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G9a drives hypoxia-mediated gene repression for breast cancer cell survival and tumorigenesis.

ABSTRACT: G9a is an epigenetic regulator that methylates H3K9, generally causing repression of gene expression, and participates in diverse cellular functions. G9a is genetically deregulated in a variety of tumor types and can silence tumor suppressor genes and, therefore, is important for carcinogenesis. Although hypoxia is recognized to be an adverse factor in tumor growth and metastasis, the role of G9a in regulating gene expression in hypoxia has not been described extensively. Here, we show that G9a protein stability is increased in hypoxia via reduced proline hydroxylation and, hence, inefficient degradation by the proteasome. This inefficiency leads to an increase in H3K9me2 at its target promoters. Blocking the methyltransferase activity of G9a inhibited cellular proliferation and migration in vitro and tumor growth in vivo. Furthermore, an increased level of G9a is a crucial factor in mediating the hypoxic response by down-regulating the expression of specific genes, including ARNTL, CEACAM7, GATA2, HHEX, KLRG1, and OGN This down-regulation can be rescued by a small molecule inhibitor of G9a. Based on the hypothesis that the changes in gene expression would influence patient outcomes, we have developed a prognostic G9a-suppressed gene signature that can stratify breast cancer patients. Together, our findings provide an insight into the role G9a plays as an epigenetic mediator of hypoxic response, which can be used as a diagnostic marker, and proposes G9a as a therapeutic target for solid cancers.

SUBMITTER: Casciello F

PROVIDER: S-EPMC5502591 | biostudies-literature | 2017 Jul

REPOSITORIES: biostudies-literature

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G9a drives hypoxia-mediated gene repression for breast cancer cell survival and tumorigenesis.

Casciello Francesco F Al-Ejeh Fares F Kelly Greg G Brennan Donal J DJ Ngiow Shin Foong SF Young Arabella A Stoll Thomas T Windloch Karolina K Hill Michelle M MM Smyth Mark J MJ Gannon Frank F Lee Jason S JS

Proceedings of the National Academy of Sciences of the United States of America 20170619 27

G9a is an epigenetic regulator that methylates H3K9, generally causing repression of gene expression, and participates in diverse cellular functions. G9a is genetically deregulated in a variety of tumor types and can silence tumor suppressor genes and, therefore, is important for carcinogenesis. Although hypoxia is recognized to be an adverse factor in tumor growth and metastasis, the role of G9a in regulating gene expression in hypoxia has not been described extensively. Here, we show that G9a ...[more]

PMID: 28630300

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Project description:Rationale: Epigenetic mechanisms are fundamental processes that can modulate gene expression, allowing cellular adaptation to environmental conditions. Hypoxia is an important factor known to initiate the metastatic cascade in cancer, activating cell motility and invasion by silencing cell adhesion genes. G9a is a histone methyltransferase previously shown to accumulate in hypoxic conditions. While its oncogenic activity has been previously reported, not much is known about the role G9a plays in the hypoxia-mediated metastatic cascade. Methods: The role of G9a in cell motility in hypoxic condition was determined by inhibiting G9a either by short-hairpin mediated knock down or pharmacologically using a small molecule inhibitor. Through gene expression profiling, we identified CDH10 to be an important G9a target that regulates breast cancer cell motility. Lung metastasis assay in mice was used to determine the physiological significance of G9a. Results: We demonstrate that, while hypoxia enhances breast cancer migratory capacity, blocking G9a severely reduces cellular motility under both normoxic and hypoxic conditions and prevents the hypoxia-mediated induction of cellular movement. Moreover, inhibition of G9a histone methyltransferase activity in mice using a specific small molecule inhibitor significantly reduced growth and colonisation of breast cancer cells in the lung. We identify the type-II cadherin CDH10 as being a novel hypoxia-dependent gene, directly repressed by G9a through histone methylation. CDH10 overexpression significantly reduces cellular movements in breast cancer cell lines and prevents the hypoxia-mediated increase in cell motility. In addition, we show that CDH10 expression is prognostic in breast cancer and that it is inversely correlated to EHMT2 (G9a) transcript levels in many tumor-types, including breast cancer. Conclusion: We propose that G9a promotes cellular motility during hypoxic stress through the silencing of the cell adhesion molecule CDH10 and we describe CDH10 as a novel prognostic biomarker for breast cancer.

Project description:BackgroundBreast cancer (BC) is a great clinical challenge because of its aggressiveness and poor prognosis. Zinc Finger Protein 64 (ZFP64), as a transcriptional factor, is responsible for the development and progression of cancers. This study aims to investigate whether ZFP64 regulates stem cell-like properties and tumorigenesis in BC by the glycolytic pathway.ResultsIt was demonstrated that ZFP64 was overexpressed in BC specimens compared to adjacent normal tissues, and patients with high ZFP64 expression had shorter overall survival and disease-free survival. The analysis of the association of ZFP64 expression with clinicopathological characteristics showed that high ZFP64 expression is closely associated with N stage, TNM stage, and progesterone receptor status. Knockdown of ZFP64 suppressed the viability and colony formation capacity of BC cells by CCK8 and colony formation assays. The subcutaneous xenograft models revealed that ZFP64 knockdown reduced the volume of formatted tumors, and decreased Ki67 expression in tumors. The opposite effects on cell proliferation and tumorigenesis were demonstrated by ZFP64 overexpression. Furthermore, we suggested that the stem cell-like properties of BC cells were inhibited by ZFP64 depletion, as evidenced by the decreased size and number of formatted mammospheres, the downregulated expressions of OCT4, Nanog, and SOX2 proteins, as well as the reduced proportion of CD44+/CD24- subpopulations. Mechanistically, glycolysis was revealed to mediate the effect of ZFP64 using mRNA-seq analysis. Results showed that ZFP64 knockdown blocked the glycolytic process, as indicated by decreasing glycolytic metabolites, inhibiting glucose consumption, and reducing lactate and ATP production. As a transcription factor, we identified that ZFP64 was directly bound to the promoters of glycolysis-related genes (ALDOC, ENO2, HK2, and SPAG4), and induced the transcription of these genes by ChIP and dual-luciferase reporter assays. Blocking the glycolytic pathway by the inhibition of glycolytic enzymes ENO2/HK2 suppressed the high proliferation and stem cell-like properties of BC cells induced by ZFP64 overexpression.ConclusionsThese data support that ZFP64 promotes stem cell-like properties and tumorigenesis of BC by activating glycolysis in a transcriptional mechanism.

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G9a drives hypoxia-mediated gene repression for breast cancer cell survival and tumorigenesis.

Publications

G9a drives hypoxia-mediated gene repression for breast cancer cell survival and tumorigenesis.

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