Subset Of Tumor Suppressor Genes Research Articles

SPOP mutation induces DNA methylation via stabilizing GLP/G9a

Mutations in SPOP E3 ligase gene are reportedly associated with genome-wide DNA hypermethylation in prostate cancer (PCa) although the underlying mechanisms remain elusive. Here, we demonstrate that SPOP binds and promotes polyubiquitination and degradation of histone methyltransferase and DNMT interactor GLP. SPOP mutation induces stabilization of GLP and its partner protein G9a and aberrant upregulation of global DNA hypermethylation in cultured PCa cells and primary PCa specimens. Genome-wide DNA methylome analysis shows that a subset of tumor suppressor genes (TSGs) including FOXO3, GATA5, and NDRG1, are hypermethylated and downregulated in SPOP-mutated PCa cells. DNA methylation inhibitor 5-azacytidine effectively reverses expression of the TSGs examined, inhibits SPOP-mutated PCa cell growth in vitro and in mice, and enhances docetaxel anti-cancer efficacy. Our findings reveal the GLP/G9a-DNMT module as a mediator of DNA hypermethylation in SPOP-mutated PCa. They suggest that SPOP mutation could be a biomarker for effective treatment of PCa with DNA methylation inhibitor alone or in combination with taxane chemotherapeutics.

YAP/TAZ and EZH2 synergize to impair tumor suppressor activity of TGFBR2 in non-small cell lung cancer.

Lung cancer is the leading cause of cancer-related deaths, worldwide. Non-small cell lung cancer (NSCLC) is the most prevalent lung cancer subtype. YAP and TAZ have been implicated in lung cancer by acting as transcriptional co-activators of oncogenes or as transcriptional co-repressors of tumor suppressor genes. Previously we reported that YAP and TAZ regulate microRNAs expression in NSCLC. Among the set of regulated miRNAs, the oncogenic miR-25, 93, and 106b, clustering within the MCM7 gene were selected for further studies. We firstly identified Transforming Growth Factor-β (TGF-β) Receptor 2 (TGFBR2), a member of the TGF-β signaling, as a target of the miRNA cluster, which exhibited prognostic value because of its tumor suppressor activity. We found that YAP/TAZ-mediated repression of TGFBR2 occurs both: post-transcriptionally through the miR-106b-25 cluster and transcriptionally by engaging the EZH2 epigenetic repressor that we reported here as a novel target gene of YAP/TAZ. Furthermore, we document that YAP/TAZ and EZH2 cooperate in lung tumorigenesis by transcriptionally repressing a specific subset of tumor suppressor genes, including TGFBR2. Our findings point to YAP/TAZ and EZH2 as potential therapeutic targets for NSCLC treatment.

Up-regulation of tumor suppressor genes by exogenous dhC16-Cer contributes to its anti-cancer activity in primary effusion lymphoma.

Primary effusion lymphoma (PEL) is a rare and highly aggressive B-cell malignancy with Kaposi's sarcoma-associated herpesvirus (KSHV) infection, while lack of effective therapies. Our recent data indicated that targeting the sphingolipid metabolism by either sphingosine kinase inhibitor or exogenous ceramide species induces PEL cell apoptosis and suppresses tumor progression in vivo. However, the underlying mechanisms for these exogenous ceramides “killing” PEL cells remain largely unknown. Based on the microarray analysis, we found that exogenous dhC16-Cer treatment affected the expression of many cellular genes with important functions within PEL cells such as regulation of cell cycle, cell survival/proliferation, and apoptosis/anti-apoptosis. Interestingly, we found that a subset of tumor suppressor genes (TSGs) was up-regulated from dhC16-Cer treated PEL cells. One of these elevated TSGs, Thrombospondin-1 (THBS1) was required for dhC16-Cer induced PEL cell cycle arrest. Moreover, dhC16-Cer up-regulation of THBS1 was through the suppression of multiple KSHV microRNAs expression. Our data demonstrate that exogenous ceramides display anti-cancer activities for PEL through regulation of both host and oncogenic virus factors.

Abstract LB-248: Protein arginine methyltransferase 5 (PRMT5) inhibition as a therapeutic strategy in B-cell lymphoma

Abstract PRMT5 is responsible for symmetric dimethylation of arginine residues in glycine and arginine rich (GAR) motifs on a variety of cytosolic and nuclear proteins including histones, spliceosome components, regulators of translation, transcription factors, kinases and others. PRMT5 driven methylation of some of these proteins has been implicated in tumorigenesis. For example, PRMT5 deposits repressive marks on histones and silences a subset of tumor suppressor genes, such as RB and ST7. PRMT5 methylation of non-histone substrates (such as E2F1 and p53) also contributes to cancer cell growth and death. PRMT5 driven methylation of spliceosome subunits and components of translational machinery has been well described but its connection to PRMT5's role in cancer has not been established. We have identified first-in-class small molecules that are highly potent, selective, reversible inhibitors of PRMT5. Cellular mechanistic studies revealed that PRMT5 inhibition decreases symmetric arginine dimethylation on a variety of cellular proteins including spliceosome components, histones and transcription factors. PRMT5 inhibition leads to gene expression and splicing changes ultimately resulting in the induction of p53 in lymphoma cell lines. In addition to impacting the p53 pathway, PRMT5 inhibition leads to attenuation of the expression of cell cycle related genes, genes involved in ribosome and spliceosome homeostasis, as well as genes important for cellular metabolism. PRMT5 inhibitor attenuates proliferation and induces cell death in a subset of mantle cell and diffuse large B-cell lymphoma cell lines and inhibits tumor growth in xenograft models of mantle cell lymphoma. These data underline the potential of PRMT5 inhibitors as a therapeutic strategy in mantle cell and diffuse large B-cell lymphoma. Citation Format: Olena Barbash, Sarah Gerhart, David Soong, Christine Thompson, Rocio Montes de Oca, Ping Zhang, Charles McHugh, Kristy Kuplast, Christina Majer, Richard Chesworth, Jesse Smith, Robert Copeland, Elayne Penebre, Kenneth Duncan, Neil Johnson, Chris Carpenter, Ryan Kruger. Protein arginine methyltransferase 5 (PRMT5) inhibition as a therapeutic strategy in B-cell lymphoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-248. doi:10.1158/1538-7445.AM2015-LB-248

Abstract 3838: Functional crosstalk between histone deacetylase 5 (HDAC5) and lysine-specific demethylase 1 (LSD1) as a novel therapeutic target in triple-negative breast cancer cells

Abstract Our previous work has shown that HDAC5 knockdown led to significant increase of H3K4me2 in the TNBC cell line, MDA-MB-231, suggesting a potential role of HDAC5 in regulating LSD1 activity in breast cancer. In this study, we demonstrated that HDAC5 physically interacts with LSD1 in MDA-MB-231 cells. Overexpression of HDAC5 increased LSD1 protein level through a reduction of LSD1 polyubiquitination. siRNA-mediated knockdown of HDAC5 mRNA, but not other class II HDACs or LSD1-containing complex members (HDAC1, HDAC2 and CoREST), significantly decreased LSD1 protein expression and half-life. HDAC5 overexpression reduced levels of H3K4me1/2, suggesting that HDAC5 acts as a positive regulator of LSD1-mediated H3K4 demethylation activity. Enhanced proliferation mediated by HDAC5 overexpression in MDA-MB-231 cells was diminished by LSD1-KD, suggesting a critical role of LSD1 in regulating oncogenic activity of HDAC5. We also observed that high-grade tumorigenic MCF10A-CA1a cells (triple negative) expressed higher levels of LSD1 and HDAC5 compared with parental counterpart MCF10A cells. By gain- and loss-of-function studies using this TNBC progression model, we observed that HDAC5 possesses a critical oncogenic function in driving TNBC development by blocking LSD1 protein degradation and re-shaping epigenetic landscape. Several HDAC inhibitors were tested for their ability to inhibit LSD1 and HDAC5 interaction. Treatment with sulforaphane, a natural HDAC inhibitor found in cruciferous vegetables, blocked the association of HDAC5 and LSD1 through down-regulation of HDAC5 mRNA and protein expression that led to LSD1 protein destabilization. Sulforaphane treatment also re-expressed a unique subset of tumor suppressor genes (RGS16, BIK, PCDH1, CDKN1a, ING1, etc), which are abnormally silenced in TNBC cells, but are expressed in other breast cancer subtypes and normal breast cells. Addition of a novel LSD1 inhibitor, HCI-2509, significantly promoted antineoplastic efficacy of sulforaphane in breast cancer cells, and TNBC cells were more susceptible than other subtypes to the combination therapy. In an in vivo study, while treatment with either sulforaphane or HCI-2509 alone each displayed significant anti-tumor effects against MDA-MB-231 xenografts, combined treatment with both compounds exhibited synergistic inhibitory effect on tumor growth in the MDA-MB-231 xenografts. Taken together, these results suggest that HDAC5 promotes LSD1 protein stabilization, leading to a dysregulated chromatin landscape which functions as an antibraking system in TNBC development. Crosstalk between LSD1 and HDAC5 may represent a novel and effective therapeutic target for this devastating disease. (Supported by DOD W81XWH-14-1-0237/W81XWH-14-1-0238, Breast Cancer Research Foundation, and UPCI Core Grant NIH P30CA047904) Citation Format: Chunyu Cao, Shauna Vasilatos, Steffi Oesterreich, Nancy E. Davidson, Yi Huang. Functional crosstalk between histone deacetylase 5 (HDAC5) and lysine-specific demethylase 1 (LSD1) as a novel therapeutic target in triple-negative breast cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3838. doi:10.1158/1538-7445.AM2015-3838

Dietary black raspberries modulate DNA methylation in dextran sodium sulfate (DSS)-induced ulcerative colitis

Ulcerative colitis (UC) is characterized by chronic inflammation of the colon. During inflammation, NF-κB is increased in colonic epithelial cells and in immune cells, leading to increases in proinflammatory cytokines. These events then increase DNA methyltransferases (DNMTs), which silence a subset of tumor suppressor genes by promoter methylation. Negative regulators of the Wnt pathway are frequently methylated in UC, leading to dysregulation of the pathway and, potentially, to colorectal cancer. We determined if black raspberries (BRBs) influence promoter methylation of suppressors in the Wnt pathway in dextran sodium sulfate (DSS)-induced UC. C57BL/6J mice received 1% DSS and were fed either control or 5% BRB diets. Mice were euthanized on days 7, 14 and 28, and their colons, spleen and bone marrow were collected. Berries reduced ulceration at day 28. This was accompanied by decreased staining of macrophages and neutrophils and decreased NF-κB p65 nuclear localization in the colon at all time points. At day 7, BRBs demethylated the promoter of dkk3, leading to its increased messenger RNA (mRNA) expression in colon, spleen and bone marrow. β-Catenin nuclear localization, c-Myc staining as well as protein expression of DNMT3B, histone deacetylases 1 and 2 (HDAC1 and HDAC2) and methyl-binding domain 2 (MBD2) were all decreased in colon; mRNA expression of these four proteins was decreased in bone marrow cells by BRBs. These results suggest that BRBs suppress colonic ulceration by correcting promoter hypermethylation of suppressor genes in the colon, as well as in the spleen and bone marrow that systematically regulate inflammation. Our results suggest that dietary BRBs suppress colonic ulceration by correcting promoter hypermethylation of suppressor genes in the colon, as well as in the spleen and bone marrow that systematically regulate inflammation in DSS-induced UC.

Characteristic patterns of altered DNA methylation predict emergence of human hepatocellular carcinoma

We aimed to identify the specific subset of tumor suppressor genes (TSGs) that are methylation-silenced during the earliest steps of hepatocarcinogenesis, and to further evaluate whether these genes can serve as predictive biomarkers of hepatocellular carcinoma (HCC) emergence. A total of 482 liver tissues including 177 pairs of HCCs and matched nontumor livers and 128 liver biopsies from chronic hepatitis C (CHC) patients were analyzed for quantitative methylation analysis in 24 TSG promoters and three MINT loci. The tumors were classified as early, less-progressed, and highly progressed HCCs using histology and radiological approaches. A subset of TSGs that harbored distinctly high levels of methylation in early HCCs were selected. Based on the methylation profiles of these genes, Kaplan-Meier analyses were performed to determine time-to-HCC occurrence in CHC patients. Subsequently, multivariate analysis was performed using age, gender, fibrosis stage, and number of methylated TSGs as covariates. Among TSGs analyzed, a subset of eight TSGs (HIC1, GSTP1, SOCS1, RASSF1, CDKN2A, APC, RUNX3, and PRDM2) demonstrated a distinct cluster by hierarchical clustering and receiver operating characteristic analyses. This subset of TSGs showed significantly higher methylation levels in the early HCCs (P < 0.0001). In the CHC patients, methylation frequencies in these TSGs were associated with shorter time-to-HCC occurrence (P < 0.0001), and number of methylated genes was an independent risk factor for HCC (hazard ratio = 5.21, 95% confidence interval = 2.25-11.76, P = 0.0002). Epigenetic inactivation of a subset of TSGs plays a critical role in the earliest steps of hepatocarcinogenesis. Furthermore, epigenetic inactivation of these genes in CHC provides a prognostic value for determining the risk for developing HCC later in life.

Genome-wide CpG island methylation analysis implicates novel genes in the pathogenesis of renal cell carcinoma

In order to identify novel candidate tumor suppressor genes (TSGs) implicated in renal cell carcinoma (RCC), we performed genome-wide methylation profiling of RCC using the HumanMethylation27 BeadChips to assess methylation at >14,000 genes. Two hundred and twenty hypermethylated probes representing 205 loci/genes were identified in genomic CpG islands. A subset of TSGs investigated in detail exhibited frequent tumor methylation, promoter methylation associated transcriptional silencing and reactivation after demethylation in RCC cell lines and down-regulation of expression in tumor tissue (e.g., SLC34A2 specifically methylated in 63% of RCC, OVOL1 in 40%, DLEC1 in 20%, TMPRSS2 in 26%, SST in 31% and BMP4 in 35%). As OVOL1, a putative regulator of c-Myc transcription, and SST (somatostatin) had not previously been linked to cancer and RCC, respectively, we (1) investigated their potential relevance to tumor growth by RNAi knockdown and found significantly increased anchorage-independent growth and (2) demonstrated that OVOL1 knockdown increased c-Myc mRNA levels.

Transient analysis for antiproliferative gene activity.

A subset of tumor suppressor genes presumably functions by the inhibition of cellular proliferation; however, antiproliferative activity after transfection with putative suppressor genes has been difficult to demonstrate and often requires lengthy selection either in nude mice or in vitro. A rapid alternative is presented here that utilizes a gene encoding a surface marker protein to identify transfectants in a transient expression assay. In this assay the labeling index, rate of DNA synthesis, cell-cycle distribution, and surface receptor display are measured by flow cytometry. Human beta-interferon, a gene with proven antiproliferative activity, was studied using the transient analysis system. The beta-interferon gene was introduced into human tumor cells along with the marker gene encoding the 55-kDa subunit of the human interleukin 2 receptor. Within a few days of transfection, analysis of transfectants by flow cytometry revealed a decrease in the fraction of cells in G2/M and an increase in the fraction of cells in G1/G0 and S phases. The distortion of the cell cycle was accompanied by as much as a 69% reduction in the rate of DNA synthesis and, in some experiments, an unanticipated increase in the labeling index. Therefore, cells accumulating in S phase were not blocked but continued to synthesize DNA although at a reduced rate. These studies on DNA synthetic rates revealed the caveat that screening for antiproliferative candidate genes with a labeling index alone could, in certain circumstances, exclude potentially interesting sequences from further consideration. Although this transient analysis system was developed for studies on cellular proliferation, it may prove suitable for phenotypic assays on other genes as well.