MED12 Expression Research Articles

MED12 loss activates endogenous retroelements to sensitise immunotherapy in pancreatic cancer

ObjectivePancreatic ductal adenocarcinoma (PDAC) stands as one of the most lethal cancers, marked by its lethality and limited treatment options, including the utilisation of checkpoint blockade (ICB) immunotherapy. Epigenetic dysregulation...

MED12 and CDK8/19 Modulate Androgen Receptor Activity and Enzalutamide Response in Prostate Cancer.

Prostate cancer progression is driven by androgen receptor (AR) activity, which is a target for therapeutic approaches. Enzalutamide is an AR inhibitor that prolongs the survival of patients with advanced prostate cancer. However, resistance mechanisms arise and impair its efficacy. One of these mechanisms is the expression of AR-V7, a constitutively active AR splice variant. The Mediator complex is a multisubunit protein that modulates gene expression on a genome-wide scale. MED12 and cyclin-dependent kinase (CDK)8, or its paralog CDK19, are components of the kinase module that regulates the proliferation of prostate cancer cells. In this study, we investigated how MED12 and CDK8/19 influence cancer-driven processes in prostate cancer cell lines, focusing on AR activity and the enzalutamide response. We inhibited MED12 expression and CDK8/19 activity in LNCaP (AR+, enzalutamide-sensitive), 22Rv1 (AR-V7+, enzalutamide-resistant), and PC3 (AR-, enzalutamide-insensitive) cells. Both MED12 and CDK8/19 inhibition reduced cell proliferation in all cell lines, and MED12 inhibition reduced proliferation in the respective 3D spheroids. MED12 knockdown significantly inhibited c-Myc protein expression and signaling pathways. In 22Rv1 cells, it consistently inhibited the AR response, prostate-specific antigen (PSA) secretion, AR target genes, and AR-V7 expression. Combined with enzalutamide, MED12 inhibition additively decreased the AR activity in both LNCaP and 22Rv1 cells. CDK8/19 inhibition significantly decreased PSA secretion in LNCaP and 22Rv1 cells and, when combined with enzalutamide, additively reduced proliferation in 22Rv1 cells. Our study revealed that MED12 and CDK8/19 regulate AR activity and that their inhibition may modulate response to enzalutamide in prostate cancer.

Abstract We143: MED12 Dysregulation Disrupts Transcriptional Programs Leading to Dilated Cardiomyopathy

The Mediator complex serves as a bridge to link transcriptional machinery and transcription factors (TFs) to control transcription, but detailed molecular mechanisms of transcriptional regulation by Mediator are not well-understood. Mediator is comprised of the head, middle, and tail submodules that make up the core Mediator, and the transiently associating kinase submodule. The CDK8 kinase submodule acts to regulate Mediator-RNA Polymerase II interaction. MED12 is an essential Mediator component within the kinase submodule and is required for CDK8 kinase activity. In aging models, dysregulated levels of Med12 mRNA are detected in both atria and ventricles by qRT-PCR. MED12 protein levels are also increased in human heart failure biopsies and in the mouse heart after transverse aortic constriction (TAC)-induced heart failure[KB1] . To test the hypothesis that increased levels of MED12 contribute to development of heart failure we generated cardiac-specific Med12 transgenic mice (cTg) by driving increased MED12 expression in cardiomyocytes using the αMHC promoter. Med12 overexpression leads to a progressive decrease in fractional shortening by serial echocardiography. Decreased cardiac function was accompanied by increased cardiac chamber size and dilation, without cardiac fibrosis. RNA sequencing of cardiomyocytes from cTg and control mice revealed that increased levels of MED12 leads to early changes in gene expression programs. Using ingenuity pathway analysis we identified MED12-regulated gene programs including those involved in calcium cycling. MED12 is a transcriptional regulator but does not directly bind DNA. To determine how MED12 regulates gene expression we screened for TFs that interact with MED12. We performed immunoprecipitation of MED12 and by mass spectrometry and determined that MED12 interacts with the TFs MEF2, CREB, and SRF. In follow-up experiments we demonstrated that MED12 interacts with the transcription factor (TF) MEF2 to regulate calcium handling genes. Current studies are focused on determining how MED12 binding coordinates gene expression through multiple TFs in cardiomyocytes. Collectively, our data demonstrate that increased MED12 expression leads to the development of heart failure, in part by dysregulation of gene expression. Therefore, precise regulation of MED12 levels is important to maintain normal cardiac function. [KB1]What about aging?

Mediator kinase module proteins, genetic alterations and expression of super-enhancer regulated genes in colorectal cancer.

Genetic alterations are well characterized as contributors to the pathogenesis of cancers. Epigenetic abnormalities can lead to perturbations of the expression of genes in cancer cells without structural defects. Deregulation of proteins of the transcription machinery may result in perturbations of target genes. Mediator, a multiprotein component of the transcription machinery facilitates the function of RNA polymerase II, which transcribes most human genes. A part of the mediator with kinase activity, called the Mediator kinase module shows genetic alterations in a sub-set of colorectal cancers. Data from publicly available genomic series of colorectal cancer patients were examined to determine alterations of Mediator kinase module component genes, including MED12, MED12L, MED13, MED13L, CDK8, CDK19, and CCNC. The prevalence of alterations in genomically defined colorectal cancer sub-sets was also interrogated. The effect of Mediator kinase module member gene expression on colorectal cancer relapse-free survival was investigated. Mutations in genes of the Mediator kinase module were present in a small percentage of colorectal cancers, ranging between 2 to 10% for MED12 and MED13 and alternative units MED12L and MED13L and below 2% for kinases CDK8 and CDK19 and cyclin C. Amplifications of the CDK8 gene were observed in 3% to 5% of colorectal cancers. The highest prevalence of mutations was observed in MSI cancers and the equivalent CMS1 group, with other genomic groups showing much lower frequency. An association of higher expression of MED12 with inferior relapse-free survival was observed. In contrast, higher expression of cyclin C was associated with improved survival. Colorectal cancer cell lines with CDK8 amplifications displayed sensitivity to several small molecule inhibitors of the KRAS/PI3K pathway but not to BET inhibitors. The Mediator kinase module is deregulated in a sub-set of colorectal cancers with differences observed in genomically defined groups. These variations may result in differences in sensitivity to targeted therapies and may have to be taken into consideration as such therapies are developed.

Novel Clinically Useful Inhibitor of Mediator Complex, RVU120, Relives Differentiation Block in MDS/AML

Background: Myelodysplastic Syndrome (MDS) and Acute Myeloid Leukemias (AML) are characterized by a block in hematopoietic differentiation that can often be traced to aberrant stage-specific transcription. The kinase module of the mediator complex, consisting of CDK8, its paralog CDK19, Cyclin C, MED12, and MED13, have been identified as a molecular switch that regulates gene expression. A variety of cancers have exploited this mechanism to maintain stemness and an undifferentiated state. RVU120 is a first-in-class, specific, and selective inhibitor of CDK8/19, currently in Phase Ib clinical trial in patients with AML or High-Risk MDS (HR-MDS) (NCT04021368). RVU120 has been shown to stimulate erythroid differentiation in transformed CD34+ cord blood (CB) cells and in CD34+ cells from Diamond-Blackfan Anemia (DBA). To evaluate the further therapeutic potential of CDK8/19 inhibition, we confirmed aberrant expression of mediator complex components and erythroid-stimulating activity in a collection of CD34+ primary samples of AML and MDS. Results: We investigated the expression of mediator complex components in a large database of MDS CD34+ marrow samples and age-matched controls. Our findings revealed that MED12, a critical component of the mediator complex, was significantly overexpressed in MDS samples from refractory anemia with excess blasts (RAEB), a higher risk subset of MDS(p=0.018) (Fig A). Furthermore, we observed that this overexpression of MED12 was associated with a higher rate of transformation to AML. To delve deeper, we examined the expression of MED12 in sorted leukemic stem cells from primary AML samples and found significantly elevated levels in Long Term Hematopoietic Stem Cells from AML samples with both normal karyotype and complex karyotypes compared to healthy controls. Next, we conducted an evaluation of the efficacy of the mediator kinase module inhibition in MDS-derived cell line (MDS-L). Inhibition of CDK8/19 with RVU120 led to increased erythroid differentiation in this model which confirmed our observations in transformed CD34+ CB cells in CB cells, these phenotypic changes were correlated with the inhibition of CDK8-dependent phosphorylation marker S726 on STAT5 and a profound increase in the expression of genes involved in erythroid commitment and hemoglobin metabolism. In order to confirm therapeutic potential of CDK8/CDK19 inhibition in MDS and AML, we performed colony-forming assay on primary samples of MDS and AML (n= 15). The treatment with RVU120 resulted in increased erythroid differentiation in a majority of samples, evident from an augmented number of colonies. Detailed analysis indicated changes in the expression of erythroid differentiation markers, including increased CD71 and Glycophorin A expression on colonies, as assessed by FACS analysis (Figure B, shows representative sample). Conclusion Our data highlight that inhibition of the overexpressed mediator complex proteins can alleviate the differentiation blocks seen in MDS/AML. These results provide a compelling rationale for further developing the CDK8/19 inhibitor RVU120 for transfusion- dependent MDS/AML patients.

Abstract P1143: Med12 “Mediates” Transcriptional Regulation In The Heart

The Mediator complex serves as a bridge to link transcriptional machinery and transcription factors (TFs) to control transcription, but the molecular mechanisms of transcriptional regulation by Mediator are not well-understood. Mediator is a large multiprotein complex organized as submodules, known as the head, body, tail, and kinase submodule. The CDK8 kinase submodule is known to regulate Mediator - RNA Polymerase II interaction. MED12 is an essential Mediator component, and within the kinase submodule, is required for CDK8 kinase activity. MED12 protein levels are increased in human heart failure biopsies and in the mouse heart after transverse aortic constriction (TAC)-induced heart failure. To test the hypothesis that increased levels of MED12 contributes to development of heart failure we generated cardiac-specific Med12 transgenic mice (cTg) by driving increased MED12 expression in cardiomyocytes using the αMHC promoter. Med12 overexpression leads to decreased cardiac function, measured as fractional shortening by serial echocardiography. Decreased cardiac function was accompanied by increased cardiac chamber size and dilation, without cardiac fibrosis. RNA sequencing of cardiomyocytes from cTg and control mice revealed that increased levels of MED12 leads to dysregulated calcium handling gene expression. MED12 is a transcriptional regulator, but does not directly bind DNA. To determine how MED12 regulates gene expression we screened for TFs that interact with MED12. We performed immunoprecipitation of MED12 and by mass spectrometry and determined that MED12 interacts with the TFs MEF2, CREB, and SRF. In follow-up experiments we demonstrated that MED12 interacts with the transcription factor (TF) MEF2 to regulate calcium handling genes. Current studies are focused on determining how MED12 binding coordinates gene expression through multiple TFs in cardiomyocytes. Collectively, our data demonstrate that increased MED12 expression leads to the development of heart failure, in part by dysregulation of gene expression. Therefore, precise regulation of MED12 levels is important to maintain normal cardiac function.

MED12 is a Transcriptional Mediator of Cardiac Contractile Function

The Mediator complex plays an essential role in transcription for nearly all genes, and functions by transmitting physiological and developmental signals to RNA polymerase II. The Mediator complex is regulated by a kinase submodule made up of four proteins: MED12, MED13, CDK8, and Cyclin C. MED12 is a pivotal member of this submodule as it is required for CDK8 activity, which regulates Polymerase II‐driven transcription. The objective of this study is to identify the role of MED12 in the heart and determine how it regulates transcription through interactions with transcription factors. MED12 protein levels are increased in human heart failure biopsies, and in the mouse heart after transverse aortic constriction (TAC)‐induced heart failure. Therefore, we hypothesize that increased levels of MED12 contributes to development of heart failure. To test this hypothesis we generated cardiac‐specific MED12 transgenic (cTg) mice by driving increased MED12 expression in cardiomyocytes using the aMHC promoter. We performed serial echocardiography and observed that increased MED12 expression leads to decreased cardiac function, measured as fractional shortening. Decreased cardiac function was accompanied by increased cardiac chamber size and dilation, without evidence of cardiac fibrosis. We performed RNA sequencing on cTg and control cardiomyocytes, to investigate mechanisms of MED12‐induced cardiac dysfunction, and found that increased MED12 levels lead to dysregulated calcium handing gene expression. MED12 is a transcriptional regulator, but does not directly bind DNA. To determine how MED12 regulates expression of calcium handling genes we used cardiomyocytes from our mouse models to screen for transcription factors that interact with MED12. We immunoprecipitated MED12 and identified interacting proteins by mass spectrometry, many of which are regulators of transcription. In follow‐up experiments we demonstrated that MED12 interacts with the transcription factor (TF) MEF2 to regulate calcium handling genes. Preliminary experiments suggest that MED12 also interacts with TFs CREB and SRF. Additional mechanistic studies are ongoing to determine how MED12 coordinates gene expression through multiple transcription factors in cardiomyocytes. Collectively, our data demonstrate that tight regulation of MED12 levels is necessary to maintain normal cardiac function and regulate gene expression in the heart, and increased MED12 expression leads to the development of heart failure.

Cyclin-dependent kinase 8 is an independent prognosticator in uterine leiomyosarcoma

Cyclin-dependent kinase 8 (CDK8) is associated with the transcriptional mediator complex and regulates several transcription factors implicated in cancer. CDK8 expression is a poor prognostic marker in colon and breast cancer by immunohistochemistry. However, somatic mutations in exon 2 of the RNA polymerase II transcriptional mediator subunit MED12 occur in 7–30% of cases of uterine leiomyosarcoma (ULMS), suggesting that these alterations contribute to tumorigenesis. Public genomic mutation data of 80 patients with ULMS were used for MED12 and CDK8 mutation analysis. The expression of MED12, CDK8 and β-catenin was evaluated by immunohistochemistry in our cohort of 60 patients with ULMS, in addition with MED12 mutation status and survival stage. Univariate analysis was performed using the log-rank test, and Cox regression was used to identify independent prognostic factors. Multivariate Cox regression analysis revealed that advanced stage (p < 0.0001) and high CDK8 expression (p = 0.0014) were independent predictors of poor prognosis. MED12 mutation status was not significantly associated with CDK8 expression (p = 0.6873) and DSS (p = 0.8075). In conclusion, our data suggest that CDK8 expression may identify a subset of ULMS patients with a poor prognosis.

MED12 Regulates Smooth Muscle Cell Functions and Participates in the Development of Aortic Dissection

Aortic dissection (AD) is a life-threatening disease with high morbidity and mortality, and effective pharmacotherapeutic remedies for it are lacking. Therefore, AD’s molecular pathogenesis and etiology must be elucidated. The aim of this study was to investigate the possible mechanism of mediator complex subunit 12 (human: MED12, mouse: Med12)involvement in AD. Firstly, we examined the expression of MED12 protein (human: MED12, mouse: Med12) in the aortic tissues of AD patients and AD mice. Subsequently, Med12 gene silencing was accomplished with RNA interference (siRNA). The effects of Med12 on AD and the possible biological mechanisms were investigated based on the proliferation, senescence, phenotypic transformation, and its involved signal pathway of mouse aortic smooth muscle cells (MOVAS), s. The results show that the expression of MED12 in the aortae of AD patients and AD mice was decreased. Moreover, the downregulation of Med12 inhibited the proliferation of MOVAS and promoted senescence. Further research found that Med12, as an inhibitor of the TGFβ1 signaling pathway, reduced the expression of Med12 and enhanced the activity of the TGFβ1 nonclassical signaling pathway, while TGFβ1 inhibited the phenotype transformation and proliferation of MOVAS by inhibiting Med12 synthesis. In conclusion, Med12 affected the phenotype, proliferation, and senescence of MOVAS through the TGFβ signaling pathway. This study provides a potential new target for the prevention and treatment of AD.

Guizhi Fuling Capsule inhibits uterine fibroids growth by modulating Med12-mediated Wnt/β-Catenin signaling pathway

Ethnopharmacological relevanceGuizhi Fuling Capsule (GFC) is a famous traditional Chinese medicine (TCM) formula recorded in Synopsis of the Golden Chamber, which has achieved obvious effects in the treatment of uterine fibroids (UFs). Aim of studyMediator complex subunit 12 (Med12) mutations were closely related to UFs in 85% of fibroid cases. The Wnt/β-Catenin signaling pathway plays an important role in the occurrence and development of UFs. This study aims to explore the pharmacological mechanism of GFC against UFs in which the Med12-mediated Wnt/β-Catenin pathway is involved. Materials and methodsMed12 was silenced in uterine fibroid cells (UFCs) using a lentivirus-based Med12 gene-specific RNA interference (RNAi) strategy. Cell proliferation was performed by CCK-8 assay, cell apoptosis and cell cycle were measured by flow cytometry. The rat model of UFs was established by injecting estradiol benzoate and progesterone. Forty-eight rats were divided into six groups, the low-dose GFC (L-GFC) group, the medium-dose GFC (M-GFC) group and the high-dose GFC (H-GFC) group were intragastrically treated with GFC solution at 0.25 g/kg, 0.50 g/kg and 1.00 g/kg per day for 8 weeks, the positive control (PC) group was administrated with mifepristone (2.70 mg/kg/day), the normal control (NC) group and the model control (MC) group were given equal volume of normal saline once a day for 8 weeks. The histopathological changes of uterine tissues were evaluated by H&E staining. The expression of Med12 in uterine tissues were detected by immunohistochemistry. The protein and mRNA levels of associated genes were evaluated by western bolt and real time-PCR, respectively. Related indicators involved in Wnt/β-Catenin pathway, such as Wnt1, β-Catenin, Cyclin D1, TCF1/TCF7 and C-myc, were compared among different groups. ResultsThe Wnt/β-Catenin signaling pathway was inhibited after Med12 gene was knocked out in UFCs. GFC-containing serum could induce cell apoptosis, make the cell cycle stagnated in G0/G1 phase to inhibiting the proliferation and reduce the expression of Wnt1, β-Catenin, Cyclin D1, TCF1/TCF7, and C-myc in control-shRNA cells, while had no significant effect on Med12-shRNA cells. Compared with the MC group, the weight, endometrial thickness, and pathological structure of the uterus in the GFC treated groups were significantly improved. The expression of Med12, Wnt1, β-Catenin, Cyclin D1, TCF1/TCF7, and C-myc that related to Wnt/β-Catenin pathway in the GFC treated groups were decreased with the increase of dosage administration. ConclusionsGFC inhibited UFs growth, which was directly associated with Med12 modulated Wnt/β-Catenin signaling pathway. This study provided new perspective to understand the therapeutic mechanism of UFs.

Natural Compounds Can Target SHH Signaling To Block MED12 Mutant Breast Cancer Oncogenesis

Current statistics show that breast cancer is the second leading cause of cancer deaths among American women. Therefore, there is a strong need to find improved treatment strategies for women suffering from this lethal disease. Though radiation and chemotherapy have been the frontline choice of treatment for cancer over the past few decades, due to the detrimental side effects, personalized treatment is rapidly rising as a superior treatment method. This is largely due to recent advances in genome-wide DNA sequencing that have allowed for the identification of cancer-related mutational landscapes. Through these studies, it has been found that up to 67% of breast cancer tumors carry a mutation in the Mediator subunit MED12 thus indicating that MED12 likely has a critical tumor suppressive role in breast cancer. Previous results from our lab, and others, have indicated that MED12 plays a critical role in restricting GLI3-dependent SHH signaling. This finding is of particular interest to this study since hyper-activated SHH signaling is known to play a major role in promoting breast cancer oncogenesis. Due to these findings, we hypothesize that mutations in MED12 cause hyper-activated SHH signaling in breast cancer to promote oncogenesis and, furthermore, that natural compounds could provide useful as a personalized treatment for MED12 mutant breast cancer. To study the effect of mutant MED12, a lentivirus carrying an shRNA against MED12 was generated and infected into MCF-7 cells. Since the vast majority of breast cancer-associated MED12 mutations lead to loss of protein function, the knockdown strategy through lentiviral shRNA is assumed to mimic the MED12 mutant setting. Proliferation assays were utilized to confirm that downregulated MED12 plays a role in the increased proliferation of breast cancer cells. Next, quantitative PCR was performed to determine the effect of downregulated MED12 on the expression of genes that are known to be regulated by GLI3. Finally, a screening strategy was employed by using a natural compound library to find potential novel treatment strategies for MED12 mutant breast cancer. Our findings confirmed that downregulation of MED12 increases proliferation of breast cancer cells in vitro thereby providing a strong argument that mutant MED12 would have the same effect. Through quantitative PCR it was confirmed that GLI3 target genes are upregulated in cancer cells when MED12 expression is low. Importantly, our natural compound screen identified several novel therapeutic compounds that specifically target MED12 downregulated breast cancer cells through a mechanism that involves SHH signaling. We therefore uncovered that MED12 mutations promote GLI3-dependent SHH signaling in breast cancer and, importantly, we identified potential novel therapeutic strategies for MED12 mutant breast cancer patients.

MED12 Mutations Promote Castration Resistant Prostate Cancer Through Hyper‐activated SHH Signaling

Prostate cancer is the most common malignancy and the second leading cause of cancer associated deaths among US men. As an androgen-driven disease, prostate cancer is critically dependent upon androgen receptor (AR) signaling. Accordingly, most prostate cancer patients respond very well to androgen deprivation therapy. The current problem lies with the fact that nearly all patients progress to a lethal form of the disease after androgen deprivation therapy known as castration resistant prostate cancer (CRPC). Once patients reach the stage of CRPC, treatment options are frustratingly limited and average survival times range from two to three years only. Thus, there is a compelling need to develop effective treatments for patients suffering from CRPC. Sonic hedgehog (SHH) signaling is a stromal-epithelial interacting pathway that is critical for prostate development and cell growth. SHH signaling is induced by androgen deprivation thereby leading to re-activation of AR target genes to promote prostate cancer cell growth in the absence of androgens. Furthermore, AR interacts with GLI proteins, downstream effectors in the SHH pathway. Therefore, GLI3-dependent SHH signaling in androgen deprived cells could promote re-activation of AR target genes and consequently lead to CRPC. It is currently unknown how GLI3-dependent SHH signaling is induced in CRPC. Several studies have found that MED12, a subunit of Mediator, is commonly mutated in prostate cancer and, interestingly, MED12 has been shown to play a critical role in restricting GLI3-dependent SHH signaling. We therefore hypothesize that MED12 mutations promote GLI3-dependent SHH signaling in prostate cancer to drive progression towards CRPC. We generated stable MED12 knockdown cells, which likely recapitulates a MED12 mutant setting, followed by proliferation and qPCR assays in androgen replete and androgen deprived conditions to test our hypothesis. Our results show that MED12 knockdown promotes prostate cancer growth specifically in an androgen deprived setting and that this hyper-active growth is dependent upon GLI3. Furthermore, we also show that GLI3 target genes are sharply upregulated when MED12 expression is low and androgens are absent. The collective results of our study therefore indicate that prostate cancer patients with MED12 mutations could relapse to CRPC after androgen deprivation therapy by hyperactivating the GLI3 dependent SHH signaling pathway. Importantly, our results thus suggest that therapeutic agents that target the SHH signaling pathway could prove to be beneficial for MED12 mutant prostate cancer patients.

Insights into the regulatory role and clinical relevance of mediator subunit, MED12, in human diseases.

Transcriptional dysregulation is central to many diseases including cancer. Mutation or deregulated expression of proteins involved in transcriptional machinery leads to aberrant gene expression that disturbs intricate cellular processes of division and differentiation. The subunits of the mediator complex are master regulators of stimuli-derived transcription and are essential for transcription by RNA polymerase II. MED12 is a part of the CDK8 kinase module of the mediator complex and is essential for kinase assembly and function. Other than its function in activation of the kinase activity of CDK8 mediator, it also brings about transcription repression or activation, in response to several signalling pathways, a function that is independent of its role as a part of kinase assembly. Accumulating evidence suggests that MED12 controls complex transcription programs that are defining in cell fate determination, differentiation, and carcinogenesis. Mutations or differential expression of MED12 manifest in several human disorders and diseases. For instance, MED12 mutations are the gold standard for the diagnosis of several X-linked intellectual disability syndromes. Further, certain MED12 mutations are categorised as driver mutations in carcinogenesis as well. This is a timely review that provides for the first time a wholesome view on the critical roles and pathways regulated by MED12, its interactions along with the implications of MED12 alterations/mutations in various cancers and nonneoplastic disorders. Based on the preclinical studies, MED12 indeed emerges as an attractive novel therapeutic target for various diseases and intellectual disorders.

MED12 Mutations Play a Pivotal Role in Breast Cancer Oncogenesis

There is a critical need for superior breast cancer treatment since this disease remains the second leading cause of cancer deaths among American women. Though radiation and chemotherapy have been the frontline choice of treatment for cancer over the past few decades, due to the detrimental side effects, personalized treatment is rapidly rising as a superior treatment method. This is largely due to recent advances in genome‐wide DNA sequencing that have allowed for the identification of cancer‐related mutational landscapes. Through these technologies, MED12, a subunit of Mediator protein, has been found to be commonly mutated in a significant number of breast cancer patients. Therefore, uncovering the mechanistic basis behind MED12 mutant breast cancer is critical in order to find a personalized treatment regimen for this subclass of breast cancer. Previous work has shown that MED12 restricts GLI3‐dependent Sonic Hedgehog (SHH) signaling, a pathway that is often hyper activated in advanced breast cancer. We therefore hypothesize that mutated MED12 promotes GLI3‐dependent SHH signaling in breast cancer cells thereby promoting tumorigenesis and that this signaling pathway could be an effective therapeutic target in MED12 mutant breast cancer. To study the effect of mutant MED12, a lentivirus carrying an shRNA against MED12 was generated and infected into MCF‐7 cells. Since the vast majority of breast cancer‐associated MED12 mutations lead to loss of protein function, the knockdown strategy through lentiviral shRNA is assumed to mimic the MED12 mutant setting. Proliferation assays were utilized to confirm that downregulated MED12 plays a role in the increased proliferation of breast cancer cells. Next, quantitative PCR was performed to determine the effect of downregulated MED12 on the expression of genes that are known to be regulated by GLI3. Finally, a screening strategy was employed by using a natural compound library to find potential novel treatment strategies for MED12 mutant breast cancer. Our findings confirmed that downregulation of MED12 increases proliferation of breast cancer cells in vitro thereby providing a strong argument that mutant MED12 would have the same effect. Through quantitative PCR it was confirmed that GLI3 target genes are upregulated in cancer cells when MED12 expression is low. Importantly, our natural compound screen identified several novel therapeutic compounds that specifically target MED12 downregulated breast cancer cells through a mechanism that involves SHH signaling. We therefore uncovered that MED12 mutations promote GLI3‐dependent SHH signaling in breast cancer and we identified potential novel therapeutic strategies for MED12 mutant breast cancer patients.

H19 lncRNA identified as a master regulator of genes that drive uterine leiomyomas

Uterine leiomyomas or fibroids (UFs) are benign tumors characterized by hyperplastic smooth muscle cells and excessive deposition of extracellular matrix (ECM). Afflicting ~80% of women, and symptomatic in 25%, UFs bring tremendous suffering and are an economic burden worldwide; they cause severe pain and bleeding, and are the leading cause of hysterectomy. Yet, UFs are severely understudied with few effective treatment options available; those that are available frequently have significant side effects such as menopausal symptoms. Recently, integrated genome-scale studies have revealed mutations and fibroid subtype-specific expression changes in key driver genes, with MED12 and HMGA2 together contributing to nearly 90% of all UFs, but their regulation of expression is poorly characterized. Here we report that the expression of H19 long noncoding RNA (lncRNA) is aberrantly increased in UFs. Using cell culture and genome-wide transcriptome and methylation profiling analyses, we demonstrate that H19 promotes expression of MED12, HMGA2, and key ECM-remodeling genes via multiple mechanisms including a new class of epigenetic modification by TET3. Our results mark the first example of an evolutionarily conserved lncRNA in pathogenesis of UFs and regulation of TET expression. Given the link between a H19 single-nucleotide polymorphism (SNP) and increased risk and tumor size of UFs, and the existence of multiple fibroid subtypes driven by key pathway genes regulated by H19, we propose a unifying mechanism for pathogenesis of uterine fibroids mediated by H19 and identify a pathway for future exploration of novel target therapies for uterine leiomyomas.

MED12 exerts an emerging role in actin-mediated cytokinesis via LIMK2/cofilin pathway in NSCLC

BackgroundMediator complex subunit 12 (MED12) is an essential hub for transcriptional regulation, in which mutations and overexpression were reported to be associated with several kinds of malignancies. Nevertheless, the role of MED12 in non-small cell lung cancer (NSCLC) remains to be elucidated.MethodsMED12 mutation was detected by Next-generation sequencing. The expression of MED12 in 179 human NSCLC tissue samples and 73 corresponding adjacent normal lung tissue samples was measured by immunohistochemistry (IHC). CRISPR-Cas9 was used to knock out MED12 in PC9 and SPC-A1 cells. MED12 rescued stable cell lines were generated by lentivirus infection. We traced cell division process by live cell imaging. The molecular mechanism of aborted cytokinesis resulted by MED12 knockout was investigated by RNA-seq. Effects of MED12 deletion on the proliferation of NSCLC cells were determined by MTT assay and Colony-formation assay in vitro and xenograft tumor model in nude mouse. Cell senescence was measured by SA-β-gal staining.ResultsIn our study, no MED12 exon mutation was detected in NSCLC samples, whereas we found that MED12 was overexpressed in human NSCLC tissues, which positively correlated with the tumor volume and adversely affected patient survival. Furthermore, knockout MED12 in NSCLC cell lines resulted in cytokinesis failure, displayed a multinuclear phenotype, and disposed to senescence, and become non-viable. Lack of MED12 decreased the proliferative potential of NSCLC cells and limited the tumor growth in vivo. Mechanism investigations revealed that MED12 knockout activated LIMK2, caused aberrant actin cytoskeleton remodeling, and disrupted the abscission of intercellular bridge, which led to the cytokinesis failure. Reconstitution of exogenous MED12 restored actin dynamics, normal cytokinesis and cell proliferation capacity in MED12 knockout cells.ConclusionsThese results revealed a novel role of MED12 as an important regulator for maintaining accurate cytokinesis and survival in NSCLC cells, which may offer a therapeutic strategy to control tumor growth for NSCLC patients especially those highly expressed MED12.

Abstract 5503: Loss of MED12 induces tumor dormancy in human ovarian cancer

Abstract Epithelial ovarian cancer (EOC) is the leading cause of death among all gynecological malignancies due to high rate of disease relapse. Disease relapse in cancer patients after clinical remission are often referred to tumor dormancy. Here we identify MED12 as an important molecular regulator of tumor dormancy. We found that MED12 knock-out could induce dormancy of EOC cells in vitro and in vivo. Mechanistically, microarray analysis showed that MED12 knock-out decreased the expression of EGFR. Restoration of EGFR expression in MED12 knock-out cells restored the proliferation ability. Additionally, we found that MED12 could bind to the promoter of EGFR, and correlation studies showed that MED12 expression positively correlated with the level of EGFR. Clinical data demonstrated that chemotherapy-resistant patients showed a lower expression of MED12. In conclusion, MED12 plays an important role in regulating dormancy of EOC through regulation of EGFR. Citation Format: Xiao-Lin Luo, Cheng-Cheng Deng, Ji-Hong Liu, Li-Wu Fu. Loss of MED12 induces tumor dormancy in human ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5503.

Abstract 4454: MED12 is recurrently mutated in Middle Eastern colorectal cancer

Abstract Colorectal cancer (CRC) is a common cancer and a leading cause of cancer deaths. Previous studies have identified a number of key steps in the evolution of CRC but our knowledge of driver mutations in CRC remains incomplete. Recognizing the potential of studying different human populations to reveal novel insights in disease pathogenesis, we conducted genomic analysis of CRC in Saudi patients. In the discovery phase of the study, we conducted whole-genome sequencing of tumor and corresponding germline DNA in 27 CRC patients. In addition to known driver mutations, we identified three MED12 somatic mutations. In the replication phase, we employed a next-generation sequencing approach to capture and sequence MED12 and other candidate genes in a larger sample of 400 CRC patients and confirmed the enrichment for recurrent MED12 mutations. In order to gain insight into a plausible biological mechanism for the potential role of MED12 mutations in CRC, we studied CRC cell lines that differ substantially in the expression level of MED12, and found the latter to be correlated inversely with TGF-beta signaling and directly with apoptosis in response to chemotherapeutic agents. Importantly, these correlations were replicated when MED12 expression was experimentally manipulated. Our data expand the recently described role of MED12 as a tumor suppressor in other cancers to include CRC, and suggest TGF-beta signaling as a potential mediator of this effect. Citation Format: Abdul K. Siraj, Tariq Masoodi, Rong Bu, Fouad Al-Dayel, Khawla S. Al-Kuraya. MED12 is recurrently mutated in Middle Eastern colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4454. doi:10.1158/1538-7445.AM2017-4454

MED12 is recurrently mutated in Middle Eastern colorectal cancer

ObjectiveColorectal cancer (CRC) is a common cancer and a leading cause of cancer deaths. Previous studies have identified a number of key steps in the evolution of CRC but our...

MED12 protein expression in breast fibroepithelial lesions: correlation with mutation status and oestrogen receptor expression.

Recent reports have identified recurrent MED12 somatic mutations in fibroadenomas and phyllodes tumours. The frequency and type of somatic mutations were noted to be similar to those of uterine leiomyomas. We aimed to investigate protein expression of MED12, correlating it to MED12 mutational status and expression of oestrogen receptors (ER). Immunohistochemistry was performed on a total of 232 fibroepithelial lesions (100 fibroadenomas, 132 phyllodes tumours) diagnosed at the Department of Pathology, Singapore General Hospital using MED12, ERα and ERβ antibodies. Expressions were evaluated in both stroma and epithelium, and correlated with MED12 mutational status. MED12 mutation was significantly associated with high MED12 protein expression (H-score >150) in the stroma (p=0.029), but not in the epithelium. It was not associated with ERα and ERβ protein expression in both stroma and epithelium. MED12 protein expression was significantly correlated with ERα in epithelial (p=0.007) and ERβ in stromal (p=0.049) components. MED12 was not significantly different between fibroadenomas and phyllodes tumours. Epithelial expression of ERα was significantly higher in fibroadenomas (p<0.001) than in phyllodes tumours. Conversely, both epithelial and stromal expression of ERβ was significantly higher in phyllodes tumours (p<0.001). Positive associations observed between MED12 and ERα, ERβ immunohistochemical expression suggest a biological interplay between the proteins. The lack of significant association of MED12 mutation with ER protein expression indicates a need to further explore the functional impact of MED12 mutations on the ER signalling pathway in breast fibroepithelial lesions.