Silencing of the retinoid response gene TIG1 by promoter hypermethylation in nasopharyngeal carcinoma

Tazarotene-induced gene 1 (TIG1) and Tazarotene-induced gene 3 (TIG3) are retinoid acid (RA) target genes as well as candidate tumor suppressor genes in human cancers. In our study, we have investigated the expression of TIG1 and TIG3 in nasopharyngeal carcinoma (NPC). Loss of TIG1 expression was found in 80% of NPC cell lines and 33% of xenografts, whereas TIG3 was expressed in all NPC samples and immortalized nasopharyngeal epithelial cells. In order to elucidate the epigenetic silencing of TIG1 in NPC, the methylation status of TIG1 promoter was examined by genomic bisulfite sequencing and methylation-specific PCR (MSP). We have detected dense methylation of TIG1 5'CpG island in the 5 TIG1-negative NPC cell lines and xenograft (C666-1, CNE1, CNE2, HONE1 and X666). Partial methylation was observed in 1 NPC cell line HK1 showing dramatic decreased in TIG1 expression. Promoter methylation was absent in 2 TIG1-expressed NPC xenografts and the normal epithelial cells. Restoration of TIG1 expression and unmethylated alleles were observed in NPC cell lines after 5-aza-2'-deoxycytidine treatment. Moreover, the methylated TIG1 sequence was detected in 39 of 43 (90.7%) primary NPC tumors by MSP. In conclusion, our results showed that TIG1 expression is lost in the majority of NPC cell lines and xenografts, while promoter hypermethylation is the major mechanism for TIG1 silencing. Furthermore, the frequent epigenetic inactivation of TIG1 in primary NPC tumors implied that it may play an important role in NPC tumorigenesis.     

Integrin α9 gene promoter is hypermethylated and downregulated in nasopharyngeal carcinoma

Epigenetic silencing of tumor suppressor genes (TSGs) by promoter methylation can be an early event in the multi-step process of carcinogenesis. Human chromosome 3 contains clusters of TSGs involved in many cancer types including nasopharyngeal carcinoma (NPC), the most common cancer in Southern China. Among ten candidate TSGs identified in chromosome 3 using NotI microarray, ITGA9 and WNT7A could be validated. 5′-aza-2′ deoxycytidine treatment restored the expression of ITGA9 and WNT7A in two NPC cell lines. Immunostaining showed strong expression of these genes in the membrane and cytoplasm of adjacent control nasopharyngeal epithelium cells, while they were weakly expressed in NPC tumor cells. The ITGA9 promoter showed marked differentially methylation between tumor and control tissue, whereas no differentially methylation could be detected for the WNT7A promoter. The expression level of ITGA9 in NPC tumors was downregulated 4.9-fold, compared to the expression in control. ITGA9 methylation was detected by methylation specific PCR (MSP) in 56% of EBV positive NPC- cases with 100% specificity. Taken together, this suggests that ITGA9 might be a TSG in NPC that is involved in tumor cell biology. The possibility of using ITGA9 methylation as a marker for early detection of NPC should further be explored.     

Epigenetic inactivation of TSLC1 gene in nasopharyngeal carcinoma

Deletion of 11q23 is a common genetic aberration in nasopharyngeal carcinoma (NPC). Multiple candidate tumor suppressor genes (TSG) were mapped to this region but few of them were investigated in NPC. TSLC1 (tumor suppressor in lung cancer) is recently reported to be a putative TSG on 11q23. This gene was found to be inactivated by promoter hypermethylation in non-small cell lung carcinoma (NSCLC), liver cancer, and breast cancer. To study the role of TSLC1 gene in NPC tumorigenesis, we screened for mutations and aberrant methylation of TSLC1 gene in 5 NPC cell lines, 3 NPC xenografts, and 38 primary NPC cases. No somatic mutations of TSLC1 were detected in the NPC samples, but a 9-bp (CCACCACCA) deletion in exon 8 was found in a primary NPC and its corresponding blood sample. Bisulfite sequencing revealed aberrant methylation of TSLC1 promoter in four NPC cell lines. Loss of TSLC1 gene expression was found in two cell lines (HK-1 and CNE-2) with dense methylation. Expression of this gene was restored in these cell lines after treatment with demethylating agent 5-aza-2'-deoxycytidine. Our results showed that silencing of TSLC1 gene expression in NPC was associated with promoter hypermethylation. Promoter hypermethylation of TSLC1 gene was further illustrated in 34.2% (13/38) of primary NPCs. No aberrant promoter methylation was found in any of the four investigated normal nasopharyngeal epithelia. Frequent epigenetic inactivation of TSLC1 gene in NPC suggested that this gene is one of the target tumor suppressor genes of this endemic cancer.     

Protocadherin8 is a functional tumor suppressor frequently inactivated by promoter methylation in nasopharyngeal carcinoma

Protocadherin8 (PCDH8), an integral membrane protein, was reported to be a tumor suppressor involved in tumorigenesis in cancers. We aimed to investigate the epigenetic inactivation of PCDH8 and its tumor-suppressor function in nasopharyngeal carcinoma (NPC). Frequent downregulation/silencing of PCDH8 was found in NPC cell lines using semiquantitative PCR. Promoter methylation of PCDH8 was observed in 100% (5/5) of cell lines and 85.3% (35/41) of primary tumors by methylation-specific PCR, but not in normal nasopharyngeal tissues. Treatment of NPC cells with 5-aza-2'-deoxycytidine and/or trichostatin A could restore PCDH8 expression. Ectopic expression of PCDH8 in silenced NPC cells significantly inhibited cell colony formation and cell migration. For the first time, our study demonstrates the epigenetic inactivation of PCDH8 by promoter methylation and its tumor-suppressive function in NPC. Thus, PCDH8 could be identified as a tumor suppressor in NPC.     

Promoter methylation of TSLC1 and tumor suppression by its gene product in human prostate cancer.

We recently identified TSLC1, a tumor suppressor gene in human lung cancer. Gene silencing by promoter methylation has been observed frequently in adenocarcinoma of the lung, liver, and pancreas. Here, we demonstrate that TSLC1 expression is also absent or markedly reduced in 3 of 4 prostate cancer cell lines. Promoter sequences of TSLC1 were heavily methylated in PPC-1 cells that lacked TSLC1 expression, supporting the idea that promoter methylation is strongly correlated with complete loss of gene expression. Promoter sequences of TSLC1 were also methylated significantly in 7 of 22 (32%) primary prostate cancers. Hypermethylation of the promoter occurred not only in advanced tumors, but also in relatively early-stage tumors. Restoration of TSLC1 expression substantially suppressed tumor formation of PPC-1 cells in nude mice. These findings indicate that alteration of TSLC1 is involved in prostate cancer.     

Role of STAT3/5 and Bcl-2/xL in 2-methoxyestradiol-induced endoreduplication of nasopharyngeal carcinoma cells

2‐methoxyestradiol (2ME2), an endogenous metabolite of 17‐β‐estradiol, has been shown to induce apoptosis and cell cycle arrest in various tumor models. We have previously shown that 2ME2 induced endoreduplication in a well‐differentiated nasopharyngeal carcinoma (NPC) HK‐1 and a poorly differentiated C666‐1 cell line. In the present study, we studied the survival factors involved in 2ME2‐induced endoreduplicating NPC cells. In the HK‐1 cells, knockdown of BcL‐xL expression by siRNA resulted in the reduction of endoreduplication and an increase in the percentage of apoptosis. Further mechanistic study revealed that 2ME2 enhanced the expression of the phosphorylated form of STAT5 (p‐STAT5‐Y694), but not p‐STAT3 (Y705) and p‐STAT3 (S727), in the nucleus of HK‐1 cells. Pre‐treatment of cells with JAK/STAT inhibitor AG490 and STAT5 inhibitor resulted not only in the reduced expression of Bcl‐xL, but also reduced the percentage of endoreduplicating cells. In contrast, 2ME2 enhanced the expression of p‐STAT3 in the poorly differentiated C666‐1 cells. Pharmacological inhibition of STAT3 or Bcl‐2/xL resulted in a decrease in endoreduplication of C666‐1 cells. Taken together, the expression of p‐STAT5 and p‐STAT3 was upregulated in 2ME2‐induced endoreduplicating HK‐1 and C666‐1 cells, respectively. Combination of 2ME2 with Bcl‐2/xL inhibitor is a novel strategy to reduce the formation of endoreduplicating cells during chemotherapeutic treatment of NPC. © 2011 Wiley Periodicals, Inc.     

Hypermethylation of the TSLC1 Gene Promoter in Primary Gastric Cancers and Gastric Cancer Cell Lines

The TSLC1 (tumor suppressor in lung cancer–1) gene is a novel tumor suppressor gene on chromosomal region 11q23.2, and is frequently inactivated by concordant promoter hypermethylation and loss of heterozygosity (LOH) in non-small cell lung cancer (NSCLC). Because LOH on 11q has also been observed frequently in other human neoplasms including gastric cancer, we investigated the promoter methylation status of TSLC1 in 10 gastric cancer cell lines and 97 primary gastric cancers, as well as the corresponding non-cancerous gastric tissues, by bisulfite-SSCP analysis followed by direct sequencing. Allelic status of the TSLC1 gene was also investigated in these cell lines and primary gastric cancers. The TSLC1 promoter was methylated in two gastric cancer cell lines, KATO-III and ECC10, and in 15 out of 97 (16%) primary gastric cancers. It was not methylated in non-cancerous gastric tissues, suggesting that this hypermethylation is a cancer-specific alteration. KATO-III and ECC10 cells retained two alleles of TSLC1 , both of which showed hypermethylation, associated with complete loss of gene expression. Most of the primary gastric cancers with promoter methylation also retained heterozygosity at the TSLC1 locus on 11q23.2. These data indicate that bi-allelic hypermethylation of the TSLC1 promoter and resulting gene silencing occur in a subset of primary gastric cancers.     

Methylation associated inactivation of RASSF1A and its synergistic effect with activated K-Ras in nasopharyngeal carcinoma

Background

Epigenetic silencing of tumor suppressor genes associated with promoter methylation is considered to be a hallmark of oncogenesis. RASSF1A is a candidate tumor suppressor gene which was found to be inactivated in many human cancers. Although we have had a prelimilary cognition about the function of RASSF1A, the exact mechanisms about how RASSF1A functions in human cancers were largely unknown. Moreover, the effect of mutated K-Ras gene on the function of RASSF1A is lacking. The aim of this study was to investigate the expression profile and methylation status of RASSF1A gene, and to explore its concrete mechanisms as a tumor suppressor gene in Nasopharyngeal Carcinoma.

Methods

We examined the expression profile and methylation status of RASSF1A in two NPC cell lines, 38 primary nasopharyngeal carcinoma and 14 normal nasopharyngeal epithelia using RT-PCR and methylated specific PCR(MSP) respectively. 5-aza-dC was then added to confirm the correlation between hypermethylation status and inactivation of RASSF1A. The NPC cell line CNE-2 was transfected with exogenous pcDNA3.1(+)/RASSF1A plasmid in the presence or absence of mutated K-Ras by liposome-mediated gene transfer method. Flow cytometry was used to examine the effect of RASSF1A on cell cycle modulation and apoptosis. Meanwhile, trypan blue dye exclusion assays was used to detect the effect of RASSF1A transfection alone and the co-transfection of RASSF1A and K-Ras on cell proliferation.

Results

Promoter methylation of RASSF1A could be detected in 71.05% (27/38) of NPC samples, but not in normal nasopharyngeal epithelia. RASSF1A expression in NPC primary tumors was lower than that in normal nasopharyngeal epithelial (p < 0.01). Expression of RASSF1A was down-regulated in two NPC cell lines. Loss of RASSF1A expression was greatly restored by the methyltransferase inhibitor 5-aza-dC in CNE-2. Ectopic expression of RASSF1A in CNE-2 could increase the percentage of G0/G1 phase cells (p < 0.01), inhibit cell proliferation and induce apoptosis (p < 0.001). Moreover, activated K-Ras could enhance the growth inhibition effect induced by RASSF1A in CNE-2 cells (p < 0.01).

Conclusion

Expression of RASSF1A is down-regulated in NPC due to the hypermethylation of promoter. Exogenous expression of RASSF1A is able to induce growth inhibition effect and apoptosis in tumor cell lines, and this effect could be enhanced by activated K-Ras.     

Promoter Methylation of TSLC1 and Tumor Suppression by Its Gene Product in Human Prostate Cancer

We recently identified TSLC1 , a tumor suppressor gene in human lung cancer. Gene silencing by promoter methylation has been observed frequently in adenocarcinoma of the lung, liver, and pancreas. Here, we demonstrate that TSLC1 expression is also absent or markedly reduced in 3 of 4 prostate cancer cell lines. Promoter sequences of TSLC1 were heavily methylated in PPC-1 cells that lacked TSLC1 expression, supporting the idea that promoter methylation is strongly correlated with complete loss of gene expression. Promoter sequences of TSLC1 were also methylated significantly in 7 of 22 (32%) primary prostate cancers. Hypermethylation of the promoter occurred not only in advanced tumors, but also in relatively early-stage tumors. Restoration of TSLC1 expression substantially suppressed tumor formation of PPC-1 cells in nude mice. These findings indicate that alteration of TSLC1 is involved in prostate cancer.     

Fine mapping of the 11q22-23 tumor suppressive region and involvement of TSLC1 in nasopharyngeal carcinoma

Previous studies transferring an intact chromosome 11 into HONE1 cells demonstrated the functional significance of chromosome regions, 11q13 and 11q22-23, in nasopharyngeal carcinoma (NPC) development. In our study the 11q22-23 region was comprehensively re-investigated by detailed microsatellite and single nucleotide polymorphism genotyping and by fluorescence in situ hybridization to map precisely the regions containing tumor suppressive activity. We observed 3 chromosomal intervals within 11q22-23 that were commonly lost in the tumor segregants derived from HONE1/chromosome 11 hybrids. One critical region of 0.36 Mb was mapped near the marker D11S2000 and a second 0.44 Mb region was located around the markers D11S1300 and D11S1391. In a third region high allelic loss was also observed at marker D11S4484, where a newly cloned tumor suppressor gene, TSLC1 (tumor suppressor in lung cancer 1), is located. The gene expression analysis showed absence or low expression levels of TSLC1 mRNA in 4 highly tumorigenic NPC cell lines. In addition, the methylation study results show that the TSLC1 promoter region was hypermethylated in all 4 NPC cell lines and re-expression of the gene occurs in HONE1 cells after 5-aza-2'-deoxycytidine treatment. Hence, the mode of silencing of this candidate TSG in NPC may be attributed to promoter hypermethylation. We have obtained functional evidence for multiple critical tumor suppressive regions in 11q22-23 by fine deletion mapping and for inactivation of TSLC1 being one of these candidate TSGs in NPC development.