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Essential role of MED1 in the transcriptional regulation of ER-dependent oncogenic miRNAs in breast cancer.

ABSTRACT: Mediator complex has been extensively shown to regulate the levels of several protein-coding genes; however, its role in the regulation of miRNAs in humans remains unstudied so far. Here we show that MED1, a Mediator subunit in the Middle module of Mediator complex, is overexpressed in breast cancer and is a negative prognostic factor. The levels of several miRNAs (miR-100-5p, -191-5p, -193b-3p, -205-5p, -326, -422a and -425-5p) were found to be regulated by MED1. MED1 induces miR-191/425 cluster in an estrogen receptor-alpha (ER-?) dependent manner. Occupancy of MED1 on estrogen response elements (EREs) upstream of miR-191/425 cluster is estrogen and ER-?-dependent and ER-?-induced expression of these miRNAs is MED1-dependent. MED1 mediates induction of cell proliferation and migration and the genes associated with it (JUN, FOS, EGFR, VEGF, MMP1, and ERBB4) in breast cancer, which is abrogated when used together with miR-191-inhibition. Additionally, we show that MED1 also regulates the levels of direct miR-191 target genes such as SATB1, CDK6 and BDNF. Overall, the results show that MED1/ER-?/miR-191 axis promotes breast cancer cell proliferation and migration and may serve as a novel target for therapy.

SUBMITTER: Nagpal N

PROVIDER: S-EPMC6081450 | biostudies-literature | 2018 Aug

REPOSITORIES: biostudies-literature

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Essential role of MED1 in the transcriptional regulation of ER-dependent oncogenic miRNAs in breast cancer.

Nagpal Neha N Sharma Shivani S Maji Sourobh S Durante Giorgio G Ferracin Manuela M Thakur Jitendra K JK Kulshreshtha Ritu R

Scientific reports 20180807 1

Mediator complex has been extensively shown to regulate the levels of several protein-coding genes; however, its role in the regulation of miRNAs in humans remains unstudied so far. Here we show that MED1, a Mediator subunit in the Middle module of Mediator complex, is overexpressed in breast cancer and is a negative prognostic factor. The levels of several miRNAs (miR-100-5p, -191-5p, -193b-3p, -205-5p, -326, -422a and -425-5p) were found to be regulated by MED1. MED1 induces miR-191/425 cluste ...[more]

PMID: 30087366

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Project description:BackgroundHeterogeneous nuclear ribonucleoprotein K (HnRNPK) is a nucleic acid-binding protein that regulates diverse biological events. Pathologically, HnRNPK proteins are frequently overexpressed and clinically correlated with poor prognosis in various types of human cancers and are therefore pursued as attractive therapeutic targets for select patients. However, both the transcriptional regulation and degradation of HnRNPK in prostate cancer remain poorly understood.MethodsqRT-PCR was used to detect the expression of HnRNPK mRNA and miRNA; Immunoblots and immunohistochemical assays were used to determine the levels of HnRNPK and other proteins. Flow cytometry was used to investigate cell cycle stage. MTS and clonogenic assays were used to investigate cell proliferation. Immunoprecipitation was used to analyse the interaction between SPOP and HnRNPK. A prostate carcinoma xenograft mouse model was used to detect the in vivo effects of HnRNPK and miRNA.ResultsIn the present study, we noted that HnRNPK emerged as an important player in the carcinogenesis process of prostate cancer. miR-206 and miR-613 suppressed HnRNPK expression by targeting its 3'-UTR in PrCa cell lines in which HnRNPK is overexpressed. To explore the potential biological function, proliferation and colony formation of PrCa cells in vitro and tumor growth in vivo were also dramatically suppressed upon reintroduction of miR-206/miR-613. We have further provided evidence that Cullin 3 SPOP is a novel upstream E3 ubiquitin ligase complex that governs HnRNPK protein stability and oncogenic functions by promoting the degradation of HnRNPK in polyubiquitination-dependent proteolysis in the prostate cancer setting. Moreover, prostate cancer-associated SPOP mutants fail to interact with and promote the destruction of HnRNPK proteins.ConclusionOur findings reveal new posttranscriptional and posttranslational modification mechanisms of HnRNPK regulation via miR-206/miR-613 and SPOP, respectively. More importantly, given the critical oncogenic role of HnRNPK and the high frequency of SPOP mutations in prostate cancer, our results provide a molecular rationale for the clinical investigation of novel strategies to combat prostate cancer based on SPOP genetic status.

Project description:Background/Aims: Mediator complex subunit 1 (MED1) is an important transcriptional co-activator involved in multiple signaling pathways. Previous studies indicated an essential role of MED1 in hair cycling and wound repair through regulating the transcription of mRNAs. Circular RNAs (circRNAs), as a novel class of non-coding RNAs, are involved in various skin biological functions. Our study aimed to investigate the circRNAs expression profile in MED1 epidermal conditional knockout mice (KO), and provide potential candidates as well as the mechanism underlying the circRNAs regulation in both hair follicles and epidermis. Method: Microarray based circRNA expression was determined in MED1 KO mice and wild type mice (WT). The expression level was further confirmed by qRT-PCR. We predicted a possible interaction network of circRNA/microRNA/mRNA by bioinformatics and constructed them with Cytoscape software. Expression of several candidate target mRNAs was verified using qRT-PCR. A TUNEL assay was performed to assess the apoptosis level of MED1 KO and WT skin. Results: Here we identified 109 (34-up, 75-down) distinct circRNAs through microarrays that are differently expressed in MED1 KO mice compared with WT mice (FC > 2 and p-value < 0.05), suggesting a potential role of circRNAs in epidermal regulation. Among these circRNAs, circRNA_004229 was found to decrease significantly after MED1 deletion. The most likely potential targets miRNA for circRNA_004229 include miR-149-5p and miR-207, which possibly further impede the expression of their target mRNA, Tnfrsf19 and Perp, respectively. Apoptosis was suppressed in MED1 KO mice, which implies a potential role of circRNAs in regulating epidermal biological processes including apoptosis. Conclusion: Our study determined the expression profile of circRNAs in MED1 KO skin, and provided hints that circRNA_004229 might be involved in the regulation of keratinocytes in both hair follicles and interfollicular epidermis through a ceRNA mechanism.

Project description:The combination of endocrine therapy and CDK4/6 inhibitors such as palbociclib is an effective and well-tolerated treatment for estrogen receptor-positive (ER+) breast cancer, yet many patients relapse with therapy-resistant disease. Determining the mechanisms underlying endocrine therapy resistance is limited by the lack of ability to fully recapitulate inter- and intratumor heterogeneity in vitro and of availability of tumor samples from women with disease progression or relapse. In this study, multiple cell line models of resistant disease were used for both two-dimensional (2D)- and three-dimensional (3D)-based inhibitor screening. The screens confirmed the previously reported role of pro-proliferative pathways, such as PI3K-AKT-mTOR, in endocrine therapy resistance and additionally identified the transcription-associated cyclin-dependent kinase CDK9 as a common hit in ER+ cell lines and patient-derived organoids modeling endocrine therapy-resistant disease in both the palbociclib-sensitive and palbociclib-resistant settings. The CDK9 inhibitor, AZD4573, currently in clinical trials for hematologic malignancies, acted synergistically with palbociclib in these ER+in vitro 2D and 3D models. In addition, in two independent endocrine- and palbociclib-resistance patient-derived xenografts, treatment with AZD4573 in combination with palbociclib and fulvestrant resulted in tumor regression. Tumor transcriptional profiling identified a set of transcriptional and cell-cycle regulators differentially downregulated only in combination-treated tumors. Together, these findings identify a clinically tractable combination strategy for overcoming resistance to endocrine therapy and CDK4/6 inhibitors in breast cancer and provide insight into the potential mechanism of drug efficacy in targeting treatment-resistant disease.SignificanceTargeting transcription-associated CDK9 synergizes with CDK4/6 inhibitor to drive tumor regression in multiple models of endocrine- and palbociclib-resistant ER+ breast cancer, which could address the challenge of overcoming resistance in patients.

Dataset Information

Essential role of MED1 in the transcriptional regulation of ER-dependent oncogenic miRNAs in breast cancer.

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Essential role of MED1 in the transcriptional regulation of ER-dependent oncogenic miRNAs in breast cancer.

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