Association of tissue promoter methylation levels of APC, TGFß2, HOXD3 and RASSF1A with prostate cancer progression.

Aberrant promoter methylation is known to silence tumor-suppressor genes in prostate cancer (PCa). We correlated quantitative promoter methylation levels of APC, TGFß2 and RASSF1A in 219 radical prostatectomies diagnosed between 1998 and 2001 with clinicopathological follow-up data available including Gleason Pattern (GP), Gleason Score (GS) and pathological stage and explored their potential in predicting biochemical recurrence using univariate and multivariate analyses. We observed that the average methylation levels of APC increased significantly from GS ≤ 6 to GS7, and pT2 to pT3a, and that of TGFß2 increased from GS ≤ 6 to GS7, but not for RASSF1A. PCa samples were also stratified into high methylation (HM) and low methylation (LM) groups based on the PMR scores of all cases analyzed for each marker. The HM frequency of APC was greater in pT3a than pT2, and in GS ≥ 8 than GS ≤ 6. The HM frequency also increased significantly from GP3 to GP4 for APC, TGFß2 and RASSF1A. APC methylation level was a significant predictor of biochemical recurrence in univariate analysis (p-value = 0.028). Finally, we combined methylation data of these three genes with the previously reported novel methylation biomarker HOXD3. Quantitative methylation assessment of a multiplex panel of markers, consisting of APC, HOXD3 and TGFß2, outperforms any single marker for the prediction of biochemical recurrence (p-value = 0.017). Our study demonstrated that quantitative increase in promoter methylation levels of APC, HOXD3 and TGFß2 are associated with PCa progression.

DNA methylation of HOXD3 as a marker of prostate cancer progression.

DNA methylation in gene promoters causes gene silencing and is a common event in cancer development and progression. The ability of aberrant methylation events to serve as diagnostic and prognostic markers is being appreciated for many cancers, including prostate cancer. Using quantitative MethyLight technology, we evaluated the relationship between HOXD3 methylation and clinicopathological parameters including biochemical recurrence, pathological stage, Gleason score (GS), and Gleason pattern in a series of 232 radical prostatectomies performed between 1998 and 2001. HOXD3 methylation was significantly greater in GS 7 cancers vs GS < or = 6 cancers (P-value <0.001) as well as pT3/pT4 vs pT2 cancers (P-value <0.001). The proportion of cases with high methylation in GS 7 vs < or = GS 6 and pT3/pT4 vs pT2 were also significantly different (P-values=0.002 and 0.005, respectively). There were also significant increases in methylation from Gleason pattern 2-3 and from pattern 3 to 4/5 (paired t-test P-values=0.01 and <0.001, respectively), whereas methylation from lymph node metastases was decreased when compared with matched tumor tissue (P-value=0.029). HOXD3 methylation was associated with biochemical recurrence in univariate analysis (P-value=0.043) and showed evidence for interaction with pathological stage as a predictor variable in Cox regression analysis (P-value=0.028). The results indicate that HOXD3 methylation distinguishes low-grade prostate cancers from intermediate and high-grade ones and may also have prognostic value when considered together with pathological stage.

Activation of tumor-specific splice variant Rac1b by dishevelled promotes canonical Wnt signaling and decreased adhesion of colorectal cancer cells.

Rac1b is a tumor-specific splice variant of the Rac1 GTPase that displays limited functional similarities to Rac1. We have shown previously a novel cross-talk between Rac1 and beta-catenin, which induces canonical Wnt pathway activation in colorectal cancer cells. This prompted us to investigate if Rac1b, frequently overexpressed in colon tumors, contributes to Wnt pathway dysregulation. We show that Rac1b overexpression stimulates Tcf-mediated gene transcription, whereas depletion of Rac1b results in decreased expression of the Wnt target gene cyclin D1. Reconstitution experiments revealed an important difference between Rac1 and Rac1b such that Rac1b was capable of functionally interacting with Dishevelled-3 (Dvl-3) but not beta-catenin to mediate synergistic induction of Wnt target genes. In agreement, Dvl-3 but not beta-catenin caused increased activation of Rac1b levels, which may explain the functional cooperativity displayed in transcription assays. Furthermore, we show that Rac1b negatively regulates E-cadherin expression and results in decreased adhesion of colorectal cancer cells. RNA interference-mediated suppression of Rac1b resulted in reduced expression of Slug, a specific transcriptional repressor of E-cadherin, and a concomitant increase in E-cadherin transcript levels was observed. Intriguingly, mutation of the polybasic region of Rac1b resulted in complete loss of Rac1b stimulatory effects on transcription and suppressive effects on adhesion, indicating the importance of nuclear and membrane localization of Rac1b. Our results suggest that Rac1b overexpression may facilitate tumor progression by enhancing Dvl-3-mediated Wnt pathway signaling and induction of Wnt target genes specifically involved in decreasing the adhesive properties of colorectal cancer cells.

Rac1 GTPase and the Rac1 exchange factor Tiam1 associate with Wnt-responsive promoters to enhance beta-catenin/TCF-dependent transcription in colorectal cancer cells.

BACKGROUND: beta-catenin is a key mediator of the canonical Wnt pathway as it associates with members of the T-cell factor (TCF) family at Wnt-responsive promoters to drive the transcription of Wnt target genes. Recently, we showed that Rac1 GTPase synergizes with beta-catenin to increase the activity of a TCF-responsive reporter. This synergy was dependent on the nuclear presence of Rac1, since inhibition of its nuclear localization effectively abolished the stimulatory effect of Rac1 on TCF-responsive reporter activity. We hypothesised that Rac1 plays a direct role in enhancing the transcription of endogenous Wnt target genes by modulating the beta-catenin/TCF transcription factor complex. RESULTS: We employed chromatin immunoprecipitation studies to demonstrate that Rac1 associates with the beta-catenin/TCF complex at Wnt-responsive promoters of target genes. This association served to facilitate transcription, since overexpression of active Rac1 augmented Wnt target gene activation, whereas depletion of endogenous Rac1 by RNA interference abrogated this effect. In addition, the Rac1-specific exchange factor, Tiam1, potentiated the stimulatory effects of Rac1 on the canonical Wnt pathway. Tiam1 promoted the formation of a complex containing Rac1 and beta-catenin. Furthermore, endogenous Tiam1 associated with endogenous beta-catenin, and this interaction was enhanced in response to Wnt3a stimulation. Intriguingly, Tiam1 was recruited to Wnt-responsive promoters upon Wnt3a stimulation, whereas Rac1 was tethered to TCF binding elements in a Wnt-independent manner. CONCLUSION: Taken together, our results suggest that Rac1 and the Rac1-specific activator Tiam1 are components of transcriptionally active beta-catenin/TCF complexes at Wnt-responsive promoters, and the presence of Rac1 and Tiam1 within these complexes serves to enhance target gene transcription. Our results demonstrate a novel functional mechanism underlying the cross-talk between Rac1 and the canonical Wnt signalling pathway.

Epigenome-Wide DNA Methylation Profiling Identifies Differential Methylation Biomarkers in High-Grade Bladder Cancer.

Epigenetic changes, including CpG island hypermethylation, occur frequently in bladder cancer (BC) and may be exploited for BC detection and distinction between high-grade (HG) and low-grade (LG) disease. Genome-wide methylation analysis was performed using Agilent Human CpG Island Microarrays to determine epigenetic differences between LG and HG cases. Pathway enrichment analysis and functional annotation determined that the most frequently methylated pathways in HG BC were enriched for anterior/posterior pattern specification, embryonic skeletal system development, neuron fate commitment, DNA binding, and transcription factor activity. We identified 990 probes comprising a 32-gene panel that completely distinguished LG from HG based on methylation. Selected genes from this panel, EOMES, GP5, PAX6, TCF4, and ZSCAN12, were selected for quantitative polymerase chain reaction-based validation by MethyLight in an independent series (n=84) of normal bladder samples and LG and HG cases. GP5 and ZSCAN12, two novel methylated genes in BC, were significantly hypermethylated in HG versus LG BC (P≤.03). We validated our data in a second independent cohort of LG and HG BC cases (n=42) from The Cancer Genome Atlas (TCGA). Probes representing our 32-gene panel were significantly differentially methylated in LG versus HG tumors (P≤.04). These results indicate the ability to distinguish normal tissue from cancer, as well as LG from HG, based on methylation and reveal important pathways dysregulated in HG BC. Our findings were corroborated using publicly available data sets from TCGA. Ultimately, the creation of a methylation panel, including GP5 and ZSCAN12, able to distinguish between disease phenotypes will improve disease management and patient outcomes.

Rac1b recruits Dishevelled and ß-catenin to Wnt target gene promoters independent of Wnt3A stimulation.

We previously reported a functional interaction between aberrant Wnt signaling and Rac1/Rac1b GTPases in tumorigenesis. In this study, we further investigated the mechanistic role of nuclear Rac1b. Using chromatin immunoprecipitation (ChIP) studies, we show that Rac1b resides at the promoters of Wnt target genes, c-Myc and Cyclin D1, in HCT116 cells with aberrant Wnt pathway. In HEK293T cells with intact Wnt signaling, Rac1b is tethered to these same gene promoters independent of Wnt3A stimulation and is further observed to recruit Dishevelled and ß-catenin in the absence of Wnt3A stimulation. Our studies suggest a novel transcriptional co-activator role of Rac1b in ß-catenin/TCF-mediated transcription.

Specific variants in the MLH1 gene region may drive DNA methylation, loss of protein expression, and MSI-H colorectal cancer.

BACKGROUND: We previously identified an association between a mismatch repair gene, MLH1, promoter SNP (rs1800734) and microsatellite unstable (MSI-H) colorectal cancers (CRCs) in two samples. The current study expanded on this finding as we explored the genetic basis of DNA methylation in this region of chromosome 3. We hypothesized that specific polymorphisms in the MLH1 gene region predispose it to DNA methylation, resulting in the loss of MLH1 gene expression, mismatch-repair function, and consequently to genome-wide microsatellite instability. METHODOLOGY/PRINCIPAL FINDINGS: We first tested our hypothesis in one sample from Ontario (901 cases, 1,097 controls) and replicated major findings in two additional samples from Newfoundland and Labrador (479 cases, 336 controls) and from Seattle (591 cases, 629 controls). Logistic regression was used to test for association between SNPs in the region of MLH1 and CRC, MSI-H CRC, MLH1 gene expression in CRC, and DNA methylation in CRC. The association between rs1800734 and MSI-H CRCs, previously reported in Ontario and Newfoundland, was replicated in the Seattle sample. Two additional SNPs, in strong linkage disequilibrium with rs1800734, showed strong associations with MLH1 promoter methylation, loss of MLH1 protein, and MSI-H CRC in all three samples. The logistic regression model of MSI-H CRC that included MLH1-promoter-methylation status and MLH1 immunohistochemistry status fit most parsimoniously in all three samples combined. When rs1800734 was added to this model, its effect was not statistically significant (P-value  = 0.72 vs. 2.3×10(-4) when the SNP was examined alone). CONCLUSIONS/SIGNIFICANCE: The observed association of rs1800734 with MSI-H CRC occurs through its effect on the MLH1 promoter methylation, MLH1 IHC deficiency, or both.

MLH1 -93G>A promoter polymorphism and the risk of microsatellite-unstable colorectal cancer.

BACKGROUND: Although up to 30% of patients with colorectal cancer have a positive family history of colorectal neoplasia, few colorectal cancers can be explained by mutations in high-penetrance genes. We investigated whether polymorphisms in DNA mismatch repair genes are associated with the risk of colorectal cancer. METHODS: We genotyped 929 case patients and 1098 control subjects from Ontario and 430 case patients and 275 control subjects from Newfoundland and Labrador for five polymorphisms in the mismatch repair genes MLH1 and MSH2 with the fluorogenic 5' nuclease assay. Tumor microsatellite instability (MSI) was determined with a polymerase chain reaction-based method; MSI status was assigned as high (MSI-H, > or = 30% unstable markers among all markers tested), low (MSI-L, <30% markers unstable), or stable (MSS, no unstable markers). We used unconditional logistic regression to evaluate the association between each polymorphism and colorectal cancer after adjusting for age and sex. The associations between polymorphisms and tumor clinicopathologic features were evaluated with a Pearson's chi-square or Fisher's exact test. All statistical tests were two-sided. RESULTS: We observed strong associations between the MLH1 -93G>A polymorphism and MSI-H tumors among case patients from Ontario (P = .001) and Newfoundland (P = .003). When compared with the control populations, homozygosity for the MLH1 -93G>A variant allele was associated with MSI-H tumors among case patients in Ontario (adjusted odds ratio [OR] = 3.23, 95% confidence interval [CI] = 1.65 to 6.30) and in Newfoundland (OR = 8.88, 95% CI = 2.33 to 33.9), as was heterozygosity among case patients in Ontario (OR = 1.84, 95% CI = 1.20 to 2.83) and in Newfoundland (OR = 2.56, 95% CI = 1.14 to 5.75). Genotype frequencies were similar among case patients with MSS and MSI-L tumors and control subjects, and the majority of homozygous variant carriers had MSS tumors. Among case patients from Ontario, an association between the MLH1 -93G>A polymorphism and a strong family history of colorectal cancer (for Amsterdam criteria I and II, P = .004 and P = .02, respectively) was observed. CONCLUSION: In two patient populations, the MLH1 -93G>A polymorphism was associated with an increased risk of MSI-H colorectal cancer.