The tumor suppressor gene rap1GAP is silenced by miR-101-mediated EZH2 overexpression in invasive squamous cell carcinoma.
ABSTRACT: Rap1GAP is a critical tumor suppressor gene that is downregulated in multiple aggressive cancers, such as head and neck squamous cell carcinoma, melanoma and pancreatic cancer. However, the mechanistic basis of rap1GAP downregulation in cancers is poorly understood. By employing an integrative approach, we demonstrate polycomb-mediated repression of rap1GAP that involves Enhancer of Zeste Homolog 2 (EZH2), a histone methyltransferase in head and neck cancers. We further demonstrate that the loss of miR-101 expression correlates with EZH2 upregulation, and the concomitant downregulation of rap1GAP in head and neck cancers. EZH2 represses rap1GAP by facilitating the trimethylation of histone 3 at lysine 27, a mark of gene repression, and also hypermethylation of rap1GAP promoter. These results provide a conceptual framework involving a microRNA-oncogene-tumor suppressor axis to understand head and neck cancer progression.
Project description:Rap1GAP is a critical tumor suppressor gene that is down-regulated in multiple aggressive cancers such as head and neck squamous cell carcinoma, melanoma and pancreatic cancer. However, the mechanistic basis of rap1GAP down-regulation in cancers is poorly understood. By employing an integrative approach, we demonstrate polycomb-mediated repression of rap1GAP that involves EZH2, a histone methyltransferase in head and neck cancers. We further concomitant down-regulation of rap1GAP in head and neck cancers. EZH2 represses rap1GAP by facilitating the trimethylation of H3K27, a mark of gene repression, and also hypermethylation of rap1GAP promoter. These results provide a conceptual framework involving a microRNA-oncogene-tumor suppressor axis to understand head and neck cancer progression. Overall design: OSCC3-siRNA Non-Targeting Vs. siRNA EZH2 with dye-swap, HOK-Adeno CMV Vs. Adeno EZH2.
Project description:Rap1GAP is a critical tumor suppressor gene that is down-regulated in multiple aggressive cancers such as head and neck squamous cell carcinoma, melanoma and pancreatic cancer. However, the mechanistic basis of rap1GAP down-regulation in cancers is poorly understood. By employing an integrative approach, we demonstrate polycomb-mediated repression of rap1GAP that involves EZH2, a histone methyltransferase in head and neck cancers. We further concomitant down-regulation of rap1GAP in head and neck cancers. EZH2 represses rap1GAP by facilitating the trimethylation of H3K27, a mark of gene repression, and also hypermethylation of rap1GAP promoter. These results provide a conceptual framework involving a microRNA-oncogene-tumor suppressor axis to understand head and neck cancer progression. OSCC3-siRNA Non-Targeting Vs. siRNA EZH2 with dye-swap, HOK-Adeno CMV Vs. Adeno EZH2.
Project description:Thyroid cancer is the most common type of endocrine malignancy, encompassing tumors with various levels of invasive growth and aggressiveness. Rap1GAP, a Rap1 GTPase-activating protein, inhibits the RAS superfamily protein Rap1 by facilitating hydrolysis of GTP to GDP. In this study, we analyzed 197 thyroid tumor samples and showed that Rap1GAP was frequently lost or downregulated in various types of tumors, particularly in the most invasive and aggressive forms of thyroid cancer. The downregulation was due to promoter hypermethylation and/or loss of heterozygosity, found in the majority of thyroid tumors. Treatment with demethylating agent 5-aza-deoxycytidine and/or histone deacetylation inhibitor trichostatin A induced gene reexpression in thyroid cells. A genetic polymorphism, Y609C, was seen in 7% of thyroid tumors but was not related to gene downregulation. Loss of Rap1GAP expression correlated with tumor invasiveness but not with specific mutations activating the mitogen-activated protein kinase pathway. Rap1GAP downregulation was required in vitro for cell migration and Matrigel invasion. Recovery of Rap1GAP expression inhibited thyroid cell proliferation and colony formation. Overall, our findings indicate that epigenetic or genetic loss of Rap1GAP is very common in thyroid cancer, where these events are sufficient to promote cell proliferation and invasion.
Project description:Rap1GAP expression is decreased in human tumors. The significance of its downregulation is unknown. We show that Rap1GAP expression is decreased in primary colorectal carcinomas. To elucidate the advantages conferred on tumor cells by loss of Rap1GAP, Rap1GAP expression was silenced in human colon carcinoma cells. Suppressing Rap1GAP induced profound alterations in cell adhesion. Rap1GAP-depleted cells exhibited defects in cell/cell adhesion that included an aberrant distribution of adherens junction proteins. Depletion of Rap1GAP enhanced adhesion and spreading on collagen. Silencing of Rap expression normalized spreading and restored E-cadherin, beta-catenin, and p120-catenin to cell/cell contacts, indicating that unrestrained Rap activity underlies the alterations in cell adhesion. The defects in adherens junction protein distribution required integrin signaling as E-cadherin and p120-catenin were restored at cell/cell contacts when cells were plated on poly-l-lysine. Unexpectedly, Src activity was increased in Rap1GAP-depleted cells. Inhibition of Src impaired spreading and restored E-cadherin at cell/cell contacts. These findings provide the first evidence that Rap1GAP contributes to cell/cell adhesion and highlight a role for Rap1GAP in regulating cell/matrix and cell/cell adhesion. The frequent downregulation of Rap1GAP in epithelial tumors where alterations in cell/cell and cell/matrix adhesion are early steps in tumor dissemination supports a role for Rap1GAP depletion in tumor progression.
Project description:MicroRNAs (miRs) play a key role in cancer etiology by coordinately repressing numerous target genes involved in cell proliferation, migration and invasion. The genomic region in chromosome 9p21 that encompasses miR-31 is frequently deleted in solid cancers including melanoma; however the expression and functional role of miR-31 has not been previously studied in melanoma. Here, we queried the expression status and performed functional characterization of miR-31 in melanoma tissues and cell lines. We found that down-regulation of miR-31 was a common event in melanoma tumors and cell lines and was associated with genomic loss in a subset of samples. Down-regulation of miR-31 gene expression was also a result of epigenetic silencing by DNA methylation, and via EZH2-mediated histone methylation. Ectopic overexpression of miR-31 in various melanoma cell lines inhibited cell migration and invasion. miR-31 targets include oncogenic kinases such as SRC, MET, NIK (MAP3K14) and the melanoma specific oncogene RAB27a. Furthermore, miR-31 overexpression resulted in down-regulation of EZH2 and a de-repression of its target gene rap1GAP; increased expression of EZH2 was associated with melanoma progression and overall patient survival. Taken together, our study supports a tumor suppressor role for miR-31 in melanoma and identifies novel therapeutic targets.
Project description:CDKN1C (encoding tumor suppressor p57(KIP2)) is a cyclin-dependent kinase (CDK) inhibitor whose family members are often transcriptionally downregulated in human cancer via promoter DNA methylation. In this study, we show that CDKN1C is repressed in breast cancer cells mainly through histone modifications. In particular, we show that CDKN1C is targeted by histone methyltransferase EZH2-mediated histone H3 lysine 27 trimethylation (H3K27me3), and can be strongly activated by inhibition of EZH2 in synergy with histone deacetylase inhibitor. Consistent with the overexpression of EZH2 in a variety of human cancers including breast cancer, CDKN1C in these cancers is downregulated, and breast tumors expressing low levels of CDKN1C are associated with a poor prognosis. We further show that assessing both EZH2 and CDKN1C expression levels as a measurement of EZH2 pathway activity provides a more predictive power of disease outcome than that achieved with EZH2 or CDKN1C alone. Taken together, our study reveals a novel epigenetic mechanism governing CDKN1C repression in breast cancer. Importantly, as a newly identified EZH2 target with prognostic value, it has implications in patient stratification for cancer therapeutic targeting EZH2-mediated gene repression.
Project description:Protein kinase A (PKA)-dependent signaling cascades play an important role in mediating the effects of dopamine and other neurotransmitters in striatal medium spiny neurons. We have identified a prominent striatal PKA substrate as Rap1-GTPase activating protein (Rap1GAP), a negative regulator of Rap1 signaling. Although present throughout the brain, Rap1GAP is enriched in striatal medium spiny neurons and is phosphorylated by PKA at Ser-441 and Ser-499 in response to activation of D1 dopamine receptors. Phosphorylation of Rap1GAP is associated with inhibition of GAP activity, as demonstrated by increased Rap1 activity in striatal neurons. Phosphorylation of Rap1GAP is also associated with decreased [corrected] dendritic spine head size in cultured neurons. These findings suggest that phosphorylation of Rap1GAP by PKA plays an important role in striatal neurons by modulating Rap1 actions.
Project description:Enhancer of zeste homolog 2 (EZH2) is a candidate oncogenic driver due to its prevalent overexpression and aberrant repression of tumor suppressor genes in diverse cancers. Therefore, blocking EZH2 enzyme activity may present a valid therapeutic strategy for the treatment of cancers with EZH2 overexpression including breast cancers. Here, we described ZLD1039 a potent, highly selective, and orally bioavailable small molecule inhibitor of EZH2, which inhibited breast tumor growth and metastasis. ZLD1039 considerably inhibited EZH2 methyltransferase activity with nanomolar potency, decreased global histone-3 lysine-27 (H3K27) methylation, and reactivated silenced tumor suppressors connected to increased survival of patients with breast cancer. Comparable to conditional silencing of EZH2, its inhibition by ZLD1039 decreased cell proliferation, cell cycle arrest, and induced apoptosis. Comparably, treatment of xenograft-bearing mice with ZLD1039 led to tumor growth regression and metastasis inhibition. These data confirmed the dependency of breast cancer progression on EZH2 activity and the usefulness of ZLD1039 as a promising treatment for breast cancer.
Project description:Enhancer of zeste homolog 2 (EZH2) is the histone lysine N-methyltransferase component of the Polycomb repressive complex 2 (PRC2), which, in conjunction with embryonic ectoderm development (EED) and suppressor of zeste 12 homolog, regulates cell lineage determination and homeostasis. Enzymatic hyperactivity has been linked to aberrant repression of tumor suppressor genes in diverse cancers. Here, we report the development of stabilized ?-helix of EZH2 (SAH-EZH2) peptides that selectively inhibit H3 Lys27 trimethylation by dose-responsively disrupting the EZH2-EED complex and reducing EZH2 protein levels, a mechanism distinct from that reported for small-molecule EZH2 inhibitors targeting the enzyme catalytic domain. MLL-AF9 leukemia cells, which are dependent on PRC2, undergo growth arrest and monocyte-macrophage differentiation upon treatment with SAH-EZH2, consistent with observed changes in expression of PRC2-regulated, lineage-specific marker genes. Thus, by dissociating the EZH2-EED complex, we pharmacologically modulate an epigenetic 'writer' and suppress PRC2-dependent cancer cell growth.
Project description:Glial cell line-derived neurotrophic factor (GDNF) was originally recognized for its ability to promote survival of midbrain dopaminergic neurons, but it has since been demonstrated to be crucial for the survival and differentiation of many neuronal subpopulations, including motor neurons, sympathetic neurons, sensory neurons and enteric neurons. To identify possible effectors or regulators of GDNF signaling, we performed a yeast two-hybrid screen using the intracellular domain of RET, the common signaling receptor of the GDNF family, as bait. Using this approach, we identified Rap1GAP, a GTPase-activating protein (GAP) for Rap1, as a novel RET-binding protein. Endogenous Rap1GAP co-immunoprecipitated with RET in neural tissues, and RET and Rap1GAP were co-expressed in dopaminergic neurons of the mesencephalon. In addition, overexpression of Rap1GAP attenuated GDNF-induced neurite outgrowth, whereas suppressing the expression of endogenous Rap1GAP by RNAi enhanced neurite outgrowth. Furthermore, using co-immunoprecipitation analyses, we found that the interaction between RET and Rap1GAP was enhanced following GDNF treatment. Mutagenesis analysis revealed that Tyr981 in the intracellular domain of RET was crucial for the interaction with Rap1GAP. Moreover, we found that Rap1GAP negatively regulated GNDF-induced ERK activation and neurite outgrowth. Taken together, our results suggest the involvement of a novel interaction of RET with Rap1GAP in the regulation of GDNF-mediated neurite outgrowth.