitivity in vivo.MATERIALS AND METHODSCell Culture and Reagents—The human colon cancer cell line SW480 was obtained from the American Type Culture Collection (Manassas, VA). SW480 cells were cultured in Dulbecco's modified Eagle's medium. Cell culture media were supplemented with 10% fetal bovine serum (FBS), 2 mm l-glutamine, penicillin (100 units/ml) and streptomycin (100 units/ml). Human recombinant human TRAIL ligand, His6 antibody, and anti-DR4 for immunofluorescence staining were obtained from R&D Systems (Minneapolis, MN). Anti-human poly(ADP-ribose) polymerase antibody was obtained from Roche Applied Science. Anti-DR4, anti-KILLER/DR5, anti-DcR1, and anti-DcR2 antibody used for flow cytometric detection and functional application (block TRAIL receptors-mediated killing) were obtained from Alexis Corp. (Lausen, Switzerland). Anti-Bax and anti-caspase-3 antibodies were obtained from BD Biosciences. Anti-caspase-8 and anti-FLIP antibodies were obtained from Cell Signaling Technology (San Diego, CA). Anti-KILLER/DR5, anti-Bcl-2, anti-Bcl-XL and anti-caspase-9 antibodies were purchased from IMGENEX (San Diego, CA). The mitochondria-selective carbocyanine dye 3,3′-dihexyloxacarbocyanine iodide was purchased from Molecular Probes, Inc. (Eugene, OR). Cycloheximide and tunicamycin were obtained from Calbiochem, The mitogen-activated protein kinase inhibitor U0126 was obtained from Cell Signaling Technology (San Diego, CA).Generation of TRAIL-resistant SW480 Cells—Selection of TRAIL-resistant cells was performed as described previously (22Landowski T.H. Shain K.H. Oshiro M.M. Buyuksal I. Painter J.S. Dalton W.S. Blood. 1999; 94: 265-274Crossref PubMed Google Scholar). In brief, 1 × 106 SW480 cells seeded in 100-mm dishes were treated with 200 ng/ml TRAIL for 24 h, resulting in ∼99% cell death. The apoptotic cells (cells floating in the medium) were then removed. Surviving cells growing in medium containing 10 ng/ml TRAIL up to 60–70% confluence were then treated with 200 ng/ml TRAIL for 24 h and allowed to regrow to 60–70% confluence again in 10 ng/ml TRAIL-containing medium. After five cycles, SW480 pooled clones resistant to TRAIL were obtained. Individual resistant clones were isolated using sterile cloning disks (Scienceware). The sensitivity of individual clones to TRAIL-induced apoptosis was subsequently examined by PI staining and flow cytometry.Colony Formation Assays—SW480 cells and TRAIL-resistant cells (5000/well) were plated in 12-well dishes and treated with TRAIL, Fas, or paclitaxel for 3 days. The cell culture medium was subsequently changed every 3 days, and after 2 weeks the resulting colonies were stained with Coomassie Blue (23Burns T.F. El-Deiry W.S. J. Biol. Chem. 2001; 276: 37879-37886Abstract Full Text Full Text PDF PubMed Google Scholar).PI Staining and Active Caspase-3 Assay—Cells were harvested at the indicated time periods (see figure legends) and prepared for detection of active caspase-3 by flow cytometry or stained with propidium iodide and analyzed by flow cytometry for sub-G1 content as described previously (30Gaeta M.L. Johnson D.R. Kluger M.S. Pober J.S. Lab. Invest. 2000; 80: 1185-1194Crossref PubMed Scopus (32) Google Scholar). The fluorescence was measured using an Epics Elite flow cytometer (Beckman Coulter).Transfections—Transfections were carried out as described previously (24McDonald 3rd, E.R. Chui P.C. Martelli P.F. Dicker D.T. El-Deiry W.S. J. Biol. Chem. 2001; 276: 14939-14945Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). In brief, before transfection, 5 × 105 cells were seeded per well in 6-well plates. SW480 cells were transfected with 2 μg of DNA with a 1:10 ratio of EGFP-spectrin co-expression vector using the Lipofectin transfection reagent (Qiagen) according to the manufacturer's specifications, and transfection efficiency was about 15–20%. Twenty-four hours after transfection, cells were collected and analyzed as indicated in the figure legends.Flow Cytometric Analysis of Mitochondrial Transmembrane Potential Change during Cell Death—Cells were washed with 2 ml of PBS, rinsed with PBS, and suspended following treatment with 0.5 ml of trypsin (trypsin-EDTA; 0.05% trypsin, 0.53 mm EDTA). Cells were transferred to 15-ml conical tubes and centrifuged at 1100 rpm for 5 min. Cells were resuspended in 300 μl of media with 100 nm 3,3′-dihexyloxacarbocyanine iodide and incubated for 30 min at 37 °C. The fluorescence was measured using an Epics Elite flow cytometer (Beckman Coulter).Flow Cytometric Detection of Death Receptor Surface Expression— The experiments were performed as described previously (25Zhang X.D. Franco A.V. Nguyen T. Gray C.P. Hersey P. J. Immunol. 2000; 164: 3961-3970Crossref PubMed Scopus (185) Google Scholar). In brief, 1 × 106 cells were rinsed and detached following the addition of 0.5 ml of trypsin (trypsin-EDTA; 0.05% trypsin, 0.53 mm EDTA·4Na). Cells were transferred to 15-ml conical tubes and centrifuged at 1100 rpm for 5 min. Cells were washed with 1 ml of PBS containing 1% FBS and then resuspended in 50 μl of fluorescence-activated cell sorting buffer (PBS with 1% FBS) containing the primary antibody (10 μg/ml) against a death receptor or a nonspecific mouse IgG1 antibody as a control. After staining on ice for 60 min, cells were washed once with PBS containing 1% FBS and incubated with FITC- or phycoerythrin-labeled goat anti-mouse secondary antibody at 4 °C for 45 min in the dark. Cells were analyzed on an Epics Elite flow cytometer (Beckman Coulter).Northern Blot Analysis—RNA was extracted from parental and TRAIL-resistant SW480 cells and then electrophoresed on a 1.5% agarose gel and transferred to a nitrocellulose membrane. The mRNA expression level of human TRAIL death receptor DR4 and KILLER/DR5 was determined through hybridization to the membrane using full-length cDNAs of the death receptors as probes. Each probe was labeled by random priming as described previously (5Wu G.S. Burns T.F. McDonald III, E.R. Jiang W. Meng R. Krantz I.D. Kao G. Gan D.D. Zhou J.Y. Muschel R. Hamilton S.R. Spinner N.B. Markowitz S. Wu G. El-Deiry W.S. Nat. Genet. 1997; 17: 141-143Crossref PubMed Scopus (937) Google Scholar). Ethidium bromide staining of 28 and 18 S RNA was performed to evaluate the integrity of the RNA and to demonstrate equal RNA loading.Reverse Transcriptase (RT)-PCR—Expression of DR4 and KILLER/DR5 RNA was also evaluated using RT-PCR. Total RNA was extracted from the parental and TRAIL-resistant SW480 cells. cDNA was prepared using an oligo(dT) primer and Superscriptase II (Invitrogen) as previously described (26Kim K. Fisher M.J. Xu S.Q. El-Deiry W.S. Clin. Cancer Res. 2000; 6: 335-346PubMed Google Scholar). Primers used in these experiments were obtained from R&D Systems.Western Blotting—Western blot analyses were performed as described previously (5Wu G.S. Burns T.F. McDonald III, E.R. Jiang W. Meng R. Krantz I.D. Kao G. Gan D.D. Zhou J.Y. Muschel R. Hamilton S.R. Spinner N.B. Markowitz S. Wu G. El-Deiry W.S. Nat. Genet. 1997; 17: 141-143Crossref PubMed Scopus (937) Google Scholar). In brief, a total of 20 μg of protein was separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore Corp., Bedford, MA). Membranes were blocked with 10% Blotto (Carnation, Los Angeles, CA), incubated with different primary antibodies, and incubated with horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG antibody used at a 1:5000 dilution. Antibody-antigen complexes were detected by the Enhanced Chemiluminescence system (Amersham Biosciences).Alanine-scanning Mutagenesis of DR4 —The DR4 cDNA was cloned in frame into the pcDNA3.1-Myc-HisA vector (Invitrogen) as an EcoRI/HindIII fragment. This C-terminally tagged Myc-His plasmid was subsequently mutagenized using the QuikChange™ site-directed mutagenesis kit following the instructions of the manufacturer (Stratagene). Mutations were verified by sequencing, and in each case the entire cDNA was checked and verified for the absence of second site mutations.TRAIL DISC Immunoprecipitation—DISC IP was performed as described previously (24McDonald 3rd, E.R. Chui P.C. Martelli P.F. Dicker D.T. El-Deiry W.S. J. Biol. Chem. 2001; 276: 14939-14945Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). In brief, 1 × 107 SW480 cells were plated to achieve 80% confluence in a T75 flask. Cells were then suspended following trypsin treatment, spun down, and resuspended in 2 ml of complete medium supplemented with 100 ng/ml His tagged-TRAIL and 1 μg/ml anti-His6 antibody (R&D Systems) for 15 min at 37 °C. For untreated control samples, the TRAIL was excluded. Cells were washed twice with ice-cold phosphate-buffered saline and lysed for 30 min on ice in TRAIL DISC IP lysis buffer (30 mm Tris, pH 7.5, 150 mm NaCl, 10% glycerol, 1% Triton X-100). The lysates were cleared twice by centrifugation at 4 °C. The supernatants were immunoprecipitated overnight with 30 μl of Protein A/G Plus-agarose (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) at 4 °C to isolate the TRAIL DISC. The complexes were subsequently washed four times with TRAIL DISC IP lysis buffer and eluted with Immunopure Gentle Ag/Ab elution buffer (Pierce) with 0.1 m dithiothreitol at room temperature for 2 h. The protein complexes were methanol/chloroform (4:1)-precipitated and resolved on 15% SDS-polyacrylamide gels. Western blots were performed to measure recruitment of specific endogenous proteins to the TRAIL DISC.Immunofluorescence Staining—This procedure was described previously (25Zhang X.D. Franco A.V. Nguyen T. Gray C.P. Hersey P. J. Immunol. 2000; 164: 3961-3970Crossref PubMed Scopus (185) Google Scholar). Briefly, SW480 or TRAIL-resistant cells were plated on coverslips. Coverslips were washed two times with PBS containing 1% FBS. To detect the intracellular level of death receptors, some samples were permeabilized with 0.25% saponin and fixed with 2% formaldehyde, and other samples were stained without permeabilization and fixation. Samples were incubated at 4 °C with anti-DR4 or KILLER/DR5 antibody overnight at 4 °C in Dulbecco's modified Eagle's medium (Invitrogen). Cells were incubated with secondary mouse anti-goat antibody conjugated to FITC (1:400; Caltag) at 4 °C for 1 h in Dulbecco's modified Eagle's medium. After three 10-min washes with PBS with 1% FBS, cells were mounted onto slides with Pro-Long (Molecular Probes) mounting medium. Cells were visualized with a ×20 objective lens using an Olympus BX60 epifluorescent microscope, and images were recorded with an Optronics CCD-camera (DEI-750).RESULTSTRAIL-resistant SW480 Colon Cancer Cells Selected after Prolonged Treatment with TRAIL Were Not Cross-resistant to Fas- or Paclitaxel-induced Apoptosis—Although TRAIL has cytotoxic effects against most tumor cells, a fraction of a given sensitive tumor cell population cannot be killed even at high doses of TRAIL. To explore how these resistant survivors escape from TRAIL-induced death, we obtained TRAIL-resistant cell lines by subjecting SW480 human colon cancer cells to repeated exposure to different doses of recombinant TRAIL (see “Materials and Methods”). After five consecutive cycles of selection, we isolated 12 individual clones from a TRAIL-resistant pool of clones. Two of them remained as sensitive as the parental SW480 cells to TRAIL, and the other 10 clones showed resistance to TRAIL-induced apoptosis. We randomly chose clones R4, R6, and R10 for further experiments. As shown in Fig. 1, less than 20% apoptosis could be detected after exposure to 100 ng/ml TRAIL for 24 h in the three resistant clones, in contrast to more than 70% cell death observed in parental SW480 cells (p < 0.01). Extended exposure at higher doses or longer times of TRAIL treatment did not increase cell death, and there remained a high percentage of surviving cells. To ensure that resistance to TRAIL-induced apoptosis was not simply due to clonal variation, we further examined six subclones of the TRAIL-resistant SW480 clone R4 derived by limiting dilution. All of them showed similar resistance to TRAIL as their parental clone R4 (data not shown). It has been reported that in vitro selection for resistance to some apoptosis-inducing stimuli may result in cross-resistance to other cytotoxic agents (20Wang G.Q. Gastman B.R. Wieckowski E. Goldstein L.A. Rabinovitz A. Yin X.M. Rabinowich H. J. Biol. Chem. 2001; 276: 3610-3619Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 22Landowski T.H. Shain K.H. Oshiro M.M. Buyuksal I. Painter J.S. Dalton W.S. Blood. 1999; 94: 265-274Crossref PubMed Google Scholar). To determine whether our resistant clones also co-selected for resistance to Fas and paclitaxel, the parental SW480 cells and the resistant clones R4, R6, and R10 were treated with TRAIL, Fas, or paclitaxel. We found differences between parental and resistant cell clones only after TRAIL treatment but not following exposure to Fas or the chemotherapeutic drug paclitaxel (Fig. 1, A and B). We found a significant change in mitochondrial membrane potential in TRAIL-sensitive SW480 cells after TRAIL exposure (p < 0.01) but not in TRAIL-resistant cells, whereas the change was similar in response following either Fas or paclitaxel when TRAIL-sensitive and TRAIL-resistant cells were compared. (Fig. 1C). To confirm that R4, R6, and R10 became stably resistant to TRAIL-induced apoptosis, we also performed long term colony formation assays. We found that TRAIL-resistant clones formed colonies in medium containing 20 ng/ml TRAIL, whereas parental SW480 cells formed very few colonies. A low number of colonies was observed following exposure of either parental or TRAIL-resistant cells to either Fas or paclitaxel (Fig. 1D). Thus, TRAIL-resistant clones selected from prolonged incubation with TRAIL did not display cross-resistance to either Fas- or paclitaxel-mediated apoptotic signals.Expression of Major Apoptosis Mediators Does Not Change in TRAIL-resistant Clones Isolated following Prolonged TRAIL Exposure—Previous studies suggested that altered expression of proapoptotic or antiapoptotic genes in tumors often results in the avoidance of cell death induced by chemotherapeutic agents or TRAIL (14Igney F.H. Krammer P.H. Nat. Rev. Cancer. 2002; 2: 277-288Crossref PubMed Scopus (1615) Google Scholar, 19LeBlanc H. Lawrence D. Varfolomeev E. Totpal K. Morlan J. Schow P. Fong S. Schwall R. Sinicropi D. Ashkenazi A. Nat. Med. 2002; 8: 274-281Crossref PubMed Scopus (482) Google Scholar). To investigate the mechanism of TRAIL resistance in the selected clones, we first examined the protein levels of molecules that are involved in the death receptor signaling pathway. We tested the expression of death receptors, including DR4, KILLER/DR5, DcR1, and DcR2, and found that protein levels were similar between TRAIL-sensitive and TRAIL-resistant cell lines. DcR1 and DcR2 were expressed in TRAIL-sensitive SW480 cells, indicating that the relative expression of decoy receptors may not efficiently block apoptosis induced by TRAIL in some tumor cell lines (Fig. 2A). IAP family members are known negative regulators of apoptosis (27Uren A.G. Pakusch M. Hawkins C.J. Puls K.L. Vaux D.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4974-4978Crossref PubMed Scopus (445) Google Scholar). Thus, we compared the expression level of IAP1 and IAP2 in the parental SW480 cells and in resistant clones and found no difference (data not shown). We also examined the mRNA levels of the TRAIL death receptors by RT-PCR and Northern blotting, and no apparent changes were observed in the resistant clones (Fig. 2C). The antiapoptotic molecules Bcl-2, Bcl-XL, and FLIP can protect some tumor cell lines from TRAIL-induced apoptosis (23Burns T.F. El-Deiry W.S. J. Biol. Chem. 2001; 276: 37879-37886Abstract Full Text Full Text PDF PubMed Google Scholar, 28Lamothe B. Aggarwal B.B. J. Interferon Cytokine Res. 2002; 22: 269-279Crossref PubMed Scopus (47) Google Scholar), and BAX is required for HCT116 cells to undergo apoptosis after treatment with TRAIL (19LeBlanc H. Lawrence D. Varfolomeev E. Totpal K. Morlan J. Schow P. Fong S. Schwall R. Sinicropi D. Ashkenazi A. Nat. Med

Proteasome-dependent degradation of regulatory proteins is a known mechanism of cell cycle control. We found that the proteasome-specific inhibitor lactacystin (LC) induced expression of the cell cycle inhibitor p21WAF1/CIP1 in human cancer cells regardless of their p53 status. Both wild-type (wt) p53 and p21 protein levels increased by two hours in wt p53 containing cells, whereas mutant (mt) p53 levels decreased and the increase in p21 levels was delayed to 6 hr following inhibition of proteolysis by LC in mt p53 expressing cells. We found that wt but not mt p53 expressing cells increased p21 mRNA and p21-promoter reporter levels following LC exposure, suggesting transcriptional induction of p21. Inhibition of protein synthesis by cycloheximide demonstrated increased p21 protein half-life in the presence of LC in mutant p53 containing cells. p21 induction was correlated with the cytostatic effects of LC. The results suggest that p21 protein expression could be increased by transcriptional mechanisms as well as inhibition of proteolysis by LC.

Journal of Cellular PhysiologyVolume 181, Issue 2 p. 231-239 Review Article The p53 pathway and apoptosis Timothy F. Burns, Timothy F. Burns Laboratory of Molecular Oncology and Cell Cycle Regulation, Howard Hughes Medical Institute, Departments of Medicine and Genetics, University of Pennsylvania School of Medicine, Philadelphia, PennsylvaniaSearch for more papers by this authorWafik S. El-Deiry, Corresponding Author Wafik S. El-Deiry Laboratory of Molecular Oncology and Cell Cycle Regulation, Howard Hughes Medical Institute, Departments of Medicine and Genetics, University of Pennsylvania School of Medicine, Philadelphia, PennsylvaniaLaboratory of Molecular Oncology and Cell Cycle Regulation, Howard Hughes Medical Institute, Departments of Medicine and Genetics, University of Pennsylvania School of Medicine, Philadelphia, PA 19104Search for more papers by this author Timothy F. Burns, Timothy F. Burns Laboratory of Molecular Oncology and Cell Cycle Regulation, Howard Hughes Medical Institute, Departments of Medicine and Genetics, University of Pennsylvania School of Medicine, Philadelphia, PennsylvaniaSearch for more papers by this authorWafik S. El-Deiry, Corresponding Author Wafik S. El-Deiry Laboratory of Molecular Oncology and Cell Cycle Regulation, Howard Hughes Medical Institute, Departments of Medicine and Genetics, University of Pennsylvania School of Medicine, Philadelphia, PennsylvaniaLaboratory of Molecular Oncology and Cell Cycle Regulation, Howard Hughes Medical Institute, Departments of Medicine and Genetics, University of Pennsylvania School of Medicine, Philadelphia, PA 19104Search for more papers by this author First published: 23 September 1999 https://doi.org/10.1002/(SICI)1097-4652(199911)181:2<231::AID-JCP5>3.0.CO;2-LCitations: 166AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume181, Issue2November 1999Pages 231-239 RelatedInformation

Type I cells have been defined to be independent of mitochondria for the induction of Fas death receptor-mediated apoptosis, whereas Type II cells are mitochondria-dependent. Knock-out studies in mice show that thymocytes are Type I and liver cells are Type II. We have previously shown that primary human hepatocytes and HCT116 human colon carcinoma cells behave like Type II cells because TRAIL-induced apoptosis can be blocked by the caspase 9 inhibitor, Z-LEHD-FMK. On the other hand, caspase 9 inhibition does not allow survival of TRAIL-treated SW480 colon cancer cells, which is predicted for Type I cells. Investigating the differences in TRAIL-induced apoptotic pathways in HCT116 and SW480 cells revealed that although FADD, BID, and procaspase 3 protein levels are higher in SW480 cells, and although procaspase 8 and FLIP processing is more efficient at the TRAIL-DISC of SW480 cells, BID, procaspase 3, XIAP, and PARP cleavages occur more rapidly in HCT116, despite the higher levels of BCL-2 and HSP70. Cytochrome c release from the mitochondria to the cytoplasm is more efficient in HCT116 cells. These results suggest BID cleavage as a possible limiting factor in the involvement of mitochondria in TRAIL-induced cell death. Thus, regulation of BID cleavage may define if a cell is mitochondria-dependent or -independent in response to TRAIL death receptor-induced apoptosis.

Loss of p53 sensitizes to antimicrotubule agents in human tumor cells, but little is known about its role during mitosis. We have identified the Polo-like kinase family member serum inducible kinase (Snk/Plk2) as a novel p53 target gene. Snk/Plk2 mutagenesis demonstrated that its kinase activity is negatively regulated by its C terminus. Small interfering RNA (siRNA)-mediated Snk/Plk2 silencing in the presence of the mitotic poisons paclitaxel (Taxol) or nocodazole significantly increased apoptosis, similar to p53 mutations, which confer paclitaxel sensitivity. Furthermore, we have demonstrated that the apoptosis due to silencing of Snk/Plk2 in the face of spindle damage occurs in mitotic cells and not in cells that have progressed to a G1-like state without dividing. Since siRNA directed against Snk/Plk2 promoted death of paclitaxel-treated cells in mitosis, we envision a mitotic checkpoint wherein p53-dependent activation of Snk/Plk2 prevents mitotic catastrophe following spindle damage. Finally, these studies suggest that disruption of Snk/Plk2 may be of therapeutic value in sensitizing paclitaxel-resistant tumors.

p53-dependent apoptosis is a major determinant of its tumor suppressor activity and can be triggered by hypoxia. No p53 target is known to be induced by p53 or to mediate p53-dependent apoptosis during hypoxia. We report that p53 can directly upregulate expression of Bnip3L, a cell death inducer. During hypoxia, Bnip3L is highly induced in wild-type p53-expressing cells, in part due to increased recruitment of p53 and CBP to Bnip3L. Apoptosis is reduced in hypoxia-exposed cells with functional p53 following Bnip3L knockdown. In vivo, Bnip3L knockdown promotes tumorigenicity of wild-type versus mutant p53-expressing tumors. Thus, Bnip3L, capable of attenuating tumorigenicity, mediates p53-dependent apoptosis under hypoxia, which provides a novel understanding of p53 in tumor suppression.

The breast and ovarian cancer susceptibility gene product BRCA1 has been reported to be expressed in a cell cycle-dependent manner; possess transcriptional activity; associate with several proteins, including the p53 tumor suppressor; and play an integral role in certain types of DNA repair. We show here that ectopic expression of BRCA1 using an adenovirus vector (Ad-BRCA1) leads to dephosphorylation of the retinoblastoma protein accompanied by a decrease in cyclin-dependent kinase activity. Flow cytometric analysis on Ad-BRCA1-infected cells revealed a G1 or G2 phase accumulation. High density cDNA array screening of colon, lung, and breast cancer cells identified several genes affected by BRCA1 expression in a p53-independent manner, including DNA damage response genes and genes involved in cell cycle control. Notable changes included induction of the GADD45 and GADD153 genes and a reduction in cyclin B1 expression. Therefore, BRCA1 has the potential to modulate the expression of genes and function of proteins involved in cell cycle control and DNA damage response pathways. The breast and ovarian cancer susceptibility gene product BRCA1 has been reported to be expressed in a cell cycle-dependent manner; possess transcriptional activity; associate with several proteins, including the p53 tumor suppressor; and play an integral role in certain types of DNA repair. We show here that ectopic expression of BRCA1 using an adenovirus vector (Ad-BRCA1) leads to dephosphorylation of the retinoblastoma protein accompanied by a decrease in cyclin-dependent kinase activity. Flow cytometric analysis on Ad-BRCA1-infected cells revealed a G1 or G2 phase accumulation. High density cDNA array screening of colon, lung, and breast cancer cells identified several genes affected by BRCA1 expression in a p53-independent manner, including DNA damage response genes and genes involved in cell cycle control. Notable changes included induction of the GADD45 and GADD153 genes and a reduction in cyclin B1 expression. Therefore, BRCA1 has the potential to modulate the expression of genes and function of proteins involved in cell cycle control and DNA damage response pathways. proliferating cell nuclear antigen multiplicity of infection fluorescence-activated cell sorting adenovirus retinoblastoma protein In inherited breast and ovarian cancer, the Brca1 tumor suppressor gene appears to be altered frequently (1.Miki Y. Swensen J. Shattuck-Eidens D. Futreal A.P. Harshman K. Tavtigian S. Liu Q. Cochran C. Bennett M.L. Ding W. Bell R. Rosenthal J. Hussey C. Tran T. McClure M. Frye C. Hattier T. Phelps R. Haugen-Strano A. Katcher H. Yakumo K. Gholami Z. Shaffer D. Stone S. Bayer S. Wray C. Bogden R. Dayananth P. Ward J. Tonin P. Narod S. Bristow P.K. Norris F.H. Helvering L. Morrison P. Rosteck P. Lai M. Barrett J.C. Lewis C. Neuhausen S. Cannon-Albright L. Goldgar D. Wiseman R. Kamb A. Skolnick M.H. Science. 1994; 266: 66-71Crossref PubMed Scopus (5304) Google Scholar). At least half of familial breast cancer cases can be linked to mutations within theBrca1 gene (2.Futreal P.A. Liu Q. Shattuck-Eidens D. Cochran C. Harshman K. Tavtigian S. Bennett L.M. Haugen-Strano A. Swensen J. Miki Y. Eddington K. McClure M. Frye C. Weaver-Feldhaus J. Ding W. Gholami Z. Soederkvist P. Terry L. Jhanwar S. Berchuck A. Iglehart J.D. Marks J. Ballinger D.G. Barrett J.C. Skolnick M.H. Kamb A. Wiseman R. Science. 1994; 266: 120-122Crossref PubMed Scopus (1139) Google Scholar). The Brca1 locus follows a classic loss-of-heterozygosity pattern in these breast cancers, with only the mutated Brca1 allele remaining (3.Smith S.A. Easton D.F. Evans D.G. Ponder B.A. Nat. Genet. 1994; 2: 128-131Crossref Scopus (391) Google Scholar, 4.Neuhausen S.L. Marshall C.J. Cancer Res. 1994; 54: 6069-6072PubMed Google Scholar). BRCA1 does not share any significant structural or sequence homology to any known proteins. There are, however, two notable domains that give clues to its overall function. A RING-finger motif is located at the extreme N-terminus, and a highly acidic domain is present for the last 100 amino acids of the protein (also known as the BRCT domain), suggesting a possible role in protein-protein interaction and transcriptional regulation, respectively (1.Miki Y. Swensen J. Shattuck-Eidens D. Futreal A.P. Harshman K. Tavtigian S. Liu Q. Cochran C. Bennett M.L. Ding W. Bell R. Rosenthal J. Hussey C. Tran T. McClure M. Frye C. Hattier T. Phelps R. Haugen-Strano A. Katcher H. Yakumo K. Gholami Z. Shaffer D. Stone S. Bayer S. Wray C. Bogden R. Dayananth P. Ward J. Tonin P. Narod S. Bristow P.K. Norris F.H. Helvering L. Morrison P. Rosteck P. Lai M. Barrett J.C. Lewis C. Neuhausen S. Cannon-Albright L. Goldgar D. Wiseman R. Kamb A. Skolnick M.H. Science. 1994; 266: 66-71Crossref PubMed Scopus (5304) Google Scholar). Indeed, several studies have recently supported a role for BRCA1 in gene transcription. First, fusion proteins consisting of BRCA1 linked to a GAL4-DNA binding domain are able to activate transcription of reporters containing GAL4 DNA-binding sites (5.Chapman M.S. Verma I.M. Nature. 1996; 382: 678-679Crossref PubMed Scopus (436) Google Scholar). Furthermore, tumor-derived mutations within the C terminus of BRCA1 are defective in the activation of reporters in the above described assay, raising the possibility that one function that is targeted in these tumors is the loss of BRCA1 transcriptional activity (6.Monteiro A.N. August A. Hanafusa H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13595-13599Crossref PubMed Scopus (428) Google Scholar). Second, BRCA1 is found as a component of the RNA polymerase holoenzyme, linked to the complex by RNA helicase A (7.Scully R. Anderson S.F. Chao D.M. Wei W. Ye L. Young R.A. Livingston D.M. Parvin J.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5605-5610Crossref PubMed Scopus (422) Google Scholar, 8.Anderson S.F. Schlegel B.P. Nakajima T. Wolpin E.S. Parvin J.D. Nat. Genet. 1998; 19: 254-256Crossref PubMed Scopus (341) Google Scholar). Third, BRCA1 is able to bind transcription factors and coactivate or corepress transcription, as is the case with p53 and c-Myc, respectively (9.Zhang H. Somasundaram K. Peng Y. Tian H. Zhang H. Bi D. Weber B.L. El-Deiry W.S. Oncogene. 1998; 16: 1713-1721Crossref PubMed Scopus (426) Google Scholar, 10.Ouchi T. Monteiro A.N. August A. Aaronson S.A. Hanafusa H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2302-2306Crossref PubMed Scopus (333) Google Scholar, 11.Wang Q. Zhang H. Kajino K. Greene M.I. Oncogene. 1998; 17: 1939-1948Crossref PubMed Scopus (193) Google Scholar). It is also able to activate the p21WAF1/Cip1 promoter both in cells that express wild-type and those that contain mutant p53 (12.Somasundaram K. Zhang H. Zeng Y.X. Houvras Y. Peng Y. Zhang H. Wu G.S. Licht J.D. Weber B.L. El-Deiry W.S. Nature. 1997; 389: 187-190Crossref PubMed Scopus (471) Google Scholar). Additionally, BRCA1 binds the CtIP transcriptional repressor that inhibits BRCA1-mediated activation of the p21WAF1/Cip1 promoter (13.Li S. Chen P.L. Subramanian T. Chinnadurai G. Tomlinson G. Osborne C.K. Sharp Z.D. Lee W.H. J. Biol. Chem. 1999; 274: 11334-11338Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). These data imply that BRCA1 is involved in transcriptional activation with a well characterized mammalian transcription factor, perhaps acting as a coactivator. Because it is also able to induce a known p53 transcriptional target in the absence of p53, the data also imply that BRCA1 can activate transcription with other yet to be identified factors. Cell cycle regulation of the BRCA1 protein has been suggested to possibly control the function of the protein in S phase (14.Scully R. Chen J. Ochs R.L. Keegan K. Hoekstra M. Feunteun J. Livingston D.M. Cell. 1997; 90: 425-435Abstract Full Text Full Text PDF PubMed Scopus (809) Google Scholar). During late G1 and early S phases, BRCA1 protein increases and becomes phosphorylated (14.Scully R. Chen J. Ochs R.L. Keegan K. Hoekstra M. Feunteun J. Livingston D.M. Cell. 1997; 90: 425-435Abstract Full Text Full Text PDF PubMed Scopus (809) Google Scholar, 15.Ruffner H. Verma I.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7138-7143Crossref PubMed Scopus (179) Google Scholar). Upon exit from mitosis, the protein becomes progressively dephosphorylated and decreases in expression. Expression of BRCA1 in SW480 and HCT116 cells results in a reduced ability to incorporate BrdU, signifying a role in S phase entry (12.Somasundaram K. Zhang H. Zeng Y.X. Houvras Y. Peng Y. Zhang H. Wu G.S. Licht J.D. Weber B.L. El-Deiry W.S. Nature. 1997; 389: 187-190Crossref PubMed Scopus (471) Google Scholar). BRCA1 has also been proposed to be tightly linked to the DNA damage response. Both BRCA1 and another gene product that segregates with breast cancer, BRCA2, are found in complexes with the human RAD51 protein (16.Scully R. Chen J. Plug A. Xiao Y. Weaver D. Feunteun J. Ashley T. Livingston D.M. Cell. 1997; 88: 265-275Abstract Full Text Full Text PDF PubMed Scopus (1326) Google Scholar, 17.Marmorstein L.Y. Ouchi T. Aaronson S.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13869-13874Crossref PubMed Scopus (239) Google Scholar). Saccharomyces cerevisiae rad51 mutants are susceptible to DNA-damaging agents. BRCA1 colocalizes with RAD51 and PCNA1 (16.Scully R. Chen J. Plug A. Xiao Y. Weaver D. Feunteun J. Ashley T. Livingston D.M. Cell. 1997; 88: 265-275Abstract Full Text Full Text PDF PubMed Scopus (1326) Google Scholar) to nuclear dot structures after the onset of DNA damage. During this process, immunoblotting has also detected a shift in the mobility of the BRCA1 protein, indicating hyperphosphorylation (14.Scully R. Chen J. Ochs R.L. Keegan K. Hoekstra M. Feunteun J. Livingston D.M. Cell. 1997; 90: 425-435Abstract Full Text Full Text PDF PubMed Scopus (809) Google Scholar). Recent evidence further implicating a role for BRCA1 in DNA repair processes has come from studies showing colocalization with MRE11/Rad50/p95 complexes (18.Zhong Q. Chen C.F. Li S. Chen Y. Wang C.C. Xiao J. Chen P.L. Sharp Z.D. Lee W.H. Science. 1999; 285: 747-750Crossref PubMed Scopus (525) Google Scholar). In addition, Brca1-null embryonic stem cells are defective in their ability to repair thymine glycols after H2O2 treatment (19.Gowen L.C. Avrutskaya A.V. Latour A.M. Koller B.H. Leadon S.A. Science. 1998; 281: 1009-1012Crossref PubMed Scopus (453) Google Scholar). This oxidative DNA damage is most quickly alleviated by transcription-coupled repair mechanisms that appear to involve BRCA1. Finally, a targeted deletion ofBrca1 exon 11 results in genetic instability, hypothesized to be due to unrepaired DNA damage (20.Xu X. Weaver Z. Linke S.P. Li C. Gotay J. Wang X.W. Harris C.C. Ried T. Deng C.-X. Mol. Cell. 1999; 3: 389-395Abstract Full Text Full Text PDF PubMed Scopus (702) Google Scholar). To examine the cellular responses to ectopic BRCA1 expression, we have used an adenovirus expressing the full-length BRCA1 transcript to deliver a high level of expression of the BRCA1 gene to >90% of cells examined. As BRCA1 protein levels have been shown to be increased at times during the cell cycle (5.Chapman M.S. Verma I.M. Nature. 1996; 382: 678-679Crossref PubMed Scopus (436) Google Scholar), we rationalized that overexpression of the protein may amplify and give insight to possible functions and effects. We report that the BRCA1 protein is capable of affecting the status, activity, and expression of cell cycle regulatory proteins and also affects the transcription of genes that are known to be modulated upon the onset of DNA damage. The culture conditions of SW480, HCT116, HCT116 p21−/−, H460, H460-Neo, H460-E6, MCF7, HeLa-GRE, HeLa-S21, and HCC1937 cells have been described previously (21.Wu G.S. El-Deiry W.S. Clin. Cancer Res. 1996; 2: 623-633PubMed Google Scholar, 22.Tomlinson G.E. Chen T.T. Stastny V.A. Virmani A.K. Spillman M.A. Tonk V. Blum J.L. Schneider N.R. Wistuba I.I. Shay J.W. Minna J.D. Gazdar A.F. Cancer Res. 1998; 58: 3237-3242PubMed Google Scholar, 23.Waldman T. Kinzler K.W. Vogelstein B. Cancer Res. 1995; 55: 5187-5190PubMed Google Scholar, 30.Kao G.D. McKenna W.G. Maity A. Blank K. Muschel R.J. Cancer Res. 1997; 57: 753-758PubMed Google Scholar). Ad-LacZ and Ad-p53 (24.El-Deiry W.S. Tokino T. Velculescu V.E. Levy D.B. Parsons R. Trent J.M. Lin D. Mercer W.E. Kinzler K.W. Vogelstein B. Cell. 1993; 75: 817-825Abstract Full Text PDF PubMed Scopus (7936) Google Scholar) were obtained from B. Vogelstein (Johns Hopkins University). Preparation of Ad-BRCA1 has been described. 2Somasundaram, K., MacLachlan, T. K., Sgagias, M., Weber, B., Cowan, K. H., and El-Deiry, W. S. (1999) Oncogene 18, 6605–6614. All viruses were propagated, titered, and amplified as described (24.El-Deiry W.S. Tokino T. Velculescu V.E. Levy D.B. Parsons R. Trent J.M. Lin D. Mercer W.E. Kinzler K.W. Vogelstein B. Cell. 1993; 75: 817-825Abstract Full Text PDF PubMed Scopus (7936) Google Scholar). The full-length sequence of the human BRCA1 cDNA (5593 bases) cloned in Ad-BRCA1 has been verified by Pfu PCR amplification in a total of 15 overlapping segments followed by direct sequencing. 3T. K. MacLachlan and W. S. El-Deiry, unpublished data. The sequences of the primers used for amplification and sequencing of the BRCA1 cDNA will be provided upon request. pCR3.1-BRCA1, CyBLuc (900 bases upstream of cyclin B1 transcriptional start site) and pCEP4-GADD45 has been previously described (12.Somasundaram K. Zhang H. Zeng Y.X. Houvras Y. Peng Y. Zhang H. Wu G.S. Licht J.D. Weber B.L. El-Deiry W.S. Nature. 1997; 389: 187-190Crossref PubMed Scopus (471) Google Scholar, 25.Hwang A. Maity A. McKenna W.G. Muschel R.J. J. Biol. Chem. 1995; 270: 28419-28424Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 26.Zhan Q.M. El-Deiry W.S. Bae I. Alamo I. Kastan M.B. Vogelstein B. Fornace Jr., A.J. Int. J. Oncol. 1995; 6: 937-946PubMed Google Scholar). pCEP4-BRCA1 was constructed by digestion of pCR3.1-BRCA1 with HindIII and XhoI endonucleases, purification of the full-length BRCA1 cDNA, and subcloning of the cDNA into HindIII/XhoI-digested pCEP4. Production of BRCA1 protein from pCEP4-BRCA1 was confirmed by transfection into SW480 cells and subsequent immunohistochemistry against BRCA1. pMV60-Bax was constructed first by amplification of Bax cDNA by PCR using the primers 5′-cgggaagatctccaccatggacgggtccggggagc-3′ and 5′-ccggaagatcttcagcccatcttcttccag-3′. The product was cloned into pMV10, subsequently released by HindIII digestion, and cloned into pMV60 as described (24.El-Deiry W.S. Tokino T. Velculescu V.E. Levy D.B. Parsons R. Trent J.M. Lin D. Mercer W.E. Kinzler K.W. Vogelstein B. Cell. 1993; 75: 817-825Abstract Full Text PDF PubMed Scopus (7936) Google Scholar). Western blotting was carried out essentially as described (12.Somasundaram K. Zhang H. Zeng Y.X. Houvras Y. Peng Y. Zhang H. Wu G.S. Licht J.D. Weber B.L. El-Deiry W.S. Nature. 1997; 389: 187-190Crossref PubMed Scopus (471) Google Scholar) using mouse anti-human p53 monoclonal (Ab-2; Oncogene Sciences), mouse anti-human BRCA1 monoclonal (Ab-1; Oncogene Sciences), mouse anti-human p21 monoclonal (Ab-1; Oncogene Sciences), and mouse anti-retinoblastoma monoclonal (Ab-5; Oncogene Sciences) antibodies and mouse anti-cyclin B1 monoclonal antibody (sc-245, Santa Cruz Biotechnology, Santa Cruz, CA). Total RNA isolation and Northern blotting were carried out as previously (24.El-Deiry W.S. Tokino T. Velculescu V.E. Levy D.B. Parsons R. Trent J.M. Lin D. Mercer W.E. Kinzler K.W. Vogelstein B. Cell. 1993; 75: 817-825Abstract Full Text PDF PubMed Scopus (7936) Google Scholar). A BamHI fragment of 1.2 kilobases in size from the plasmid pCEP4 carrying the GADD45 coding sequence was used as probe for gadd45 northerns (26.Zhan Q.M. El-Deiry W.S. Bae I. Alamo I. Kastan M.B. Vogelstein B. Fornace Jr., A.J. Int. J. Oncol. 1995; 6: 937-946PubMed Google Scholar). A 1.3-kilobaseHindIII fragment from the plasmid pMV60 carrying the Bax coding sequence was used as probe for Bax northerns. For the GADD153 Northern blotting, a PCR amplified fragment of 500 base pairs corresponding to the 3′ coding sequence of GADD153 was used. The primers used were 5′-ATGGCAGCTGAGTCATTG-3′ and 5′-CATGCTTGGTGCAGATTC-3′. The GADD153 PCR amplified fragment was sequenced to verify the insert. Cyclin B1 and Pin1 cDNAs were obtained as ESTs from Genome Systems (St. Louis, MO) (GenBankTM accession numbers AI142325 and AI138849, respectively) and cut with NotI and EcoRI to release the fragments. The CLONTECH(Mountainveiw, CA) Atlas human cancer array was hybridized with32P-labeled cDNA probes derived from total RNA from SW480, H460, HCT116, and HCC1937 cells infected with 50, 10, 20, and 20 MOI of Ad-LacZ or Ad-BRCA1, respectively, as per the manufacturer's instructions. Infection of these cell lines at the indicated MOIs has been justified by infection with Ad-LacZ at the same MOIs and finding >90% of cells staining for β-galactosidase activity. Quantitation of hybridization intensity of each dot doublet on the array was performed using Imagequant software (Molecular Dynamics). Adenovirus infections were performed at the appropriate MOIs (defined in text) in 1% fetal bovine serum/phosphate-buffered saline. Preparation of cells for fluorescence-activated cell sorting was performed essentially as described (27.van den Heuvel S. Harlow E. Science. 1993; 262: 2050-2054Crossref PubMed Scopus (976) Google Scholar). Cell sorting was performed on a Coulter Epics Elite counter. DNA content analysis was performed using MacCycle software (Phoenix Flow Systems, San Diego, CA) SW480 cells were transfected using Lipofectin with 0.5 μg of reporter, 2 μg of test plasmid, and 0.1 μg of pCMVβ (internal transfection efficiency control). Luciferase assays were performed as described (28.Zeng Y.X. Somasundaram K. El-Deiry W.S. Nat. Genet. 1997; 15: 78-82Crossref PubMed Scopus (260) Google Scholar). Assessment of transfection efficiency was determined by β-galactosidase activity. 20 μl of lysate was incubated in 353 μl of ONPG solution (45 μmβ-mercaptoethanol, 1 mm MgCl2, 100 mm sodium phosphate, 0.6 mg/ml o-nitrophenyl-β-d-galactopyranoside) at 37° C for 20 min. Reactions were stopped by addition of 500 ml of 1 M Na2CO3. Amount of activity was determined by measurement of absorbance at 420 nm. A total of 200 μg of H460 and MCF7 protein lysate was precleared with protein A-agarose beads (Santa Cruz Biotechnology) and subsequently incubated with cyclin B1 monoclonal antibody. Antibody was precipitated with 30 μl of protein A-agarose beads. Precipitated beads were washed in lysis buffer (50 mm Tris, 5 mm EDTA, 250 mm NaCl, 50 mm NaF, 0.1% Triton, 0.1 mmNa3VO4, 10 μg/ml each of pepstatin A, chymostatin, leupeptin and antipain protease inhibitors) and then in kinase buffer (20 mm HEPES, pH 7.4, 10 mmmagnesium acetate) and incubated with 20 μl of reaction buffer (50 mm HEPES, pH 7.4, 25 mm magnesium acetate, 50 mm ATP, 2.5 mm dithiothreitol, 5 mCi of [γ-32P]ATP, 1 μg of substrate (purified histone H1 protein (Roche Molecular Biochemicals)) at 30 °C for 20 min. 30 μl of 2× Laemmli sample buffer was added to stop the reaction, and 30 μl of the reaction was separated on a denaturing a 10% polyacrylamide gel. The H460 lung cancer cell line, MCF7 breast cancer cell line and SW480 colon cancer cell line, lines that express wild-type, wild-type, and mutant p53, respectively, were infected with an adenovirus expressing the full open reading frame of BRCA1 or the LacZ gene as control. The use of H460, MCF7, and SW480 cells infected at 10, 10, and 50 MOI, respectively, with an adenovirus expressing β-galactosidase (LacZ) results in >90% of cells staining blue (data not shown). Infection at these MOIs yielded full-length BRCA1 protein as detected by immunoblotting (Fig. 1). In the H460 cell line, this was also accompanied by an accumulation of p53 and p21WAF1/Cip1 protein as previously reported (9.Zhang H. Somasundaram K. Peng Y. Tian H. Zhang H. Bi D. Weber B.L. El-Deiry W.S. Oncogene. 1998; 16: 1713-1721Crossref PubMed Scopus (426) Google Scholar).2 Infection with Ad-BRCA1 also resulted in a hypophosphorylation of the retinoblastoma protein, regardless of the status of p53 protein in these cells. pRb is known to be phosphorylated by a number of kinases, one of which is cdk2 (29.Akiyama T. Ohuchi T. Sumida S. Matsumoto K. Toyoshima K. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7900-7904Crossref PubMed Scopus (198) Google Scholar). BRCA1 is capable of inducing the p21WAF1/Cip1protein, a potent inhibitor of cdk2 (12.Somasundaram K. Zhang H. Zeng Y.X. Houvras Y. Peng Y. Zhang H. Wu G.S. Licht J.D. Weber B.L. El-Deiry W.S. Nature. 1997; 389: 187-190Crossref PubMed Scopus (471) Google Scholar). We performed experiments to determine whether BRCA1 may be causing the dephosphorylation of pRb through inhibition of cdk2. Lysates from H460 and SW480 cells infected with Ad-LacZ, p53, or BRCA1 or mock-infected cells were immunoprecipitated using an anti-cdk2 antibody. A kinase assay using histone H1 as a substrate revealed a reduction of cdk2 activity in cells infected with Ad-p53 or Ad-BRCA1 (Fig.2). Cdk2 protein levels were similar in all cases (Fig. 2). Therefore, BRCA1 overexpression may, directly or indirectly, modify cell cycle proteins that control the G1to S phase transition. BRCA1 has been shown to be regulated in a cell cycle-dependent manner (14.Scully R. Chen J. Ochs R.L. Keegan K. Hoekstra M. Feunteun J. Livingston D.M. Cell. 1997; 90: 425-435Abstract Full Text Full Text PDF PubMed Scopus (809) Google Scholar, 15.Ruffner H. Verma I.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7138-7143Crossref PubMed Scopus (179) Google Scholar). Additionally, cell cycle phase changes are known to occur upon cellular stresses such as DNA damage, the response to which has been proposed to involve BRCA1 (14.Scully R. Chen J. Ochs R.L. Keegan K. Hoekstra M. Feunteun J. Livingston D.M. Cell. 1997; 90: 425-435Abstract Full Text Full Text PDF PubMed Scopus (809) Google Scholar, 19.Gowen L.C. Avrutskaya A.V. Latour A.M. Koller B.H. Leadon S.A. Science. 1998; 281: 1009-1012Crossref PubMed Scopus (453) Google Scholar). In order to determine whether ectopic expression of BRCA1 affects cell cycle control, we infected various cell lines with LacZ, p53, or BRCA1-expressing adenoviruses and subsequently investigated cellular DNA content by FACS analysis. H460 cells underwent a G1 and a minor G2 phase arrest when infected with Ad-p53 or Ad-BRCA1 (Fig.3 A). The wild-type p53-expressing cell line MCF7 also arrested in both G1 and G2 phases with a significant decrease in cells within S phase (Fig. 3 B). HCT116, a colon carcinoma cell line expressing wild-type p53 protein, arrested mainly in the G1phase when infected with Ad-p53. As BRCA1 has been shown to coactivate the endogenous wild-type p53 in these cells (9.Zhang H. Somasundaram K. Peng Y. Tian H. Zhang H. Bi D. Weber B.L. El-Deiry W.S. Oncogene. 1998; 16: 1713-1721Crossref PubMed Scopus (426) Google Scholar), Ad-BRCA1-infected cells were hypothesized to possess a cell cycle profile like that of Ad-p53-infected cells, which appears to be the case in H460 and MCF7 cells (Fig. 3, A and B). In contrast to other cell lines, the cell cycle profile of Ad-BRCA1-infected HCT116 cells displayed a large increase in G2 phase as compared with mock-infected cells, with a small fraction of cells blocked in G1 phase (Fig. 3 C). As p21WAF1/Cip1has been proposed to be essential for blockage of HCT116 cells into S phase by BRCA1 (12.Somasundaram K. Zhang H. Zeng Y.X. Houvras Y. Peng Y. Zhang H. Wu G.S. Licht J.D. Weber B.L. El-Deiry W.S. Nature. 1997; 389: 187-190Crossref PubMed Scopus (471) Google Scholar), we compared the cell cycle profiles of parental HCT116 and HCT116 p21−/− cells after Ad-BRCA1 infection. HCT116 p21−/− cells arrested upon infection with Ad-BRCA1 but appeared to accumulate entirely in G2, as opposed to a mixture of G1 and G2 in the parental HCT116 cells (Fig.3 D). This suggests a p21WAF1/Cip1 dependence on BRCA1-mediated responses, as the G1 population of HCT116 p21−/− cells is almost completely lost. In accordance with previous results (12.Somasundaram K. Zhang H. Zeng Y.X. Houvras Y. Peng Y. Zhang H. Wu G.S. Licht J.D. Weber B.L. El-Deiry W.S. Nature. 1997; 389: 187-190Crossref PubMed Scopus (471) Google Scholar), more cells are able to pass through S phase after BRCA1 overexpression in the absence of p21WAF1Cip1 (Fig.3 D). Whereas a decrease in the S phase population of cells was observed in wild-type p53-expressing cells, a different pattern was observed in cells not expressing wild-type p53. Mutant p53-expressing cells SW480, DLD1 and DLD1 p21−/− and the H460 cell line expressing human papilloma virus E6 protein, which degrades endogenous wild-type p53, did not appear to have a reduced S phase content upon Ad-BRCA1 infection (Fig. 3, E–H). SW480 cells also shifted to an increased number of cells in G2/M phase. Additionally, whereas p53 was able to cause a rapid apoptosis in lines depicted in Fig. 3, E–H, Ad-BRCA1 infection did not result in a significant apoptotic response within 24 h of infection. The effect of BRCA1 on cell cycle phase changes also was time-dependent (as an example, HCT116 cells are shown in Fig. 3 I). The appearance of G2 phase accumulation was coincident in HCT116 cells with the typical timing of exogenous gene expression. Infection with Ad-LacZ had no effect over 18 h on the cell cycle distribution of this cell type, indicating that the changes seen in cell cycle phase are likely due not to exposure to adenovirus but by the protein, BRCA1, being expressed. Previously published data suggest that BRCA1 may be involved in cellular gene transcription, which may in part contribute to the cellular changes described above (5.Chapman M.S. Verma I.M. Nature. 1996; 382: 678-679Crossref PubMed Scopus (436) Google Scholar, 6.Monteiro A.N. August A. Hanafusa H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13595-13599Crossref PubMed Scopus (428) Google Scholar, 8.Anderson S.F. Schlegel B.P. Nakajima T. Wolpin E.S. Parvin J.D. Nat. Genet. 1998; 19: 254-256Crossref PubMed Scopus (341) Google Scholar, 9.Zhang H. Somasundaram K. Peng Y. Tian H. Zhang H. Bi D. Weber B.L. El-Deiry W.S. Oncogene. 1998; 16: 1713-1721Crossref PubMed Scopus (426) Google Scholar, 10.Ouchi T. Monteiro A.N. August A. Aaronson S.A. Hanafusa H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2302-2306Crossref PubMed Scopus (333) Google Scholar, 11.Wang Q. Zhang H. Kajino K. Greene M.I. Oncogene. 1998; 17: 1939-1948Crossref PubMed Scopus (193) Google Scholar, 12.Somasundaram K. Zhang H. Zeng Y.X. Houvras Y. Peng Y. Zhang H. Wu G.S. Licht J.D. Weber B.L. El-Deiry W.S. Nature. 1997; 389: 187-190Crossref PubMed Scopus (471) Google Scholar). BRCA1 is known to coactivate the p53 transcription factor as well as activate transcription of the p21WAF1/Cip1 gene either in the presence or absence of functional p53 (9.Zhang H. Somasundaram K. Peng Y. Tian H. Zhang H. Bi D. Weber B.L. El-Deiry W.S. Oncogene. 1998; 16: 1713-1721Crossref PubMed Scopus (426) Google Scholar, 10.Ouchi T. Monteiro A.N. August A. Aaronson S.A. Hanafusa H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2302-2306Crossref PubMed Scopus (333) Google Scholar, 12.Somasundaram K. Zhang H. Zeng Y.X. Houvras Y. Peng Y. Zhang H. Wu G.S. Licht J.D. Weber B.L. El-Deiry W.S. Nature. 1997; 389: 187-190Crossref PubMed Scopus (471) Google Scholar, 13.Li S. Chen P.L. Subramanian T. Chinnadurai G. Tomlinson G. Osborne C.K. Sharp Z.D. Lee W.H. J. Biol. Chem. 1999; 274: 11334-11338Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar).2 In order to identify additional downstream targets of BRCA1 independent of wild-type p53 coactivation, we screened a high density cDNA array with labeled cDNA derived from SW480 cells infected with either Ad-LacZ or Ad-BRCA1 at 50 MOI. A list of 45 major targets out of a total of 588 genes represented on the array, the expression of which was either induced or repressed are described in TableI. Of note are effects on cell cycle controlling genes (p21WAF1/Cip1, cyclin B1, PIN1, PCNA, etc.), apoptosis regulators (BAX, BAK, etc.), and DNA damage response genes (GADD45, GADD153, Ku60, Ku70, etc.), whereas housekeeping genes, such as glyceraldehyde-3-phosphate dehydrogenase and β-actin, were minimally changed. Additional array screens were carried out utilizing Ad-LacZ and Ad-BRCA1 infection into the BRCA1-deficient and p53-mutant cell line, HCC1937 (22.Tomlinson G.E. Chen T.T. Stastny V.A. Virmani A.K. Spillman M.A. Tonk V. Blum J.L. Schneider N.R. Wistuba I.I. Shay J.W. Minna J.D. Gazdar A.F. Cancer Res. 1998; 58: 3237-3242PubMed Google Scholar) as well as the wild-type p53-expressing lines H460 and HCT116. Interestingly, several of the genes identified in the SW480 screen that were affected by BRCA1 ectopic expression were also found modulated in abundance in the other cell lines as well, including but not limited to the GADD genes, Ku genes, the human homolog of yeast rad6, PCNA, and cyclin B1 (indicated in Table I).Table IMajor target genesInduced genesRepressed genesGeneFold inductionGeneFold reductionCDC34aAlso found in other cell lines.2.14GRB2 adaptor2.05PCTAIRE1aAlso found in other cell lines.1.59grb105.50cdk42.18PIN1aAlso found in other cell lines.5.30p21WAF1/Cip1aAlso found in other cell lines.1.68Cyclin B1aAlso found in other cell lines.1.50PCNAaAlso found in other cell lines.3.40Cytokeratin 41.23Cytokeratin 2E1.23Cytokeratin 51.30RhoAaAlso found in other cell lines.3.42BAXaAlso found in other cell lines.3.80METaAlso found in other cell lines.2.70BAKaAlso found in other cell lines.3.70Superoxide dismutaseaAlso found in other cell lines.1.10Frizzled-relatedaAlso found in other cell lines.4.30IGFBP22.08Frizzled homolog8.30IGFBP43.30Dishevelled homolog2.00Ku70aAlso found in other c

Deficiency in p53-mediated cell death is common in human cancer, contributing to both tumorigenesis and chemoresistance. In an attempt to restore p53, we evaluated in vitro infectivity and cytotoxicity of a wild type (w.t.) p53-expressing adenovirus (Ad-p53) toward a panel of human cancer cell lines (n = 19). At a multiplicity of infection of 30, both Ad-p53 and adenovirus expressing β-galactosidase (Ad-LacZ) infected greater than 99% of cells derived from brain, lung, breast, ovarian, colon, and prostate cancer, but failed to infect leukemia or lymphoma cells. Ad-p53, but not Ad-LacZ, infection of cancer cells was followed by nuclear accumulation of the CDK inhibitor p21WAF1/CIP1, cell cycle arrest and loss of viability. Ad-p53 induced apoptotic death in cancer cells that express mutant p53, including multi-drug resistant cells, but fewer deaths were observed in some w.t. p53 expressing cells. Ad-p53-infected SKBr3 breast cancer cells were more sensitive to cytotoxicity of the DNA damaging drugs mitomycin C or Adriamycin, but not the M-phase specific drug vincristine. Our results suggest that Ad-p53 is capable of infecting and killing cancer cells of diverse tissue origins (including multi-drug resistant cancer cells), that p21WAF1/CIP1 may be a useful marker of p53 infectivity and that there may be synergy between Ad-p53 and either mitomycin C or Adriamycin induced cell death in tumors with p53 mutations. © 1996 Wiley-Liss, Inc.

Recent studies have shown that paclitaxel leads to activation of Raf-1 kinase and have suggested that this activation is essential for bcl-2 phosphorylation and apoptosis. In the present study, we demonstrate that, in addition to paclitaxel, other agents that interact with tubulin and microtubules also induce Raf-1/bcl-2 phosphorylation, whereas DNA-damaging drugs, antimetabolites, and alkylating agents do not. Activation of Raf-1 kinase by paclitaxel is linked to tubulin polymerization; the effect is blunted in paclitaxel-resistant cells, the tubulin of which does not polymerize following the addition of paclitaxel. In contrast, vincristine and vinblastine, drugs to which the paclitaxel-resistant cells retain sensitivity were able to bring about Raf-1 phosphorylation. The requirement for disruption of microtubules in this signaling cascade was strengthened further using paclitaxel analogues by demonstrating a correlation between tubulin polymerization, Raf-1/bcl-2 phosphorylation, and cytotoxicity. Inhibition of RNA or protein synthesis prevents Raf-1 activation and bcl-2 phosphorylation, suggesting that an intermediate protein(s) acts upstream of Raf-1 in this microtubule damage-activating pathway. A model is proposed that envisions a pathway of Raf-1 activation and bcl-2 phosphorylation following disruption of microtubular architecture, serving a role similar to p53 induction following DNA damage.

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL or Apo2L) is a potent inducer of death of cancer but not normal cells, which suggests its potential use as a tumor-specific antineoplastic agent. TRAIL binds to the proapoptotic death receptors DR4 and the p53-regulated proapoptotic KILLER/DR5 as well as to the decoy receptors TRID and TRUNDD. In the present studies, we identified a subgroup of TRAIL-resistant cancer cell lines characterized by low or absent basal DR4 or high expression of the caspase activation inhibitor FLIP. Four of five TRAIL-sensitive cell lines expressed high levels of DR4 mRNA and protein, whereas six of six TRAIL-resistant cell lines expressed low or undetectable levels of DR4 (chi 2; P < 0.01). FLIP expression appeared elevated in five of six (83%) TRAIL-resistant cell lines and only one of five (20%) TRAIL-sensitive cells (chi 2; P < 0.05). Two TRAIL-resistant lines that expressed DR4 contained an A-to-G alteration in the death domain encoding arginine instead of lysine at codon 441. The K441R polymorphism is present in 20% of the normal population and can inhibit DR4-mediated cell killing in a dominant-negative fashion. The expression level of KILLER/DR5, TRID, TRUNDD or TRID, and TRUNDD did not correlate with TRAIL sensitivity (P > 0.05). These results suggest that the major determinants for TRAIL sensitivity may be the expression level of DR4 and FLIP. TRAIL-resistant cells became susceptible to TRAIL-mediated apoptosis in the presence of doxorubicin. In TRAIL-sensitive cells, caspases 8, 9, and 3 were activated after TRAIL treatment, but in TRAIL-resistant cells, they were activated only by the combination of TRAIL and doxorubicin. Our results suggest: (a) evaluation of tumor DR4 and FLIP expression and host DR4 codon 441 status could be potentially useful predictors of TRAIL sensitivity, and (b) doxorubicin, in combination with TRAIL, may effectively promote caspase activation in TRAIL-resistant tumors.

The death receptor (DR) KILLER/DR5 gene has recently been identified as a doxorubicin-regulated transcript that was also induced by exogenous wild-type p53 in p53-negative cells. KILLER/DR5 gene encodes a DR containing cell surface protein that is highly homologous to DR4, another DR of the tumor necrosis factor (TNF) receptor family. Both DR4 and KILLER/DR5 independently bind to their specific ligand TRAIL and engage the caspase cascade to induce apoptosis. TRID (also known as TRAIL-R3) is an antiapoptotic decoy receptor that lacks the cytoplasmic death domain and competes with KILLER/DR5 and DR4 for binding to TRAIL. In this study, we demonstrate that the DR KILLER/DR5 gene is regulated in a p53-dependent and -independent manner during genotoxic and nongenotoxic stress-induced apoptosis. Just like other p53-regulated genes, ionizing radiation induction of KILLER/DR5 occurs in p53 wild-type cells, whereas methyl methanesulfonate regulation of KILLER/DR5 occurs in a p53-dependent and -independent manner. However, unlike other p53-regulated genes, KILLER/DR5 is not regulated following UV irradiation. TNF-alpha, a nongenotoxic cytokine, also induced the expression of KILLER/DR5 in a number of cancer cell lines, irrespective of p53 status. TNF-alpha did not alter the KILLER/DR5 mRNA stability, suggesting that the TNF-alpha regulation of KILLER/DRS expression appears transcriptional. We also provide evidence that KILLER/DR5 is regulated in a trigger and cell type-specific manner and that its induction by TNF-alpha, p53, or DNA damage is not the consequence of apoptosis induced by these agents. Unlike KILLER/DR5, none of the other KILLER/DR5 family members, including DR4, TRID, or the ligand TRAIL, displayed genotoxic stress or TNF-alpha regulation in a p53 transcription-dependent manner. Thus, KILLER/DR5 appears a bona fide downstream target of p53 that is also regulated in a cell type-specific, trigger-dependent, and p53-independent manner.

Toxic bile salts induce hepatocyte apoptosis by both Fas-dependent and -independent mechanisms. In this study, we examined the cellular mechanisms responsible for Fas-independent, bile acid-mediated apoptosis. HuH-7 cells, which are known to be Fas deficient, were stably transfected with the sodium-dependent bile acid transporting polypeptide. The toxic bile acid glycochenodeoxycholate (GCDC)-induced apoptosis in these cells in a time- and concentration-dependent manner. Apoptosis and mitochondrial cytochrome <i>c</i> release were inhibited by transfection with dominant negative FADD, CrmA transfection, or treatment with the selective caspase 8 inhibitor IETD-CHO. These observations suggested the Fas-independent apoptosis was also death receptor mediated. Reverse transcriptase-polymerase chain reaction demonstrated tumor necrosis factor-R1, tumor necrosis factor-related apoptosis inducing ligand (TRAIL)-R1/DR4, -R2/DR5, and TRAIL, but not tumor necrosis fator-α expression by these cells. GCDC treatment increased expression of TRAIL-R2/DR5 mRNA and protein 10-fold while expression of TRAIL-R1 was unchanged. Furthermore, aggregation of TRAIL-R2/DR5, but not TRAIL-R1/DR4 was observed following GCDC treatment of the cells. Induction of TRAIL-R2/DR5 expression and apoptosis by bile acids provides new insights into the mechanisms of hepatocyte apoptosis and the regulation of TRAIL-R2/DR5 expression.

Esophageal cancer is a prototypic squamous cell cancer that carries a poor prognosis, primarily due to presentation at advanced stages. We used human esophageal epithelial cells as a platform to recapitulate esophageal squamous cell cancer, thereby providing insights into the molecular pathogenesis of squamous cell cancers in general. This was achieved through the retroviral-mediated transduction into normal, primary human esophageal epithelial cells of epidermal growth factor receptor (EGFR), the catalytic subunit of human telomerase (hTERT), and p53(R175H), genes that are frequently altered in human esophageal squamous cell cancer. These cells demonstrated increased migration and invasion when compared with control cells. When these genetically altered cells were placed within the in vivo-like context of an organotypic three-dimensional (3D) culture system, the cells formed a high-grade dysplastic epithelium with malignant cells invading into the stromal extracellular matrix (ECM). The invasive phenotype was in part modulated by the activation of matrix metalloproteinase-9 (MMP-9). Using pharmacological and genetic approaches to decrease MMP-9, invasion into the underlying ECM could be suppressed partially. In addition, tumor differentiation was influenced by the type of fibroblasts within the stromal ECM. To that end, fetal esophageal fibroblasts fostered a microenvironment conducive to poorly differentiated invading tumor cells, whereas fetal skin fibroblasts supported a well-differentiated tumor as illustrated by keratin "pearl" formation, a hallmark feature of well-differentiated squamous cell cancers. When inducible AKT was introduced into fetal skin esophageal fibroblasts, a more invasive, less-differentiated esophageal cancer phenotype was achieved. Invasion into the stromal ECM was attenuated by genetic knockdown of AKT1 as well as AKT2. Taken together, alterations in key oncogenes and tumor suppressor genes in esophageal epithelial cells, the composition and activation of fibroblasts, and the components of the ECM conspire to regulate the physical and biological properties of the stroma.

Abstract The p53 tumor suppressor gene controls cellular growth after DNA damage through mechanisms involving growth arrest and apoptosis. Mutations that inactivate p53 occur commonly in virtually all human malignancies and can be detected by sequencing of the p53 gene, immunohistochemical staining of tumor tissue with anti-p53 antibodies, single-strand conformation polymorphisms, or other biological assays. Identification of p53 mutation in the germ line is diagnostic of the cancer-prone Li-Fraumeni syndrome. Alterations of the p53 gene result in defective cellular responses after DNA damage and predispose cells to dysregulated growth, tumor formation and progression, and potential resistance (of tumor cells) to certain chemotherapeutic agents or ionizing radiation. A variety of tumors involving mutant p53 have a worse prognosis than tumors of the same type containing no p53 mutations. New diagnostic and therapeutic strategies are evolving as the p53 pathways of cell-cycle arrest and apoptosis become elucidated.

Cellular transformation by the adenovirus E1A oncoprotein requires its p300/CBP- and Rb-binding domains. We mapped inhibition of p53-mediated transactivation to the p300/CBP-binding region of E1A. An E1A mutant incapable of physically interacting with Rb retained the capacity to inhibit transactivation by p53, whereas E1A mutants of the p300/CBP-interacting domain failed to inhibit p53. The inhibitory effect of the p300/CBP-binding region of E1A on p53 was demonstrated with p53-activated reporters and endogenous p53 targets such as p21(WAF1/CIP1) or MDM2. E1A lacking the capacity to interact with Rb, but capable of p300/CBP interaction, was competent in suppression of a DNA-damage activated p53-dependent cell cycle checkpoint. Exogenous CBP and p300 were able to individually relieve E1A's inhibitory effect on p53-mediated transcription. Mutants of E1A that are not capable of interacting with p300 or CBP were found to efficiently stabilize endogenous p53 but were not competent in repression of p21 expression thus dissociating these two effects of E1A. Our results suggest that the p300/CBP-binding domain of E1A inhibits a p53-dependent cellular response which normally inhibits DNA replication following Adenovirus infection.

Preclinical data support the potential of the death-signaling receptors for TRAIL as targets for cancer therapy. However, it is unclear whether these death-signaling receptors suppress the emergence and growth of malignant tumors in vivo. Herein we show that TNF-related apoptosis-inducing ligand receptor (TRAIL-R), the only proapoptotic death-signaling receptor for TRAIL in the mouse, suppresses inflammation and tumorigenesis. Loss of a single TRAIL-R allele on the lymphoma-prone Emu-myc genetic background significantly reduced median lymphoma-free survival. TRAIL-R-deficient lymphomas developed with equal frequency irrespective of mono- or biallelic loss of TRAIL-R, had increased metastatic potential, and showed apoptotic defects relative to WT littermates. In addition, TRAIL-R-/- mice showed decreased long-term survival following a sublethal dose of ionizing radiation. Histological evaluation of moribund irradiated TRAIL-R-/- animals showed hallmarks of bronchopneumonia as well as tumor formation with increased NF-kappaB p65 expression. TRAIL-R also suppressed diethylnitrosamine-induced (DEN-induced) hepatocarcinogenesis, as an increased number of large tumors with apoptotic defects developed in the livers of DEN-treated TRAIL-R-/- mice. Thus TRAIL-R may function as an inflammation and tumor suppressor in multiple tissues in vivo.

Tumor suppressor p53 is a transcription factor that guards the genome stability and normal cell growth. Stresses like DNA damage, oncogenic assault will turn on p53 function which leads to cell cycle arrest for DNA repair, senescence for permanent growth arrest or apoptosis for programmed cell death. At the late stage of cancer progression, p53 is hijacked in all forms of tumors either trapped in the negative regulator such as MDM2/viral proteins or directly mutated/deleted. Re-introduction of a functional p53 alone has been proven to induce tumor regression robustly. Also, an active p53 pathway is essential for effective chemo- or radio-therapy. The emerging cyclotherapy in which p53 acts as a chemoprotectant of normal tissues further expands the utility of p53 activators. Functionally, it is unquestionable that drugging p53 will render tumor-specific intervention. One direct method is to deliver the functional wild-type (wt) p53 to tumors via gene therapy. The small molecule strategies consist of activation of p53 family member such as p73, manipulating p53 posttranslational modulators to increase wt p53 protein levels, protein-protein interaction inhibitors to free wt p53 from MDM2 or viral protein, and restoring p53 function to mutant p53 by direct modulation of its conformation. Although most of the current pre-clinical leads are in microM range and need further optimization, the success in proving that small molecules can reactivate p53 marks the beginning of the clinical development of p53-based cancer therapy.

TNF-related apoptosis-inducing ligand (TRAIL) is a member of the TNF family of cytokines, which can induce apoptotic cell death in a variety of tumor cells by engaging specific death receptors, TRAIL-R1 and TRAIL-R2, while having low toxicity towards normal cells. There is interest in cancer therapy inducing cell death by activation of the death-receptor-mediated apoptotic pathway while avoiding decoy-receptor-mediated neutralization of the signal. This has led to the development of a number of receptor-specific TRAIL-variants and agonistic antibodies. Some of these soluble recombinant TRAIL and agonist antibodies targeting TRAIL-R1 and/or TRAIL-R2 are progressing in clinical trials. In addition, TRAIL-resistant tumors can be sensitized to TRAIL by a combination of TRAIL or agonistic antibodies with chemotherapeutic agents, targeted small molecules or irradiation.Recent advances in developing TRAIL or its agonist receptor antibodies in cancer therapy. We also discuss combination therapies in overcoming TRAIL resistance in cancer cells.Knowledge of current clinical trials, the promise and obstacles in the future development of therapies affecting TRAIL signaling pathways.Cancer therapeutics targeting the TRAIL/TRAIL receptor signaling pathway hold great promise for molecularly targeted pro-apoptotic anti-cancer therapy.

The dissemination of circulating tumor cells (CTCs) that cause metastases in distant organs accounts for the majority of cancer-related deaths. CTCs have been established as a cancer biomarker of known prognostic value. The enrichment of viable CTCs for ex vivo analysis could further improve cancer diagnosis and guide treatment selection. We designed a new flexible micro spring array (FMSA) device for the enrichment of viable CTCs independent of antigen expression.Unlike previous microfiltration devices, flexible structures at the micro scale minimize cell damage to preserve viability, while maximizing throughput to allow rapid enrichment directly from whole blood with no need for sample preprocessing. Device performance with respect to capture efficiency, enrichment against leukocytes, viability, and proliferability was characterized. CTCs and CTC microclusters were enriched from clinical samples obtained from breast, lung, and colorectal cancer patients.The FMSA device enriched tumor cells with 90% capture efficiency, higher than 10(4) enrichment, and better than 80% viability from 7.5-mL whole blood samples in <10 min on a 0.5-cm(2) device. The FMSA detected at least 1 CTC in 16 out of 21 clinical samples (approximately 76%) compared to 4 out of 18 (approximately 22%) detected with the commercial CellSearch® system. There was no incidence of clogging in over 100 tested fresh whole blood samples.The FMSA device provides a versatile platform capable of viable enrichment and analysis of CTCs from clinically relevant volumes of whole blood.

Abstract Purpose: ONC201 is a small-molecule selective antagonist of the G protein–coupled receptor DRD2 that is the founding member of the imipridone class of compounds. A first-in-human phase I study of ONC201 was conducted to determine its recommended phase II dose (RP2D). Experimental Design: This open-label study treated 10 patients during dose escalation with histologically confirmed advanced solid tumors. Patients received ONC201 orally once every 3 weeks, defined as one cycle, at doses from 125 to 625 mg using an accelerated titration design. An additional 18 patients were treated at the RP2D in an expansion phase to collect additional safety, pharmacokinetic, and pharmacodynamic information. Results: No grade &amp;gt;1 drug-related adverse events occurred, and the RP2D was defined as 625 mg. Pharmacokinetic analysis revealed a Cmax of 1.5 to 7.5 μg/mL (∼3.9–19.4 μmol/L), mean half-life of 11.3 hours, and mean AUC of 37.7 h·μg/L. Pharmacodynamic assays demonstrated induction of caspase-cleaved keratin 18 and prolactin as serum biomarkers of apoptosis and DRD2 antagonism, respectively. No objective responses by RECIST were achieved; however, radiographic regression of several individual metastatic lesions was observed along with prolonged stable disease (&amp;gt;9 cycles) in prostate and endometrial cancer patients. Conclusions: ONC201 is a selective DRD2 antagonist that is well tolerated, achieves micromolar plasma concentrations, and is biologically active in advanced cancer patients when orally administered at 625 mg every 3 weeks. Clin Cancer Res; 23(15); 4163–9. ©2017 AACR.

Abstract Self-renewing colorectal cancer stem/progenitor cells (CSC) contribute to tumor maintenance and resistance to therapy. Therapeutic targeting of CSCs could improve treatment response and prolong patient survival. ONC201/TIC10 is a first-in-class antitumor agent that induces TRAIL pathway–mediated cell death in cancer cells without observed toxicity. We have previously described that ONC201/TIC10 exposure leads to transcriptional induction of the TRAIL gene via transcription factor Foxo3a, which is activated by dual inactivation of Akt and ERK. The Akt and ERK pathways serve as important targets in CSCs. Foxo3a is a key mediator of Akt and ERK-mediated CSC regulation. We hypothesized that the potent antitumor effect of ONC201/TIC10 in colorectal cancer involves targeting CSCs and bulk tumor cells. ONC201/TIC10 depletes CD133+, CD44+, and Aldefluor+ cells in vitro and in vivo. TIC10 significantly inhibits colonosphere formation of unsorted and sorted 5-fluorouracil–resistant CSCs. ONC201/TIC10 significantly reduces CSC-initiated xenograft tumor growth in mice and prevents the passage of these tumors. ONC201/TIC10 treatment also decreased xenograft tumor initiation and was superior to 5-fluorouracil treatment. Thus, ONC201/TIC10 inhibits CSC self-renewal in vitro and in vivo. ONC201/TIC10 inhibits Akt and ERK, consequently activating Foxo3a and significantly induces cell surface TRAIL and DR5 expression in both CSCs and non-CSCs. ONC201/TIC10-mediated anti-CSC effect is significantly blocked by the TRAIL sequestering antibody RIK-2. Overexpression of Akt, DR5 knockdown, and Foxo3a knockdown rescues ONC201/TIC10-mediated depletion of CD44+ cells and colonosphere inhibition. In conclusion, ONC201/TIC10 is a promising agent for colorectal cancer therapy that targets both non-CSCs and CSCs in an Akt–Foxo3a–TRAIL–dependent manner. Cancer Res; 75(7); 1423–32. ©2015 AACR.

ONC201 is the founding member of a novel class of anti-cancer compounds called imipridones that is currently in Phase II clinical trials in multiple advanced cancers. Since the discovery of ONC201 as a p53-independent inducer of TRAIL gene transcription, preclinical studies have determined that ONC201 has anti-proliferative and pro-apoptotic effects against a broad range of tumor cells but not normal cells. The mechanism of action of ONC201 involves engagement of PERK-independent activation of the integrated stress response, leading to tumor upregulation of DR5 and dual Akt/ERK inactivation, and consequent Foxo3a activation leading to upregulation of the death ligand TRAIL. ONC201 is orally active with infrequent dosing in animals models, causes sustained pharmacodynamic effects, and is not genotoxic. The first-in-human clinical trial of ONC201 in advanced aggressive refractory solid tumors confirmed that ONC201 is exceptionally well-tolerated and established the recommended phase II dose of 625 mg administered orally every three weeks defined by drug exposure comparable to efficacious levels in preclinical models. Clinical trials are evaluating the single agent efficacy of ONC201 in multiple solid tumors and hematological malignancies and exploring alternative dosing regimens. In addition, chemical analogs that have shown promise in other oncology indications are in pre-clinical development. In summary, the imipridone family that comprises ONC201 and its chemical analogs represent a new class of anti-cancer therapy with a unique mechanism of action being translated in ongoing clinical trials.

Nonalcoholic steatohepatitis is characterized by hepatic steatosis, elevated levels of circulating free fatty acids (FFA), endoplasmic reticulum (ER) stress, and hepatocyte lipoapoptosis. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) death receptor 5 (DR5) is significantly elevated in patients with nonalcoholic steatohepatitis, and steatotic hepatocytes demonstrate increased sensitivity to TRAIL-mediated cell death. Nonetheless, a role for TRAIL and/or DR5 in mediating lipoapoptotic pathways is unexplored. Here, we examined the contribution of DR5 death signaling to lipoapoptosis by free fatty acids. The toxic saturated free fatty acid palmitate induces an increase in DR5 mRNA and protein expression in Huh-7 human hepatoma cells leading to DR5 localization into lipid rafts, cell surface receptor clustering with subsequent recruitment of the initiator caspase-8, and ultimately cellular demise. Lipoapoptosis by palmitate was not inhibited by a soluble human recombinant DR5-Fc chimera protein suggesting that DR5 cytotoxic signaling is ligand-independent. Hepatocytes from murine TRAIL receptor knock-out mice (DR−/−) displayed reduced palmitate-mediated lipotoxicity. Likewise, knockdown of DR5 or caspase-8 expression by shRNA technology attenuated palmitate-induced Bax activation and apoptosis in Huh-7 cells, without altering induction of ER stress markers. Similar observations were verified in other cell models. Finally, knockdown of CHOP, an ER stress-mediated transcription factor, reduced DR5 up-regulation and DR5-mediated caspase-8 activation upon palmitate treatment. Collectively, these results suggest that ER stress-induced CHOP activation by palmitate transcriptionally up-regulates DR5, likely resulting in ligand-independent cytotoxic signaling by this death receptor. Nonalcoholic steatohepatitis is characterized by hepatic steatosis, elevated levels of circulating free fatty acids (FFA), endoplasmic reticulum (ER) stress, and hepatocyte lipoapoptosis. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) death receptor 5 (DR5) is significantly elevated in patients with nonalcoholic steatohepatitis, and steatotic hepatocytes demonstrate increased sensitivity to TRAIL-mediated cell death. Nonetheless, a role for TRAIL and/or DR5 in mediating lipoapoptotic pathways is unexplored. Here, we examined the contribution of DR5 death signaling to lipoapoptosis by free fatty acids. The toxic saturated free fatty acid palmitate induces an increase in DR5 mRNA and protein expression in Huh-7 human hepatoma cells leading to DR5 localization into lipid rafts, cell surface receptor clustering with subsequent recruitment of the initiator caspase-8, and ultimately cellular demise. Lipoapoptosis by palmitate was not inhibited by a soluble human recombinant DR5-Fc chimera protein suggesting that DR5 cytotoxic signaling is ligand-independent. Hepatocytes from murine TRAIL receptor knock-out mice (DR−/−) displayed reduced palmitate-mediated lipotoxicity. Likewise, knockdown of DR5 or caspase-8 expression by shRNA technology attenuated palmitate-induced Bax activation and apoptosis in Huh-7 cells, without altering induction of ER stress markers. Similar observations were verified in other cell models. Finally, knockdown of CHOP, an ER stress-mediated transcription factor, reduced DR5 up-regulation and DR5-mediated caspase-8 activation upon palmitate treatment. Collectively, these results suggest that ER stress-induced CHOP activation by palmitate transcriptionally up-regulates DR5, likely resulting in ligand-independent cytotoxic signaling by this death receptor.

In this work, we demonstrate an integrated, single-layer, miniature flow cytometry device that is capable of multi-parametric particle analysis. The device integrates both particle focusing and detection components on-chip, including a "microfluidic drifting" based three-dimensional (3D) hydrodynamic focusing component and a series of optical fibers integrated into the microfluidic architecture to facilitate on-chip detection. With this design, multiple optical signals (i.e., forward scatter, side scatter, and fluorescence) from individual particles can be simultaneously detected. Experimental results indicate that the performance of our flow cytometry chip is comparable to its bulky, expensive desktop counterpart. The integration of on-chip 3D particle focusing with on-chip multi-parametric optical detection in a single-layer, mass-producible microfluidic device presents a major step towards low-cost flow cytometry chips for point-of-care clinical diagnostics.

We have developed 3D-tumoroids and tumor slice in vitro culture systems from surgical tumor specimens derived from patients with colorectal cancer (CRC) or lung cancer to evaluate immune cell populations infiltrating cultured tissues. The system incorporates patient's peripherally and tumor-derived immune cells into tumoroid in vitro cultures to evaluate the ability of the culture to mimic an immunosuppressive tumor microenvironment (ITM). This system enables analysis of tumor response to standard therapy within weeks of surgical resection. Here we show that tumoroid cultures from a CRC patient are highly sensitive to the thymidylate synthase inhibitor 5-fluorouracil (adrucil) but less sensitive to the combination of nucleoside analog trifluridine and thymidine phosphorylase inhibitor tipiracil (Lonsurf). Moreover, re-introduction of isolated immune cells derived from surrounding and infiltrating tumor tissue as well as CD45+ tumor infiltrating hematopoietic cells displayed prolonged (>10 days) survival in co-culture. Established tumor slice cultures were found to contain both an outer epithelial and inner stromal cell compartment mimicking tumor structure in vivo. Collectively, these data suggest that, 3D-tumoroid and slice culture assays may provide a feasible in vitro approach to assess efficacy of novel therapeutics in the context of heterogeneous tumor-associated cell types including immune and non-transformed stromal cells. In addition, delineating the impact of therapeutics on immune cells, and cell types involved in therapeutic resistance mechanisms may be possible in general or for patient-specific responses.

Activation of hypoxia-inducible factor 1 (HIF-1) signaling is observed in a broad range of human cancers due to tumor hypoxia and epigenetic mechanisms. HIF-1 activation leads to the transcription of a plethora of target genes that promote physiological changes associated with therapeutic resistance, including the inhibition of apoptosis and senescence and the activation of drug efflux and cellular metabolism. As a result, targeting HIF-1 represents an attractive strategy to enhance the efficacy of current therapies as well as reduce resistance to chemotherapy in tumors. Approaches to inhibit HIF-1 signaling have primarily focused on reducing HIF-1α protein levels, by inducing its degradation or inhibiting its transcription, inhibiting HIF-1-mediated transcription, or disrupting the formation of the HIF-1 transcription factor complex. To date, multiple preclinical and clinical agents have been identified that effectively inhibit HIF-1 activity through various mechanisms, likely accounting for a portion of their anti-tumor efficacy. This review aims to provide an overview of our current understanding of the role of HIF-1 in therapeutic resistance and discuss the ongoing effort to develop HIF-1 inhibitors as an anti-cancer strategy.

ONC201 was originally discovered as TNF-Related Apoptosis Inducing Ligand (TRAIL)-inducing compound TIC10. ONC201 appears to act as a selective antagonist of the G protein coupled receptor (GPCR) dopamine receptor D2 (DRD2), and as an allosteric agonist of mitochondrial protease caseinolytic protease P (ClpP). Downstream of target engagement, ONC201 activates the ATF4/CHOP-mediated integrated stress response leading to TRAIL/Death Receptor 5 (DR5) activation, inhibits oxidative phosphorylation via c-myc, and inactivates Akt/ERK signaling in tumor cells. This typically results in DR5/TRAIL-mediated apoptosis of tumor cells; however, DR5/TRAIL-independent apoptosis, cell cycle arrest, or antiproliferative effects also occur. The effects of ONC201 extend beyond bulk tumor cells to include cancer stem cells, cancer associated fibroblasts and immune cells within the tumor microenvironment that can contribute to its efficacy. ONC201 is orally administered, crosses the intact blood brain barrier, and is under evaluation in clinical trials in patients with advanced solid tumors and hematological malignancies. ONC201 has single agent clinical activity in tumor types that are enriched for DRD2 and/or ClpP expression including specific subtypes of high-grade glioma, endometrial cancer, prostate cancer, mantle cell lymphoma, and adrenal tumors. Synergy with radiation, chemotherapy, targeted therapy and immune-checkpoint agents has been identified in preclinical models and is being evaluated in clinical trials. Structure-activity relationships based on the core pharmacophore of ONC201, termed the imipridone scaffold, revealed novel potent compounds that are being developed. Imipridones represent a novel approach to therapeutically target previously undruggable GPCRs, ClpP, and innate immune pathways in oncology.

Hypoxia-inducible factor 1 (HIF-1) is a major mediator of tumor physiology, and its activation is correlated with tumor progression, metastasis, and therapeutic resistance. HIF-1 is activated in a broad range of solid tumors due to intratumoral hypoxia or genetic alterations that enhance its expression or inhibit its degradation. As a result, decreasing HIF-1α expression represents an attractive strategy to sensitize hypoxic tumors to anticancer therapies. Here, we show that cyclin-dependent kinase 1 (CDK1) regulates the expression of HIF-1α, independent of its known regulators. Overexpression of CDK1 and/or cyclin B1 is sufficient to stabilize HIF-1α under normoxic conditions, whereas inhibition of CDK1 enhances the proteasomal degradation of HIF-1α, reducing its half-life and steady-state levels. In vitro kinase assays reveal that CDK1 directly phosphorylates HIF-1α at a previously unidentified regulatory site, Ser668. HIF-1α is stabilized under normoxic conditions during G 2/M phase via CDK1-mediated phosphorylation of Ser668. A phospho-mimetic construct of HIF-1α at Ser668 (S668E) is significantly more stable under both normoxic and hypoxic conditions, resulting in enhanced transcription of HIF-1 target genes and increased tumor cell invasion and migration. Importantly, HIF-1α (S668E) displays increased tumor angiogenesis, proliferation, and tumor growth in vivo compared with wild-type HIF-1α. Thus, we have identified a novel link between CDK1 and HIF-1α that provides a potential molecular explanation for the elevated HIF-1 activity observed in primary and metastatic tumors, independent of hypoxia, and offers a molecular rationale for the clinical translation of CDK inhibitors for use in tumors with constitutively active HIF-1.

While apoptosis is critical for maintaining homeostasis in normal cells, defective apoptosis contributes to the survival of cancer cells. TNF-related apoptosis-inducing ligand (TRAIL)-targeted therapy has attracted significant effort for treating cancer, but the clinical results have revealed limitations. The authors review the current status of development of TRAIL-targeted therapy with an outlook towards the future.Recombinant human proteins, small molecules and agonistic monoclonal antibodies targeting death receptors that trigger TRAIL-mediated apoptosis are covered in this article. The authors review both intrinsic and extrinsic apoptotic pathways, highlighting how the apoptosis serves as a promising therapeutic target. They also review different categories of TRAIL pathway targeting agents and provide a brief overview of clinical trials using these agents. The authors discuss the limitations of conventional approaches for targeting the TRAIL pathway as well as future directions.The development of better combination partners for pro-apoptotic TRAIL pathway modulators including novel agents inhibiting anti-apoptotic molecules or targeting alternative resistance pathways may improve the chances for anti-tumor responses in the clinic. Developing predictive biomarkers via circulating tumor cells/DNA, apoptosis signal products, and genetic signatures/protein biomarkers from tumor tissue are also suggested as future directions.

ONC201/TIC10 is a first-in-class small molecule inducer of TRAIL that causes early activation of the integrated stress response. Its promising safety profile and broad-spectrum efficacy in vitro have been confirmed in Phase I/II trials in several advanced malignancies. Binding and reporter assays have shown that ONC201 is a selective antagonist of the dopamine D2-like receptors, specifically, DRD2 and DRD3. We hypothesized that ONC201's interaction with DRD2 plays a role in ONC201's anticancer effects. Using cBioportal and quantitative reverse-transcription polymerase chain reaction analyses, we confirmed that DRD2 is expressed in different cancer cell types in a cell type-specific manner. On the other hand, DRD3 was generally not detectable. Overexpressing DRD2 in cells with low DRD2 levels increased ONC201-induced PARP cleavage, which was preceded and correlated with an increase in ONC201-induced CHOP mRNA expression. On the other hand, knocking out DRD2 using CRISPR/Cas9 in three cancer cell lines was not sufficient to abrogate ONC201's anticancer effects. Although ONC201's anticancer activity was not dependent on DRD2 expression in the cancer cell types tested, we assessed the cytotoxic potential of DRD2 blockade. Transient DRD2 knockdown in HCT116 cells activated the integrated stress response and reduced cell number. Pharmacological antagonism of DRD2 significantly reduced cell viability. Thus, we demonstrate in this study that disrupting dopamine receptor expression and activity can have cytotoxic effects that may at least be in part due to the activation of the integrated stress response. On the other hand, ONC201's anticancer activity goes beyond its ability to antagonize DRD2, potentially due to ONC201's ability to activate other pathways that are independent of DRD2. Nevertheless, blocking the dopamine D1-like receptor DRD5 via siRNA or the use of a pharmacological antagonist promoted ONC201-induced anticancer activity.

The tumor-suppressor p53 prevents cancer development via initiating cell-cycle arrest, cell death, repair, or antiangiogenesis processes. Over 50% of human cancers harbor cancer-causing mutant p53. p53 mutations not only abrogate its tumor-suppressor function, but also endow mutant p53 with a gain of function (GOF), creating a proto-oncogene that contributes to tumorigenesis, tumor progression, and chemo- or radiotherapy resistance. Thus, targeting mutant p53 to restore a wild-type p53 signaling pathway provides an attractive strategy for cancer therapy. We demonstrate that small-molecule NSC59984 not only restores wild-type p53 signaling, but also depletes mutant p53 GOF. NSC59984 induces mutant p53 protein degradation via MDM2 and the ubiquitin-proteasome pathway. NSC59984 restores wild-type p53 signaling via p73 activation, specifically in mutant p53-expressing colorectal cancer cells. At therapeutic doses, NSC59984 induces p73-dependent cell death in cancer cells with minimal genotoxicity and without evident toxicity toward normal cells. NSC59984 synergizes with CPT11 to induce cell death in mutant p53-expressing colorectal cancer cells and inhibits mutant p53-associated colon tumor xenograft growth in a p73-dependent manner in vivo. We hypothesize that specific targeting of mutant p53 may be essential for anticancer strategies that involve the stimulation of p73 in order to efficiently restore tumor suppression. Taken together, our data identify NSC59984 as a promising lead compound for anticancer therapy that acts by targeting GOF-mutant p53 and stimulates p73 to restore the p53 pathway signaling.

<h3>Background & Aims</h3> Low-grade chronic inflammation is a cardinal feature of the metabolic syndrome, yet its pathogenesis is not well defined. The purpose of this study was to examine the role of TRAIL receptor (TR) signaling in the pathogenesis of obesity-associated inflammation using mice with the genetic deletion of TR. <h3>Methods</h3> TR knockout (<i>TR^−/−</i>) mice and their littermate wild-type (WT) mice were fed a diet high in saturated fat, cholesterol and fructose (FFC) or chow. Metabolic phenotyping, liver injury, and liver and adipose tissue inflammation were assessed. Chemotaxis and activation of mouse bone marrow-derived macrophages (BMDMϕ) was measured. <h3>Results</h3> Genetic deletion of TR completely repressed weight gain, adiposity and insulin resistance in FFC-fed mice. Moreover, <i>TR^−/−</i> mice suppressed steatohepatitis, with essentially normal serum ALT, hepatocyte apoptosis and liver triglyceride accumulation. Gene array data implicated inhibition of macrophage-associated hepatic inflammation in the absence of the TR. In keeping with this, there was diminished accumulation and activation of inflammatory macrophages in liver and adipose tissue. <i>TR^−/−</i> BMDMϕ manifest reduced chemotaxis and diminished activation of nuclear factor-κ B signaling upon activation by palmitate and lipopolysaccharide. <h3>Conclusions</h3> These data advance the concept that macrophage-associated hepatic and adipose tissue inflammation of nutrient excess requires TR signaling.

Metastatic colorectal cancer (mCRC) carries a poor prognosis with an overall 5-year survival of 13.1%. Therapies guided by tumor profiling have suggested benefit in advanced cancer. We used a multiplatform molecular profiling (MP) approach to identify key molecular changes that may provide therapeutic options not typically considered in mCRC. We evaluated 6892 mCRC referred to Caris Life Sciences by MP including sequencing (Sanger/NGS), immunohistochemistry (IHC) and in-situ hybridization (ISH). mCRC metastases to liver, brain, ovary or lung (n = 1507) showed differential expression of markers including high protein expression of TOPO1 (52%) and/or low RRM1 (57%), TS (71%) and MGMT (39%), suggesting possible benefit from irinotecan, gemcitabine, 5FU/capecitabine and temozolomide, respectively. Lung metastases harbored a higher Her2 protein expression than the primary colon tumors (4% vs. 1.8%, p = 0.028). Brain and lung metastases had higher KRAS mutations than other sites (65% vs 59% vs 47%, respectively, p = 0.07, <0.01), suggesting poor response to anti-EGFR therapies. BRAF-mutated CRC (n = 455) showed coincident high protein expression of RRM1 (56%), TS (53%) and low PDGFR (22%) as compared with BRAF wild-type tumors. KRAS-mutated mCRC had higher protein expression of c-MET (47% vs. 36%) and lower MGMT (56% vs. 63%), suggesting consideration of c-MET inhibitors and temozolomide. KRAS-mutated CRC had high TUBB3 (42% vs. 33%) and low Her2 by IHC (0.5%) and HER2 by FISH (3%, p <0.05). CRC primaries had a lower incidence of PIK3CA and BRAF mutations in rectal cancer versus colon cancer (10% and 3.3%, respectively). MP of 6892 CRCs identified significant differences between primary and metastatic sites and among BRAF/KRAS sub-types. Our findings are hypothesis generating and need to be examined in prospective studies. Specific therapies may be considered for different actionable targets in mCRC as revealed by MP.

LBA2501 Background: Larotrectinib is the first selective small-molecule pan-TRK inhibitor. TRK fusions appear oncogenic independent of tumor lineage, are widely distributed across cancers, and affect all ages. We present an integrated dataset from 3 studies intended to support regulatory approval. Methods: All NTRK fusion pts with RECIST measurable disease enrolled to the adult (NCT02122913, n=8) and pediatric (NCT02637687, n=12) phase I trials and adult/adolescent phase 2 trial (NCT02576431, n=35) were analyzed. TRK fusion status was determined by local testing prior to enrollment. Pts were dosed predominantly at 100mg BID on a continuous 28-day schedule. Primary objective was investigator-assessed overall response rate (ORR) per RECIST v1.1. Secondary endpoints included duration of response (DOR) and safety. Data were cut on 31-JAN-2017. Results: 55 TRK fusion pts (12 peds, 43 adult, range: 4 mo.-76 yrs) were enrolled (median priors=2). Fusions involved NTRK1 (n=25), NTRK2 (n=1), and NTRK3 (n=29), and 14 unique partners. 13 discrete tumor types were treated: salivary (12), sarcoma (10), infantile fibrosarcoma (7), lung (5), thyroid (5), colon (4), melanoma (4), cholangio (2), GIST (2), and other (4). For the 46 pts evaluated to date, the ORR was 78% (95% CI: 64%–89%) with responses in 12 unique tumor types. Responses are ongoing in 29/33 (88%) pts, excluding 3 peds pts whose DOR was censored at attempted curative resection. A median DOR has not been reached as the majority of responders remain on treatment without progression. The longest responder remains on treatment at 23 mos., 8 pts remain in response at &gt;12 mos., and 16 pts at &gt;6 mos. NTRK solvent front mutations were detected in all 4 pts to develop acquired resistance. The most common TEAEs were fatigue (30%), dizziness (28%), and nausea (28%). 5 (11%) pts required dose reductions. Conclusions: Larotrectinib has demonstrated consistent and durable antitumor activity in TRK fusion cancers, across a wide range of ages and tumor types, and was well-tolerated. Larotrectinib could be the first targeted therapy developed in a tissue type-agnostic manner, and the first developed simultaneously in adults and pediatrics. Clinical trial information: NCT02576431, NCT02122913, NCT02637687.

IntroductionPrognosis of biliary tract cancers (BTC) remains dismal and novel treatment strategies are needed to improve survival. BRCA mutations are known to occur in BTC but their frequency and the molecular landscape in which they are observed in distinct sites of BTC remain unknown.Material and methodsTumour samples from 1292 patients with BTC, comprising intrahepatic cholangiocarcinoma (IHC, n=746), extrahepatic cholangiocarcinoma (EHC, n=189) and gallbladder cancer (GBC, n=353), were analysed using next-generation sequencing (NGS). Tumour mutational burden (TMB) was calculated based on somatic non-synonymous missense mutations. Determination of tumour mismatch repair (MMR) or microsatellite instability (MSI) status was done by fragment analysis, immunohistochemistry and the evaluation of known microsatellite loci by NGS. Programmed death ligand 1 expression was analysed using immunohistochemistry.ResultsOverall, BRCA mutations were detected in 3.6% (n=46) of samples (BRCA1: 0.6%, BRCA2: 3%) with no significant difference in frequency observed based on tumour site. In GBC and IHC, BRCA2 mutations (4.0% and 2.7%) were more frequent than BRCA1 (0.3% and 0.4%, p<0.05) while in EHC, similar frequency was observed (2.6% for BRCA2 vs 2.1% for BRCA1). BRCA mutations were associated with a higher rate in subjects with MSI-H/deficient mismatch repair (19.5% vs 1.7%, p<0.0001) and tumours with higher TMB, regardless of the MMR or MSI status (p<0.05).ConclusionsBRCA mutations are found in a subgroup of patients with BTC and are characterised by a distinct molecular profile. These data provide a rationale testing poly(ADP-ribose)polymeraseinhibitors and other targeted therapies in patients with BRCA-mutant BTC.

TP53 is a tumor suppressor gene that encodes a sequence-specific DNA-binding transcription factor activated by stressful stimuli; it upregulates target genes involved in growth suppression, cell death, DNA repair, metabolism, among others. TP53 is the most frequently mutated gene in tumors, with mutations not only leading to loss-of-function (LOF), but also gain-of-function (GOF) that promotes tumor progression, and metastasis. The tumor-specific status of mutant p53 protein has suggested it is a promising target for cancer therapy. We summarize the current progress of targeting wild-type and mutant p53 for cancer therapy through biotherapeutic and biopharmaceutical methods for (1) boosting p53 activity in cancer, (2) p53-dependent and p53-independent strategies for targeting p53 pathway functional restoration in p53-mutated cancer, (3) targeting p53 in immunotherapy, and (4) combination therapies targeting p53, p53 checkpoints, or mutant p53 for cancer therapy.

Though early-stage colorectal cancer has a high 5 year survival rate of 65-92% depending on the specific stage, this probability drops to 13% after the cancer metastasizes. Frontline treatments for colorectal cancer such as chemotherapy and radiation often produce dose-limiting toxicities in patients and acquired resistance in cancer cells. Additional targeted treatments are needed to improve patient outcomes and quality of life. Immunotherapy involves treatment with peptides, cells, antibodies, viruses, or small molecules to engage or train the immune system to kill cancer cells. Preclinical and clinical investigations of immunotherapy for treatment of colorectal cancer including immune checkpoint blockade, adoptive cell therapy, monoclonal antibodies, oncolytic viruses, anti-cancer vaccines, and immune system modulators have been promising, but demonstrate limitations for patients with proficient mismatch repair enzymes. In this review, we discuss preclinical and clinical studies investigating immunotherapy for treatment of colorectal cancer and predictive biomarkers for response to these treatments. We also consider open questions including optimal combination treatments to maximize efficacy, minimize toxicity, and prevent acquired resistance and approaches to sensitize mismatch repair-proficient patients to immunotherapy.

Pancreatic cancer is a highly aggressive malignancy with a climbing incidence. The majority of cases are detected late, with incurable locally advanced or metastatic disease. Even in individuals who undergo resection, recurrence is unfortunately very common. There is no universally accepted screening modality for the general population and diagnosis, evaluation of treatment response, and detection of recurrence relies primarily on the use of imaging. Identification of minimally invasive techniques to help diagnose, prognosticate, predict response or resistance to therapy, and detect recurrence are desperately needed. Liquid biopsies represent an emerging group of technologies which allow for non-invasive serial sampling of tumor material. Although not yet approved for routine use in pancreatic cancer, the increasing sensitivity and specificity of contemporary liquid biopsy platforms will likely change clinical practice in the near future. In this review, we discuss the recent technological advances in liquid biopsy, focusing on circulating tumor DNA, exosomes, microRNAs, and circulating tumor cells.

In 2018, a patient received capecitabine without prior testing for dihydropyrimidine dehydrogenase (DPYD) and later presented with vomiting, rash, and diarrhea.The hospital failed to provide uridine triacetate in a timely fashion, and the patient died.The patient's widow filed a wrongful death lawsuit against Oregon Health Sciences University (OHSU) and assisted in the formation of a nonprofit organization to advocate for DPYD testing for fluoropyrimidines.A settlement for $1 million US dollars was reached requiring OHSU oncologists to undergo education about DPYD testing and inform their patients about its availability. 1Clinical practice guidelines from the National Comprehensive Cancer Network (NCCN) and ASCO still do not support testing for DPYD genetic variants before fluoropyrimidine chemotherapy.The US Food and Drug Administration (FDA) package inserts for capecitabine and fluorouracil (FU) acknowledge patients with dihydropyrimidine dehydrogenase protein (DPD) deficiency have increased risk of life-threatening toxicity; however, instead of recommending preemptive testing, they posit an unlikely scenario in which patients who have known DPD deficiency should discuss it with their physicians. 2,3The European Medicines Agency, the French National Agency for the Safety of Medicines and Health Products, and the Medicines and Healthcare products Regulatory Agency have each approved guidelines for preemptive DPYD testing for patients treated with fluoropyrimidines.

The discovery of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) along with its potent and selective antitumor effects initiated a decades-long search for therapeutic strategies to target the TRAIL pathway. First-generation approaches were focused on the development of TRAIL receptor agonists (TRAs), including recombinant human TRAIL (rhTRAIL) and TRAIL receptor-targeted agonistic antibodies. While such TRAIL pathway-targeted therapies showed promise in preclinical data and clinical trials have been conducted, none have advanced to FDA approval. Subsequent second-generation approaches focused on improving upon the specific limitations of first-generation approaches by ameliorating the pharmacokinetic profiles and agonistic abilities of TRAs as well as through combinatorial approaches to circumvent resistance. In this review, we summarize the successes and shortcomings of first- and second-generation TRAIL pathway-based therapies, concluding with an overview of the discovery and clinical introduction of ONC201, a compound with a unique mechanism of action that represents a new generation of TRAIL pathway-based approaches. We discuss preclinical and clinical findings in different tumor types and provide a unique perspective on translational directions of the field.

Advancements in cutting-edge molecular profiling techniques, such as next-generation sequencing and bioinformatic analytic tools, have allowed researchers to examine tumour biology in detail and stratify patients based on factors linked with clinical outcome and response to therapy. This manuscript highlights the most relevant prognostic and predictive biomarkers in kidney, bladder, prostate and testicular cancers with recognised impact in clinical practice. In bladder and prostate cancer, new genetic acquisitions concerning the biology of tumours have modified the therapeutic scenario and led to the approval of target directed therapies, increasing the quality of patient care. Thus, it has become of paramount importance to choose adequate molecular tests, i.e., FGFR screening for urothelial cancer and BRCA1-2 alterations for prostate cancer, to guide the treatment plan for patients. While no tissue or blood-based biomarkers are currently used in routine clinical practice for renal cell carcinoma and testicular cancers, the field is quickly expanding. In kidney tumours, gene expression signatures might be the key to identify patients who will respond better to immunotherapy or anti-angiogenic drugs. In testicular germ cell tumours, the use of microRNA has outperformed conventional serum biomarkers in the diagnosis of primary tumours, prediction of chemoresistance, follow-up monitoring, and relapse prediction.

Prostate cancer (PCa) is a common malignancy in males. The pathology review of PCa is crucial for clinical decision-making, but traditional pathology review is labor intensive and subjective to some extent. Digital pathology and whole-slide imaging enable the application of artificial intelligence (AI) in pathology. This review highlights the success of AI in detecting and grading PCa, predicting patient outcomes, and identifying molecular subtypes. We propose that AI-based methods could collaborate with pathologists to reduce workload and assist clinicians in formulating treatment recommendations. We also introduce the general process and challenges in developing AI pathology models for PCa. Importantly, we summarize publicly available datasets and open-source codes to facilitate the utilization of existing data and the comparison of the performance of different models to improve future studies.

Chemotherapeutic drug resistance is a major clinical problem and cause for failure in the therapy of human cancer. One of the goals of molecular oncology is to identify the underlying mechanisms, with the hope that more effective therapies can be developed. Several mechanisms have been suggested to contribute to chemoresistance: 1) amplification or overexpression of the P-glycoprotein family of membrane transporters (eg, MDR1, MRP, LRP) which decrease the intracellular accumulation of chemotherapy; 2) changes in cellular proteins involved in detoxification (eg, glutathione S-transferase π, metallothioneins, human MutT homologue, bleomycin hydrolase, dihydrofolate reductase) or activation of the chemotherapeutic drugs (DT-diaphorase, nicotinamide adenine dinucleotide phosphate:cytochrome P-450 reductase); 3) changes in molecules involved in DNA repair (eg, O6-methylguanine-DNA methyltransferase. DNA topoisomerase II, hMLH1, p21WAF1/CIP1; 4) activation of oncogenes such as Her-2/neu, bcl-2, bcl-XL, c-myc, ras, c-jun, c-fos. MDM2, p210 BCR-abl, or mutant p53. An overview of these resistance mechanisms is presented, with a particular focus on the role of oncogenes. Some current strategies attempting to reverse their effects are discussed.

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent inducer of apoptosis in tumor cell lines, whereas normal cells appear to be protected from its cytotoxic effects. Therefore TRAIL holds promise as a potential therapeutic agent against cancer. To elucidate some of the critical factors that contribute to TRAIL resistance, we performed a genetic screen in the human colon carcinoma cell line SW480 by infecting this TRAIL-sensitive cell line with a human placental cDNA retroviral library and isolating TRAIL-resistant clones. Characterization of the resulting clones for inhibitors of TRAIL-induced death (ITIDs) led to the isolation of c-FLIP_S, Bax inhibitor 1, and Bcl-X_L as candidate suppressors of TRAIL signaling. We have demonstrated that c-FLIP_S and Bcl-X_L are sufficient when overexpressed to convey resistance to TRAIL treatment in previously sensitive cell lines. Furthermore both c-FLIP_S and Bcl-X_L protected against overexpression of the TRAIL receptors DR4 and KILLER/DR5. When c-FLIP_S and Bcl-X_L were overexpressed together in SW480 and HCT 116, an additive inhibitory effect was observed after TRAIL treatment suggesting that these two molecules function in the same pathway in the cell lines tested. Furthermore, we have demonstrated for the first time that a proapoptotic member of the Bcl-2 family, Bax, is required for TRAIL-mediated apoptosis in HCT 116 cells. Surprisingly, we have found that the serine/threonine protein kinase Akt, which is an upstream regulator of both c-FLIP_S and Bcl-X_L, is not sufficient when overexpressed to protect against TRAIL in the cell lines tested. These results suggest a key role for c-FLIP_S, Bcl-X_L, and Bax in determining tumor cell sensitivity to TRAIL.

The pathway leading to BRCA1-dependent tumor suppression is not yet clear but appears to involve activities in DNA repair as well as gene transcription.Moreover, it has been shown that BRCA1 can regulate p53dependent transcription.Because BRCA1 overexpression stabilizes wild-type p53 but does not lead to apoptosis of most cell lines, we investigated the selectivity of BRCA1 for p53-dependent target gene activation.We find that BRCA1-stabilized p53 regulates transcription of DNA repair and growth arrest genes while p53 stabilized by DNA-damaging agents induces a wide array of genes, including those involved in apoptosis.This differential expression profile was reflected in the treatment outcome-apoptosis following DNA damage and growth arrest after expression of BRCA1.Depletion of BRCA1 in wild-type-p53-expressing cells abolished the induction of such repair genes as p53R2, while the expression of PIG3, an apoptosis-inducing gene, was still induced.BRCA1 also conferred diminished cell death in a p53-dependent manner in response to adriamycin compared to that conferred by controls.These results suggest that BRCA1 selectively coactivates the p53 transcription factor towards genes that direct DNA repair and cell cycle arrest but not towards those that direct apoptosis.

Little is known about how a cell's apoptotic threshold is controlled after exposure to chemotherapy, although the p53 tumor suppressor has been implicated. We identified executioner caspase-6 as a transcriptional target of p53. The mechanism involves DNA binding by p53 to the third intron of the caspase-6 gene and transactivation. A p53-dependent increase in procaspase-6 protein level allows for an increase in caspase-6 activity and caspase-6-specific Lamin A cleavage in response to Adriamycin exposure. Specific inhibition of caspase-6 blocks cell death in a manner that correlates with caspase-6 mRNA induction by p53 and enhances long-term survival in response to a p53-mediated apoptotic signal. Caspase-6 is an executioner caspase found directly regulated by p53, and the most downstream component of the death pathway controlled by p53. The induction of caspase-6 expression lowers the cell death threshold in response to apoptotic signals that activate caspase-6. Our results provide a potential mechanism of lowering the death threshold, which could be important for chemosensitization.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) exhibits specific tumoricidal activity and is under development for cancer therapy. Mismatch-repair-deficient colonic tumors evade TRAIL-induced apoptosis through mutational inactivation of Bax, but chemotherapeutics including Camptosar (CPT-11) restore TRAIL sensitivity. However, the signaling pathways in restoring TRAIL sensitivity remain to be elucidated. Here, we imaged p53 transcriptional activity in Bax -/- carcinomas by using bioluminescence, in vivo , and find that p53 is required for sensitization to TRAIL by CPT-11. Small interfering RNAs directed at proapoptotic p53 targets reveal TRAIL receptor KILLER/DR5 contributes significantly to TRAIL sensitization, whereas Bak plays a minor role. Caspase 8 inhibition protects both CPT-11 pretreated wild-type and Bax -/- HCT116 cells from TRAIL-induced apoptosis, whereas caspase 9 inhibition only rescued the wild-type HCT116 cells from death induced by TRAIL. The results suggest a conversion in the apoptotic mechanism in HCT116 colon carcinoma from a type II pathway involving Bax and the mitochondria to a type I pathway involving efficient extrinsic pathway caspase activation. In contrast to Bax -/- cells, Bak-deficient human cancers undergo apoptosis in response to TRAIL or CPT-11, implying that these proteins have nonoverlapping functions. Our studies elucidate a mechanism for restoration of TRAIL sensitivity in MMR-deficient Bax -/- human cancers through p53-dependent activation of KILLER/DR5 and reconstitution of a type I death pathway. Efforts to identify agents that up-regulate DR5 may be useful in cancer therapies restoring TRAIL sensitivity.