Soo‐Youl Kim

Dynamic regulation of glucose flux between aerobic glycolysis and the pentose phosphate pathway (PPP) during epithelial-mesenchymal transition (EMT) is not well-understood. Here we show that Snail (SNAI1), a key transcriptional repressor of EMT, regulates glucose flux toward PPP, allowing cancer cell survival under metabolic stress. Mechanistically, Snail regulates glycolytic activity via repression of phosphofructokinase, platelet (PFKP), a major isoform of cancer-specific phosphofructokinase-1 (PFK-1), an enzyme involving the first rate-limiting step of glycolysis. The suppression of PFKP switches the glucose flux towards PPP, generating NADPH with increased metabolites of oxidative PPP. Functionally, dynamic regulation of PFKP significantly potentiates cancer cell survival under metabolic stress and increases metastatic capacities in vivo. Further, knockdown of PFKP rescues metabolic reprogramming and cell death induced by loss of Snail. Thus, the Snail-PFKP axis plays an important role in cancer cell survival via regulation of glucose flux between glycolysis and PPP.

Proteinaceous aggregates containing α-synuclein represent a feature of neurodegenerative disorders such as Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy. Despite extensive research, the mechanisms underlying α-synuclein aggregation remain elusive. Previously, tissue transglutaminase (tTGase) was found to contribute to the generation of aggregates by cross-linking pathogenic substrate proteins in Huntington's and Alzheimer's diseases. In this article, the role of tTGase in the formation of α-synuclein aggregates was investigated. Purified tTGase catalyzed α-synuclein cross-linking, leading to the formation of high molecular weight aggregates in vitro , and overexpression of tTGase resulted in the formation of detergent-insoluble α-synuclein aggregates in cellular models. Immunocytochemical studies demonstrated the presence of α-synuclein-positive cytoplasmic inclusions in 8% of tTGase-expressing cells. The formation of these aggregates was significantly augmented by the calcium ionophore A23187 and prevented by the inhibitor cystamine. Immunohistochemical studies on postmortem brain tissue confirmed the presence of transglutaminase-catalyzed ɛ(γ-glutamyl)lysine cross-links in the halo of Lewy bodies in Parkinson's disease and dementia with Lewy bodies, colocalizing with α-synuclein. These findings, taken together, suggest that tTGase activity leads to α-synuclein aggregation to form Lewy bodies and perhaps contributes to neurodegeneration.

The transglutaminase (TGase) family of enzymes, of which seven different members are known in the human genome, participate in many biological processes involving cross-linking proteins into large macromolecular assemblies. The TGase 2 enzyme is known to be present in neuronal tissues and may play a role in neuronal degenerative diseases such as Alzheimer's disease (AD) by aberrantly cross-linking proteins. In this paper, we demonstrate by reverse transcriptase-polymerase chain reaction and immunological methods with specific antibodies that in fact three members, the TGase 1, TGase 2, and TGase 3 enzymes, and are differentially expressed in various regions of normal human brain tissues. Interestingly, the TGase 1 and 3 enzymes and their proteolytically processed forms are involved in terminal differentiation programs of epithelial cell development and barrier function. In addition, we found that the levels of expression and activity of the TGase 1 and 2 enzymes were both increased in the cortex and cerebellum of AD patients. Furthermore, whereas normal brain tissues contain ≈1 residue of cross-link/10,000 residues, AD patient cortex and cerebellum tissues contain 30–50 residues of cross-link/10,000 residues. Together, these findings suggest that multiple TGase enzymes are involved in normal neuronal structure and function, but their elevated expression and cross-linking activity may also contribute to neuronal degenerative disease.

Transglutaminase 2 (TGase 2) expression is increased in inflammatory diseases. We demonstrated previously that inhibitors of TGase 2 reduce nitric oxide (NO) generation in a lipopolysaccharide (LPS)-treated microglial cell line. However, the precise mechanism by which TGase 2 promotes inflammation remains unclear. We found that TGase 2 activates the transcriptional activator nuclear factor (NF)-κB and thereby enhances LPS-induced expression of inducible nitric-oxide synthase. TGase 2 activates NF-κB via a novel pathway. Rather than stimulating phosphorylation and degradation of the inhibitory subunit α of NF-κB (I-κBα), TGase2 induces its polymerization. This polymerization results in dissociation of NF-κB and its translocation to the nucleus, where it is capable of up-regulating a host of inflammatory genes, including inducible nitric-oxide synthase and tumor necrosis factor α (TNF-α). Indeed, TGase inhibitors prevent depletion of monomeric I-κBα in the cytosol of cells overexpressing TGase 2. In an LPS-induced rat brain injury model, TGase inhibitors significantly reduced TNF-α synthesis. The findings are consistent with a model in which LPS-induced NF-κB activation is the result of phosphorylation of I-κBα by I-κB kinase as well as I-κBα polymerization by TGase 2. Safe and stable TGase2 inhibitors may be effective agents in diseases associated with inflammation.

In 1923, Dr. Warburg had observed that tumors acidified the Ringer solution when 13 mM glucose was added, which was identified as being due to lactate.When glucose is the only source of nutrient, it can serve for both biosynthesis and energy production.However, a series of studies revealed that the cancer cell consumes glucose for biosynthesis through fermentation, not for energy supply, under physiological conditions.Recently, a new observation was made that there is a metabolic symbiosis in which glycolytic and oxidative tumor cells mutually regulate their energy metabolism.Hypoxic cancer cells use glucose for glycolytic metabolism and release lactate which is used by oxygenated cancer cells.This study challenged the Warburg effect, because Warburg claimed that fermentation by irreversible damaging of mitochondria is a fundamental cause of cancer.However, recent studies revealed that mitochondria in cancer cell show active function of oxidative phosphorylation although TCA cycle is stalled.It was also shown that blocking cytosolic NADH production by aldehyde dehydrogenase inhibition, combined with oxidative phosphorylation inhibition, resulted in up to 80% decrease of ATP production, which resulted in a significant regression of tumor growth in the NSCLC model.This suggests a new theory that NADH production in the cytosol plays a key role of ATP production through the mitochondrial electron transport chain in cancer cells, while NADH production is mostly occupied inside mitochondria in normal cells.

The transglutaminase 1 (TGase 1) enzyme is required for the formation of a cornified cell envelope in epidermal keratinocytes. We show here that in addition to its membrane-anchored form, soluble forms of it are also important in keratinocytes. Proliferating cells contain soluble full-length enzyme of 106 kDa, but terminally differentiating cells contain a soluble 67-kDa form often complexed with a 33-kDa protein as well. The amino terminus of the 67 kDa form is residue 93 of the TGase 1 protein, corresponding to the site of proteolytic activation of the factor XIIIa TGase. The amino terminus of the 33-kDa protein is residue 573, corresponding to the site of a second proteolytic cleavage site of factor XIIIa, and of the site for proteolytic activation of the TGase 3 enzyme. The specific activity of the 67/33-kDa soluble complex is twice that of the soluble 67-kDa form and 10 times that of full-length TGase 1. The half-lives of the 67/33- and 106-kDa forms are about 7 or 20 h, respectively. Thus the TGase 1 enzyme is complex, since it exists in keratinocytes as multiple soluble forms, either intact or proteolytically processed at conserved sites, and which have varying specific activities and likely functions. The transglutaminase 1 (TGase 1) enzyme is required for the formation of a cornified cell envelope in epidermal keratinocytes. We show here that in addition to its membrane-anchored form, soluble forms of it are also important in keratinocytes. Proliferating cells contain soluble full-length enzyme of 106 kDa, but terminally differentiating cells contain a soluble 67-kDa form often complexed with a 33-kDa protein as well. The amino terminus of the 67 kDa form is residue 93 of the TGase 1 protein, corresponding to the site of proteolytic activation of the factor XIIIa TGase. The amino terminus of the 33-kDa protein is residue 573, corresponding to the site of a second proteolytic cleavage site of factor XIIIa, and of the site for proteolytic activation of the TGase 3 enzyme. The specific activity of the 67/33-kDa soluble complex is twice that of the soluble 67-kDa form and 10 times that of full-length TGase 1. The half-lives of the 67/33- and 106-kDa forms are about 7 or 20 h, respectively. Thus the TGase 1 enzyme is complex, since it exists in keratinocytes as multiple soluble forms, either intact or proteolytically processed at conserved sites, and which have varying specific activities and likely functions. Of the six known active transglutaminases (TGases)1 1The abbreviations used are: TGase(s)transglutaminase(s)NHEKnormal human epidermal keratinocytesPVDFpolyvinylidene difluorideTBSTris-buffered salineTGase 1suggested new nomenclature for the membrane-associated enzyme (K)TGase 2suggested new nomenclature for the tissue enzyme (C)TGase 3suggested new nomenclature for pro-enzyme (E)FPLCfast protein liquid chromatographyTricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. in humans, three are expressed during terminal differentiation in stratified squamous epithelia such as the epidermis. These are the membrane-associated TGase 1 of about 92 kDa(1Rice R.H. Green H. Cell. 1977; 11: 417-422Google Scholar, 2Thacher S.M. Rice R.H. Cell. 1985; 40: 685-695Google Scholar, 3Chang S.-K. Chung S.-I. J. Biol. Chem. 1986; 261: 8112-8121Google Scholar, 4Jetten A.M. Shirley J.E. J. Biol. Chem. 1986; 261: 15097-15101Google Scholar, 5Schmidt R. Michel S. Shroot B. Reichert U. J. Invest. Dermatol. 1988; 90: 475-479Google Scholar, 6Rice R.H. Rong X. Chakravarty R. Biochem. J. 1990; 265: 351-357Google Scholar, 7Kim H.-C. Idler W.W. Kim I.-G. Han J.-H. Chung S.-I. Steinert P.M. J. Biol. Chem. 1991; 266: 536-539Google Scholar, 8Kim I.-G. McBride O.W. Wang M. Kim S.-Y. Idler W.W. Steinert P.M. J. Biol. Chem. 1992; 267: 7710-7717Google Scholar), the ubiquitous soluble tissue type TGase 2 of 80 kDa (9Chung S.-I. Ann. N. Y. Acad. Sci. 1972; 202: 240-255Google Scholar, 10Curtis C.G. Stenberg P. Brown K.L. Baron A. Chen K. Gray A. Simpson I. Lorand L. Biochemistry. 1974; 13: 3257-3262Google Scholar, 11Ikura K. Nasu T. Yokota H. Tsuchiya Y. Sasaki R. Chiba H. Biochemistry. 1988; 27: 2898-2905Google Scholar, 12Gentile V. Saydak M. Chiocca E.A. Akande O. Birckbichler P.J. Lee K.-N. Stein J.P. Davies P.J. J. Biol. Chem. 1991; 266: 478-483Google Scholar), and the soluble pro-enzyme TGase 3 of 77 kDa(13Chung S.-I. Folk J.E. Proc. Natl. Acad. Sci. U. S. A. 1972; 69: 303-308Google Scholar, 14Negi M. Colbert M.C. Goldsmith L.A. J. Invest. Dermatol. 1985; 85: 75-78Google Scholar, 15Park S.-C. Kim S.-Y. Kim H.-C. Thacher S.M. Chung S.-I. J. Cell Biol. 1988; 107: 139Google Scholar, 16Kim H.-C. Lewis M.S. Gorman J.J. Park S.-C. Girard J.E. Folk J.E. Chung S.-I. J. Biol. Chem. 1990; 265: 21971-21978Google Scholar, 17Kim I.-G. Gorman J.J. Lee S.-C. Park S.-C. Chung S.-I. Steinert P.M. J. Biol. Chem. 1993; 268: 12682-12690Google Scholar). These enzymes are thought to be responsible at least in part for the assembly of a cornified cell envelope, which provides a vitally important barrier function for the tissue(18Greenberg C.S. Birckbichler P.J. Rice R.H. FASEB J. 1991; 5: 3071-3077Google Scholar, 19Reichert U. Michel S. Schmidt R. Darmon M. Blumenberg M. Molecular Biology of the Skin: The Keratinocyte. Academic Press Inc., New York1993: 107-150Google Scholar). However, the mechanism of assembly of this structure and the substrate preferences, if any, of these three TGases in cornified cell envelope formation remains to be resolved. Such studies are complicated by the fact that the TGase 1 enzyme is perhaps the most difficult to work with because of its lability during isolation and purification(2Thacher S.M. Rice R.H. Cell. 1985; 40: 685-695Google Scholar, 20Thacher S.M. J. Invest. Dermatol. 1989; 92: 578-584Google Scholar, 21Kim S.-Y. Kim I.-G. Chung S.-I. Steinert P.M. J. Biol. Chem. 1994; 269: 27979-27986Google Scholar). However, we have recently demonstrated that a recombinant TGase 1 enzyme can be expressed in bacteria, which has an activity comparable with that isolated from cultured keratinocytes(21Kim S.-Y. Kim I.-G. Chung S.-I. Steinert P.M. J. Biol. Chem. 1994; 269: 27979-27986Google Scholar). Furthermore, by deletion cloning, removal of the first 36-98 residues results in large increases in specific activity as well as changes in reactivities toward and kinetic efficiencies with a number of potential cornified cell envelope substrates, but removal of up to 240 residues from the carboxyl-terminal end has little affect on activity(21Kim S.-Y. Kim I.-G. Chung S.-I. Steinert P.M. J. Biol. Chem. 1994; 269: 27979-27986Google Scholar). Thus an important question arises as to whether smaller highly active forms of the TGase 1 system exist in cells. transglutaminase(s) normal human epidermal keratinocytes polyvinylidene difluoride Tris-buffered saline suggested new nomenclature for the membrane-associated enzyme (K) suggested new nomenclature for the tissue enzyme (C) suggested new nomenclature for pro-enzyme (E) fast protein liquid chromatography N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. In addition, a truncated recombinant form of 467 amino acid residues, which retained half of the activity of the full-length TGase 1 expressed form or native enzyme from cultured cells(21Kim S.-Y. Kim I.-G. Chung S.-I. Steinert P.M. J. Biol. Chem. 1994; 269: 27979-27986Google Scholar), has been used to make a TGase 1 polyclonal antibody. This antibody decorates the entire epidermis, including the basal layers and epidermal derivative organs such as the hair follicle(22Kim S.-Y. Yoneda K. Chung S.-I. Steinert P.M. J. Invest. Dermatol. 1995; 103: 211-217Google Scholar, 23Steinert P.M. Lee S.-C. Kim S.-Y. Yoneda K. Chonnam J. Med. Sci. 1994; 6: 78-87Google Scholar). In contrast, a widely used commercially available TGase 1 monoclonal antibody (B.C1) decorates only the granular layer of the epidermis(20Thacher S.M. J. Invest. Dermatol. 1989; 92: 578-584Google Scholar, 24Michel S. Bernerd F. Jetten A.M. Floyd E.E. Shroot D. Reichert U. J. Invest. Dermatol. 1992; 98: 364-368Abstract Full Text PDF Google Scholar, 25Schroeder W.T. Thacher S.M. Stewart-Galetka S. Annarella M. Chema D. Siciliano M.J. Davies P.J. Tang H.-Y. Sowa B.A. Duvic M. J. Invest. Dermatol. 1992; 99: 27-34Abstract Full Text PDF Google Scholar, 26Duvic M. Nelson D.C. Annarella M. Cho M. Esgleyes-Ribot T. Remenyik E. Ulmer R. Rapini R.P. Sacks P.G. Clayman G.L. Davies P.J.A. Thacher S. J. Invest. Dermatol. 1994; 102: 462-469Abstract Full Text PDF Google Scholar). By Western blotting methods, the monoclonal antibody recognizes a band of about 90 kDa(20Thacher S.M. J. Invest. Dermatol. 1989; 92: 578-584Google Scholar, 26Duvic M. Nelson D.C. Annarella M. Cho M. Esgleyes-Ribot T. Remenyik E. Ulmer R. Rapini R.P. Sacks P.G. Clayman G.L. Davies P.J.A. Thacher S. J. Invest. Dermatol. 1994; 102: 462-469Abstract Full Text PDF Google Scholar), thought to be the size of the full-length TGase 1 enzyme in cultured epithelial cells and epidermal tissue extracts, but the major proteins recognized and immunoprecipitated by it have molecular masses of 10-20 kDa, which we have recently demonstrated are the SPR1 and SPR2 proteins also expressed in the epidermis(22Kim S.-Y. Yoneda K. Chung S.-I. Steinert P.M. J. Invest. Dermatol. 1995; 103: 211-217Google Scholar). However, our new antibody recognizes a major band of 106 kDa, which is apparently the true full-length size of the TGase 1 enzyme in keratinocytes(21Kim S.-Y. Kim I.-G. Chung S.-I. Steinert P.M. J. Biol. Chem. 1994; 269: 27979-27986Google Scholar, 22Kim S.-Y. Yoneda K. Chung S.-I. Steinert P.M. J. Invest. Dermatol. 1995; 103: 211-217Google Scholar). This 15% increase in size may be due to postsynthetic modifications of a basic core protein of 92 kDa(7Kim H.-C. Idler W.W. Kim I.-G. Han J.-H. Chung S.-I. Steinert P.M. J. Biol. Chem. 1991; 266: 536-539Google Scholar, 8Kim I.-G. McBride O.W. Wang M. Kim S.-Y. Idler W.W. Steinert P.M. J. Biol. Chem. 1992; 267: 7710-7717Google Scholar, 27Chakravarty R. Rice R.H. J. Biol. Chem. 1989; 264: 625-629Google Scholar). In addition to the band of 106 kDa, our antibody recognized several other minor bands of lower molecular weight(21Kim S.-Y. Kim I.-G. Chung S.-I. Steinert P.M. J. Biol. Chem. 1994; 269: 27979-27986Google Scholar, 22Kim S.-Y. Yoneda K. Chung S.-I. Steinert P.M. J. Invest. Dermatol. 1995; 103: 211-217Google Scholar), also reported earlier(2Thacher S.M. Rice R.H. Cell. 1985; 40: 685-695Google Scholar), that may be due to degradation of the TGase 1 protein or cross-reactivity with other TGase proteins of the keratinocytes. In the process of a systematic analysis of these possibilities by use of immunoprecipitation and Western blotting experiments, we have found to our surprise that the TGase 1 system in cultured epidermal keratinocytes and foreskin epidermal cells is far more complicated than heretofore described. It has been thought that most of the “soluble” (that is, cytosolic) TGase activity in cultured epidermal cells is due to the TGase 2 enzyme(2Thacher S.M. Rice R.H. Cell. 1985; 40: 685-695Google Scholar, 16Kim H.-C. Lewis M.S. Gorman J.J. Park S.-C. Girard J.E. Folk J.E. Chung S.-I. J. Biol. Chem. 1990; 265: 21971-21978Google Scholar, 18Greenberg C.S. Birckbichler P.J. Rice R.H. FASEB J. 1991; 5: 3071-3077Google Scholar, 19Reichert U. Michel S. Schmidt R. Darmon M. Blumenberg M. Molecular Biology of the Skin: The Keratinocyte. Academic Press Inc., New York1993: 107-150Google Scholar, 28Connellan J.M. Chung S.-I. Whetzel N.K. Bradley L.M. Folk J.E. J. Biol. Chem. 1970; 246: 1093-1098Google Scholar), whereas most of the TGase 1 activity is anchored to membranes(2Thacher S.M. Rice R.H. Cell. 1985; 40: 685-695Google Scholar, 18Greenberg C.S. Birckbichler P.J. Rice R.H. FASEB J. 1991; 5: 3071-3077Google Scholar, 19Reichert U. Michel S. Schmidt R. Darmon M. Blumenberg M. Molecular Biology of the Skin: The Keratinocyte. Academic Press Inc., New York1993: 107-150Google Scholar). We describe here that most of the soluble TGase activity in cultured keratinocytes is in fact due to soluble full-length or smaller more active forms of the TGase 1 enzyme, generated by proteolytic processing of the full-length protein at specific sequence sites comparable with the sites of activation of other TGases. Normal human foreskin epidermal keratinocytes (NHEK) (Clonetics Corp., San Diego, CA) were seeded at a density of 5.103 cells/cm2 in 15-cm dishes and grown in low Ca2+ KGM medium (0.05 mM CaCl2) as recommended by the manufacturer. In some cases, at confluence (about 3 days) the medium CaCl2 concentration was raised to 0.6 mM (high Ca2+), previously determined to be optimal for induction of terminal differentiation in human epidermal keratinocytes in cell culture(29Yuspa S.H. Kilkenny A.E. Steinert P.M. Roop D.R. J. Cell Biol. 1989; 109: 1207-1218Google Scholar). In other high Ca2+ experiments, the calcium ionophore A23187 (Calbiochem) (30Cline P.R. Rice R.H. Cancer Res. 1983; 43: 3203-3207Google Scholar) was also added to cultures at confluence (25 μg/ml, final concentration). In most experiments, the cultures were metabolically labeled with a mixture of [35S]cysteine and [35S]methionine (1 μCi each/ml of medium) (Amersham Corp.). This was added either (i) 4 h before planned harvesting of cells, or (ii) in pulse-chase experiments, for 4 h in 2 day post-confluent cultures grown in the presence of high Ca2+, followed by replacement with unlabeled medium. As required, the cells were harvested by scraping and sonicated in a buffer containing 0.1 M Tris acetate, 0.15 M NaCl, 1 mM EDTA (pH 7.5) (TBS) (4 × 108 cells/ml) in the presence (or absence in the case of one control set of experiments) of a mixture of protease inhibitors leupeptin (1 mM), 4-(2-aminoethyl)-benzenesulfonyl fluoride (0.2 mM), calpain inhibitor (10 μM), and aprotinin (0.1 unit/ml) (Boehringer Mannhiem). The lysate was clarified by centrifugation at 10,000 × g for 20 min at 4°C to obtain the cytosolic (soluble) fraction. Freshly excised foreskins were cut open and cultured for 4 h in suspension organ culture (5 ml/tissue) in Dulbecco's modified Eagle's medium in the absence or presence of 100 μCi each of [35S]cysteine and [35S]methionine. Following thorough washing in phosphate-buffered saline, the tissues were floated on trypsin (Difco) overnight at 4°C to separate the epidermis, from which a total epidermal cell suspension was recovered by standard procedures. These cells were then plated on plastic for 4 h exactly as described previously(31Yuspa S.H. Harris C.C. Exp. Cell Res. 1974; 86: 95-105Google Scholar), during which time essentially only the basal cells attached(32Jones P.H. Harper S. Watt F.M. Cell. 1995; 80: 83-93Google Scholar). Both the attached and unattached suprabasal cells committed to terminal differentiation were then harvested, sonicated in the TBS buffer, and pelleted to recover the cytosolic fraction as above. Total 35S-labeled foreskin tissues (epidermis + dermis) were used to prepare human actin exactly as described(33Steinert P.M. Peck G.L. Yuspa S.H. McGuire J.S. DiPasquale A. J. Invest. Dermatol. 1976; 66: 276Google Scholar). In some experiments, aliquots of the total cell lysates or cytosolic fractions were boiled in polyacrylamide gel loading buffer containing 2% SDS and 2% 2-mercaptoethanol, and proteins were resolved on 10% linear or 10-20% gradient gels. Following transfer to PVDF membranes, bands were identified by Western blotting with specific TGase antibodies and developed with the Bio-Rad reagent(16Kim H.-C. Lewis M.S. Gorman J.J. Park S.-C. Girard J.E. Folk J.E. Chung S.-I. J. Biol. Chem. 1990; 265: 21971-21978Google Scholar). Six recombinant deletion constructs of the human TGase 1 enzyme that produced particularly stable active enzymes were prepared exactly as described previously (21Kim S.-Y. Kim I.-G. Chung S.-I. Steinert P.M. J. Biol. Chem. 1994; 269: 27979-27986Google Scholar) and were constructs 1 (full length, 92 kDa), 2 (ΔN37), 3 (ΔN52), 4 (ΔN61), 5 (ΔN97), and 20 (ΔN108ΔC575). [35S]Cysteine-labeled construct 1 was routinely used as a marker for autoradiography of SDS gels. These recombinant proteins, as well as the native full-length 106-kDa enzyme (prepared from cultured NHEK cells as described below), were substrates for proteolysis using 0.01 unit/ml of dispase (Boehringer Mannhiem) (about 1:20 protease to TGase ratio). The three specific affinity-purified antibodies used were: our new polyclonal anti-human TGase 1 made in goats(22Kim S.-Y. Yoneda K. Chung S.-I. Steinert P.M. J. Invest. Dermatol. 1995; 103: 211-217Google Scholar), polyclonal anti-guinea pig liver TGase 2 made in rabbits which cross-reacts with the human enzyme(34Folk J.E. Chung S.-I. Methods Enzymol. 1985; 113: 358-375Google Scholar), and polyclonal anti-guinea pig skin TGase 3 made in rabbits which cross-reacts with the human enzyme (16Kim H.-C. Lewis M.S. Gorman J.J. Park S.-C. Girard J.E. Folk J.E. Chung S.-I. J. Biol. Chem. 1990; 265: 21971-21978Google Scholar). Each of the three antibodies used in this study was serially diluted 10-1 to 10-4 in TBS to explore optimal precipitation of enzyme activity. In control experiments, we found that essentially complete immunoprecipitation of the respective 35S-labeled antigens and activity occurred with a 1:50 dilution of each antibody(21Kim S.-Y. Kim I.-G. Chung S.-I. Steinert P.M. J. Biol. Chem. 1994; 269: 27979-27986Google Scholar, 22Kim S.-Y. Yoneda K. Chung S.-I. Steinert P.M. J. Invest. Dermatol. 1995; 103: 211-217Google Scholar). Cell lysate fractions of NHEK cells or separated foreskin epidermal cells (200 μl) were incubated with the antibodies for 1 h at 4°C by gentle shaking. Protein A-conjugated agarose beads (ImmunoPure Plus, Pierce) were pre-equilibrated in TBS buffer, and resuspended as a 1:1 slurry in TBS, of which 20 μl was then added to the cytosolic primary antibody mixtures. Following incubation for 1 h at 4°C in a rotary shaker, the conjugated beads of each reaction were collected by centrifugation at 10,000 × g for 2 min and washed twice with TBS to remove unabsorbed proteins. In most experiments, the absorbed antigens and primary antibodies were harvested by boiling the washed beads in 50 μl of SDS sample buffer containing 10% 2-mercaptoethanol, resolved by electrophoresis on 10% linear or 10-20% gradient polyacrylamide gels, dried, and autoradiographed. Gels were exposed to x-ray film for 1-15 days. In some autoradiograms, selected bands were quantitated by scanning in a computing densitometer with ImageQuant software version 3.0 (Molecular Dynamics). Standard protein markers (Life Technologies, Inc.) were used. In some experiments, the absorbed active TGase proteins were recovered from the washed beads with 100 μl of elution buffer (Pierce). After 30 s, the suspension was neutralized with 25 μl of 1 M Tris acetate buffer (pH 7.5) and then pelleted to remove the beads. The time of exposure to the low pH (~2.8) elution buffer was strictly limited to ~30 s. Control experiments showed that (i) >95% of the 35S-labeled TGase protein antigens were eluted within 30 s, and (ii) the half-life of TGase 1 activity in the low pH buffer is about 4 min. The TGase 1, 2, and 3 enzymes were recovered from cytosolic fractions of unlabeled or 35S-labeled foreskin epidermal “suprabasal” cells (for the TGase 3 proenzyme) or “basal” cells (for the soluble TGase 1 and TGase 2 enzymes). Samples were chromatographed by FPLC on a 0.5 × 5 cm mono-Q FPLC column equilibrated in a buffer of 50 mM Tris acetate (pH 7.5) containing 1 mM EDTA using 60 ml of a 0-0.5 M NaCl linear gradient, and collected into 0.5-ml fractions, essentially as described(16Kim H.-C. Lewis M.S. Gorman J.J. Park S.-C. Girard J.E. Folk J.E. Chung S.-I. J. Biol. Chem. 1990; 265: 21971-21978Google Scholar). Peaks of TGase activities were ascertained by assays of every second fraction. The TGase 3 eluted in the column wash, and the TGase 1 and 2 enzymes eluted at about 0.2 or 0.3 M NaCl, as expected(2Thacher S.M. Rice R.H. Cell. 1985; 40: 685-695Google Scholar, 16Kim H.-C. Lewis M.S. Gorman J.J. Park S.-C. Girard J.E. Folk J.E. Chung S.-I. J. Biol. Chem. 1990; 265: 21971-21978Google Scholar). 35S-Labeled TGases from immunoprecipitation reactions of cytosolic fractions with either the TGase 1 (22Kim S.-Y. Yoneda K. Chung S.-I. Steinert P.M. J. Invest. Dermatol. 1995; 103: 211-217Google Scholar) or TGase 2 (34Folk J.E. Chung S.-I. Methods Enzymol. 1985; 113: 358-375Google Scholar) antibody were resolved similarly and detected by counting every second fraction. In this case, the eluted 35S-labeled TGases were neutralized with 1 M Tris acetate (pH 7.8) and diluted to 1 ml before loading. TGase enzyme forms were also recovered from the cytosolic fraction of 3-day post-confluent NHEK cells that had been grown in high Ca2+ and in the presence of the calcium ionophore. The 35S-labeled cytosolic fraction was immunoprecipitated with antibodies, eluted, neutralized, and the products chromatographed as described above and counted. An alternative method to recover the full-length 106-kDa TGase 1 enzyme was to fractionate the unlabeled cytosolic fraction from 1-day post-confluent NHEK cells grown in low Ca2+. The specificities of the TGase antibodies used was also examined in double immunoprecipitation reactions. In this case, the three 35S-labeled TGases purified from foreskin epidermal cells as described above were used for a second round of immunoprecipitations with various combinations of antibodies (see Fig. 2A). Alternatively, TGase 1 or TGase 2 antigens of 3-day post-confluent NHEK cells grown in high Ca2+ were first harvested from separate affinity columns, eluted, neutralized, and concentrated, exactly as described before(21Kim S.-Y. Kim I.-G. Chung S.-I. Steinert P.M. J. Biol. Chem. 1994; 269: 27979-27986Google Scholar, 22Kim S.-Y. Yoneda K. Chung S.-I. Steinert P.M. J. Invest. Dermatol. 1995; 103: 211-217Google Scholar). Standard TGase assays were performed by measurement of the incorporation of [3H]putrescine (Amersham) into succinylated casein(21Kim S.-Y. Kim I.-G. Chung S.-I. Steinert P.M. J. Biol. Chem. 1994; 269: 27979-27986Google Scholar). Protein assays were performed colorimetrically (Bio-Rad)(35Bradford M.M. Anal. Biochem. 1976; 72: 248-254Google Scholar). Samples of unlabeled active TGase species from the Mono Q FPLC experiments were used for titrations with 5 mM [14C]iodoacetamide to measure the amount of active TGase protein(13Chung S.-I. Folk J.E. Proc. Natl. Acad. Sci. U. S. A. 1972; 69: 303-308Google Scholar, 21Kim S.-Y. Kim I.-G. Chung S.-I. Steinert P.M. J. Biol. Chem. 1994; 269: 27979-27986Google Scholar). Subsequent immunoprecipitation of the inactivated [14C]methylcarboxamide cysteine-TGase(s) with the polyclonal TGase 1, 2, or 3 antibodies enabled measurement of radioactivity incorporated, from which the amount of each TGase protein and its specific activity was then calculated(21Kim S.-Y. Kim I.-G. Chung S.-I. Steinert P.M. J. Biol. Chem. 1994; 269: 27979-27986Google Scholar). While it was possible in the present work to purify 35S-labeled TGases, direct specific activity measurements were hampered by the overlap of energies of β decay of the [35S]methionine/cysteine and [3H]putrescine isotopes. Specific activities were not measured on TGase proteins previously exposed to the low pH antibody elution buffer of either ImmunoPure beads or affinity columns. The products of some immunoprecipitation reactions using the TGase 1 antibody from the cytosolic fractions of NHEK cells, as described above, were resolved on a 10% polyacrylamide gel run in Tricine buffers and transferred to PVDF membranes. The bands containing 2-10 pmol of the 106-, 72 (minor)-, 67-, 65 (minor)-, and 33-kDa proteins were excised, placed in a LF3500 gas-liquid phase sequencer (Porton), and run for 15 Edman degradation cycles. Released phenylthiohydantoin-derivatized amino acids were resolved and quantitated by on-line analytical high performance liquid chromatography (Beckmam Instruments, using System Gold software). The 106-kDa band initially did not give a sequence. Another sample of this band on PVDF membrane was boiled in 5.7 N HCl at 106°C in vacuo for 2 h to hydrolyze off the probable NH2-terminal blocking adduct. The acid solution was dried, redissolved in 5 μl of 50% aqueous acetonitrile, and covalently attached to a PVDF solid support (Sequelon-AA, Millipore) for sequencing. The purpose of the present experiments was to better characterize the properties of the TGase 1 system than has heretofore been possible. Our earlier work has documented significant differences in the published expression properties of the TGase 1 enzyme system using our new polyclonal antibody (21Kim S.-Y. Kim I.-G. Chung S.-I. Steinert P.M. J. Biol. Chem. 1994; 269: 27979-27986Google Scholar, 22Kim S.-Y. Yoneda K. Chung S.-I. Steinert P.M. J. Invest. Dermatol. 1995; 103: 211-217Google Scholar) as compared with use of a commercial monoclonal antibody (B.C1)(24Michel S. Bernerd F. Jetten A.M. Floyd E.E. Shroot D. Reichert U. J. Invest. Dermatol. 1992; 98: 364-368Abstract Full Text PDF Google Scholar, 25Schroeder W.T. Thacher S.M. Stewart-Galetka S. Annarella M. Chema D. Siciliano M.J. Davies P.J. Tang H.-Y. Sowa B.A. Duvic M. J. Invest. Dermatol. 1992; 99: 27-34Abstract Full Text PDF Google Scholar, 26Duvic M. Nelson D.C. Annarella M. Cho M. Esgleyes-Ribot T. Remenyik E. Ulmer R. Rapini R.P. Sacks P.G. Clayman G.L. Davies P.J.A. Thacher S. J. Invest. Dermatol. 1994; 102: 462-469Abstract Full Text PDF Google Scholar). In addition, Western blots of cytosolic fractions from NHEK cells using the polyclonal antibody recognized a major band of TGase 1 protein of 106 kDa, as well as minor bands between 70 and 90 kDa and below 50 kDa (21Kim S.-Y. Kim I.-G. Chung S.-I. Steinert P.M. J. Biol. Chem. 1994; 269: 27979-27986Google Scholar, 22Kim S.-Y. Yoneda K. Chung S.-I. Steinert P.M. J. Invest. Dermatol. 1995; 103: 211-217Google Scholar). However, because these bands were relatively weak, in the present work we have metabolically labeled NHEK cells or human foreskin epidermis with [35S]methionine and [35S]cysteine and immunoprecipitated the TGase proteins in order to better visualize them by autoradiography. The amount of 35S label incorporated during 4 h in cell culture was in the range of 0.5-3 × 104 dpm/108 cells. When cultured for several days post-confluence in low Ca2+ medium, under which conditions the cells do not differentiate to a significant extent(29Yuspa S.H. Kilkenny A.E. Steinert P.M. Roop D.R. J. Cell Biol. 1989; 109: 1207-1218Google Scholar), most of the TGase 1 immunoprecipitable protein was a major band of 106 kDa, as expected, as well as minor bands of about 67 and 33 kDa, and other very minor species of 70-95 and 45-65 kDa (Fig. 1A). When confluent cultures were changed to high Ca2+ medium (not shown, but see Fig. 2B, lane 1), and in the presence of the calcium ionophore A23187 (Fig. 1B), the 67- and 33-kDa forms were major products of the immunoprecipitation reaction, which increased in time as the cells differentiated. However, in order to ascertain that these bands are not due to cross-reactivity of our polyclonal TGase 1 antibody with other proteins or TGases, a series of control experiments was performed. First, we purified active 35S-labeled TGase 1, 2, and 3 enzymes from labeled foreskin epidermal cells as described (16Kim H.-C. Lewis M.S. Gorman J.J. Park S.-C. Girard J.E. Folk J.E. Chung S.-I. J. Biol. Chem. 1990; 265: 21971-21978Google Scholar) (see Figure 4:, Figure 5:, Figure 6:, Figure 7:) and used them for double immunoprecipitation reactions (Fig. 2A). In the case of our polyclonal TGase 1 antibody, a second immunoprecipitation reaction was able to reprecipitate 98% of the 35S-labeled 106-kDa band from its first precipitation reaction (Fig. 2A, lane 2), but the TGase 2 antibody could only precipitate 2% (lane 3), and the TGase 3 antibody, <1% (lane 4). Likewise, the TGase 2 antibody used could reprecipitate 97% of its first reaction of 80 kDa (lane 7), but the TGase 1 and TGase 3 antibodies could precipitate only 1% or <1% (lanes 6 and 8, respectively). The TGase 3 antibody displayed a similar degree of specificity: <1% of its immunoprecipitation product of about 77 kDa could be reprecipitated by the antibodies for TGases 1 and 2 (lanes 10 and 11, respectively). Two internal controls were done: (i) the second antibody (ImmunoPure beads) used as the primary antibody bound <2% of the 35S label in each case, and (ii) each of these three antibodies could precipitate <1% of 35S-labeled actin either in the first or second precipitation reactions (data not shown). In a second series of control experiments, the TGase 1 and 2 antibodies were used harvest antigens from separate affinity columns (21Kim S.-Y. Kim I.-G. Chung S.-I. Steinert P.M. J. Biol. Chem. 1994; 269: 27979-27986Google Scholar, 22Kim S.-Y. Yoneda K. Chung S.-I. Steinert P.M. J. Invest. Dermatol. 1995; 103: 211-217Google Scholar) using the cytosolic fractions of 35S-labeled confluent NHEK cells cultured in the high Ca2+ for 3 days. Then in immunoreprecipitation reactions, the TGase 1 antibody reprecipitated >95% of its first reaction of the 106, 67, and 33 kDa bands (Fig. 2B, compare lane 2 with lane 1), but the TGase 2 and 3 antibodies reprecipitated <1% and about 2% (lanes 3 and 4, respectively). Likewise, the TGase 2 antibody reprecipitated most of its first reaction, but <2% could be reprecipitated by the TGase 1 antibody (compare lanes 6 and 8 with lane 7). These controls show that the three antibodies are highly specific, since they display only trace amounts of cross-reactivity, in confirmation of our earlier data(16Kim H.-C. Lewis M.S. Gorman J.J. Park S.-C. Girard J.E. Folk J.E. Chung S.-I. J. Biol. Chem. 1990; 265: 21971-21978Google Scholar, 22Kim

Many reports have shown that the expression of transglutaminase 2 (TG 2) is increased in inflammatory diseases. Although during the last several decades multiple physiological roles for TG 2 have been demonstrated in various cell types, its role in the inflammatory process is not yet clear. TG 2 is a crosslinking enzyme that is widely used in many biological systems for tissue stabilization purposes and immediate defense against injury or infection. Aberrant activation of TG 2 activity in tissues contributes to a variety of diseases including neurodegenerative diseases, autoimmune diseases, and cancers. In most cases, TG 2 appears to form an inappropriate protein aggregate that may be cytotoxic enough to trigger inflammation and/or apoptosis. In some cases, such as celiac disease and rheumatoid arthritis, TG 2 is also associated with the pathogenic progression, as well as in the generation of autoantibodies. Recently, we discovered that increased TG 2 activity triggers NF-kappaB activation without I-kappaBalpha kinase signaling. TG 2 induces the polymerization of I-kappaBalpha rather than stimulating I-kappaBalpha kinase. This polymerization of I-kappaB results in the direct activation of NF-kappaB in various cell lines. We also found that TG inhibition reverses NF-kappaB activation. Interestingly, this coincides with the reversal of inflammation in conjunctivitis models by treatment with TG 2 inhibitors. Here, I introduce a new role for TG 2 as a signal modulator, which may suggest a new paradigm for the inflammatory process.

We found that non-small-cell lung cancer (NSCLC) cells express high levels of multiple aldehyde dehydrogenase (ALDH) isoforms via an informatics analysis of metabolic enzymes in NSCLC and immunohistochemical staining of NSCLC clinical tumor samples. Using a multiple reaction-monitoring mass spectrometry analysis, we found that multiple ALDH isozymes were generally abundant in NSCLC cells compared with their levels in normal IMR-90 human lung cells. As a result of the catalytic reaction mediated by ALDH, NADH is produced as a by-product from the conversion of aldehyde to carboxylic acid. We hypothesized that the NADH produced by ALDH may be a reliable energy source for ATP production in NSCLC. This study revealed that NADH production by ALDH contributes significantly to ATP production in NSCLC. Furthermore, gossypol, a pan-ALDH inhibitor, markedly reduced the level of ATP. Gossypol combined with phenformin synergistically reduced the ATP levels, which efficiently induced cell death following cell cycle arrest. The role of aldehyde dehydrogenase enzymes (ALDH) in the most common type of lung cancer cells offers new opportunities for treatment. 'Non-small-cell lung cancer' cells are found in around 80-90 per cent of lung cancers and are relatively insensitive to chemotherapy. The cancer cells are sustained by high levels of the energy-supplying molecule adenosine triphosphate (ATP). Researchers in South Korea led by Soo-Youl Kim at the National Cancer Center, Goyang, detected high levels of several forms of ALDH in the cancer cells. They uncovered a mechanism whereby the ALDH significantly contributed to the high ATP levels. A known inhibitor of ALDH enzymes called gossypol, combined with the drug phenformin, stopped cultured cancer cells from multiplying by ATP depletion and induced cell death through cell cycle arrest. The therapeutic potential of this new insight into the cancer cells' metabolism warrants further investigation.

Abstract Phosphorylation-dependent YAP translocation is a well-known intracellular mechanism of the Hippo pathway; however, the molecular effectors governing YAP cytoplasmic translocation remains undefined. Recent findings indicate that oncogenic YAP paradoxically suppresses Wnt activity. Here, we show that Wnt scaffolding protein Dishevelled (DVL) is responsible for cytosolic translocation of phosphorylated YAP. Mutational inactivation of the nuclear export signal embedded in DVL leads to nuclear YAP retention, with an increase in TEAD transcriptional activity. DVL is also required for YAP subcellular localization induced by E-cadherin, α-catenin, or AMPK activation. Importantly, the nuclear-cytoplasmic trafficking is dependent on the p53-Lats2 or LKB1-AMPK tumor suppressor axes, which determine YAP phosphorylation status. In vivo and clinical data support that the loss of p53 or LKB1 relieves DVL-linked reciprocal inhibition between the Wnt and nuclear YAP activity. Our observations provide mechanistic insights into controlled proliferation coupled with epithelial polarity during development and human cancer.

Targeted approaches for treating glioblastoma (GBM) attempted to date have consistently failed, highlighting the imperative for treatment strategies that operate on different mechanistic principles. Bioenergetics deprivation has emerged as an effective therapeutic approach for various tumors. We have previously found that cancer cells preferentially utilize cytosolic NADH supplied by aldehyde dehydrogenase (ALDH) for ATP production through oxidative phosphorylation (OxPhos). This study is aimed at examining therapeutic responses and underlying mechanisms of dual inhibition of ALDH and OxPhos against GBM. For inhibition of ALDH and OxPhos, the corresponding inhibitors, gossypol and phenformin were used. Biological functions, including ATP levels, stemness, invasiveness, and viability, were evaluated in GBM tumorspheres (TSs). Gene expression profiles were analyzed using microarray data. In vivo anticancer efficacy was examined in a mouse orthotopic xenograft model. Combined treatment of GBM TSs with gossypol and phenformin significantly reduced ATP levels, stemness, invasiveness, and cell viability. Consistently, this therapy substantially decreased expression of genes associated with stemness, mesenchymal transition, and invasion in GBM TSs. Supplementation of ATP using malate abrogated these effects, whereas knockdown of ALDH1L1 mimicked them, suggesting that disruption of ALDH-mediated ATP production is a key mechanism of this therapeutic combination. In vivo efficacy confirmed remarkable therapeutic responses to combined treatment with gossypol and phenformin. Our findings suggest that dual inhibition of tumor bioenergetics is a novel and effective strategy for the treatment of GBM.

Steroidal anti-inflammatory drugs induce proteins that inhibit phospholipase A 2 (PLA 2 ), including uteroglobin and lipocortin-1 (annexin I).Uteroglobin and lipocortin-1 retain several conserved sequences.Based on these sequences, several nonapeptides (antiflammins) were synthesized.These nonapeptides were shown to have anti-inflammatory effects in vitro and in vivo, possibly by inhibiting PLA 2 .Subsequent research showed that PLA 2 is activated by transglutaminase 2 (TGase 2).We hypothesize here that TGase 2 inhibitors may increase the anti-inflammatory efficacy of inhibiting PLA 2 activity.To test this theory, we constructed recombinant peptides containing sequences from pro-elafin (for inhibition of TGase 2), and from lipocortin-1, lipocortin-5, and uteroglobin (for inhibition of PLA 2 ).The recombinant peptides, which had dual inhibitory effects on purified TGase 2 and PLA 2 , reversed the inflammation of allergic conjunctivitis to ragweed in a guinea pig model.The present work suggests that novel recombinant peptides may be safe and effective agents for the treatment of various inflammatory diseases.

The decision between survival and death in cells exposed to TNF relies on a highly regulated equilibrium between proapoptotic and antiapoptotic factors. The TNF-activated antiapoptotic response depends on several transcription factors, including NF-κB and its RelA/p65 subunit, that are activated through phosphorylation-mediated degradation of IκB inhibitors, a process controlled by the IκB kinase complex. Genetic studies in mice have identified the IκB kinase-related kinase TANK-binding kinase 1 (TBK1; also called NAK or T2K) as an additional regulatory molecule that promotes survival downstream of TNF, but the mechanism through which TBK1 exerts its survival function has remained elusive. Here we show that TBK1 triggers an antiapoptotic response by controlling a specific RelA/p65 phosphorylation event. TBK1-induced RelA phosphorylation results in inducible expression of plasminogen activator inhibitor-2 (PAI-2), a member of the serpin family with known antiapoptotic activity. PAI-2 limits caspase-3 activation through stabilization of transglutaminase 2 (TG2), which cross-links and inactivates procaspase-3. Importantly, Tg2(-/-) mice were found to be more susceptible to apoptotic cell death in two models of TNF-dependent acute liver injury. Our results establish PAI-2 and TG2 as downstream mediators in the antiapoptotic response triggered upon TBK1 activation.

Transglutaminase 2 (TG2) is a calcium-dependent multifunctional protein associated with various human diseases. We determined the crystal structure of human TG2 in complex with adenosine triphosphate (ATP). The ATP molecule binds to the previously identified guanosine diphosphate (GDP) binding pocket but has different hydrogen bonds and ion interaction with protein. The four residues Arg476, Arg478, Val479 and Tyr583, all of which are involved in both ATP and GDP binding by hydrogen bonds, might play important roles in the stabilization of TG2 by ATP or GDP. However, Ser482 and Arg580, which are involved in GDP binding, do not form hydrogen bond with ATP. Additionally, we newly discovered an intramolecular disulfide bond between Cys230 and Cys370, which formation might regulate the enzymatic activity of TG2.

Renal cell carcinoma (RCC), the predominant form of kidney cancer, is characterized by high resistance to radiation and chemotherapy. This study shows that expression of protein cross-linking enzyme transglutaminase 2 (TGase 2) is markedly increased in 7 renal cell carcinoma (RCC) cell lines in comparison to HEK293 and other cancer cell lines, such as NCI 60. However, the key role of TGase 2 in RCC was not clear. The down-regulation of TGase 2 was found to stabilize p53 expression, thereby inducing a 3- to 10-fold increase in apoptosis for 786-O, A498, CAKI-1, and ACHN cell lines by DAPI staining. MEF cells from TGase 2(-/-) mice showed stabilized p53 under apoptotic stress to compare to MEFs from wild-type mice. TGase 2 directly cross links the DNA binding domain of p53, leading to p53 depletion via autophagy in RCC. TGase 2 and p53 expression showed an inverse relationship in RCC cells. This finding implies that induced expression of TGase 2 promotes tumor cell survival through p53 depletion in RCC.

Among ALDH isoforms, ALDH1L1 in the folate pathway showed highly increased expression in non-small-cell lung cancer cells (NSCLC). Based on the basic mechanism of ALDH converting aldehyde to carboxylic acid with by-product NADH, we suggested that ALDH1L1 may contribute to ATP production using NADH through oxidative phosphorylation. ALDH1L1 knockdown reduced ATP production by up to 60% concomitantly with decrease of NADH in NSCLC. ALDH inhibitor, gossypol, also reduced ATP production in a dose dependent manner together with decrease of NADH level in NSCLC. A combination treatment of gossypol with phenformin, mitochondrial complex I inhibitor, synergized ATP depletion, which efficiently induced cell death. Pre-clinical xenograft model using human NSCLC demonstrated a remarkable therapeutic response to the combined treatment of gossypol and phenformin.

Diabetic retinopathy is predominantly caused by vascular endothelial growth factor (VEGF)-induced vascular leakage; however, the underlying mechanism is unclear. Here we designed an in vivo transglutaminase (TGase) activity assay in mouse retina and demonstrated that hyperglycemia induced vascular leakage by activating TGase2 in diabetic retina. VEGF elevated TGase2 activity through sequential elevation of intracellular Ca(2+) and reactive oxygen species (ROS) concentrations in endothelial cells. The TGase inhibitors cystamine and monodansylcadaverin or TGase2 small interfering RNA (siRNA) prevented VEGF-induced stress fiber formation and vascular endothelial (VE)-cadherin disruption, which play a critical role in modulating endothelial permeability. Intravitreal injection of two TGase inhibitors or TGase2 siRNA successfully inhibited hyperglycemia-induced TGase activation and microvascular leakage in the retinas of diabetic mice. C-peptide or ROS scavengers also inhibited TGase activation in diabetic mouse retinas. The role of TGase2 in VEGF-induced vascular leakage was further supported using diabetic TGase2(-/-) mice. Thus, our findings suggest that ROS-mediated activation of TGase2 plays a key role in VEGF-induced vascular leakage by stimulating stress fiber formation and VE-cadherin disruption.

Transglutaminase 2 (TGase2, TG2) activity has been implicated in the pathogenesis of a number of unrelated disorders, including celiac, neurological, and renal diseases, and various forms of cancer. It has been suggested that TGase2 activity, such as cross-linking, deamidation, and GTP-related activity, is associated with each disease. Continuing efforts to develop small molecule TG2 inhibitors are ongoing. To develop a new class of TG2 inhibitors, the factors impeding the development of TG2 inhibitors have been identified. Additionally, the conformational effect of TG2 enzyme in regard to its pathological roles, in vitro screening methods, recently discovered TG2 inhibitors, and preclinical evaluations are discussed with a brief summary of current TG2 inhibitor pipelines under the clinical setting.

Several metabolic pathways for the supply of adenosine triphosphate (ATP) have been proposed; however, the major source of reducing power for ADP in cancer remains unclear. Although glycolysis is the source of ATP in tumors according to the Warburg effect, ATP levels do not differ between cancer cells grown in the presence and absence of glucose. Several theories have been proposed to explain the supply of ATP in cancer, including metabolic reprograming in the tumor microenvironment. However, these theories are based on the production of ATP by the TCA-OxPhos pathway, which is inconsistent with the Warburg effect. We found that blocking fatty acid oxidation (FAO) in the presence of glucose significantly decreased ATP production in various cancer cells. This suggests that cancer cells depend on fatty acids to produce ATP through FAO instead of glycolysis. We observed that cancer cell growth mainly relies on metabolic nutrients and oxygen systemically supplied through the bloodstream instead of metabolic reprogramming. In a spontaneous mouse tumor model (KrasG12D; Pdx1-cre), tumor growth was 2-fold higher in mice fed a high-fat diet (low-carbo diet) that caused obesity, whereas a calorie-balanced, low-fat diet (high-carbo diet) inhibited tumor growth by 3-fold compared with that in mice fed a control/normal diet. This 5-fold difference in tumor growth between mice fed low-fat and high-fat diets suggests that fat-induced obesity promotes cancer growth, and tumor growth depends on fatty acids as the primary source of energy.

In previous work, we showed that cancer cells do not depend on glycolysis for ATP production, but they do on fatty acid oxidation. However, we found some cancer cells induced cell death after glucose deprivation along with a decrease of ATP production. We investigated the different response of glucose deprivation with two types of cancer cells including glucose insensitive cancer cells (GIC) which do not change ATP levels, and glucose sensitive cancer cells (GSC) which decrease ATP production in 24 h. Glucose deprivation-induced cell death in GSC by more than twofold after 12 h and by up to tenfold after 24 h accompanied by decreased ATP production to compare to the control (cultured in glucose). Glucose deprivation decreased the levels of metabolic intermediates of the pentose phosphate pathway (PPP) and the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) in both GSC and GIC. However, glucose deprivation increased reactive oxygen species (ROS) only in GSC, suggesting that GIC have a higher tolerance for decreased NADPH than GSC. The twofold higher ratio of reduced/oxidized glutathione (GSH/GSSG) in GIS than in GSC correlates closely with the twofold lower ROS levels under glucose starvation conditions. Treatment with N-acetylcysteine (NAC) as a precursor to the biologic antioxidant glutathione restored ATP production by 70% and reversed cell death caused by glucose deprivation in GSC. The present findings suggest that glucose deprivation-induced cancer cell death is not caused by decreased ATP levels, but rather triggered by a failure of ROS regulation by the antioxidant system. Conclusion is clear that glucose deprivation-induced cell death is independent from ATP depletion-induced cell death.

The transglutaminase 1 (TGase 1) enzyme is involved in the formation of a cornified cell envelope in terminally differentiating epidermal keratinocytes. The enzyme is present in proliferating cells but is more abundantly expressed in differentiating cells and exists in several intact or proteolytically processed cytosolic or membrane-anchored forms. We show here that the equilibrium partitioning of TGase 1 between the cytosol and membranes is controlled by variable modification by myristate and palmitate. During synthesis, it is constitutively <i>N</i>-myristoylated. Later, it is modified by an average of two <i>S</i>-myristoyl adducts in proliferating cells or one <i>S</i>-palmitoyl adduct in differentiating cells. The three myristoyl adducts of the former provide more robust anchorage to membranes than the one myristoyl and one palmitoyl adduct of the latter. The half-lives of the <i>S</i>-myristoyl and especially the <i>S</i>-palmitoyl adducts are less than that of the TGase 1 protein, suggesting a mechanism for cycling off membranes. In <i>in vitro</i> overlay assays, the <i>S</i>-acylated 10-kDa anchorage fragment facilitates binding of TGase 1 forms, supporting a mechanism of cycling back onto membranes <i>in vivo</i>. We conclude that differential acylation increases the repertoire of functional TGase 1 forms, depending on the differentiation state of epidermal keratinocytes.

Abstract The major source of ATP in cancer cells remains unclear. Here, we examined energy metabolism in gastric cancer cells and found increased fatty acid oxidation and increased expression of ALDH3A1. Metabolic analysis showed that lipid peroxidation by reactive oxygen species led to spontaneous production of 4-hydroxynonenal, which was converted to fatty acids with NADH production by ALDH3A1, resulting in further fatty acid oxidation. Inhibition of ALDH3A1 by knock down using siRNA of ALDH3A1 resulted in significantly reduced ATP production by cancer cells, leading to apoptosis. Oxidative phosphorylation by mitochondria in gastric cancer cells was driven by NADH supplied via fatty acid oxidation. Therefore, blockade of ALDH3A1 together with mitochondrial complex I using gossypol and phenformin led to significant therapeutic effects in a preclinical gastric cancer model.

BackgroundFast growing cancer cells require greater amounts of ATP than normal cells. Although glycolysis was suggested as a source of anabolic metabolism based on lactate production, the main source of ATP to support cancer cell metabolism remains unidentified.MethodsWe have proposed that the oxoglutarate carrier SLC25A11 is important for ATP production in cancer by NADH transportation from the cytosol to mitochondria as a malate. We have examined not only changes of ATP and NADH but also changes of metabolites after SLC25A11 knock down in cancer cells.FindingsThe mitochondrial electron transport chain was functionally active in cancer cells. The cytosolic to mitochondrial NADH ratio was higher in non-small cell lung cancer (NSCLC) and melanoma cells than in normal cells. This was consistent with higher levels of the oxoglutarate carrier SLC25A11. Blocking malate transport by knockdown of SLC25A11 significantly impaired ATP production and inhibited the growth of cancer cells, which was not observed in normal cells. In in vivo experiments, heterozygote of SLC25A11 knock out mice suppressed KRASLA2 lung tumor formation by cross breeding.InterpretationCancer cells critically depended on the oxoglutarate carrier SLC25A11 for transporting NADH from cytosol to mitochondria as a malate form for the purpose of ATP production. Therefore blocking SLC25A11 may have an advantage in stopping cancer growth by reducing ATP production.FundThe Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Science and ICT to SYK (NRF-2017R1A2B2003428).

Glycolysis is known as the main pathway for ATP production in cancer cells. However, in cancer cells, glucose deprivation for 24 h does not reduce ATP levels, whereas it does suppress lactate production. In this study, metabolic pathways were blocked to identify the main pathway of ATP production in pancreatic ductal adenocarcinoma (PDAC). Blocking fatty acid oxidation (FAO) decreased ATP production by 40% in cancer cells with no effect on normal cells. The effects of calorie balanced high- or low-fat diets were tested to determine whether cancer growth is modulated by fatty acids instead of calories. A low-fat diet caused a 70% decrease in pancreatic preneoplastic lesions compared with the control, whereas a high-fat diet caused a two-fold increase in preneoplastic lesions accompanied with increase of ATP production in the Kras (G12D)/Pdx1-cre PDAC model. The present results suggest that ATP production in cancer cells is dependent on FAO rather than on glycolysis, which can be a therapeutic approach by targeting cancer energy metabolism.

Abstract Lung-resident neutrophils need to be tightly regulated to avoid degranulation- and cytokine-associated damage to fragile alveolar structures that can lead to fatal outcomes. Here we show that lung neutrophils (LNs) express distinct surface proteins and genes that distinguish LNs from bone marrow and blood neutrophils. Functionally, LNs show impaired migratory activity toward chemoattractants and produce high levels of interleukin-6 (IL-6) at steady state and low levels of tumor necrosis factor-α in response to lipopolysaccharide (LPS) challenge. Treating bone marrow neutrophils with bronchoalveolar lavage fluid or prostaglandin E2 induces LN-associated characteristics, including the expression of transglutaminase 2 (Tgm2) and reduced production of inflammatory cytokines upon LPS challenge. Neutrophils from Tgm2−/− mice release high levels of inflammatory cytokines in response to LPS. Lung damage is significantly exacerbated in Tgm2−/− mice in an LPS-induced acute respiratory distress syndrome model. Collectively, we demonstrate that prostaglandin E2 is a key factor for the generation of LNs with unique immune suppressive characteristics, acting through protein kinase A and Tgm2, and LNs play essential roles in protection of the lungs against pathogenic inflammation.

The transglutaminase 1 enzyme is important for the formation of a cornified cell envelope in terminally differentiating keratinocytes. We show here that it is present in low levels in proliferating foreskin or cultured epidermal cells as an inactive zymogen full length form of 106 kDa, of which >95% is attached to membranes. In terminally differentiating keratinocytes, there is a > or = 100-fold induction of mRNA and protein. In addition to some cytosolic protein, most of the newly expressed protein is attached to membranes, of which about half exist in the zymogen form. Other protein consists of a 67/33/10 kDa complex formed by proteolytic processing at specific sites, and is anchored by way of the 10 kDa fragment. This processed form is very highly active and thus accounts for almost all transglutaminase 1 activity in keratinocytes.

Several active transglutaminase (TGase) isoforms are known to be present in human and rodent tissues, at least three of which, namely, TGase 1, TGase 2 (tissue transglutaminase), and TGase 3, are present in the brain. TGase activity is known to be present in the cytosolic, nuclear, and extracellular compartments of the brain. Here, we show that highly purified mouse brain nonsynaptosomal mitochondria and mouse liver mitochondria and mitoplast fractions derived from these preparations possess TGase activity. Western blotting and experiments with TGase 2 knock-out (KO) mice ruled out the possibility that most of the mitochondrial/mitoplast TGase activity is due to TGase 2, the TGase isoform responsible for the majority of the activity ([14C]putrescine-binding assay) in whole brain and liver homogenates. The identity of the mitochondrial/mitoplast TGase(s) is not yet known. Possibly, the activity may be due to one of the other TGase isoforms or perhaps to a protein that does not belong to the classical TGase family. This activity may play a role in regulation of mitochondrial function both in normal physiology and in disease. Its nature and regulation deserve further study.

Background: Individuals with active celiac disease (CD+) have an increased incidence of thyroid dysfunction, which improves on a gluten-free diet (CD−). We investigated whether tissue transglutaminase-2 IgA antibodies (anti-TGase II) present in sera of patients with celiac disease react with thyroid tissue and possibly contribute to thyroid disease. Methods: Serum from 40 active celiac patients taken before a gluten-free diet (CD+), 46 patients on a gluten-free diet (CD−), 40 normal controls (NC), and 25 with Crohn's disease (CROHN) was used. All sera were screened for antithyroperoxidase antibodies (TPO-AB) and thyroglobulin antibodies (TG-AB), and indirect immunofluorescence (IIF) was performed on primate thyroid tissue sections using TPO-AB– and TG-AB–negative sera. Results: IIF with thyroid seronegative, anti-TGase II–positive CD+ sera (n = 23) demonstrated staining of thyroid follicular cells and extracellular matrix, in an identical pattern with monoclonal anti-human TGase II antibody. Evidence of TGase II as the antigen in thyroid tissue was supported by elimination of the IIF pattern when sera were depleted of anti-TGase II by pretreatment with human recombinant TGase II. No staining of thyroid tissue was observed when sera from CD+ patients that were negative for TGase II antibodies, or sera from NC subjects were used. Thyroid antibodies were found in 43% of CD+ patients, significantly higher than NC and CROHN patients (p < 0.0001). In addition, a positive correlation was observed between anti-TGase II and TPO-AB titers (p = 0.0001; r = 0.63). Conclusions: Anti-TGase II antibodies bind to TGase II in thyroid tissue, and titers correlate with TPO antibody titers. These findings suggest that anti-TGase II antibodies could contribute to the development of thyroid disease in celiac disease.

Protein kinase CK2 is a ubiquitous kinase that can phosphorylate hundreds of cellular proteins and plays important roles in cell growth and development. Deregulation of CK2 is related to a variety of human cancers, and CK2 is regarded as a suppressor of apoptosis; therefore, it is a target of anticancer therapy. Nucleolar phosphoprotein 140 (Nopp140), which is an intrinsically disordered protein, interacts with CK2 and inhibits the latter's catalytic activity in vitro. Interestingly, the catalytic activity of CK2 is recovered in the presence of d-myo-inositol 1,2,3,4,5,6-hexakisphosphate (IP6). IP6 is widely distributed in animal cells, but the molecular mechanisms that govern its cellular functions in animal cells have not been completely elucidated. In this study, the crystal structure of CK2 in complex with IP6 showed that the lysine-rich cluster of CK2 plays an important role in binding to IP6. The biochemical experiments revealed that a Nopp140 fragment (residues 568-596) and IP6 competitively bind to the catalytic subunit of CK2 (CK2α), and phospho-Ser574 of Nopp140 significantly enhances its interaction with CK2α. Substitutions of K74E, K76E, and K77E in CK2α significantly reduced the interactions of CK2α with both IP6 and the Nopp140-derived peptide. Our study gives an insight into the regulation of CK2. In particular, our work suggests that CK2 activity is inhibited by Nopp140 and reactivated by IP6 by competitive binding at the substrate recognition site of CK2.

Drug development groups are close to discovering another pot of gold-a therapeutic target-similar to the success of imatinib (Gleevec) in the field of cancer biology.Modern molecular biology has improved cancer therapy through the identification of more pharmaceutically viable targets, and yet major problems and risks associated with late-phase cancer therapy remain.Presently, a growing number of reports have initiated a discussion about the benefits of metabolic regulation in cancers.The Warburg effect, a great discovery approximately 70 years ago, addresses the "universality" of cancer characteristics.For instance, most cancer cells prefer aerobic glycolysis instead of mitochondrial respiration.Recently, cancer metabolism has been explained not only by metabolites but also through modern molecular and chemical biological techniques.Scientists are seeking context-dependent universality among cancer types according to metabolic and enzymatic pathway signatures.This review presents current cancer metabolism studies and discusses future directions in cancer therapy targeting bio-energetics, bio-anabolism, and autophagy, emphasizing the important contribution of cancer metabolism in cancer therapy.

Despite the importance of mitochondrial fatty acid oxidation (FAO) in cancer metabolism, the biological mechanisms responsible for the FAO in cancer and therapeutic intervention based on catabolic metabolism are not well defined. In this study, we observe that Snail (SNAI1), a key transcriptional repressor of epithelial–mesenchymal transition, enhances catabolic FAO, allowing pro-survival of breast cancer cells in a starved environment. Mechanistically, Snail suppresses mitochondrial ACC2 (ACACB) by binding to a series of E-boxes located in its proximal promoter, resulting in decreased malonyl-CoA level. Malonyl-CoA being a well-known endogenous inhibitor of fatty acid transporter carnitine palmitoyltransferase 1 (CPT1), the suppression of ACC2 by Snail activates CPT1-dependent FAO, generating ATP and decreasing NADPH consumption. Importantly, combinatorial pharmacologic inhibition of pentose phosphate pathway and FAO with clinically available drugs efficiently reverts Snail-mediated metabolic reprogramming and suppresses in vivo metastatic progression of breast cancer cells. Our observations provide not only a mechanistic link between epithelial–mesenchymal transition and catabolic rewiring but also a novel catabolism-based therapeutic approach for inhibition of cancer progression.

Anti-PD-1/PD-L1 immunotherapy, as a treatment for many tumors, has shown good efficacy. However, responses to immunotherapy did not always occur or last long., i.e. primary or acquired resistance, even tumors were PD-L1 positive. Several oncogenic pathways, including PI3K/AKT activation by PTEN loss and NF-κB activation, induce PD-L1 expression and PD-L1 inhibitor-resistance. They also induce expression of CCL2, an inhibitory chemokine that blocks T cell tracking into the tumor by binding to CCR2 on the T cell surface. In this study, we showed that transglutaminase 2 (TG2), a post-translational modification enzyme, induced ubiquitin-proteasome dependent degradation of tumor suppressors including PTEN and IκBα by peptide cross-linking, inducing CCL2 as well as PD-L1 expression via PI3K/AKT and NF-κB activation. It also induced PD-L1 inhibitor-resistance because CCL2 was expressed despite increased PD-L1, which was blocked by PD-L1 inhibitor. We also revealed that inhibition of TG2, instead of PD-L1, restored T cell-dependent killing effect by blocking expression of both PD-L1 and CCL2 in PD-L1(+) triple negative breast cancer (TNBC) cells. In addition, the TG2-expressing TNBC patient group showed higher PD-L1 expression incidence than did the TG2-negative TNBC patient group. In conclusion, TG2 induces primary PD-1/PD-L1 inhibitor-resistance by inducing CCL2 expression. TG2 blockade can be utilized as an excellent therapeutic strategy to overcome PD-L1 inhibitor-resistance in PD-L1(+) TNBC patients. Our study suggested that PD-L1 expression alone might not always be a predictive biomarker for PD-L1(+) TNBC, but TG2 could be a useful predictive marker to select PD-L1 inhibitor-resistant TNBC patients.

Abstract Background Pancreatic cancer (PC) has a grim prognosis, and an early diagnostic biomarker has been highly desired. The molecular link between diabetes and PC has not been well established. Methods Bioinformatics screening was performed for a serum PC marker. Experiments in cell lines (5 PC and 1 normal cell lines), mouse models, and human tissue staining (37 PC and 10 normal cases) were performed to test asprosin production from PC. Asprosin’s diagnostic performance was tested with serums from multi-center cohorts (347 PC, 209 normal, and 55 additional diabetic patients) and evaluated according to PC status, stages, and diabetic status, which was compared with that of CA19-9. Results Asprosin, a diabetes-related hormone, was found from the bioinformatics screening, and its production from PC was confirmed. Serum asprosin levels from multi-center cohorts yielded an age-adjusted diagnostic area under the curve (AUC) of 0.987 (95% confidence interval [CI] = 0.961 to 0.997), superior to that of CA19-9 (AUC = 0.876, 95% CI = 0.847 to 0.905), and a cut-off of 7.18 ng/mL, at which the validation set exhibited a sensitivity of 0.957 and a specificity of 0.924. Importantly, the performance was maintained in early-stage and non-metastatic PC, consistent with the tissue staining. A slightly lower performance against additional diabetic patients (n = 55) was restored by combining asprosin and CA19-9 (AUC = 0.985, 95% CI = 0.975 to 0.995). Conclusions Asprosin is presented as an early-stage PC serum marker that may provide clues for PC-induced diabetes. Larger prospective clinical studies are warranted to solidify its utility.

C-peptide has a beneficial effect against diabetic complications, but its role in hyperglycemia-induced metastasis is unknown. We investigated hyperglycemia-mediated pulmonary vascular leakage and metastasis and C-peptide inhibition of these molecular events using human pulmonary microvascular endothelial cells (HPMVECs) and streptozotocin-induced diabetic mice. VEGF, which is elevated in the lungs of diabetic mice, activated transglutaminase 2 (TGase2) in HPMVECs by sequential elevation of intracellular Ca2+ and reactive oxygen species (ROS) levels. VEGF also induced vascular endothelial (VE)-cadherin disruption and increased the permeability of endothelial cells, both of which were prevented by the TGase inhibitors monodansylcadaverine and cystamine or TGM2-specific small interfering RNA. C-peptide prevented VEGF-induced VE-cadherin disruption and endothelial cell permeability through inhibiting ROS-mediated activation of TGase2. C-peptide supplementation inhibited hyperglycemia-induced ROS generation and TGase2 activation and prevented vascular leakage and metastasis in the lungs of diabetic mice. The role of TGase2 in hyperglycemia-induced pulmonary vascular leakage and metastasis was further demonstrated in diabetic Tgm2-/- mice. These findings demonstrate that hyperglycemia induces metastasis, and C-peptide prevents the hyperglycemia-induced metastasis in the lungs of diabetic mice by inhibiting VEGF-induced TGase2 activation and subsequent vascular leakage.-Jeon, H.-Y., Lee, Y.-J., Kim, Y.-S., Kim, S.-Y., Han, E.-T., Park, W. S., Hong, S.-H., Kim, Y.-M., Ha, K.-S. Proinsulin C-peptide prevents hyperglycemia-induced vascular leakage and metastasis of melanoma cells in the lungs of diabetic mice.

Angiogenesis and the expression of vascular endothelial growth factor (VEGF) are increased in renal cell carcinoma (RCC). Transglutaminase 2 (TGase 2), which promotes angiogenesis in endothelial cells during wound healing, is upregulated in RCC. Tumor angiogenesis involves three domains: cancer cells, the extracellular matrix, and endothelial cells. TGase 2 stabilizes VEGF in the extracellular matrix and promotes VEGFR-2 nuclear translocation in endothelial cells. However, the role of TGase 2 in angiogenesis in the cancer cell domain remains unclear. Hypoxia-inducible factor (HIF)-1α-mediated VEGF production underlies the induction of angiogenesis in cancer cells. In this study, we show that p53 downregulated HIF-1α in RCC, and p53 overexpression decreased VEGF production. Increased TGase 2 promoted angiogenesis by inducing p53 degradation, leading to the activation of HIF-1α. The interaction of HIF-1α and p53 with the cofactor p300 is required for stable transcriptional activation. We found that TGase 2-mediated p53 depletion increased the availability of p300 for HIF-1α-p300 binding. A preclinical xenograft model suggested that TGase 2 inhibition can reverse angiogenesis in RCC.

Transglutaminase 2 (tissue transglutaminase, TGase 2) was recently identified as an endomysial autoantigen in celiac disease (CD). Identification of how TGase 2 expression is increased may allow a better understanding of this autoimmune disease. Certain inflammatory cytokines, tumor necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta (TGF-beta), and the Th type I cytokine interferon-gamma (INF-gamma) are abundant in CD. We have investigated whether these play a role in the regulation of TGase 2 expression in a model rat small intestinal epithelial cell line (IEC-6). After treatment for 24 h, TNF-alpha did not significantly alter TGase 2 mRNA or activity, but TGF-beta decreased mRNA and activity by 4-5-fold. IFN-gamma increased mRNA and TGase 2 activity by about 2-fold in 24 h and 5-fold by 5 days. Our new data suggest that increased TGase 2 expression in the upper small intestine of CD patients may be due to increased IFN-gamma expression, loss of TGF-beta signaling, or both.

Increased levels of transglutaminase 2 (TGase 2) expression have been reported in many inflammatory diseases, as well as in drug resistant cancer cells. Previous reports have shown that TGase 2 is capable of inducing nuclear factor-kappaB (NF-kappaB) activation via depletion of inhibitor of kappaB (I-kappaB)alpha through polymerization in the absence of I-kappaBalpha kinase activation. This raises the question of whether increased expression of TGase 2 can extend NF-kappaB activation mediated by a canonical activation pathway. In the TGase 2-inducible EcR23/TG cell line, TGase 2 over-expression resulted in sustained activation of NF-kappa B in the presence of TNF-alpha, for up to 24 hrs, while in the absence of TGase 2 induction, NF-kappaB activity was restored to basal levels within 6 hrs of TNF-alpha treatment. In mice injected with an adenovirus vector expressing TGase 2, NF-kappaB was constitutively activated for up to 5 days, whereas Adeno/GFP-injected mice exhibited attenuated activation of NF-kappaB in response to TNF-alpha stress. Thus, the presence of increased levels of TGase 2 may exacerbate NF-kappa B activation in inflammatory states.

We found that non-small cell lung cancer (NSCLC) is remarkably sensitive to the regulation of glutamine supply by testing the metabolic dependency of 11 cancer cell lines against regulation of glycolysis, autophagy, fatty acid synthesis, and glutamine supply. Glutamine is known as a key supplement of cancer cell growth that is converted to α-ketoglutarate for anabolic biogenesis via glutamate by glutaminase 1 (GLS1). GLS1 inhibition using 10 μM of bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide (BPTES) showed about 50% cell growth arrest by SRB assay. By testing the synergistic effects of conventional therapeutics, BPTES combined with 5-fluorouracil (5-FU), an irreversible inhibitor of thymidylate synthase, significant effects were observed on cell growth arrest in NSCLC. We found that GLS1 inhibition using BPTES reduced metabolic intermediates including thymidine and carbamoyl phosphate. Reduction of thymidine and carbamoyl-phosphate synthesis by BPTES treatment exacerbated pyrimidine supply by combination with 5-FU, which induced cell death synergistically in NSCLC.

Transglutaminase 2 (TGase 2) plays a key role in p53 regulation, depleting p53 tumor suppressor through autophagy in renal cell carcinoma. We found that microtubule-associated protein 1A/1B-light chain 3 (LC3), a hallmark of autophagy, were tightly associated with the level of TGase 2 in cancer cells. TGase 2 overexpression increased LC3 levels, and TGase 2 knockdown decreased LC3 levels in cancer cells. Transcript abundance of LC3 was inversely correlated with level of wild type p53. TGase 2 knockdown using siRNA, or TGase 2 inhibition using GK921 significantly reduced autophagy through reduction of LC3 transcription, which was followed by restoration of p53 levels in cancer cells. TGase 2 overexpression promoted the autophagy process by LC3 induction, which was correlated with p53 depletion in cancer cells. Rapamycin-resistant cancer cells also showed higher expression of LC3 compared to the rapamycin-sensitive cancer cells, which was tightly correlated with TGase 2 levels. TGase 2 knockdown or TGase 2 inhibition sensitized rapamycin-resistant cancer cells to drug treatment. In summary, TGase 2 induces drug resistance by potentiating autophagy through LC3 induction via p53 regulation in cancer.

Oral keratinocytes of buccal and gingival tissues undergo a terminal differentiation program to form a protective epithelial barrier as non‐keratinized or parakeratinized stratified cells. We have examined the protein composition of cell envelopes (CEs) from normal human buccal and gingival tissues as well as keratinocytes from normal human gingival cells grown in culture. Biochemical and sequencing analyses reveal that the CEs contain 60–70% small proline‐rich protein 1a/b (SPR1a/b), together with smaller amounts of involucrin, annexin I and several other known CE proteins. The data imply a specialized role for SPR1 proteins in the unique barrier function requirements of oral epithelia.

A hallmark of brain inflammation is the activation of microglia. Excessive production of nitric oxide (NO), as a consequence of increased inducible nitric oxide synthase (iNOS) in glia, contributes to neurodegeneration. Transglutaminase 2 (TGase 2) is a cross-linking enzyme, which is increased in neurodegeneration. TGase 2 is also considered to be a useful and reliable marker for activation levels in resident and inflammatory macrophages. Therefore, an increase of TGase 2 expression may contribute to activation of microglia. To test this hypothesis, we analyzed the expression of TGase 2 in BV-2 microglia activated with lipopolysaccharide (LPS). Total TGase activity was increased about 5-fold after 24 h exposure to LPS. The increase of NO synthesis is correlated with increase of TGase 2 expression. Secretion of NO was reduced between 40 and 80% by TGase inhibition in a dose-dependent manner. This suggests that TGase 2 appears to control iNOS transcription.

Recent studies have shown that glucosamine inhibits the proliferation of various human cancer cell lines and downregulates the activity of COX-2, HIF-1α, p70S6K, and transglutaminase 2. Because the IGF-1R/Akt pathway is a common upstream regulator of p70S6K, HIF-1α, and COX-2, we hypothesized that glucosamine inhibits cancer cell proliferation through this pathway.We used various in vitro assays including flow cytometry assays, small interfering RNA (siRNA) transfection, western blot analysis, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays, reverse transcription-polymerase chain reaction, and in vivo xenograft mouse model to confirm anticancer activities of glucosamine and to investigate the molecular mechanism.We found that glucosamine inhibited the growth of human non-small cell lung cancer (NSCLC) cells and negatively regulated the expression of IGF-1R and phosphorylation of Akt. Glucosamine decreased the stability of IGF-1R and induced its proteasomal degradation by increasing the levels of abnormal glycosylation on IGF-1R. Moreover, picropodophyllin, a selective inhibitor of IGF-1R, and the IGF-1R blocking antibody IMC-A12 induced significant cell growth inhibition in glucosamine-sensitive, but not glucosamine-resistant cell lines. Using in vivo xenograft model, we confirmed that glucosamine prohibits primary tumor growth through reducing IGF-1R signalling and increasing ER-stress.Taken together, our results suggest that targeting the IGF-1R/Akt pathway with glucosamine may be an effective therapeutic strategy for treating some type of cancer.

Transglutaminase 2 (EC 2.3.2.13; TG2 or TGase 2) plays important roles in the pathogenesis of many diseases, including cancers, neurodegeneration, and inflammatory disorders. Under normal conditions, however, mice lacking TGase 2 exhibit no obvious abnormal phenotype. TGase 2 expression is induced by chemical, physical, and viral stresses through tissue-protective signaling pathways. After stress dissipates, expression is normalized by feedback mechanisms. Dysregulation of TGase 2 expression under pathologic conditions, however, can potentiate pathogenesis and aggravate disease severity. Consistent with this, TGase 2 knockout mice exhibit reversal of disease phenotypes in neurodegenerative and chronic inflammatory disease models. Accordingly, TGase 2 is considered to be a potential therapeutic target. Based on structure⁻activity relationship assays performed over the past few decades, TGase 2 inhibitors have been developed that target the enzyme's active site, but clinically applicable inhibitors are not yet available. The recently described the small molecule GK921, which lacks a group that can react with the active site of TGase 2, and efficiently inhibits the enzyme's activity. Mechanistic studies revealed that GK921 binds at an allosteric binding site in the N-terminus of TGase 2 (amino acids (a.a.) 81⁻116), triggering a conformational change that inactivates the enzyme. Because the binding site of GK921 overlaps with the p53-binding site of TGase 2, the drug induces apoptosis in renal cell carcinoma by stabilizing p53. In this review, we discuss the possibility of developing TGase 2 inhibitors that target the allosteric binding site of TGase 2.

Overexpression of transglutaminase 2 (TGase 2; TG2) has been implicated in the progression of renal cell carcinoma (RCC) through the inactivation of p53 by forming a protein complex. Because most p53 in RCC has no mutations, apoptosis can be increased by inhibiting the binding between TG2 and p53 to increase the stability of p53. In the present study, a novel TG2 inhibitor was discovered by investigating the structure of 1H-benzo[d]imidazole-4,7-dione as a simpler chemotype based on the amino-1,4-benzoquinone moiety of streptonigrin, a previously reported inhibitor. Through structure-activity relationship (SAR) studies, compound 8j (MD102) was discovered as a potent TG2 inhibitor with an IC50 value of 0.35 µM, p53 stabilization effect and anticancer effects in the ACHN and Caki-1 RCC cell lines with sulforhodamine B (SRB) GI50 values of 2.15 µM and 1.98 µM, respectively. The binding property of compound 8j (MD102) with TG2 was confirmed to be reversible in a competitive enzyme assay, and the binding interaction was expected to be formed at the β-sandwich domain, a p53 binding site, in the SPR binding assay with mutant proteins. The mode of binding of compound 8j (MD102) to the β-sandwich domain of TG2 was analyzed by molecular docking using the crystal structure of the active conformation of human TG2. Compound 8j (MD102) induced a decrease in the downstream signaling of p-AKT and p-mTOR through the stabilization of p53 by TG2 inhibition, resulting in tumor cell apoptosis. In a xenograft animal model using ACHN cancer cells, oral administration and intraperitoneal injection of compound 8j (MD102) showed an inhibitory effect on tumor growth, confirming increased levels of p53 and decreased levels of Ki-67 in tumor tissues through immunohistochemical (IHC) tissue staining. These results indicated that the inhibition of TG2 by compound 8j (MD102) could enhance p53 stabilization, thereby ultimately showing anticancer effects in RCC. Compound 8j (MD102), a novel TG2 inhibitor, can be further applied for the development of an anticancer candidate drug targeting RCC.

Abstract Co-occurring mutations in KEAP1 in LKB1-mutant NSCLC activate NRF2 to compensate losing LKB1-AMPK activity during metabolic adaptation and survival. Here, we investigated the regulatory crosstalk between LKB1-AMPK and KEAP1-NRF2 pathways during metabolic stress. We found that metabolic stress activates NRF2 through the expression and phosphorylation of p62, causing the autophagic degradation of KEAP1. Intriguingly, the induction of p62 during metabolic stress is also required to activate AMPK by promoting AXIN-LKB1-AMPK complex formation and recruiting it to the lysosomal membrane. Importantly, the p62-driven dual-activation of AMPK and NRF2 was critical for tumour growth by synergizing antioxidant defences. In turn, the induction of p62 also required LKB1-AMPK activity, suggesting a double positive feedback loop between AMPK and p62. Mechanistically, the increase in lysosomal pH caused by low glucose metabolism and AMPK-dependent reduction of proton generation induced PP2A-dependent dephosphorylation of TFEB/TFE3 which increased the expression of p62. The increase of ROS caused by metabolic stress induced lysosomal MCOLN1-Ca2+ dependent activation of TAK1 which increased p62 phosphorylation. Protons provided by lactic acid abrogated all the effects caused by metabolic stress. This positive feedback loop between AMPK and p62 that activates AMPK and NRF2 can potentially explain why co-occurring mutations in LKB1 and KEAP1 occur and further provide promising therapeutic strategies for lung cancer. Citation Format: Eun-Ji Choi, Hyun-Taek Oh, Seon-Hyeong Lee, Chen-Song Zhang, Mengqi Li, Soo-Youl Kim, Sunghyouk Park, Tong-Shin Chang, Byung-Hoon Lee, Sheng-Cai Lin, Sang-Min Jeon. Metabolic stress induces a double positive feedback loop between AMPK and p62 conferring dual activation of AMPK and NRF2 to synergize antioxidant defense [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4455.

Calpain I (mu-calpain) and II (m-calpain) are well known calcium-activated neutral cysteine proteases. Many reports have shown that activation of calpain is related to cataract formation, neuronal degeneration, blood clotting, ischemic injuries, muscular dystrophy and cornified cell envelope (CE) formation. Here, we report that insoluble CE formation was reduced after treatment with calpain I inhibitor (N-acetyl-leucyl-leucyl-norleucinal) on normal human epidermal keratinocytes (NHEK), whereas serine and thiol protease inhibitors had no effect on the reduction of CE. When NHEK cells were confluent, keratinocytes were treated with various concentrations (0.5 microM-0.5 mM) of calpain I inhibitor or serine and thiol protease inhibitors under calcium induced differentiation. Insoluble CE formation was reduced about 90% in the 50 microM calpain inhibitor I treated group by day 9 of culture, whereas insoluble CE was reduced only 10% in the same condition. Interestingly TGase activity was blocked by 90% in the 0.5 mM calpain inhibitor treated group within 72 h, whereas TGase activity was retained by 80% in the 0.5 mM serine protease inhibitor treated group at 7 day treatment. Therefore it can be suggested that cysteine protease calpains might be responsible for the activation of the TGase 1 enzyme to complete insoluble CE formation during epidermal differentiation.

Transglutaminase 2 (TGase 2) is a calcium-dependent cross-linking enzyme that catalyzes a covalent iso-peptide bond between two proteins. Interestingly, this catalysis can activate the nuclear factor-kappaB (NF-kappaB) through the polymerization of the inhibitory protein of NF-kappaB (I-kappaB). The objective of the present study was to investigate the expression of TGase 2 in the human atherosclerotic human coronary artery, and the possible roles of TGase 2 in NF-kappaB activation.We explored whether expressions of TGase 2 and NF-kappaB are associated in atherosclerosis. Using human samples, we found that TGase 2 was markedly higher than normal in the neointimal tissue of atherosclerotic coronary arteries with atherosclerosis progression. TGase 2 activity was also increased approximately two-fold in the atherosclerotic vascular wall. In immunofluorescence analysis, NF-kappaB, COX-2, and TNF-alpha were co-localized at TGase 2-positive neointimal smooth muscle cells. A promoter assay test showed that NF-kappaB activity increased in both the human monocyte and human breast carcinoma cell by TGase 2, and that TGase 2-mediated NF-kappaB activation was reversed by TGase 2 siRNA.According to these results, we suggest that TGase 2 may function as an activator in the NF-kappaB pathway; this effect may occur in the atherosclerotic vessel wall.

This chapter contains sections titled: Introduction Revisiting Diseases in Relation to the TGase 2-NF-κB Mechanism Conclusion Acknowledgements References

Lung adenocarcinoma cells express high levels of ALDH1L1, an enzyme of the one-carbon pathway that catalyzes the conversion of 10-formyltetrahydrofolate into tetrahydrofolate and NAD(P)H. In this study, we evaluated the potential of ALDH1L1 as a therapeutic target by deleting the Aldh1l1 gene in KrasLA2 mice, a model of spontaneous non-small cell lung cancer (NSCLC). Reporter assays revealed KRAS-mediated upregulation of the ALDH1L1 promoter in human NSCLC cells. Aldh1l1-/- mice exhibited a normal phenotype, with a 10% decrease in Kras-driven lung tumorigenesis. By contrast, the inhibition of oxidative phosphorylation inhibition using phenformin in Aldh1l1-/-; KrasLA2 mice dramatically decreased the number of tumor nodules and tumor area by up to 50%. Furthermore, combined treatment with pan-ALDH inhibitor and phenformin showed a decreased number and area of lung tumors by 70% in the KrasLA2 lung cancer model. Consistent with this, previous work showed that the combination of ALDH1L1 knockdown and phenformin treatment decreased ATP production by as much as 70% in NSCLS cell lines. Taken together, these results suggest that the combined inhibition of ALDH activity and oxidative phosphorylation represents a promising therapeutic strategy for NSCLC.

Long‐term survivors of harlequin ichthyosis (HI) have raised a controversy over the differences between HI and lamellar ichthyosis (LI). Abnormal lamellar granules and the failure of conversion from profilaggrin to filaggrin have been reported in HI. On the other hand, malformation of the cornified cell envelope as a result of mutation of keratinocyte transglutaminase has been found in LI. In the present study, we analyzed the distribution of keratins, filaggrin/profilaggrin and cornified cell envelope proteins in the epidermis in HI. We studied a newborn Japanese male with typical clinical features of HI. Electron microscopic observation of a skin biopsy specimen taken from the trunk revealed the presence of lipid inclusions within the cornified cells, the absence of lamellar granules in the granular layer keratinocytes, and a lack of extracellular lamellar structures between the first cornified cell and the granular cell. Immunohistochemical labeling showed a normal distribution of keratins (keratins 1,5, 10, and 14), filaggrin/profilaggrin and cornified cell envelope proteins (involucrin, small proline‐rich proteins, and loricrin) in the epidermis of lesional skin. The present observations of the patient's skin verified that keratins and cornified cell envelope proteins are normally expressed in HI, thus demonstrating a different pathogenesis between HI and LI.

Post-transcriptional regulation of mRNA stability by Hu proteins is an important mechanism for tumorigenesis. We focused on the molecular interactions between the HuC protein and AU-rich elements (AREs) to find chemical inhibitors of RNA-protein interactions using RNA electrophoretic mobility shift assay with non-radioactive probes. Screening of 52 natural compounds identified 14 candidate compounds that displayed potent inhibitory activity. Six (quercetin, myricetin, (-)-epigallocatechin gallate, ellagic acid, (-)-epicatechin gallate, and rhamnetin) were categorized as phytochemicals, and their IC(50) values were low (0.2-1.8 microM). [BMB reports 2009; 42(1): 41-46].

Octapeptide R2 (KVLDGQDP), which has anti-transglutaminas (TGase) activity, decreases inflammation in allergic conjunctivitis model in guinea pigs. The authors examined the effect of R2 on lipopolysaccharide (LPS)-induced lung injury in BALB/c mice. R2 inhalation significantly decreased neutrophil count and cytokine mRNA expression in the lungs of LPS (25 mg/kg)-treated mice (P < .05). It also showed a tendency for decreased tumor necrosis factor (TNF)-α–immunoreactive protein in lung homogenates and significantly decreased TNF-α–immunoreactive protein in the serum of LPS-injected mice (P < .05). These results indicate that TGase may be a new therapeutic target in LPS-induced lung inflammation.

Our goal was to investigate the effects of inhibition of transglutaminase 2 (TGase 2) on endotoxin-induced uveitis (EIU) METHODS: EIU was induced in female Lewis rats by single footpad injections of 200 microg of lipopolysaccharide (LPS). TGase 2 inhibitors were administered intraperitoneally 30 minutes before and at the time of LPS administration. Rats were sacrificed 24 hours after injection, and the effects of the TGase 2 inhibitors were evaluated by the number of intraocular inflammatory cells present on histologic sections and by measuring the TGase 2 activity and TGase products in the aqueous humor (AqH). TGase 2 substrates were also assayed in AqH from uveitis patients.Clinical indications of EIU, the number of cells present on histologic sections, and TGase 2 activity in AqH increased in a time-dependent manner, peaking 24 hours after LPS injection. Inflammation in EIU was significantly reversed by treatment with TGase inhibitors. A 23-kDa cross-linked TGase substrate was identified in the AqH from EIU rats and uveitis patients. MALDI-TOF analysis showed that this substrate in uveitis patients was human Ig kappa chain C region.TGase 2 activity and its catalytic product were increased in the AqH of EIU rats. TGase 2 inhibition attenuated the degree of inflammation in EIU. Safe and stable TGase inhibitors may have great potential for the treatment of inflammatory uveitis.

Cancer cells are characterized by an abnormal cell cycle. Therefore, the cell cycle has been a potential target for cancer therapeutic agents. We developed a new lead compound, DGG200064 (7c) with a 2-alkoxythieno [2,3-b]pyrazine-3-yl)-4-arylpiperazine-1-carboxamide core skeleton. To evaluate its properties, compound DGG200064 was tested in vivo through a xenograft mouse model of colorectal cancer using HCT116 cells. The in vivo results showed high cell growth inhibition efficacy. Our results confirmed that the newly synthesized DGG200064 inhibits the growth of colorectal cancer cells by inducing G2/M arrest. Unlike the known cell cycle inhibitors, DGG200064 (GI50 = 12 nM in an HCT116 cell-based assay) induced G2/M arrest by selectively inhibiting the interaction of FBXW7 and c-Jun proteins. Additionally, the physicochemical properties of the lead compounds were analyzed. Based on the results of the study, we suggested further development of DGG200064 as a novel oral anti-colorectal cancer drug.

Annually, millions of new cancer cases are reported, leading to millions of deaths worldwide. Among the newly reported cases, breast and colon cancers prevail as the most frequently detected variations. To effectively counteract this rapid increase, the development of innovative therapies is crucial. Small molecules possessing pyridine and urea moieties have been reported in many of the currently available anticancer agents, especially VEGFR2 inhibitors. With this in mind, a rational design approach was employed to create hybrid small molecules combining urea and pyridine. These synthesized compounds underwent in vitro testing against breast and colon cancer cell lines, revealing potent submicromolar anticancer activity. Compound 8a, specifically, exhibited an impressive GI50 value of 0.06 μM against the MCF7 cancer cell line, while compound 8h displayed the highest cytotoxic activity against the HCT116 cell line, with a GI50 of 0.33 ± 0.042 μM. Notably, compounds 8a, 8h, and 8i demonstrated excellent safety profiles when tested on normal cells. Molecular docking, dynamic studies, and free energy calculations were employed to validate the affinity of these compounds as VEGFR2 inhibitors.

Transglutaminase (TGase) is a calcium-dependent enzyme which catalyzes the iso-peptide cross-link between peptide-bound glutamine and lysinein vivo.Though the cross-link is developed as a barrier function in the skin system, overexpression of this could invoke skin hyperkeratosis in psoriasis and roughness in aged skin. In former research, many strong irreversible TGase inhibitors failed application because of high cytotoxicity. We selected one peptide after primary screening of six synthetic peptides designed from domains of known TGase substrates. Then we attempted to reduce the size and finally obtained two tetrameric peptides. When we treated keratinocyte with these TGase inhibitors under calcium-induced differentiation, the formation of a cornified cell envelope (CE) was decreased to the same level of CE under proliferating conditions without cytotoxic effect. Therefore, we propose that these TGase inhibitors may be useful for solving the physiological hypercross-linking problems for pharmaceutical or cosmetic purposes.

Recent evidence suggests that aberrant transglutaminase activity is associated with a wide variety of diseases. Tissue transglutaminase is the most widely distributed of the six well-characterized transglutaminases in humans. We describe a method for expressing hexahistidine-tagged human tissue transglutaminase in Escherichia coli BL21(DE3) using the pET-30 Ek/LIC expression vector. Purification of the expressed enzyme from suspensions of E. coli cells treated with CelLytic B Bacterial Cell Lysis/Extraction Reagent was accomplished by immobilized metal (Ni2+) affinity column chromatography. The procedure typically yields highly purified and highly active recombinant human tissue transglutaminase in about 1 day (about 0.6 mg/from a 1-liter culture).

Pruritus is one of cardinal symptoms of atopic dermatitis, the control of itching is important in its treatment. Its molecular mechanisms remain largely unexplained. Certainly, an itch–scratch vicious cycle, in which scratch irritation enhances itch, is at work in atopic patients [ [1] Yosipovitch G. Greaves M.W. Schmelz M. Itch. Lancet. 2003; 361: 690-694 Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar ]. A defective epidermal barrier due to a marked decrease of ceramide in atopic dermatitis allows the penetration of allegens through the skin, facilitating the interaction of these allergens with the local antigen-presenting cells and immune-effector cells [ [2] Imokawa G. A possible mechanism underlying the ceramide deficiency in atopic dermatitis: expression of a deacylase enzyme that cleaves the N-acyl linkage of sphingomyelin and glucosylceramide. J Dermatol Sci. 2009; 55: 1-9 Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar ]. Disturbed skin-barrier function in atopic dermatitis is at least partly related to a disturbed lipid composition of the stratum corneum. A significant reduction in ceramides has been found in lesional as well as non-lesional skin of atopic dermatitis patients [ [3] Imokawa G. Abe A. Jin K. Higaki Y. Kawashima M. Hidano A. Decreased level of ceramides in stratum corneum of atopic dermatitis: an etiologic factor in atopic dry skin?. J Invest Dermatol. 1991; 96: 523-526 Abstract Full Text PDF PubMed Scopus (0) Google Scholar ]. A novel enzyme moreover, glucosylceramide/sphingomyelin (GCer-SM) deacylase, which cleaves the N-acyl linkage sphingomyelin and glucosylceramide, has been found in atopic dermatitis patients. Due to this enzyme's activity, the level of sphingosylphosphorylcholine in the stratum corneum of atopic dermatitis patients is high in comparison with that in normal skin [ [2] Imokawa G. A possible mechanism underlying the ceramide deficiency in atopic dermatitis: expression of a deacylase enzyme that cleaves the N-acyl linkage of sphingomyelin and glucosylceramide. J Dermatol Sci. 2009; 55: 1-9 Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar ]. Recently, we reported that SPC, a breakdown product of sphingomyelin, induced the itch–scratch response in mice [ [4] Kim H.J. Kim H. Han E.S. Park S.M. Koh J.Y. Kim K.M. et al. Characterizations of sphingosylphosphorylcholine-induced scratching responses in ICR mice using naltrexon, capsaicin, ketotifen and Y-27632. Eur J Pharmacol. 2008; 583: 92-96 Crossref PubMed Scopus (23) Google Scholar ].

Recent findings indicate that (a) mitochondria in proliferating cancer cells are functional, (b) cancer cells use more oxygen than normal cells for oxidative phosphorylation, and (c) cancer cells critically rely on cytosolic NADH transported into mitochondria via the malate-aspartate shuttle (MAS) for ATP production. In a spontaneous lung cancer model, tumor growth was reduced by 50% in heterozygous oxoglutarate carrier (OGC) knock-out mice compared with wild-type counterparts. To determine the mechanism through which OGC promotes tumor growth, the effects of the OGC inhibitor N-phenylmaleimide (NPM) on mitochondrial activity, oxygen consumption, and ATP production were evaluated in melanoma cell lines. NPM suppressed oxygen consumption and decreased ATP production in melanoma cells in a dose-dependent manner. NPM also reduced the proliferation of melanoma cells. To test the effects of NPM on tumor growth and metastasis in vivo, NPM was administered in a human melanoma xenograft model. NPM reduced tumor growth by approximately 50% and reduced melanoma invasion by 70% at a dose of 20 mg/kg. Therefore, blocking OGC activity may be a useful approach for cancer therapy.

In a recent report, no significance of transglutaminase 2 (TGase 2) was noted in the analyses of expression differences between normal and clear cell renal cell carcinoma (ccRCC), although we found that knock down of TGase 2 induced significant p53-mediated cell death in ccRCC. Generally, to find effective therapeutic targets, we need to identify targets that belong specifically to a cancer phenotype that can be differentiated from a normal phenotype. Here, we offer precise reasons why TGase 2 may be the first therapeutic target for ccRCC, according to several lines of evidence. TGase 2 is negatively regulated by von Hippel-Lindau tumor suppressor protein (pVHL) and positively regulated by hypoxia-inducible factor 1-α (HIF-1α) in renal cell carcinoma (RCC). Therefore, most of ccRCC presents high level expression of TGase 2 because over 90% of ccRCC showed VHL inactivity through mutation and methylation. Cell death, angiogenesis and drug resistance were specifically regulated by TGase 2 through p53 depletion in ccRCC because over 90% of ccRCC express wild type p53, which is a cell death inducer as well as a HIF-1α suppressor. Although there have been no detailed studies of the physiological role of TGase 2 in multi-omics analyses of ccRCC, a life-long study of the physiological roles of TGase 2 led to the discovery of the first target as well as the first therapeutic treatment for ccRCC in the clinical field.

More than 50% of human cancers harbor TP53 mutations and increased expression of Mouse double minute 2 homolog (MDM2), which contribute to cancer progression and drug resistance. Renal cell carcinoma (RCC) has an unusually high incidence of wild-type p53, with a mutation rate of less than 4%. MDM2 is master regulator of apoptosis in cancer cells, which is triggered through proteasomal degradation of wild-type p53. Recently, we found that p53 protein levels in RCC are regulated by autophagic degradation. Transglutaminase 2 (TGase 2) was responsible for p53 degradation through this pathway. Knocking down TGase 2 increased p53-mediated apoptosis in RCC. Therefore, we asked whether depleting p53 from RCC cells occurs via MDM2-mediated proteasomal degradation or via TGase 2-mediated autophagic degradation. In vitro gene knockdown experiments revealed that stability of p53 in RCC was inversely related to levels of both MDM2 and TGase 2 protein. Therefore, we examined the therapeutic efficacy of inhibitors of TGase 2 and MDM2 in an in vivo model of RCC. The results showed that inhibiting TGase 2 but not MDM2 had efficient anticancer effects.

Ultraviolet radiation (UVR) stimulates cellular mitosis, which leads to epidermal hyperplasia. On the basis of hypothesis that chronic UVR may modulate differentiation as well as epidermal hyperplasia, we evaluated the modulation of markers of epidermal differentiation, such as transglutaminase 1 (TGase 1), filaggrin and loricrin, by chronic UVR in vivo.Total TGase activities assay or in situ TGase activities were measured in human and mouse skin. TGase 1 expression was identified by immunohistochemical staining in human skin. In the human, the pre-auricular skin of face was used for samples of chronic UVR, and the post-auricular skin was selected as non-UVR control. The changes of filaggrin and loricrin were identified by western immunoblots.In human and mouse epidermis, chronic UVR induced the increase of in situ TGase activities or total TGase activities as it up-regulated TGase 1 expression in the epidermis. As the substrates of TGase 1, chronic UVR induced the up-regulation of filaggrin and loricrin in mouse epidermis as well. At the same time, chronic UVR induced the marked epidermal hyperplasia in human and mouse skin.Chronic UVR stimulates epidermal differentiation as it up-regulates TGase 1 and its substrates. The modified epidermal differentiation is balanced with epidermal hyperplasia, leading to the maintenance of epidermal homeostasis in the UV-irradiated epidermis.

Abstract We recently reported that increased transglutaminase 2 (TGase 2) expression correlates with increased resistance to the cancer drug doxorubicin in breast‐cancer cell lines. Interestingly, high‐molecular‐weight (HMW) proteins also increased with increased TGase 2 expression in the drug‐resistant cell lines. TGase 2 is likely to be responsible for the formation of HMW proteins, because TGase 2 catalyzes cross‐linking between proteins. Although the role of the HMW proteins is unclear, we demonstrated that TGase 2 inhibition increases drug sensitivity in breast‐cancer cells. Herein we find that TGase 2 inhibition by cystamine dramatically reduces the level of HMW proteins. Identification of the HMW proteins may suggest the mechanism of cancer drug resistance associated with aberrant TGase 2 function. To explore the identities of HMW proteins, we performed in‐gel tryptic digestions of unresolved HMW proteins and analyzed the resulting peptides using LC‐MALDI‐MS/MS. Most of the identified proteins were associated with gene regulation, such as polyadenylate‐binding proteins, translation initiation factors, and ribonucleoproteins. This finding suggests that TGase 2 may participate in gene regulation, in addition to its role in cell adhesion.

In the process of optimization, we developed a novel core skeleton of thieno[3,4-b]pyrazine viaGK-13. The derivatives synthesized were shown to inhibit TGase 2 activity in cancer cells. Some of the hit compounds such as the arylethynyl group-coupled thieno[3,4-b]pyrazine derivatives were shown to exhibit promising activity for use as potential therapeutic small-molecules in renal cancer by inhibiting TGase 2 activity.

The non-small cell lung cancer (NSCLC) patients with EGFR-sensitive mutations can be therapeutically treated by EGFR-TKI such as erlotinib and gefitinib. However, about 40% of individuals harboring EGFR-TKI sensitive mutations are still resistant to EGFR-TKI. And, it has been reported that both PTEN loss and NF-κB activation contribute to intrinsic EGFR-TKI resistance in EGFR-mutant lung cancer. Transglutaminse 2 (TG2) is post-translational modification enzyme and known to induce degradation of tumor suppressors including PTEN and IκBα with peptide cross-linking activity. Because TG2 was known as a regulator of PTEN and IκBα (NF-κB inhibitor) level in cytosol, we have explored if TG2 can be another key regulator to the intrinsic resistance of EGFR-TKI in the intrinsic EGFR-TKI resistant NSCLC cell. We first found that higher TG2 expression level and lower PTEN and IκBα expression levels in the intrinsic EGFR-TKI resistant NSCLC compare with EGFR-TKI sensitive NSCLC. TG2 stably expressing EGFR-TKI sensitive NSCLC cells harboring EGFR mutations showed reduction of both PTEN and IκBα and exhibited EGFR-TKI resistance. In reverse, When TG2 is downregulated by TG2 inhibitor in H1650, intrinsic EGFR-TKI resistant NSCLC cell harboring EGFR sensitive mutation, reversed EGFR-TKI resistance via IκBα restoration. Moreover, combination treatment of TG2 inhibitor and EGFR-TKI decreased the tumor growth in mouse xenograft models of EGFR mutant NSCLCs. Therefore, we have demonstrated that TG2 elicits the intrinsic EGFR-TKI resistance via PTEN loss and activation of NF-κB pathway. These results suggest that TG2 may be a useful predictive marker and also be a target for overcoming the resistance.

Nopp140 is a highly phosphorylated protein that resides in the nucleolus of mammalian cell and is involved in the biogenesis of the nucleolus. It interacts with a variety of proteins related to the synthesis and assembly of the ribosome. It also can bind to a ubiquitous protein kinase CK2 that mediates cell growth and prevents apoptosis. We found that Nopp140 is an intrinsically unfolded protein (IUP) lacking stable secondary structures over its entire sequence of 709 residues. We discovered that mitoxantrone, an anticancer agent, was able to enhance the interaction between Nopp140 and CK2 and maintain suppressed activity of CK2. Surface plasma resonance studies on different domains of Nopp140 show that the C-terminal region of Nopp140 is responsible for binding with mitoxantrone. Our results present an interesting example where a small chemical compound binds to an intrinsically unfolded protein (IUP) and enhances protein-protein interactions.

Abstract Background: Glycolysis is known as the main pathway for ATP production in cancer cells. However, in pancreatic ductal adenocarcinoma (PDAC) cells, glucose deprivation for 24 h did not reduce ATP levels, whereas it suppressed lactate production. We found that ATP production in PDAC cells critically depended on fatty acid oxidation (FAO) while normal cells showed no dependency on fatty acid. Therefore, FAO inhibition significantly decrease oxygen consumption rate as well as ATP production only in cancer cells. Methods: To test whether cancer depends on fatty acid for energy metabolism, high- or low-fat diet was tested in spontaneous PDAC cancer model (KPC mouse: Kras(G12D)/p53(R172H)/Pdx1-Cre model). To test whether FAO is absolute requirement for ATP production in cancer, overall survival (OS) was monitored in KPC mice by crossbreeding with FAO gene knock out mice. Results: First, the effects of calorie balanced high- or low-fat diets were tested to determine whether cancer growth is modulated by fatty acid instead of calories. A low-fat diet caused a 70 % decrease in pancreatic preneoplastic lesions accompanying decrease of body weight 20% compared with the control, whereas high-fat diet caused a 2-fold increase in preneoplastic lesions accompanied with 25% increase of body mass index in Kras(G12D)/p53(R172H)/Pdx1-Cre model (KPC model; spontaneous PDAC model). Second, the effect of FAO gene knock down was tested to determine whether OS is increased in KPC model. The crossbreeding of FAO gene knockout (+/-) and KPC mice resulted in 2-fold increase of OS. Conclusions: The two results suggest that controlling obesity by diet as well as targeting FAO can enhance anti-cancer effect of cancer therapeutics including conventional anti-cancer drug or immune-oncology drug. Clinical Trial Identification: IND for PDAC therapy with drugs targeting FAO will be submitted in 2022. Legal Entity Responsible for the Study: This study is legally under review by IRB in National Cancer Center, Korea, and sponsored by National Research Foundation of Korea (NRF) and NCC-Bio Co. Funding: Funding source: Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT (NRF-2019M3A9G1104345) Citation Format: Sang M. Woo, Sung-Sik Han, Ho Lee, Soo-Youl Kim. Blocking fatty acid oxidation suggests a potential new therapeutic approach for pancreatic cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3686.

Transglutaminase 2 is a calcium-dependent multi-func tional protein that catalyzes the formation of Ne-(户glutamyl)lysine isopeptide bond between lysine and glutamine residues.1 Transglutaminase 2 induces Nuclear Factor-kB (NF-kB) activation through the polymerization of I-kBo, which results in anti-apoptotic cellular function.2,3It is highly associated with inflammatory diseases and various cancers.4,5Recently, three types of missense mutations (M330R, 1331N, N333S) in transglutaminase 2 were found in patients with early-onset type 2 diabetes.Mutated residues, more over, were located near the catalytic site, and the mutations resulted in loss of the transamidation activity of trans glutaminase 2 from the in vitro analysis.6To explain why these mutants do not have transamidation activity in vitro, we analyzed the structural basis of functional loss in mutants using the molecular modeling method.The in vivo analysis of functional loss of mutants was studied by monitoring the substrate I-KBa level in cytosolic fraction of PANC-1 cells.Structural basis of mutation and loss of function by molecular modeling.Poirzio et al. reported that three mutations in early-onset type 2 diabetes lacked their transamidation activity.6 The structural basis of the loss of function was analyzed by the molecular modeling method.A five-amino-acid sequence around the glutamine 182 (SQ182SKV) of 四casein was used as substrate for model-7

FANCD2 is a pivotal molecule in the pathogenesis of Fanconi anemia (FA), an autosomal recessive human syndrome with diverse clinical phenotypes, including cancer predisposition, short stature, and hematological abnormalities. In our previous study, we detected the functional association of FANC proteins, whose mutations are responsible for the onset of FA, with AMPK in response to DNA interstrand crosslinking lesions. Because AMPK is well known as a critical sensing molecule for cellular energy levels, we checked whether FANCD2 activation occurs after treatments affecting AMPK and/or cellular energy status. Among the treatments tested, AMPK-activating 5-aminoimidazole-4-carboxamide-ribonucleoside (AICAR) induced monoubiquitination and nuclear foci formation of FANCD2, which are biomarkers of FANCD2 activation. FANCD2 activation was abolished by treatments with Compound C, an AMPK inhibitor, or after AMPKα1 knockdown, substantiating the involvement of AMPK in AICAR-induced FANCD2 activation. Similarly, FANCA protein, which is a component of the FA core complex monoubiquitinating FANCD2, was required for this event. Furthermore, FANCD2 repression enhanced cell death upon AICAR treatments in transformed fibroblasts and cell cycle arrest in the renal cell carcinoma cell line Caki-1. Overall, this study showed FANCD2 involvement in response to AICAR, a chemical modulating cellular energy metabolism.

Glioblastoma (GBM), the most common primary brain tumor, continues to be associated with poor prognosis despite the best treatment modalities currently available. Targeted approaches for treating GBM attempted to date have consistently failed, highlighting the imperative for treatment strategies that operate on different mechanistic principles. Bioenergetics deprivation has emerged as an effective therapeutic approach for various tumors. We have previously found that cancer cells preferentially utilize cytosolic NADH supplied by aldehyde dehydrogenase (ALDH) for ATP production through oxidative phosphorylation (OxPhos). This study is aimed to examine therapeutic responses and underlying mechanisms of dual inhibition of ALDH and OxPhos against GBM. For inhibition of ALDH and OxPhos, the corresponding inhibitors, gossypol and phenformin were used. Biological functions, including ATP levels, stemness, invasiveness, and viability, were evaluated in GBM tumorspheres (TSs). Gene expression profiles were analyzed using microarray data. In vivo anticancer efficacy was examined in a mouse orthotopic xenograft model. Combined treatment of GBM TSs with gossypol and phenformin significantly reduced ATP levels, stemness, invasiveness, and cell viability. Consistently, this therapy substantially decreased expression of genes associated with stemness, mesenchymal transition, and invasion in GBM TSs. Supplementation of ATP using malate reversed these effects, whereas knockdown of ALDH1L1 mimicked them, suggesting that disruption of ALDH-mediated ATP production is a key mechanism of this therapeutic combination. In vivoefficacy confirmed remarkable therapeutic responses to combined treatment with gossypol and phenformin. In this study, we showed that combined treatment with gossypol and phenformin induces dual inhibition of bioenergetics by targeting ALDH and OxPhos, causing ATP depletion in GBM TSs. This regimen subsequently attenuated stemness, mesenchymal transition, and invasion, which are prominent features of GBM TSs, ultimately leading to a decrease in cell viability. Our findings suggest that dual inhibition of tumor bioenergetics is a novel and effective strategy for the treatment of GBM.

In the Cancers paper, we observed the increase ALDH1L1 protein expression following oncogenesis, as well as a therapeutic effect, by deleting the Aldh1l1 gene in KrasLA2 mice, a model of spontaneous non-small cell lung cancer (NSCLC) [...]

It is my great pleasure to open the first special issue of BMB Reports for Korean Society for Biochemistry and Molecular Biology (KSBMB) Winter Workshop. The 5th KSBMB Winter Workshop was held in ‘High 1 Ski Resort’ with about 400 attendees. This year has 6 specialized sessions including Stem cell, Proteomics, Drug development, Molecular probes, Translational research, and Young Scientists with 22 outstanding speakers. In this special issue, we have 8 invited reviews from speakers in the 2013 and 2014 KSBMB Winter Workshop. Some people may wonder why we need to make a special issue of KSBMB Winter Workshop. The purpose of this issue is for updating scientific theories and technical advances in the specialized professional fields. I wish that this special issue will be continued annually by the leading professionals in science. Since I took the job of the Chairman of the 4th KSBMB Winter Workshop in 2013, I realized that we needed to be identified ourselves as “Who are we?” and “What are we doing in the Ski Resort?”. It looks very funny raising a question in the Ski resort, but we are born Scientists. A series of meetings with committee members proposed the Winter Program that must be run by professional sessions. We are, in fact, professional scientists and doing science workshop with leading scientists. This special issue invites 8 reviews including two reviews for Proteomics, two reviews for Drug development, one review of each for Stem cell research, Molecular probes, Translational research, and Young scientist. The Proteomics review of “Small-molecule probes elucidate global enzyme activity in a proteomic context” by Prof. Lee Jun-Seok (Korea Institute of Science and Technology) summarizes the unique roles of small molecule probes in proteomics studies and highlights some recent examples in which this principle has been applied. Another review of “Proteomics Approaches for the Studies of Bone Metabolism” by Prof. Cho Je-Yoel (Seoul National University) briefly reviews recent major advances in the application of proteomics for bone biology especially in the aspect of cellular signaling. The Drug development reviews are focused on trends in cancer research such as “Structural insights into the transcription-independent apoptotic pathway of p53” by Prof. Chi Seung-Wook (Korea Research Institute of Bioscience and Biotechnology), which introduces structural basis for the transcription-independent apoptotic pathway of p53 and discusses its potential application to anticancer therapy. Another review introduces a new concept of cancer metabolism as “Tumor bioenergetics: An emerging Avenue for cancer metabolism targeted therapy” by Prof. Cheong Jae-Ho (Yonsei University College of Medicine) discusses the focused review of cancer energy metabolism and the therapeutic exploitation of glycolysis and OXPHOS as a novel anti-cancer strategy which reveal unexpected complexity and context-dependent metabolic adaptability complicating the development of effective strategies. A review for Stem cell research “The potential of mesenchymal stem cells derived from amniotic membrane and amniotic fluid for neuronal regenerative therapy” by Prof. Kim Min Kyu (Chungnam National University) suggests potential of the mesenchymal stem cells identified from the amnionic membrane and amniotic fluid focus on cure of neuronal degenerative diseases. A review for Molecular probe session may cover immunology research as well as nanotechnology. The review “In vitro and in vivo application of anti-cotinine antibody and cotinineconjugated compounds” by Prof. Chung Junho (Seoul National University College of Medicine) introduces the anti-cotinine IgG molecule that could be complexed with aptamers to form a novel affinity unit, and extended the in vivo half-life of aptamers, opening up the possibility of applying the same strategy to therapeutic peptides and chemical compounds. A review for translational research invites the successful story of anti-cancer therapy using 4-1BB (CD137) as a Specific Target for Cancer Therapy{4-1BB (CD137; TNFRS9), an activation-induced costimulatory}. Dr. Kwon Byoung Se (National Cancer Center) introduces the various aspects of 4-1BB-mediated anti-tumor responses and the basis of such responses. A review from Young scientists introduces “Short-chain fatty acid receptors and their therapeutic perspectives by Prof. Kim Sunhong (Korea Research Institute of Bioscience and Biotechnology). This review discusses about the synthetic modulators of GPR41 and GPR43 that are critical to understand the functions of the short chain fatty acid receptors as a therapeutic approach. With the 5th Winter Workshop having been a great success, I am very proud to have served as a Chairman for the 4th and 5th KSBMB Winter Workshop. I am sure that Professor Cho Je-Yoel (Seoul National University) will be a successful Chairman of the 6th KSBMB Winter Workshop.

Protein kianse CK2 is a ubiquitous kinase that can phosphorylate more than hundreds of cellular proteins, and has important roles in cell growth and development. The interaction of the catalytic subunit of CK2 (CK2alpha) with inositol hexakisphosphate (IP6) and an intrinsically disordered protein, Nopp140, has been analyzed to elucidate the IP6 and Nopp140-dependent regulation mechanism of CK2. X-ray crystallography analysis of the complex of CK2alpha and IP6 showed that lysine rich domain of CK2alpha which locates near the active site was important for the binding to IP6. One of the interaction site of Nopp140 to CK2alpha was identified at the amino acid residues 560∼580 by measuring the interactions between the peptides representing different regions of Nopp140. Particularly, the phosphorylation at Ser568 of Nopp140 significantly enhanced its interaction with CK2alpha. These results suggested a regulatory model of Nopp140 and IP6 on CK2alpha in which CK2alpha activity is inhibited by Nopp140 and re-activated by IP6 by competitive binding at the substrate recognition site of CK2alpha.

Abstract Background: Recent studies have shown that glucosamine inhibits the proliferation of various human cancer cell lines and downregulates the activity of COX-2, HIF-1α, p70S6K, and transglutaminase 2. Objective: Because the IGF-1R/Akt pathway is a common upstream regulator of p70S6K, HIF-1α, and COX-2, we hypothesized that glucosamine inhibits cancer cell proliferation through this pathway. Methods: Cell viability was assayed by MTT assay. Total RNA was isolated and reverse transcribed to cDNA for real-time PCR quantification of genes. Western blotting as performed for analyzing expression of proteins. Flow cytometry was performed for apoptosis and cell progression. Balbc/nu mice used in the study for validating results in vivo. Results: We found that glucosamine inhibited the growth of human non-small cell lung cancer cells in vitro and in vivo and negatively regulated the expression of IGF-1R and phosphorylation of Akt. In other types of cancer cells, including head and neck, breast, prostate, and colon carcinoma cell lines, glucosamine-sensitive cell lines exhibited a more significant decrease in IGF-1R and pAkt levels than glucosamine-resistant cell lines. Interestingly, most of the glucosamine-resistant cell lines have “hot-spot” mutations in PIK3CA, the p110α subunit of PI3K, or loss of PTEN, a negative regulator of Akt activation. In contrast, most of the glucosamine-sensitive cell lines have normal PIK3CA and PTEN genes. Glucosamine decreased the pAkt level through activation of IGF-1R more efficiently in the glucosamine-sensitive than in the glucosamine-resistant cell lines. Furthermore, co-treatment of cells with glucosamine and LY294002, a specific inhibitor of PI3K, significantly enhanced the anticancer effect of glucosamine in the glucosamine-resistant cell lines, whereas transient inhibition of PTEN by siRNA partially decreased the glucosamine sensitivity in the resistant and sensitive cell lines. Conclusions: Our results indicate that glucosamine is an effective inhibitor of the IGF-1R/Akt pathway and that glucosamine sensitivity of each cell line was affected by the mutation status of PIK3CA and PTEN. Citation Format: Ju-Hee Kang, Ki-Hoon Song, Jeong-Seok Nam, Hye-Young Min, Ho-Young Lee, Sung-Dae Cho, Soo-Youl Kim, Seung Hyun Oh. The novel IGF-IR/Akt-dependent anticancer activities of glucosamine are affected by PIK3CA hot-spot mutations and PTEN deletion. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-99. doi:10.1158/1538-7445.AM2013-LB-99

The number of cancer patients has increased due to longer life spans and treatment has become a universal problem. Since molecular-targeted therapies were introduced as a new developmental strategy, certain targets have been examined hundreds of times, with developers overlapping their research efforts. We need to focus our energy and resources on novel drug candidate identification and optimization, in order to enhance the entry of early-stage drug candidates into the therapeutics pipeline. This presents a major opportunity for Korea to jump the decades-old development gap between our programs and those that are more advanced in other countries. Although this country does not have a specific center for validation and development of cancer therapeutics, we do have cutting-edge scientists performing research in many institutions. In this paper, I will review cancer drug development in Korea and suggest future directions, while urging colleagues to utilize their networking expertise so we can move toward a new paradigm of novel therapeutics development. An example of such efforts has begun with the Drug Development Consortium, which was described in the KSBMB chapter. This consortium was launched in 2010 by biochemists, chemists, cell and molecular biologists and pharmacologists. It is clear that effective cancer therapeutics will be developed more efficiently when we all strive for the same goal.

Abstract Background: A few recent studies have demonstrated a possible role of transglutaminase 2 (TG2) in tumorigenesis or progression of renal cell carcinoma (RCC). The aim of this study was to examine TG2 expression and its clinical significance in patients with metastatic clear cell RCCs (ccRCCs). Methods: We analyzed 206 metastatic ccRCC patients who received the first-line vascular endothelial growth factor (VEGF) targeted therapy including sunitinib (n = 33), pazopanib (n = 120), and sorafenib (n = 53) between 2006 and 2016. The expression of TG2 was determined by immunohistochemistry and categorized into four groups, according to membranous staining intensity: negative (0), mild (1+), moderate (2+), and strong (3+). Results: TG2 staining intensity was negative in 20.9% of ccRCC (n = 43), 1+ in 46.6% (n = 96), 2+ in 9.71% (n = 20), and 3+ in 22.8% (n = 47). The survival analysis showed a significant association between stronger TG2 expression (≥ 2+) and worse progression-free survival (PFS) (P = 0.032) in the 1st line VEGF-targeted therapy. On multivariate analysis including previous nephrectomy, number of metastatic organs, and International Metastatic RCC Database Consortium Risk Score, stronger TG2 expression was a significant independent predictive indicator for poor PFS (P = 0.004). Conclusions: Our study is the first to demonstrate the significance of TG2 expression on clinical outcome in metastatic ccRCCs. TG2 expression was a significant negative predictive marker for PFS in 1st line VEGF-targeted therapy and targeting TG2 may be a new therapeutic approach to metastatic ccRCC. Citation Format: Seung-Hoon Beom, Sejung Park, Woo Sun Kwon, Sang Joon Shin, Soo-Youl Kim, Nam Hoon Cho, Sun Young Rha. Significance of Transglutaminase 2 expression on clinical outcome in metastatic renal cell carcinoma [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A053. doi:10.1158/1535-7163.TARG-19-A053

Soo-Youl Kim, Hong-Yeoul Kim, Jae-Dong Lee Department of Neurology and Neuroscience, Weill Medical College of Cornell University and Burke Medical Research Institute, White Plains, NY, U.S.A. Dept. of Biological Sciences of Oriental Medicine, Graduate School of Interdepartmental Studies, Institute of Oriental Medicines, Kyung Hee University, Seoul, Korea. Department of Acupuncture and Moxibustion, Kyung Hee Oriental Medical Hospital, Kyung Hee University.