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M6A mRNA methylation regulates AKT activity to promote the proliferation and tumorigenicity of endometrial cancer.

ABSTRACT: N6-methyladenosine (m6A) messenger RNA methylation is a gene regulatory mechanism affecting cell differentiation and proliferation in development and cancer. To study the roles of m6A mRNA methylation in cell proliferation and tumorigenicity, we investigated human endometrial cancer in which a hotspot R298P mutation is present in a key component of the methyltransferase complex (METTL14). We found that about 70% of endometrial tumours exhibit reductions in m6A methylation that are probably due to either this METTL14 mutation or reduced expression of METTL3, another component of the methyltransferase complex. These changes lead to increased proliferation and tumorigenicity of endometrial cancer cells, likely through activation of the AKT pathway. Reductions in m6A methylation lead to decreased expression of the negative AKT regulator PHLPP2 and increased expression of the positive AKT regulator mTORC2. Together, these results reveal reduced m6A mRNA methylation as an oncogenic mechanism in endometrial cancer and identify m6A methylation as a regulator of AKT signalling.

SUBMITTER: Liu J

PROVIDER: S-EPMC6245953 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

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m6A mRNA methylation regulates AKT activity to promote the proliferation and tumorigenicity of endometrial cancer.

Liu Jun J Eckert Mark A MA Harada Bryan T BT Liu Song-Mei SM Lu Zhike Z Yu Kangkang K Tienda Samantha M SM Chryplewicz Agnieszka A Zhu Allen C AC Yang Ying Y Huang Jing-Tao JT Chen Shao-Min SM Xu Zhi-Gao ZG Leng Xiao-Hua XH Yu Xue-Chen XC Cao Jie J Zhang Zezhou Z Liu Jianzhao J Lengyel Ernst E He Chuan C

Nature cell biology 20180827 9

N6-methyladenosine (m6A) messenger RNA methylation is a gene regulatory mechanism affecting cell differentiation and proliferation in development and cancer. To study the roles of m6A mRNA methylation in cell proliferation and tumorigenicity, we investigated human endometrial cancer in which a hotspot R298P mutation is present in a key component of the methyltransferase complex (METTL14). We found that about 70% of endometrial tumours exhibit reductions in m ...[more]

PMID: 30154548

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Project description:BackgroundN6-Methyladenosine (m6A) modification has been implicated in many biological processes. It is important for the regulation of messenger RNA (mRNA) stability, splicing, and translation. However, its role in cancer has not been studied in detail. Here we investigated the biological role and underlying mechanism of m6A modification in hepatoblastoma (HB).MethodsWe used Reverse transcription quantitative real-time PCR (RT-qPCR) and Western blotting to determine the expression of m6A related factors. And we clarified the effects of these factors on HB cells using cell proliferation assay, colony formation, apoptotic assay. Then we investigated of methyltransferase-like 13 (METTL3) and its correlation with clinicopathological features and used xenograft experiment to check METTL3 effect in vivo. m6A-Seq was used to profiled m6A transcriptome-wide in hepatoblastoma tumor tissue and normal tissue. Finally, methylated RNA immunoprecipitation (MeRIP) assay, RNA remaining assay to perform the regulator mechanism of MEETL3 on the target CTNNB1 in HB.ResultsIn this research, we discovered that m6A modifications are increased in hepatoblastoma, and METTL3 is the main factor involved with aberrant m6A modification. We also profiled m6A across the whole transcriptome in hepatoblastoma tumor tissues and normal tissues. Our findings suggest that m6A is highly expressed in hepatoblastoma tumors. Also, m6A is enriched not only around the stop codon, but also around the coding sequence (CDS) region. Gene ontology analysis indicates that m6A mRNA methylation contributes significantly to regulate the Wnt/β-catenin pathway. Reduced m6A methylation can lead to a decrease in expression and stability of the CTNNB1.ConclusionOverall our findings suggest enhanced m6A mRNA methylation as an oncogenic mechanism in hepatoblastoma, METTL3 is significantly up-regulated in HB and promotes HB development. And identify CTNNB1 as a regulator of METTL3 guided m6A modification in HB.

Project description:ObjectiveTCP1 is one of the eight subunits of the TCP1 ring complex (TRiC) or the multi-protein mammalian cytosolic chaperone complex. TRiC participates in protein folding and regulates the expression of multiple signaling proteins and cytoskeletal components in cells. Although the clinical importance of its subunits has been clarified in various carcinomas, the function of TCP1 in ovarian cancer (OC) remains unclear. We aimed to identify the association between the expression of TCP1 and the development of epithelial OC (EOC) and patient prognosis, and explore the underlying mechanisms of TCP1 on the tumor progression of OC cells.MethodsTCP1 protein expression was tested in various ovarian tissues by immunohistochemistry, and the correlation between TCP1 expression and clinical physiologic or pathologic parameters of patients with EOC was analyzed. The relationship between TCP1 expression and the prognosis of patients with OC was investigated and analyzed using the Kaplan-Meier (KM) plotter online database. The expression level of TCP1 was then tested in different OC cell lines by Western blotting. Further, a model using OC cell line A2780 was constructed to study the functions of TCP1 in growth, migration, and invasion of human EOC cells. Finally, the possible regulating signaling pathways were discussed.ResultsTCP1 protein expression in OC or borderline tissues was significantly higher than that in benign ovarian tumors and normal ovarian tissue. The upregulated expression of TCP1 in OC was positively associated with the differentiation grade and FIGO stage of tumors and predicted poor clinical outcomes. Compared with IOSE-80 cells, TCP1 protein was overexpressed in A2780 cells. TCP1 knockdown using shRNA lentivirus inhibited the viability of A2780 cells. Western blotting showed that the phosphatidylinositol-3 kinase (PI3K) signaling pathway was activated in the tumor invasion in EOC driven by TCP1.ConclusionUpregulated TCP1 is correlated with the poor prognosis of patients with OC. The mechanism of cancer progression promoted by TCP1 upregulation may be linked to the activation of the PI3K signaling pathway, and TCP1 may serve as a novel target for the treatment of OC.

Project description:Macroautophagy/autophagy is indispensable for testosterone synthesis in Leydig cells (LCs), and here we report a negative association between m6A modification and autophagy in LCs during testosterone synthesis. A gradual decrease of METTL14 (methyltransferase like 14) and an increase of ALKBH5 (alkB homolog 5, RNA demethylase) were observed in LCs during their differentiation from stem LCs to adult LCs. These events led to reduced mRNA methylation levels of N6-methyladenosine (m6A) and enhanced autophagy in LCs. Similar regulation of METTL14, ALKBH5, and m6A was also observed in LCs upon treatment with human chorionic gonadotropin (HsCG). Mechanistically, m6A modification promoted translation of PPM1A (protein phosphatase 1A, magnesium dependent, alpha isoform), a negative AMP-activated protein kinase (AMPK) regulator, but decreased expression of CAMKK2 (calcium/calmodulin-dependent protein kinase kinase 2, beta), a positive AMPK regulator, by reducing its RNA stability. Thus, m6A modification resulted in reduced AMPK activity and subsequent autophagy inhibition. We further demonstrated that ALKBH5 upregulation by HsCG was dependent on enhanced binding of the transcriptional factor CEBPB (CCAAT/enhancer binding protein [C/EBP], beta) and the TFEB (transcription factor EB) to its gene promoter. Moreover, HsCG treatment decreased METTL14 by reducing its stability. Collectively, this study highlights a vital role of m6A RNA methylation in the modulation of testosterone synthesis in LCs, providing insight into novel therapeutic strategies by exploiting m6A RNA methylation as targets for treating azoospermatism and oligospermatism patients with reduction in serum testosterone.Abbreviations: 3-MA: 3-methyladenine; ACTB: Actin, beta; ALKBH5: alkB homolog 5, RNA demethylase; AMPK: AMP-activated protein kinase; BafA1: bafilomycin A1; CAMKK2: calcium/calmodulin-dependent protein kinase kinase 2, beta; CEBPB: CCAAT/enhancer-binding protein (C/EBP), beta; ChIP: chromatin immunoprecipitation; FTO: fat mass and obesity associated; HsCG: human chorionic gonadotropin; HSD3B: 3β-hydroxysteroid dehydrogenase; LCs: Leydig cells; m6A: N6-methyladenosine; METTL14: methyltransferase like 14; METTL3: methyltransferase like 3; MTOR: mechanistic target of rapamycin kinase; PPM1A: protein phosphatase 1A, magnesium dependent, alpha isoform; PRKAA: 5'-AMP-activated protein kinase catalytic subunit alpha; SQSTM1: sequestosome 1; STK11/LKB1: serine/threonine kinase 11; TFEB: transcription factor EB; ULK1: unc-51-like kinase 1; WTAP: Wilms tumor 1-associating protein; YTHDF: YTH N6-methyladenosine RNA binding protein.

Project description:Endometrial cancer is the most commonly diagnosed gynecologic malignancy worldwide; yet the tumor microenvironment, especially the fibroblast cells surrounding the cancer cells, is poorly understood. We established four primary cultures of fibroblasts from human endometrial cancer tissues (cancer-associated fibroblasts, CAFs) using antibody-conjugated magnetic bead isolation. These relatively homogenous fibroblast cultures expressed fibroblast markers (CD90, vimentin and alpha-smooth muscle actin) and hormonal (estrogen and progesterone) receptors. Conditioned media collected from CAFs induced a dose-dependent proliferation of both primary cultures and cell lines of endometrial cancer in vitro (175%) when compared to non-treated cells, in contrast to those from normal endometrial fibroblast cell line (51%) (P<0.0001). These effects were not observed in fibroblast culture derived from benign endometrial hyperplasia tissues, indicating the specificity of CAFs in affecting endometrial cancer cell proliferation. To determine the mechanism underlying the differential fibroblast effects, we compared the activation of PI3K/Akt and MAPK/Erk pathways in endometrial cancer cells following treatment with normal fibroblasts- and CAFs-conditioned media. Western blot analysis showed that the expression of both phosphorylated forms of Akt and Erk were significantly down-regulated in normal fibroblasts-treated cells, but were up-regulated/maintained in CAFs-treated cells. Treatment with specific inhibitors LY294002 and U0126 reversed the CAFs-mediated cell proliferation (P<0.0001), suggesting for a role of these pathways in modulating endometrial cancer cell proliferation. Rapamycin, which targets a downstream molecule in PI3K pathway (mTOR), also suppressed CAFs-induced cell proliferation by inducing apoptosis. Cytokine profiling analysis revealed that CAFs secrete higher levels of macrophage chemoattractant protein (MCP)-1, interleukin (IL)-6, IL-8, RANTES and vascular endothelial growth factor (VEGF) than normal fibroblasts. Our data suggests that in contrast to normal fibroblasts, CAFs may exhibit a pro-tumorigenic effect in the progression of endometrial cancer, and PI3K/Akt and MAPK/Erk signaling may represent critical regulators in how endometrial cancer cells respond to their microenvironment.

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M6A mRNA methylation regulates AKT activity to promote the proliferation and tumorigenicity of endometrial cancer.

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m<sup>6</sup>A mRNA methylation regulates AKT activity to promote the proliferation and tumorigenicity of endometrial cancer.

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