Our study shows a clear correlation between TMS1/ASC
methylation and silencing of the gene suggesting a role in prostate cancer cell lines. Re-expression accompanied by partial demethylation of TMS1/ASC
following 5-AZA-2'-deoxycytidine confirms that methylation is responsible for transcriptional silencing of this gene. Lack of response to TSA treatment suggests that histone acetylation does not play a role in downregulating the expression of TMS1/ASC
. This finding is similar to the results obtained by Stimson et al[23
]. Over expression of TMS1/ASC
was shown to inhibit cellular proliferation and induce DNA fragmentation which can be blocked by a caspase inhibitor [24
]. In addition, forced reduction in TMS1/ASC
promotes cell survival perhaps in a NFκ-B dependent pathway [25
]. This makes it a therapeutic target by use of demethylating agents alone or in combination with additional apoptosis inducing drugs.
ChIP analysis showed binding predominantly with MBD3 and only minimal enrichment of the other MBDs and MeCP2.
Earlier studies have demonstrated that MBD3 does not bind to methylated DNA alone [26
]. However, it has a definite role in maintaining methylation. Recent studies have shown binding of MBD3 to several genes like cox6c, leng6, bat5 etc. [28
]. Wade et al showed that MBD3 is a subunit of the NuRD complex that has nucleosome remodeling and histone deacetylase activities [29
]. MBD3 forms a part of the multiprotein NuRD complex and probably has a role as a transcriptional co-repressor. Our finding that MBD3 binds to methylated TMS1/ASC
is contrary to the known pattern of MBD binding by MBD proteins to methylated DNA. This could be because MBD3-containing NuRD complexes bind more specifically to the methylated TMS1/ASC
A noteworthy finding in our study was the statistically significant difference in the methylation of TMS1/ASC
among the cases and controls in Whites compared to Blacks. We observed an age adjusted odds-ratio of 7.6 (95% CI 2.1–27.3) in Whites as compared to only 1.1 (95% CI 0.2–5.5) among Blacks. Whether this finding reflects involvement of different pathogenetic pathways in different races will be interesting to study. In the US, the incidence and mortality of prostate cancer is about two-fold higher among Blacks compared to Whites, suggesting racial differences in prostate tumor occurrence and aggressiveness [30
]. The reason for these racial differences is not well understood. It is possible that promoter-region gene hypermethylation may be influenced by environmental exposures.
There have been only a few studies on differences in gene methylation between different races. Two studies by Woodson et al showed a differential methylation pattern and expression of CD44 in Blacks and Whites [31
]. When we examined 5 genes frequently methylated in prostate cancer (GSTP1, CD44, ECAD, RASSF1A and EBR) in the same patient population we did not find any significant differences in the gene specific methylation pattern between the different races (Table ). Ethnic group related differences in hypermethylation of promoter region of GSTP1 gene were reported in a recent paper [33
]. The authors observed higher hazard ratio (HR) for pathogenesis among African Americans as compared to Caucasians. In contrast to the above observation, we found the hazard ratio of TMS1/ASC
methylation (prostate cancer versus BPH) to be lower in Blacks as compared to Whites. Ethnic origin is an important determinant of prostate cancer risk, incidence, and disease progression. In the US, the African-American male group has the highest incidence rate for prostate cancer [30
]. Differences in diet, socioeconomic environment, lifestyle between the two ethnic groups have been implicated as causative factors for the striking ethnic differences in the incidence and clinical behavior of prostate cancer. However, molecular mechanisms underlying the racial diversity are not well understood. The recent report by Fang et. al., shows that Genistein leads to reversal of hypermethylation and reactivation of p16INK4a, RARβ, and MGMT genes [34
]. Thus, diet seems to be an important factor in affecting the methylation status of different genes implicated in cancer. It is also likely that genes are differentially methylated in different ethnic groups owing to the lifestyle and dietary differences. Methylation of promoter in controls (BPH) may reflect that epigenetic alteration of the gene has already occurred and that they have acquired epigenetic malignant potential even though the pathological diagnosis classifies them as benign [33
]. Our results indicate that differences in methylation pattern of TMS1/ASC
in BPH among ethnic groups might explain the differences among different racial groups in susceptibility to prostate cancer. Thus it seems that the epigenetic make up of different ethnic groups would determine the risk to prostate cancer pathogenesis.
Risk of prostate cancer in relation to gene methylation, total patients and by race.
There did not appear to be a significant relationship between TMS1/ASC methylation status and patient's age. On our limited dataset there was a trend towards association between TMS1/ASC methylation and Gleason score 7 or higher (odds-ratio 2.3). Though the relationship was not statistically significant it possibly suggests a worse prognosis as patients with Gleason score 7 do worse that those with Gleason score 6. Many of our patient samples showed partial methylation status – this could be due to presence of normal fibroblasts, endothelial cells, inflammatory cells and non malignant prostate tissue surrounding the tumor. Tumors where TMS1/ASC was not found to be methylated could involve other genes in the apoptotic pathway. Use of pathway specific cDNA microarrays may help in determining the individual genes affected.
In summary our study has shown that TMS1/ASC, a pro-apoptotic gene, is silenced by hypermethylation of the CpG islands in the promoter region. This transcriptional repression is relieved by treatment with a demethylating agent (5-Aza-2'-deoxycytidine). Frequent methylation of TMS1/ASC in prostate cancer suggests this gene may be important in pathogenesis of prostate cancer and can be a target of pharmacologic demethylation in clinical trials. TMS1/ASC methylation patterns show significant ethnic differences. Our findings provide a novel insight into the molecular determinants of tumor growth that may underlie the ethnic differences in prostate cancer incidence and clinical behavior. Further studies are needed to find out if this has any significant clinical implications in the development of novel diagnostic approaches for biologically aggressive prostate cancer from diverse racial origin.