Xianwei Su

The Ubiquitin Peptidase UCHL1 Induces G0/G1 Cell Cycle Arrest and Apoptosis Through Stabilizing p53 and Is Frequently Silenced in Breast Cancer

Background Breast cancer (BrCa) is a complex disease driven by aberrant gene alterations and environmental factors. Recent studies reveal that abnormal epigenetic gene regulation also plays an important role in its pathogenesis. Ubiquitin carboxyl- terminal esterase L1 (UCHL1) is a tumor suppressor silenced by promoter methylation in multiple cancers, but its role and alterations in breast tumorigenesis remain unclear. Methodology/Principal Findings We found that UCHL1 was frequently downregulated or silenced in breast cancer cell lines and tumor tissues, but readily expressed in normal breast tissues and mammary epithelial cells. Promoter methylation of UCHL1 was detected in 9 of 10 breast cancer cell lines (90%) and 53 of 66 (80%) primary tumors, but rarely in normal breast tissues, which was statistically correlated with advanced clinical stage and progesterone receptor status. Pharmacologic demethylation reactivated UCHL1 expression along with concomitant promoter demethylation. Ectopic expression of UCHL1 significantly suppressed the colony formation and proliferation of breast tumor cells, through inducing G0/G1 cell cycle arrest and apoptosis. Subcellular localization study showed that UCHL1 increased cytoplasmic abundance of p53. We further found that UCHL1 induced p53 accumulation and reduced MDM2 protein level, and subsequently upregulated the expression of p21, as well as cleavage of caspase3 and PARP, but not in catalytic mutant UCHL1 C90S-expressed cells. Conclusions/Significance UCHL1 exerts its tumor suppressive functions by inducing G0/G1cell cycle arrest and apoptosis in breast tumorigenesis, requiring its deubiquitinase activity. Its frequent silencing by promoter CpG methylation may serve as a potential tumor marker for breast cancer.

Src-like adaptor protein 2 (SLAP2) binds to and inhibits FLT3 signaling

Fms-like tyrosine kinase (FLT3) is a frequently mutated oncogene in acute myeloid leukemia (AML). FLT3 inhibitors display promising results in a clinical setting, but patients relapse after short-term treatment due to the development of resistant disease. Therefore, a better understanding of FLT3 downstream signal transduction pathways will help to identify an alternative target for the treatment of AML patients carrying oncogenic FLT3. Activation of FLT3 results in phosphorylation of FLT3 on several tyrosine residues that recruit SH2 domain-containing signaling proteins. We screened a panel of SH2 domain-containing proteins and identified SLAP2 as a potent interacting partner of FLT3. We demonstrated that interaction occurs when FLT3 is activated, and also, an intact SH2 domain of SLAP2 is required for binding. SLAP2 binding sites in FLT3 mainly overlap with those of SRC. SLAP2 over expression in murine proB cells or myeloid cells inhibited oncogenic FLT3-ITD-mediated cell proliferation and colony formation in vitro, and tumor formation in vivo. Microarray analysis suggests that higher SLAP2 expression correlates with a gene signature similar to that of loss of oncogene function. Furthermore, FLT3-ITD positive AML patients with higher SLAP2 expression displayed better prognosis compared to those with lower expression of SLAP2. Expression of SLAP2 blocked FLT3 downstream signaling cascades including AKT, ERK, p38 and STAT5. Finally, SLAP2 accelerated FLT3 degradation through enhanced ubiquitination. Collectively, our data suggest that SLAP2 acts as a negative regulator of FLT3 signaling and therefore, modulation of SLAP2 expression levels may provide an alternative therapeutic approach for FLT3-ITD positive AML.

Protocadherin 17 acts as a tumour suppressor inducing tumour cell apoptosis and autophagy, and is frequently methylated in gastric and colorectal cancers

Gastric and colorectal cancers are among the most common cancers worldwide and cause serious cancer mortality. Both epigenetic and genetic disruptions of tumour suppressor genes (TSGs) are frequently involved in their pathogenesis. Here, we studied the epigenetic and genetic alterations of a novel TSG–PCDH17 and its functions in the pathogenesis of these tumours. We found that PCDH17 was frequently silenced and methylated in almost all gastric and colorectal tumour cell lines as well as in ∼95% of primary tumours, but not in normal gastric and colonic mucosa. Moreover, its deletion was detected in only 18% of gastric and 12% of colorectal cancer tissues, suggesting that epigenetic and genetic inactivation of PCDH17 are both involved in gastric and colorectal tumourigenesis. PCDH17 protein expression was significantly correlated with low tumour stage and less lymph node metastasis of gastric and colorectal cancer patients, indicating its potential as a tumour marker. Restoring PCDH17 expression inhibited tumour cell growth in vitro and in vivo through promoting apoptosis, as evidenced by increased TUNEL staining and caspase‐3 activation. Furthermore, PCDH17‐induced autophagy, along with increased numbers of autophagic vacuoles and up‐regulated autophagic proteins Atg‐5, Atg‐12 and LC3B II. Thus, PCDH17 acts as a tumour suppressor, exerting its anti‐proliferative activity through inducing apoptosis and autophagy, and is frequently silenced in gastric and colorectal cancers. PCDH17 methylation is a tumour‐specific event that could serve as an epigenetic biomarker for these tumours.

DACT1, an antagonist to Wnt/β-catenin signaling, suppresses tumor cell growth and is frequently silenced in breast cancer

IntroductionAberrant activation of Wnt/β-catenin signaling plays an important role in the pathogenesis of breast cancer. DACT1 (Dapper/Frodo) has been identified as involved in antagonizing Wnt/β-catenin signaling through interacting with Dishevelled (Dvl), a central mediator of Wnt signaling, whereas its role in breast tumorigenesis remains unclear.MethodsWe examined DACT1 expression in breast cancer cell lines and primary tumors with semiquantitative or quantitative RT-PCR and immunochemistry, and further evaluated the promoter methylation of DACT1 with methylation-specific PCR (MSP). We also explored the tumor-suppressive functions of DACT1 in vivo and in vitro, and its related mechanism in breast cancer.ResultsWe identified DACT1 as a methylated target in our breast cancer epigenome study. Here, we further investigated DACT1 expression in multiple breast cell lines and primary tumors, and further studied its function and molecular mechanisms. We found that DACT1 expression was silenced in eight (88.9%) of nine breast cancer cell lines, and its protein levels were obviously reduced in breast tumors compared with paired surgical-margin tissues. Promoter CpG methylation of DACT1 was detected in five (55.6%) of nine breast cancer cell lines and 40 (29.9%) of 134 primary tumors, but not in surgical-margin tissues and normal breast tissues. Demethylation treatment of breast cancer cell lines restored DACT1 expression along with promoter demethylation, suggesting that an epigenetic mechanism mediates DACT1 silencing in breast cancer. Functional assays showed that ectopic expression of DACT1 could inhibit breast tumor cell proliferation in vivo and in vitro through inducing apoptosis, and further suppress tumor cell migration through antagonizing the Wnt/β-catenin signaling pathway.ConclusionsOur study demonstrates that DACT1 could function as a tumor suppressor but was frequently downregulated in breast cancer.

The metalloprotease ADAMTS8 displays antitumor properties through antagonizing EGFR-MEK-ERK signaling and is silenced in carcinomas by CpG methylation.

A disintegrins and metalloproteinases with thrombospondin motifs (ADAMTS) family members have been reported dysregulated in various cancers. Through refining a loss of heterozygosity locus at 11q25 by array-CGH, we identified ADAMTS8 as a novel candidate tumor suppressor gene. Although ADAMTS8 downregulation has been reported in several tumors, its biologic function and underlying mechanism remain largely unknown. Here, we found that ADAMTS8 is broadly expressed in normal tissues but frequently downregulated or silenced by promoter methylation in common carcinoma cell lines, including nasopharyngeal, esophageal squamous cell, gastric, and colorectal carcinomas. Pharmacologic or genetic demethylation restored ADAMTS8 expression, indicating that promoter methylation mediates its silencing. Aberrant methylation of ADAMTS8 was also detected in several types of primary tumors but rarely in normal tissues. Further functional studies showed that restoring ADAMTS8 expression suppressed tumor cell clonogenicity through inducing apoptosis. ADAMTS8 as a secreted protease inhibited epidermal growth factor receptor (EGFR) signaling along with decreased levels of phosphorylated MEK and ERK. We further found that ADAMTS8 disrupted actin stress fiber organization and inhibited tumor cell motility. Thus, our data demonstrate that ADAMTS8 metalloprotease acts as a functional tumor suppressor through antagonizing EGFR–MEK–ERK signaling, in addition to its previously reported anti-angiogenesis function, and is frequently methylated in common tumors. Implications: This study uncovers the tumor suppressive function of ADAMTS8, one of the ADAMTS family members, and its frequent methylation in certain tumors could be developed as a potential biomarker. Mol Cancer Res; 12(2); 228–38. ©2013 AACR.

Methylation profiling of Epstein-Barr virus immediate-early gene promoters, BZLF1 and BRLF1 in tumors of epithelial, NK- and B-cell origins

BackgroundEpstein-Barr virus (EBV) establishes its latency in EBV-associated malignancies, accompanied by occasionally reactivated lytic cycle. Promoter CpG methylation of EBV genome plays an essential role in maintaining viral latency. Two immediate-early (IE) genes, BZLF1 and BRLF1, induce the switch from latent to lytic infection. Studies of methylation-dependent binding of BZLF1 and BRLF1 to EBV promoters have been well reported, but little is known about the methylation status of BZLF1 and BRLF1 promoters (Zp and Rp) in tumor samples.MethodsWe evaluated the methylation profiles of Zp and Rp by methylation-specific PCR (MSP) and bisulfite genomic sequencing (BGS), as well as BZLF1 and BRLF1 expression by semiquantitative reverse transcription (RT)-PCR in tumors of epithelial, NK- and B-cell origins.ResultsWe found that both Zp and Rp were hypermethylated in all studied EBV-positive cell lines and tumors of lymphoid (B- or NK cell) or epithelial origin, while unmethylated Zp and Rp alleles were detected in cell lines expressing BZLF1 and BRLF1. Following azacytidine treatment or combined with trichostatin A (TSA), the expression of BZLF1 and BRLF1 was restored along with concomitant promoter demethylation, which subsequently induced the reactivation of early lytic gene BHRF1 and late lytic gene BLLF1.ConclusionsHypermethylation of Zp and Rp mediates the frequent silencing of BZLF1 and BRLF1 in EBV-associated tumors, which could be reactivated by demethylation agent and ultimately initiated the EBV lytic cascade.

Characterization of the nasopharyngeal carcinoma methylome identifies aberrant disruption of key signaling pathways and methylated tumor suppressor genes.

AIMS Nasopharyngeal carcinoma (NPC) is a common tumor consistently associated with Epstein-Barr virus infection and prevalent in South China, including Hong Kong, and southeast Asia. Current genomic sequencing studies found only rare mutations in NPC, indicating its critical epigenetic etiology, while no epigenome exists for NPC as yet. MATERIALS & METHODS We profiled the methylomes of NPC cell lines and primary tumors, together with normal nasopharyngeal epithelial cells, using methylated DNA immunoprecipitation (MeDIP). RESULTS We observed extensive, genome-wide methylation of cellular genes. Epigenetic disruption of Wnt, MAPK, TGF-β and Hedgehog signaling pathways was detected. Methylation of Wnt signaling regulators (SFRP1, 2, 4 and 5, DACT2, DKK2 and DKK3) was frequently detected in tumor and nasal swab samples from NPC patients. Functional studies showed that these genes are bona fide tumor-suppressor genes for NPC. CONCLUSION The NPC methylome shows a special high-degree CpG methylation epigenotype, similar to the Epstein-Barr virus-infected gastric cancer, indicating a critical epigenetic etiology for NPC pathogenesis.

A novel 3p22.3 gene CMTM7 represses oncogenic EGFR signaling and inhibits cancer cell growth

Deletion of 3p12-22 is frequent in multiple cancer types, indicating the presence of critical tumor-suppressor genes (TSGs) at this region. We studied a novel candidate TSG, CMTM7, located at the 3p22.3 CMTM-gene cluster, for its tumor-suppressive functions and related mechanisms. The three CMTM genes, CMTM6, 7 and 8, are broadly expressed in human normal adult tissues and normal epithelial cell lines. Only CMTM7 is frequently silenced or downregulated in esophageal and nasopharyngeal cell lines, but uncommon in other carcinoma cell lines. Immunostaining of tissue microarrays for CMTM7 protein showed its downregulation or absence in esophageal, gastric, pancreatic, liver, lung and cervix tumor tissues. Promoter CpG methylation and loss of heterozygosity were both found contributing to CMTM7 downregulation. Ectopic expression of CMTM7 in carcinoma cells inhibits cell proliferation, motility and tumor formation in nude mice, but not in immortalized normal cells, suggesting a tumor inhibitory role of CMTM7. The tumor-suppressive function of CMTM7 is associated with its role in G1/S cell cycle arrest, through upregulating p27 and downregulating cyclin-dependent kinase 2 (CDK2) and 6 (CDK6). Moreover, CMTM7 could promote epidermal growth factor receptor (EGFR) internalization, and further suppress AKT signaling pathway. Thus, our findings suggest that CMTM7 is a novel 3p22 tumor suppressor regulating G1/S transition and EGFR/AKT signaling during tumor pathogenesis.

A Novel 19q13 Nucleolar Zinc Finger Protein Suppresses Tumor Cell Growth through Inhibiting Ribosome Biogenesis and Inducing Apoptosis but Is Frequently Silenced in Multiple Carcinomas

Epigenetic disruption of tumor suppressor genes is frequently involved in tumorigenesis. We identified a novel 19q13 KRAB domain-containing zinc finger protein, ZNF545/ZFP82, broadly expressed in normal tissues but downregulated in multiple tumor cell lines. The ZNF545 promoter contains a CpG island, which is frequently methylated in cell lines. The transcriptional silencing of ZNF545 could be reversed by pharmacologic or genetic demethylation, indicating direct epigenetic silencing. ZNF545 was also frequently methylated in multiple primary tumors of nasopharyngeal, esophageal, lung, gastric, colon, and breast, but rarely in normal epithelial tissues and paired normal tissues. ZNF545 is located in the nucleus and mainly sequestered in nucleoli, functioning as a repressor. ZNF545 is able to repress NF-κB and AP-1 signaling pathways, whereas ectopic expression of ZNF545 in silenced tumor cells significantly inhibited their growth and induced apoptosis. Functional studies showed that ZNF545 was involved in ribosome biogenesis through inhibiting the activity of rDNA promoter and decreasing cellular protein translation efficiency. Thus, we identified ZNF545 as a novel tumor suppressor inducing tumor cell apoptosis, repressing ribosome biogenesis and target gene transcription. The tumor-specific methylation of ZNF545 could be an epigenetic biomarker for cancer diagnosis. Mol Cancer Res; 10(7); 925–36. ©2012 AACR.

Epigenetic identification of receptor tyrosine kinase-like orphan receptor 2 as a functional tumor suppressor inhibiting β-catenin and AKT signaling but frequently methylated in common carcinomas

Abstract Through subtraction of tumor-specific CpG methylation, we identified receptor tyrosine kinase-like orphan receptor 2 (ROR2) as a candidate tumor suppressor gene (TSG). ROR2 is a specific receptor or co-receptor for WNT5A, involved in canonical and non-canonical WNT signaling, with its role in tumorigenesis controversial. We characterized its functions and related cell signaling in common carcinomas. ROR2 was frequently silenced by promoter CpG methylation in multiple carcinomas including nasopharyngeal, esophageal, gastric, colorectal, hepatocellular, lung, and breast cancers, while no direct correlation of ROR2 and WNT5A expression was observed. Ectopic expression of ROR2 resulted in tumor suppression independent of WNT5A status, through inhibiting tumor cell growth and inducing cell cycle arrest and apoptosis. ROR2 further suppressed epithelial-mesenchymal transition and tumor cell stemness through repressing β-catenin and AKT signaling, leading to further inhibition of tumor cell migration/invasion and increased chemo-sensitivity. Thus ROR2, as an epigenetically inactivated TSG, antagonizes both β-catenin and AKT signaling in multiple tumorigenesis. Its epigenetic silencing could be a potential tumor biomarker and therapeutic target for carcinomas.