Identification of epithelial-specific ETS-1 (ESE-1) as a tumor suppressor and molecular target of green tea compound, EGCG.

Epithelial specific ETS-1 (ESE-1) belongs to the E26 transformation-specific transcription factor superfamily and is of great interest as a potential target for managing several types of cancer. Despite its clinical significance, the documented effects of ESE-1 on cancer development and progression are contradictory and its underlying biological mechanism of action remains elusive. The objectives of this study are to investigate whether ESE-1 is a tumor suppressor and to identify dietary anti-cancer compound to activate ESE-1 expression in human colon cancer model. ESE-1 knockout and xenograft mouse models were used to examine the effect of ESE-1 in colon tumorigenesis. Stable human colon cancer cell lines were used for in vitro mechanistic studies. ESE-1 knockout in mice increased azoxymethane (AOM)-induced and dextran sulfate sodium (DSS)-promoted formation of aberrant crypt foci (ACF). Conversely, overexpression of ESE-1 suppressed tumorigenicity in a xenograft mouse study, and repressed anchorage-independent growth and migration/invasion in human colon cancer cells. Full length ESE-1 localized abundantly in the nucleus, and internal deletion of nuclear localization sequence 2 (NLS2) reduced nuclear ESE-1. Three lysine residues (318 KKK320 ) in the NLS2 determine its nuclear localization. We identified epigallocatechin-3-gallate (EGCG) that acts as a transcriptional activator of ESE-1 in human colon cancer cells. These findings propose a novel and promising molecular target of dietary anti-cancer compounds for prevention of colon cancer.

XAF1 directs apoptotic switch of p53 signaling through activation of HIPK2 and ZNF313.

X-linked inhibitor of apoptosis (XIAP)-associated factor 1 (XAF1) is a tumor suppressor that is frequently inactivated in many human cancers. However, the molecular mechanism underlying its growth-inhibitory function remains largely unknown. Here, we report that XAF1 forms a positive feedback loop with p53 and acts as a molecular switch in p53-mediated cell-fate decisions favoring apoptosis over cell-cycle arrest. XAF1 binds directly to the N-terminal proline-rich domain of p53 and thus interferes with E3 ubiquitin ligase MDM2 binding and ubiquitination of p53. XAF1 stimulates homeodomain-interacting protein kinase 2 (HIPK2)-mediated Ser-46 phosphorylation of p53 by blocking E3 ubiquitin ligase Siah2 interaction with and ubiquitination of HIPK2. XAF1 also steps up the termination of p53-mediated cell-cycle arrest by activating zinc finger protein 313 (ZNF313), a p21(WAF1)-targeting ubiquitin E3 ligase. XAF1 interacts with p53, Siah2, and ZNF313 through the zinc finger domains 5, 6, and 7, respectively, and truncated XAF1 isoforms preferentially expressed in cancer cells fail to form a feedback loop with p53. Together, this study uncovers a novel role for XAF1 in p53 stress response, adding a new layer of complexity to the mechanisms by which p53 determines cell-fate decisions.

Antisense transcription from lentiviral gene targeting linked to an integrated stress response in colorectal cancer cells.

Advances in gene therapy research have resulted in the successful development of new therapies for clinical use. Here, we explored a gene targeting approach to deplete ephrinB2 from colorectal cancer cells using an inducible lentiviral vector. EphrinB2, a transmembrane ephrin ligand, promotes colorectal cancer cell growth and viability and predicts poor patient survival when expressed at high levels in colorectal cancer tissues. We discovered that lentiviral vector integration and expression in the host DNA frequently drive divergent host gene transcription, generating antisense reads coupled with splicing events and generation of chimeric vector/host transcripts. Antisense transcription of host DNA was linked to development of an integrated stress response and cell death. Despite recent successes, off-target effects remain a concern in genetic medicine. Our results provide evidence that divergent gene transcription is a previously unrecognized off-target effect of lentiviral vector integration with built-in properties for regulation of gene expression.

Targeting the SHP2 phosphatase promotes vascular damage and inhibition of tumor growth.

The tyrosine phosphatase SHP2 is oncogenic in cancers driven by receptor-tyrosine-kinases, and SHP2 inhibition reduces tumor growth. Here, we report that SHP2 is an essential promoter of endothelial cell survival and growth in the remodeling tumor vasculature. Using genetic and chemical approaches to inhibit SHP2 activity in endothelial cells, we show that SHP2 inhibits pro-apoptotic STAT3 and stimulates proliferative ERK1/2 signaling. Systemic SHP2 inhibition in mice bearing tumor types selected for SHP2-independent tumor cell growth promotes degeneration of the tumor vasculature and blood extravasation; reduces tumor vascularity and blood perfusion; and increases tumor necrosis. Reduction of tumor growth ensues, independent of SHP2 targeting in the tumor cells, blocking immune checkpoints, or recruiting macrophages. We also show that inhibiting the Angiopoietin/TIE2/AKT cascade magnifies the vascular and anti-tumor effects of SHP2 inhibition by blocking tumor endothelial AKT signaling, not a target of SHP2. Since the SHP2 and Ang2/TIE2 pathways are active in vascular endothelial cells of human melanoma and colon carcinoma, SHP2 inhibitors alone or with Ang2/TIE2 inhibitors hold promise to effectively target the tumor endothelium.

Epigenetic inactivation of the NORE1 gene correlates with malignant progression of colorectal tumors.

BACKGROUND: NORE1 (RASSF5) is a newly described member of the RASSF family with Ras effector function. NORE1 expression is frequently inactivated by aberrant promoter hypermethylation in many human cancers, suggesting that NORE1 might be a putative tumor suppressor. However, expression and mutation status of NORE1 and its implication in colorectal tumorigenesis has not been evaluated. METHODS: Expression, mutation, and methylation status of NORE1A and NORE1B in 10 cancer cell lines and 80 primary tumors were characterized by quantitative PCR, SSCP, and bisulfite DNA sequencing analyses. Effect of NORE1A and NORE1B expression on tumor cell growth was evaluated using cell number counting, flow cytometry, and colony formation assays. RESULTS: Expression of NORE1A and NORE1B transcript was easily detectable in all normal colonic epithelial tissues, but substantially decreased in 7 (70%) and 4 (40%) of 10 cancer cell lines and 31 (38.8%) and 25 (31.3%) of 80 primary carcinoma tissues, respectively. Moreover, 46 (57.6%) and 38 (47.5%) of 80 matched tissue sets exhibited tumor-specific reduction of NORE1A and NORE1B, respectively. Abnormal reduction of NORE1 was more commonly observed in advanced stage and high grade tumors compared to early and low grade tumors. While somatic mutations of the gene were not identified, its expression was re-activated in all low expressor cells after treatment with the demethylating agent 5-aza-dC. Bisulfite DNA sequencing analysis of 31 CpG sites within the promoter region demonstrated that abnormal reduction of NORE1A is tightly associated with promoter CpG sites hypermethylation. Moreover, transient expression and siRNA-mediated knockdown assays revealed that both NORE1A and NORE1B decrease cellular growth and colony forming ability of tumor cells and enhance tumor cell response to apoptotic stress. CONCLUSION: Our data indicate that epigenetic inactivation of NORE1 due to aberrant promoter hypermethylation is a frequent event in colorectal tumorigenesis and might be implicated in the malignant progression of colorectal tumors.

Frequent inactivation of hSRBC in ovarian cancers by promoter CpG island hypermethylation.

OBJECTIVE: To explore the implication of human SRBC gene [serum deprivation response factor-related gene product that binds to the c-kinase (hSRBC)] abnormality in ovarian tumorigenesis. DESIGN: Retrospective study. SETTING: Medical center. SAMPLE: Twenty-two epithelial ovarian cancer and six normal ovary tissues. MEASURES: Mutation and altered expression of hSRBC gene. METHODS: hSRBC expression was characterized by polymerase chain reaction (PCR) analysis. Promoter CG dinucleotide (CpG) site methylation was determined using methylation specific PCR and bisulfite sequencing. RESULTS: Expression of hSRBC transcript was easily detectable in all normal tissues we examined, but 50% (two of four) of cancer cell lines and 41% (nine of 22) of primary carcinomas exhibited undetectable or substantially decreased expression. While genomic deletion or somatic mutations of the gene was not identified, its expression was reactivated in tumor cells by 5-aza-2'-deoxycytidine treatment, suggesting epigenetic inactivation of the gene in tumors. Promoter methylation was detected in all nine tumors with low expression but in only one of 13 (7.7%) tumors with normal expression. Bisulfite DNA sequencing analysis of 23 CpG sites within the promoter region revealed that the CpG sites are highly methylated in low-expressing tumors. In addition, promoter CpG sites methylation status showed a tight association with gene expression level. CONCLUSION: Our data demonstrate that epigenetic inactivation of hSRBC due to aberrant promoter hypermethylation is a common event and might be implicated in human ovarian tumorigenesis.

NF-kappaB activates transcription of the RNA-binding factor HuR, via PI3K-AKT signaling, to promote gastric tumorigenesis.

BACKGROUND & AIMS: HuR is a RNA-binding factor whose expression is commonly upregulated in some human tumor types. We explored the molecular mechanism underlying HuR elevation and its role in gastric cancer tumorigenesis. METHODS: HuR expression and subcellular localization were determined by polymerase chain reaction, immunoblot, and immunohistochemical analyses. Its effect on tumor growth was characterized using flow cytometry, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling, and soft agar analyses. Luciferase reporter, chromatin immunoprecipitation, and electrophoretic mobility shift assays were used to measure transcriptional activation by nuclear factor kappaB (NF-kappaB) signaling. RESULTS: Compared with normal gastric tissues, HuR was expressed at higher levels in gastric tumors, particularly in advanced versus early tumors; this increase was associated with enhanced cytoplasmic translocation of HuR. HuR overexpression increased proliferation of tumor cells, activating the G(1) to S transition of the cell cycle, DNA synthesis, and anchorage-independent growth. Small interfering RNA-mediated knockdown of HuR expression reduced tumor cell proliferation and response to apoptotic stimuli. No genetic or epigenetic alterations of HuR were observed in gastric tumor cell lines or primary tumors; overexpression depended on phosphatidylinositol 3-kinase/AKT signaling and NF-kappaB activity. AKT activation increased p65/RelA binding to a putative NF-kappaB binding site in the HuR promoter, the stability of HuR target transcripts, and the cytoplasmic import of HuR. CONCLUSIONS: HuR is a direct transcription target of NF-kappaB; its activation in gastric cancer cell lines depends on phosphatidylinositol 3-kinase/AKT signaling. HuR activation by this pathway has proliferative and antiapoptotic effects on gastric cancer cells.

DLC1 deficiency and YAP signaling drive endothelial cell contact inhibition of growth and tumorigenesis.

Deleted in Liver Cancer 1 (DLC1) is a tumor suppressor gene deleted in many cancers, including angiosarcoma, an aggressive malignancy of endothelial cell derivation. DLC1-deficiency in primary endothelial cells causes the loss of cell contact inhibition of growth through incompletely defined mechanisms. We report that DLC1 is a regulator of YAP, a transcriptional coactivator of proliferation-promoting and tumor-promoting genes; when confluent, active/nuclear YAP was significantly more abundant in DLC1-deficient endothelial cells compared with control cells. We also found that YAP is a required effector of the loss of cell contact inhibition of growth manifested by DLC1-deficient endothelial cells, as the silencing of YAP prevents this loss. Consistently, human angiosarcomas specimens contained a significantly greater proportion of DLC1- tumor cells with nuclear YAP compared with the DLC1+ normal cells in the adjacent tissue. Verteporfin, an inhibitor of YAP, significantly reduced angiosarcoma growth in mice. These results identify YAP as a previously unrecognized effector of DLC1 deficiency-associated loss of cell contact growth inhibition in endothelial cells and a potential therapeutic target in angiosarcoma.

Identification of Eph receptor signaling as a regulator of autophagy and a therapeutic target in colorectal carcinoma.

Advanced colorectal carcinoma is currently incurable, and new therapies are urgently needed. We report that phosphotyrosine-dependent Eph receptor signaling sustains colorectal carcinoma cell survival, thereby uncovering a survival pathway active in colorectal carcinoma cells. We find that genetic and biochemical inhibition of Eph tyrosine kinase activity or depletion of the Eph ligand EphrinB2 reproducibly induces colorectal carcinoma cell death by autophagy. Spautin and 3-methyladenine, inhibitors of early steps in the autophagic pathway, significantly reduce autophagy-mediated cell death that follows inhibition of phosphotyrosine-dependent Eph signaling in colorectal cancer cells. A small-molecule inhibitor of the Eph kinase, NVP-BHG712 or its regioisomer NVP-Iso, reduces human colorectal cancer cell growth in vitro and tumor growth in mice. Colorectal cancers express the EphrinB ligand and its Eph receptors at significantly higher levels than numerous other cancer types, supporting Eph signaling inhibition as a potential new strategy for the broad treatment of colorectal carcinoma.

ESE­1 suppresses the growth, invasion and migration of human NSCLC cells and tumor formation in vivo.

Lung cancer is the first leading cause of cancer­related death in the United States. Non­small cell lung cancer (NSCLC) is the most common type of lung cancer and is associated with a poor patient prognosis. Identification of promising molecular targets is required for the effective prevention and therapy of NSCLC. Epithelial­specific ETS­1 (ESE­1) belongs to the superfamily of ETS transcription factors. The effect of ESE­1 on tumorigenesis is controversial in several types of cancer while its role in lung cancer remains unknown. The present study was designed to investigate whether ESE­1 expression affects tumorigenic activity using human NSCLC cells and a mouse xenograft model. ESE­1 expression suppressed anchorage­independent growth in soft agar assay and led to an increase in G1 arrest and apoptosis in human NSCLC cells. ESE­1 expression suppressed the invasion and migration of human NSCLC cells. Western blot analysis, RT­PCR and promoter assay indicated that ESE­1 expression was transcriptionally downregulated by treatment of transforming growth factor (TGF)­ß, an EMT (epithelial­mesenchymal transition) stimulator. The xenograft study indicated that ESE­1 expression inhibited tumor formation and development. Our data demonstrated that ESE­1 plays a key role as a tumor suppressor in human NSCLC.

Effects of DLC1 Deficiency on Endothelial Cell Contact Growth Inhibition and Angiosarcoma Progression.

Background: Deleted in Liver Cancer 1 (DLC1) is a tumor suppressor gene frequently deleted in cancer. However, DLC1 is not known to be deleted in angiosarcoma, an aggressive malignancy of endothelial cell derivation. Additionally, the physiologic functions of DLC1 protein in endothelial cells are poorly defined. Methods: We investigated the effects of shRNA-induced DLC1 depletion in endothelial cells. Cell growth was measured by 3H thymidine incorporation, IncuCyte imaging, and population doublings; cell death by cell cycle analysis; gene expression by Affimetrix arrays and quantitative polymerase chain reaction; NF-κB activity by reporter assays; and protein levels by immunoblotting and immunofluorescence staining. We tested Tanespimycin/17-AAG and Fasudil treatment in groups of nine to 10 mice bearing ISOS-1 angiosarcoma. All statistical tests were two-sided. Results: We discovered that DLC1 is a critical regulator of cell contact inhibition of proliferation in endothelial cells, promoting statistically significant (P < .001) cell death when cells are confluent (mean [SD] % viability: control DLC1 = 15.6 [19.3]; shDLC1 = 73.4 [13.1]). This prosurvival phenotype of DLC1-depleted confluent endothelial cells is attributable to a statistically significant and sustained increase of NF-κB activity (day 5, P = .001; day 8, P = .03) associated with increased tumor necrosis factor alpha-induced protein 3 (TNFAIP3/A20) signaling. Consistently, we found that DLC1 is statistically significantly reduced (P < .001 in 5 of 6) and TNFAIP3/A20 is statistically significantly increased (P < .001 in 2 of 3 and P = 0.02 in 1 of 3) in human angiosarcoma compared with normal adjacent endothelium. Treatment with the NF-κB inhibitor Tanespimycin/17-AAG statistically significantly reduced angiosarcoma tumor growth in mice (treatment tumor weight vs control, 0.50 [0.19] g vs 0.91 [0.21] g, P = .001 experiment 1; 0.66 [0.26] g vs 1.10 [0.31] g, P = .01 experiment 2). Conclusions: These results identify DLC1 as a previously unrecognized regulator of endothelial cell contact inhibition of proliferation that is depleted in angiosarcoma and support NF-κB targeting for the treatment of angiosarcoma where DLC1 is lost.

Potential Dual Role of Activating Transcription Factor 3 in Colorectal Cancer.

BACKGROUND/AIM: Activating transcription factor 3 (ATF3) is a member of the ATF/CREB transcription factor family and has been proposed as a molecular target for cancer therapy. The present study was undertaken in order to investigate whether ATF3 influences cancer-related phenotypes in colorectal cancer. MATERIALS AND METHODS: ATF3 was overexpressed in human colorectal cancer cells and the effects of ATF3 on apoptosis, cell cycle, cell migration and epithelial mesenchymal transition (EMT) were investigated. B-cell lymphoma-2 (Bcl-2) promoter was cloned and used for luciferase assay in cells transfected with control or ATF3 expression vector. RESULTS: ATF3 down-regulated the expression of Bcl-2 and promoter activity of the Bcl-2 gene. ATF3 increased collective cell migration and expression of cluster of differentiation 44 (CD44) and decreased retinoblastoma (Rb) expression. In addition, ATF3 down-regulated EMT-inducing transcription factors and ß-catenin. CONCLUSION: ATF3 may play a dichotomous role in apoptosis and metastasis in human colorectal cancer cells.

Tolfenamic acid downregulates ß-catenin in colon cancer.

Tolfenamic acid is one of the fenamic acid-derived non-steroid anti-inflammatory drugs (NSAIDs) and has been shown to exhibit anti-cancer activities in several types of cancer. Both mutations and aberrant expression of ß-catenin are highly associated with progression of cancer. Therefore, ß-catenin is considered to be a promising molecular target for cancer prevention and treatment. The current study investigates the role of tolfenamic acid on ß-catenin expression in colon cancer. Treatment with tolfenamic acid led to inhibition of cell growth and down-regulation of ß-catenin expression in a dose- and time-dependent manner in human colon cancer cell lines. Reduction of ß-catenin upon tolfenamic acid treatment was associated with ubiquitin-mediated proteasomal degradation, without affecting mRNA level and promoter activity of ß-catenin. In addition, treatment with tolfenamic acid downregulated Smad2 and Smad3 expression, while overexpression of Smad2, but not Smad3, blocked tolfenamic acid-induced suppression of ß-catenin expression. Tolfenamic acid also decreased expression of ß-catenin target genes, including vascular endothelial growth factor (VEGF). Compared to adjacent normal tissue, intestinal tumor tissues of Apc(Min/+) mice exhibited increased expression of ß-catenin, Smad2, Smad3, and VEGF, which were down-regulated with tolfenamic acid treatment at a dose of 50mg/kg body weight. In conclusion, our findings suggest that tolfenamic acid inhibits growth of colon cancer cells through downregulation of Smad2 and, subsequently, facilitating ubiquitin-proteasome-mediated ß-catenin degradation in colon cancer.

An ab Initio Study of Intramolecular Hydrogen Bondings in α-Hydroxy Ketomethylene Dipeptide Isostere.

Ab initio calculations of the representative α-hydroxy ketomethylene dipeptide isostere (2S,5S)-5-amino-2-hydroxy-4-oxohexanoic acid (1) are described. All calculations including full geometry optimizations were performed at the MP2/6-31G* level. In the gas phase, 12 low-energy conformers are located by minimizing geometries assembled from stable molecular fragments. Among these conformers, six structurally similar conformers, in which the 2-hydroxyl group forms hydrogen bondings with both the O atom of the 4-carbonyl group in 1,3-fashion and the O atom of 1-carboxylic acid in 1,2-fashion simultaneously, are found to be particularly stable. Thus, the conformational preference of 1 appears to be governed by arrangements and strength of intramolecular hydrogen bondings. To examine conformational natures of 1 in solutions more accurately, we corrected the thermochemical properties and carried out self-consistent reaction field calculations. Going from the gas phase to solutions, the basic features of the conformational preferences in 1 also appear to be maintained in solutions including a highly polar aqueous medium, despite slight changes in the population of each conformer.

PPARδ promotes oncogenic redirection of TGF-ß1 signaling through the activation of the ABCA1-Cav1 pathway.

TGF-ß1 plays biphasic functions in prostate tumorigenesis, inhibiting cell growth at early stages but promoting malignant progression at later stages. However, the molecular basis for the oncogenic conversion of TGF-ß1 function remains largely undefined. Here, we demonstrate that PPARδ is a direct transcription target of TGF-ß1 and plays a critical role in oncogenic redirection of TGF-ß1 signaling. Blockade of PPARδ induction enhances tumor cell response to TGF-ß1-mediated growth inhibition, while its activation promotes TGF-ß1-induced tumor growth, migration and invasion. PPARδ-mediated switch of TGF-ß1 function is associated with down- and upregulation of Smad and ERK signaling, respectively, and tightly linked to its function to activate ABCA1 cholesterol transporter followed by caveolin-1 (Cav1) induction. Intriguingly, TGF-ß1 activation of the PPARδ-ABCA1-Cav1 pathway facilitates degradation of TGF-ß receptors (TßRs) and attenuates Smad but enhances ERK response to TGF-ß1. Expression of PPARδ and Cav1 is tightly correlated in both prostate tissues and cell lines and significantly higher in cancer vs. normal tissues. Collectively, our study shows that PPARδ is a transcription target of TGF-ß1 and contributes to the oncogenic conversion of TGF-ß1 function through activation of the ABCA1-Cav1-TßR signaling axis.

CAV1/caveolin 1 enhances aerobic glycolysis in colon cancer cells via activation of SLC2A3/GLUT3 transcription.

Although elevated expression of CAV1/caveolin 1 is associated with the malignant progression of various human cancers, the molecular mechanism underlying its oncogenic functions is largely unknown. We found that CAV1 is frequently overexpressed in advanced colorectal tumors due to aberrant promoter CpG site hypomethylation, and its elevation is implicated in enhanced aerobic glycolysis of tumor cells. Depletion of elevated CAV1 downregulates glucose uptake, intracellular ATP level and lactate accumulation, and triggers autophagy through activation of AMPK-TP53/p53 signaling. CAV1 elevation increases glucose uptake and ATP production by stimulating transcription of the glucose transporter SLC2A3/GLUT3 via an HMGA1-binding site within the promoter. Collectively, our study suggests that elevated CAV1 expression may contribute to colorectal tumor progression by providing tumor cells growth and survival advantages under nutritional stress conditions.

Caveolin-1 increases aerobic glycolysis in colorectal cancers by stimulating HMGA1-mediated GLUT3 transcription.

Caveolin-1 (CAV1) acts as a growth suppressor in various human malignancies, but its expression is elevated in many advanced cancers, suggesting the oncogenic switch of its role during tumor progression. To understand the molecular basis for the growth-promoting function of CAV1, we characterized its expression status, differential roles for tumor growth, and effect on glucose metabolism in colorectal cancers. Abnormal elevation of CAV1 was detected in a substantial fraction of primary tumors and cell lines and tightly correlated with promoter CpG sites hypomethylation. Depletion of elevated CAV1 led to AMPK activation followed by a p53-dependent G1 cell-cycle arrest and autophagy, suggesting that elevated CAV1 may contribute to ATP generation. Furthermore, CAV1 depletion downregulated glucose uptake, lactate accumulation, and intracellular ATP level, supporting that aerobic glycolysis is enhanced by CAV1. Consistently, CAV1 was shown to stimulate GLUT3 transcription via an HMGA1-binding site within the GLUT3 promoter. HMGA1 was found to interact with and activate the GLUT3 promoter and CAV1 increased the HMGA1 activity by enhancing its nuclear localization. Ectopic expression of HMGA1 increased glucose uptake, whereas its knockdown caused AMPK activation. In addition, GLUT3 expression was strongly induced by cotransfection of CAV1 and HMGA1, and its overexpression was observed predominantly in tumors harboring high levels of CAV1 and HMGA1. Together, these data show that elevated CAV1 upregulates glucose uptake and ATP production through HMGA1-mediated GLUT3 transcription, suggesting that CAV1 may render tumor cells growth advantages by enhancing aerobic glycolysis.

Epigenetic alteration of PRKCDBP in colorectal cancers and its implication in tumor cell resistance to TNFα-induced apoptosis.

PURPOSE: PRKCDBP is a putative tumor suppressor in which alteration has been observed in several human cancers. We investigated expression and function of PRKCDBP in colorectal cells and tissues to explore its candidacy as a suppressor in colorectal tumorigenesis. EXPERIMENTAL DESIGN: Expression and methylation status of PRKCDBP and its effect on tumor growth were evaluated. Transcriptional regulation by NF-κB signaling was defined by luciferase reporter and chromatin immunoprecipitation assays. RESULTS: PRKCDBP expression was hardly detectable in 29 of 80 (36%) primary tumors and 11 of 19 (58%) cell lines, and its alteration correlated with tumor stage and grade. Promoter hypermethylation was commonly found in cancers. PRKCDBP expression induced the G(1) cell-cycle arrest and increased cellular sensitivity to various apoptotic stresses. PRKCDBP was induced by TNFα, and its level correlated with tumor cell sensitivity to TNFα-induced apoptosis. PRKCDBP induction by TNFα was disrupted by blocking NF-κB signaling while it was enhanced by RelA transfection. The PRKCDBP promoter activity was increased in response to TNFα, and this response was abolished by disruption of a κB site in the promoter. PRKCDBP delayed the formation and growth of xenograft tumors and improved tumor response to TNFα-induced apoptosis. CONCLUSIONS: PRKCDBP is a proapoptotic tumor suppressor which is commonly altered in colorectal cancer by promoter hypermethylation, and its gene transcription is directly activated by NF-κB in response to TNFα. This suggests that PRKCDBP inactivation may contribute to tumor progression by reducing cellular sensitivity to TNFα and other stresses, particularly under chronic inflammatory microenvironment.