[Show abstract][Hide abstract] ABSTRACT: PGP9.5 is a controversial molecule from an oncologic point of view. We recently identified frequent methylation of PGP9.5 gene exclusively in primary head and neck squamous cell carcinoma (HNSCC), suggesting that it could be a tumor suppressor gene. On the other hand, PGP9.5 was reported to be overexpressed in a subset of human cancers presumably due to intrinsic oncogenic properties or as a result of transformation. To demonstrate that PGP9.5 possesses tumor suppressive activity, we examined forced expression by stable transfection of PGP9.5 in 4 HNSCC cell lines. Although all 4 cell lines demonstrated reduced log growth rates in culture after transfection, only 2 cell lines with wild type p53 (011, 022) demonstrated decreased growth in soft agar. In 2 cell lines with mutant p53 (013, 019), we observed no altered growth in soft agar and increased sensitivity to UV irradiation. We then tested for and found a high frequency of promoter methylation in a larger panel of primary tumors including HNSCC, esophageal SCC, gastric, lung, prostate and hepatocellular carcinoma. Our data support the notion that PGP9.5 is a tumor suppressor gene that is inactivated by promoter methylation or gene deletion in several types of human cancers.
Full-text · Article · Aug 2008 · International Journal of Cancer
[Show abstract][Hide abstract] ABSTRACT: Deleted in Colorectal Cancer (DCC) is a putative tumor suppressor gene, whose loss has been implicated in colorectal tumorigenesis. Decreased or loss of DCC expression has been demonstrated in a number of human cancers, including esophageal cancer. In this study, we analyzed esophageal squamous cell carcinoma (ESCC) cell lines and primary ESCCs as well as normal esophageal tissues for DCC methylation by bisulfite sequencing, methylation-specific PCR (MSP) and/or quantitative methylation-specific PCR (qMSP). When a qMSP cut-off value for positivity was set to 1.0, DCC methylation was detected in 10 of 12 ESCC cell lines tested, 74% of primary ESCCs (n = 70), 0% of corresponding normal esophageal tissues (n = 20) and 0% of normal esophagus from healthy individuals (n = 19). DCC expression was undetectable in the majority of ESCC cell lines, and treatment with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine reactivated gene expression. DCC overexpression suppressed colony formation in ESCC cell lines, suggesting that DCC may function as a tumor suppressor gene in the esophagus. However, DCC methylation was not associated with any clinical or pathologic parameters measured. We have demonstrated that DCC methylation is a frequent and cancer-specific event in primary ESCCs, suggesting that DCC and associated pathways may represent a new diagnostical therapeutic target.
Full-text · Article · Jun 2008 · International Journal of Cancer
[Show abstract][Hide abstract] ABSTRACT: N-methyl-D-aspartate receptors (NMDARs) are the predominant excitatory neurotransmitter receptors in the mammalian brain. We found that among the three NMDARs examined (NMDAR1, NMDAR2A, NMDAR2B), only NMDAR2A was silenced in colorectal carcinoma (CRC) cell lines at basal line and reactivated by the demethylating agent, 5-aza-2'-deoxycytidine. NMDAR2A was expressed in normal colon epithelium, while expression was hardly detectable in colon cancer tissues. Promoter methylation of NMDAR2A was confirmed by bisulfite sequencing and combined bisulfite restriction analysis in the CRC cell lines and primary tumors. Quantitative methylation-specific PCR demonstrated NMDAR2A promoter hypermethylation in 82 of 100 primary human CRC, 15 of 100 normal corresponding epithelial tissues and 1 of 11 (9%) normal colon mucosa samples obtained from patients without cancer. Moreover, forced expression of full-length NMDAR2A in CRC cell lines induced apoptosis and almost abolished the ability of the cells to form colonies in culture, while NMDAR2A knockdown increased cell growth. Thus, NMDAR2A is commonly hypermethylated in primary human CRC and possesses tumor-suppressive activity.
[Show abstract][Hide abstract] ABSTRACT: Promoter DNA hypermethylation with gene silencing is a common feature of human cancer, and cancer-prone methylation is believed to be a landmark of tumor suppressor genes (TSG). Identification of novel methylated genes would not only aid in the development of tumor markers but also elucidate the biological behavior of human cancers. We identified several epigenetically silenced candidate TSGs by pharmacologic unmasking of esophageal squamous cell carcinoma (ESCC) cell lines by demethylating agents (5-aza-2'-deoxycitidine and trichostatin A) combined with ESCC expression profiles using expression microarray. HOP/OB1/NECC1 was identified as an epigenetically silenced candidate TSG and further examined for (a) expression status, (b) methylation status, and (c) functional involvement in cancer cell lines. (a) The HOP gene encodes two putative promoters (promoters A and B) associated with two open reading frames (HOPalpha and HOPbeta, respectively), and HOPalpha and HOPbeta were both down-regulated in ESCC independently. (b) Promoter B harbors dense CpG islands, in which we found dense methylation in a cancer-prone manner (55% in tumor tissues by TaqMan methylation-specific PCR), whereas promoter A does not harbor CpG islands. HOPbeta silencing was associated with DNA methylation of promoter B in nine ESCC cell lines tested, and reactivated by optimal conditions of demethylating agents, whereas HOPalpha silencing was not reactivated by such treatments. Forced expression of HOP suppressed tumorigenesis in soft agar in four different squamous cell carcinoma cell lines. More convincingly, RNA interference knockdown of HOP in TE2 cells showed drastic restoration of the oncogenic phenotype. In conclusion, HOP is a putative TSG that harbors tumor inhibitory activity, and we for the first time showed that the final shutdown process of HOP expression is linked to promoter DNA hypermethylation under the double control of the discrete promoter regions in cancer.
Preview · Article · Feb 2008 · Molecular Cancer Research
[Show abstract][Hide abstract] ABSTRACT: We report here that human MFGE8 encoding milk fat globule-EGF factor 8 protein (MFG-E8), also termed 46 kDa breast epithelial antigen and lactadherin, is transcriptionally activated by p63, or TP63, a p53 (TP53) family protein frequently overexpressed in head-and-neck squamous cell carcinomas, mammary carcinomas and so on. Despite that human MFG-E8 was originally identified as a breast cancer marker, and has recently been reported to provide peptides for cancer immunotherapy, its transcriptional control remains an open question. Observations in immunohistochemical analyses, a tetracycline-induced p63 expression system and keratinocyte cultures suggested a physiological link between p63 and MFGE8. By reporter assays with immediately upstream regions of MFGE8, we determined that the trans-activator (TA) isoforms of p63 activate MFGE8 transcription though a p53/p63 motif at -370, which was confirmed by a chromatin immunoprecipitation experiment. Upon siRNA-mediated p63 silencing in a squamous cell carcinoma line, MFG-E8 production decreased to diminish Saos-2 cell adhesion. Interestingly, the DeltaN-p63 isoform lacking the TA domain enhanced the MFGE8-activating function of TA-p63, if DeltaN-p63 was dominant over TA-p63 as typically observed in undifferentiated keratinocytes and squamous cell carcinomas, implying a self-regulatory mechanism of p63 by the TA:DeltaN association. MFG-E8 may provide a novel pathway of epithelial-nonepithelial cell interactions inducible by p63, probably in pathological processes.
[Show abstract][Hide abstract] ABSTRACT: To investigate whether the promoter methylation pattern in N-methyl-d-aspartate receptor 2B (NMDAR2B) is correlated with clinical features of human esophageal squamous cell carcinoma (ESCC), the methylation status of the gene was examined at three different sites (P1, P2, and P3) where two CpG islands reside within 1 kb upstream of the transcription start site.
Three independent modalities for methylation analysis (bisulfite sequencing, combined bisulfite restriction analysis, and TaqMan methylation-specific PCR) were done to analyze total 67 ESCC tissues that included 43 primary tumors with well-characterized clinicopathologic variables including patient outcome.
Using an optimized cutoff value based on quantitative methylation-specific PCR, we found that patients with higher NMDAR2B methylation ratio in the proximal region (P1) showed a worse 5-year disease-specific survival rate than those without NMDAR2B methylation (P < 0.006). A significant correlation was also seen between NMDAR2B promoter methylation and the presence of vascular permeation (P = 0.03).
NMDAR2B promoter methylation could be a clinically applicable marker in ESCC.
No preview · Article · Nov 2007 · Clinical Cancer Research
[Show abstract][Hide abstract] ABSTRACT: GDF15 is a transcriptional target gene for p53 and its family members, p63 and p73. Its promoter region contains two p53-type response elements, RE1 and RE2, and RE2 confers p53-specific transactivation. RE2 contains several mismatches from the canonical p53 response element (RRRCWWGYYY). Two mismatches in the RRR span and T base of the RE2 core sequence in the most 3' quarter-site are critical for inhibiting the binding affinity to p63 and p73 and corresponding promoter activity. Our results strongly suggest that differential DNA-binding affinities between p53 family member proteins act, at least in part, to confer specific target gene activation.
Full-text · Article · Apr 2007 · Biochemical and Biophysical Research Communications
[Show abstract][Hide abstract] ABSTRACT: p63 plays a more complex role than initially thought in cancer and development. As a p53 homolog, p63 encodes transcription factors that primarily functions through regulation of downstream gene expression. However, p63 is also involved in RNA processing and activation of beta-catenin signaling. A number of genes activated by TAp63 support the notion that p63 is involved in tight transcriptional control of epithelial differentiation, cell adhesion, and tumorigenesis via cell cycle arrest, apoptosis, and other cellualr functions. In addition, DeltaNp63 isotypes retain a rather short transactivation domain and were found to transcriptionally regulate a specific set of downstream gene targets. We found that p63 is capable of activating gene expression through binding to specific cis-elements, RE1 and RE2, with the latter being more specific for p63 than for p53. Differences in p53 family members DNA binding may help to explain key differences in their function and biology.
[Show abstract][Hide abstract] ABSTRACT: Based on the oncogenic role of phosphatidylinositol glycan (PIG) class U in human tumors, we explored the role of two additional subunits of the glycosylphosphatidylinositol (GPI) transamidase complex in human breast cancer. We found that PIG class T (PIG-T) and GPI anchor attachment 1 (GPAA1) were overexpressed in breast cancer cell lines and primary tumors. Forced expression of PIG-T and GPAA1 transformed NIH3T3 cells in vitro and increased tumorigenicity and invasion of these cells in vivo. Suppression of PIG-T expression in breast cancer cell lines led to inhibition of anchorage-independent growth. Moreover, we found that PIG-T and GPAA1 expression levels positively correlated with paxillin phosphorylation in invasive breast cancer cell lines. Furthermore, suppression of PIG-T and GPAA1 expression led to a decrease in paxillin phosphorylation with a concomitant decrease in invasion ability. These results suggest that the GPI transamidase complex is composed of a group of proto-oncogenes that individually or as a group contribute to breast cancer growth. This aberrant growth is mediated, at least partially, by phosphorylation of paxillin, contributing to invasion and progression of breast cancer.
[Show abstract][Hide abstract] ABSTRACT: Promoter hypermethylation accompanied by gene silencing is a common feature of human cancers. We identified previously several new tumor suppressor genes based on pharmacologic unmasking of the promoter region and detection of reexpression on microarray analysis. In this study, we modified the selection of candidates from our previous microarray data by excluding genes that showed basal expression in cancer cell lines. With the new method, we found novel methylated genes with 90% accuracy. Among these 33 novel methylated genes that we identified in esophageal squamous cell carcinoma (ESCC) cell lines, N-methyl-D-aspartate receptor type 2B (NMDAR2B) was of particular interest. NMDAR2B was methylated in 95% of primary human ESCC tissue specimens and 12 ESCC cell lines by sequence analysis. NMDAR2B expression was silenced in all 12 ESCC cell lines and was reactivated by the demethylating agent 5-aza-2'-deoxycytidine. Moreover, reintroduction of the gene was accompanied by marked Ca(2+)-independent apoptosis in ESCC cell lines, suggesting that NMDAR2B can suppress tumor growth. Thus, NMDAR2B promoter methylation is common in ESCC, abrogating gene transcription and leading to cellular resistance to apoptosis.
[Show abstract][Hide abstract] ABSTRACT: Diffuse-type gastric cancer (DGC) is the most deadly form of gastric cancer and is frequently accompanied by peritoneal dissemination and metastasis. The specific molecular events involved in DGC pathogenesis remain elusive. Accumulating evidence of epigenetic inactivation in tumor suppressor genes led us to conduct a comprehensive screen to identify novel methylated genes in human cancers using pharmacologic unmasking and subsequent microarray analysis. We compared differential RNA expression profiles of DGC and intestinal-type gastric cancer (IGC) cell lines treated with 5-aza-2'-deoxycytidine using microarrays containing 22,284 genes. We identified 16 methylated genes, including many novel genes, in DGC cell lines and studied PGP9.5 with particular interest. In primary gastric cancers, PGP9.5 was found to be more frequently methylated in DGCs (78%) than in IGCs (36%; DGC versus IGC, P < 0.05). Furthermore, real-time methylation-specific PCR analysis of PGP9.5 showed relatively higher methylation levels in DGC than in IGC. Our data thus implicate a molecular event common in the DGC phenotype compared with IGC.
[Show abstract][Hide abstract] ABSTRACT: Recent evidence indicates that in vitro p53 augments base excision repair (BER) activities in mammalian cells. To understand the role of p53 in BER, we analyzed the repair activity of hOgg1 in isogenic cell lines HCT116p53+/+ and HCT116p53-/-. We found that hOgg1 activity was significantly decreased in HCT116p53-/- cells as compared with HCT116p53+/+ cells, indicating a functional role for p53 in the regulation of hOGG1. Using gel-shift assays, we showed that p53 binds to its putative cis-elements within the hOGG1 promoter. In addition we demonstrated that supplementing p53 in HCT116p53-/- cells enhanced the transcription of hOGG1. To further strengthen our findings, we used p53-RNAi to study the effects of decreased p53 levels on hOgg1 activity. We observed that p53-RNAi resulted in decreased hOGG1 expression both at the mRNA and protein levels. This decrease in hOGG1 expression was associated with reduced cell viability upon oxidative damage and reduced hOgg1 activity as evidenced by the 8-oxoG incision assay. Taken together, our results indicate that loss of p53 function can lead to decreased hOgg1 repair activity.
No preview · Article · Feb 2006 · The FASEB Journal
[Show abstract][Hide abstract] ABSTRACT: The p53 tumor suppressor gene family consists of three genes, p53, p63, and p73. p53 family proteins share high homology in their DNA-binding domains but exhibit diverse biological functions. In this study, we demonstrated differential target gene activation by specific p53, p63, and p73 induction in Saos2 cells by oligonucleotide microarray expression analysis. We further analyzed the WNT4 promoter, which was induced by p63 and p73 but not p53, in order to clarify the mechanism of differential target gene activation between the three family members. Luciferase analysis showed that the WNT4 promoter harbors two p63/p73 response elements, designated RE1 and RE2. RE1 resembles the canonical p53 response element (tandem repeats of RRRCWWGYYY), located between -141 and -121, while RE2 consists of a GC-rich sequence further downstream. Neither response element alone was able to confer transcriptional activity. It is thus likely that both RE1 and RE2 are necessary in rendering p63/p73-specific activation of the WNT4 promoter.
No preview · Article · Feb 2006 · Biochemical and Biophysical Research Communications
[Show abstract][Hide abstract] ABSTRACT: p63 is a member of the p53 tumor suppressor gene family, which regulates downstream target gene expression by binding to sequence-specific response elements similar to those of p53. By using oligonucleotide expression microarray analysis and analyzing the promoters of p63-induced genes, we have identified novel p63-specific response elements (p63-REs) in the promoter regions of EVPL and SMARCD3. These p63-REs exhibit characteristic differences from the canonical p53-RE (RRRCWWGYYY) in both the core-binding element (CWWG) as well as the RRR and/or YYY stretches. Luciferase assays on mutagenized promoter constructs followed by electromobility shift analysis showed that p53 preferentially activates and binds to the RRRCATGYYY sequence, whereas p63 preferentially activates RRRCGTGYYY. Whereas EVPL protein is highly expressed in epithelial cells of the skin and pharynx in the p63+/+ mouse, it is undetectable in these tissues in the p63-/- mouse. Our results indicate that p63 can regulate expression of specific target genes such as those involved in skin, limb, and craniofacial development by preferentially activating distinct p63-specific response elements.
Full-text · Article · Aug 2005 · Molecular and Cellular Biology
[Show abstract][Hide abstract] ABSTRACT: p63, a member of the p53 superfamily, is an essential cell fate determinant for stratified epithelium. Deficiency of p63 leads to lack of differentiated epithelium from the skin and the presence of trace undifferentiated cells left in the dermis. We found that transcriptionally active isoforms of p63, TAp63beta and TAp63gamma, activated the skin-specific promoter of bullous pemphigoid antigen 1 (BPAG-1). The p63-response element was localized between bases -177 and -153 upstream of exon 1 in the BPAG-1e promoter, whereas regions surrounding the response element suppressed transcriptional responses to p53 and TAp73beta, resulting in p63-specific activation of the promoter. This represents a novel molecular mechanism by which target gene induction by p63 is distinguished from induction by other p53 family members.
No preview · Article · Aug 2005 · Journal of Investigative Dermatology
[Show abstract][Hide abstract] ABSTRACT: PGP9.5/UCHL1 is a member of the carboxyl-terminal ubiquitin hydrolase family with a potential role in carcinogenesis. We previously identified PGP9.5 as a putative tumor-suppressor gene and methylation of the promoter as a cancer-specific event in primary cancer tissues. In this current study, we analyzed PGP9.5 methylation in 50 esophageal squamous cell carcinoma (ESCC) primary tumors with well characterized clinicopathologic variables including patient outcome. Two independent modalities for methylation analysis (TaqMan methylation-specific PCR and combined bisulfite restriction analysis) were used to analyze these samples. The two data sets were consistent with each other, as the 21 patients (42%) with highest methylation levels by TaqMan analysis all showed visible combined bisulfite restriction analysis bands on acrylamide gels. Using an optimized cutoff value by TaqMan quantitation, we found that patients with higher PGP9.5 methylation ratios in the primary tumor showed poorer 5-year survival rates than those without PGP9.5 methylation (P = 0.01). A significant correlation was also seen between PGP9.5 promoter methylation and the presence of regional lymph node metastases (P = 0.03). Multivariate analysis subsequently revealed that PGP9.5 methylation was an independent prognostic factor for ESCC survival (P = 0.03). These results suggest that PGP9.5 promoter methylation could be a clinically applicable marker for ESCC progression.
[Show abstract][Hide abstract] ABSTRACT: HSP70, a stress response protein, is known to be a determinant of cell death and cell transformation. We show that different isoforms of p63 have different transcriptional activities on hsp70 genes. DeltaNp63alpha, an abundantly expressed isoform of p63, activates (in vitro and in vivo), whereas TAp63gamma down-regulates the expression of hsp70. We further show that the transactivation domain at the NH(2) terminus of p63 represses, whereas the COOH terminus activates hsp70 transcription. In addition, DeltaNp63alpha regulates transcription of the hsp70 gene through its interaction with the CCAAT binding factor and NF-Y transcription factors which are known to form a complex with the CCAAT box located in the hsp70 promoter. Moreover, DeltaNp63alpha expression correlates with HSP70 expression in all head and neck cancer cell lines. Finally, we show colocalization of DeltaNp63alpha and HSP70 in the epithelium and coexpression of both proteins in 41 primary head and neck cancers. Our study provides strong evidence for the physiologic association between DeltaNp63alpha and hsp70 in human cancer, thus further supporting the oncogenic potential of DeltaNp63alpha.
[Show abstract][Hide abstract] ABSTRACT: p63 mutations have been identified in several developmental abnormalities, including split-hand/foot malformation (SHFM). In this study, we demonstrate that the C-terminal domain of p63alpha associates with the E2 ubiquitin conjugating enzyme, Ubc9. A p63alpha mutation, Q634X, which naturally occurs in SHFM modulated the interaction of p63alpha with Ubc9 in yeast genetic assay. Furthermore, Ubc9 catalyzed the conjugation of p63alpha with small ubiquitin modifier-1 (SUMO-1), which covalently modified p63alpha in vitro and in vivo at two positions (K549E and K637E), each situated in a SUMO-1 modification consensus site (phiKXD/E). In addition, p63alpha mutations (K549E and K637E) abolished sumoylation of p63alpha, dramatically activated transactivation properties of TAp63alpha, and inhibited the dominant-negative effect of DeltaNp63alpha. These p63alpha mutations also affected the transcriptional regulation of gene targets involved in bone and tooth development (e.g., RUNX2 and MINT) and therefore might contribute to the molecular mechanisms underlying the SHFM phenotype.
[Show abstract][Hide abstract] ABSTRACT: p51/p63, a member of the tumor suppressor p53 gene family, is crucial for skin development. We describe here identification of ITGA3 encoding integrin α3 as a target of its trans-activating function, proposing that p51/p63 allows epidermal stem cells to express laminin receptor α3β1 for anchorage to the basement membrane. When activated by genotoxic stress or overexpressed ectopically in non-adherent cells,
p51/p63 transduced a phenotype to attach to extracellular matrices, which was accompanied by expression of ITGA3. Motifs matching the p53-binding consensus sequence were located in a scattered form in intron 1 of human ITGA3, and served as p51/p63-responsive elements in reporter assays. In addition to the trans-activating ability of the TA isoform, we detected a positive effect of the ΔN isoform on ITGA3. The high level α3 production in human keratinocyte stem cells diminished upon elimination of p51/p63 by small interfering RNA or by Ca2+-induced differentiation. Furthermore, a chromatin immunoprecipitation experiment indicated a physical interaction of p51/p63
with intron 1 of ITGA3. This study provides a molecular basis for the standing hypothesis that p51/p63 is essential for epidermal-mesenchymal interactions.
Preview · Article · Dec 2004 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: p53 family members with a transactivation (TA) domain induce cell cycle arrest and promote apoptosis. However, DeltaNp63 isotypes lacking the TA-domain promote cell proliferation and tumorigenesis in vitro and in vgammavo. Although p53, TAp63 or TAp73 are stabilized upon DNA damage, we found that the genotoxic stress agents induced a dramatic decrease and phosphorylation of DeltaNp63alpha in squamous cell carcinoma cells. Further work revealed that RACK1 physically associated with the p63alpha C-terminal domain through its WD40 domain. However, stratifin binds with phosphorylated DeltaNp63alpha in response to cisplatin. Upon DNA damage induced by cisplatin, stratifin mediated a nuclear export of DeltaNp63alpha into cytoplasm and then RACK1 targeted latter into a proteasome degradation pathway possibly serving as an E3 ubiquitin ligase. Moreover, siRNA knockdown of both stratifin and RACK1 inhibited a nuclear export and protein degradation of DeltaNp63alpha, respectively. Our data suggest that modification and down regulation of DeltaNp63alpha is one of the major determinants of the cellular response to DNA damage in human head and neck cancers.