Hormone-induced enhancer assembly requires an optimal level of hormone receptor multivalent interactions.

Transcription factors (TFs) activate enhancers to drive cell-specific gene programs in response to signals, but our understanding of enhancer assembly during signaling events is incomplete. Here, we show that androgen receptor (AR) forms condensates through multivalent interactions mediated by its N-terminal intrinsically disordered region (IDR) to orchestrate enhancer assembly in response to androgen signaling. AR IDR can be substituted by IDRs from selective proteins for AR condensation capacity and its function on enhancers. Expansion of the poly(Q) track within AR IDR results in a higher AR condensation propensity as measured by multiple methods, including live-cell single-molecule microscopy. Either weakening or strengthening AR condensation propensity impairs its heterotypic multivalent interactions with other enhancer components and diminishes its transcriptional activity. Our work reveals the requirement of an optimal level of AR condensation in mediating enhancer assembly and suggests that alteration of the fine-tuned multivalent IDR-IDR interactions might underlie AR-related human pathologies.

A Non-canonical Role of YAP/TEAD Is Required for Activation of Estrogen-Regulated Enhancers in Breast Cancer.

YAP/TEAD are nuclear effectors of the Hippo pathway, regulating organ size and tumorigenesis largely through promoter-associated function. However, their function as enhancer regulators remains poorly understood. Through an in vivo proximity-dependent labeling (BioID) technique, we identified YAP1 and TEAD4 protein as co-regulators of ERα on enhancers. The binding of YAP1/TEAD4 to ERα-bound enhancers is augmented upon E2 stimulation and is required for the induction of E2/ERα target genes and E2-induced oncogenic cell growth. Furthermore, their enhancer binding is a prerequisite for enhancer activation marked by eRNA transcription and for the recruitment of the enhancer activation machinery component MED1. The binding of TEAD4 on active ERE-containing enhancers is independent of its DNA-binding behavior, and instead, occurs through protein-tethering trans-binding. Our data reveal a non-canonical function of YAP1 and TEAD4 as ERα cofactors in regulating cancer growth, highlighting the potential of YAP/TEAD as possible actionable drug targets for ERα+ breast cancer.

miR-137 is a tumor suppressor in endometrial cancer and is repressed by DNA hypermethylation.

Endometrial cancer is the most common gynecological cancer in the United States. We wanted to identify epigenetic aberrations involving microRNAs (miRNAs), whose genes become hypermethylated in endometrial primary tumors. By integrating known miRNA sequences from the miRNA database (miRBase) with DNA methylation data from methyl-CpG-capture sequencing, we identified 111 differentially methylated regions (DMRs) associated with CpG islands (CGIs) and miRNAs. Among them, 22 DMRs related to 29 miRNAs and within 8 kb of CGIs were hypermethylated in endometrial tumors but not in normal endometrium. miR-137 was further validated in additional endometrial primary tumors. Hypermethylation of miR-137 was found in both endometrioid and serous endometrial cancer (P < 0.01), and it led to the loss of miR-137 expression. Treating hypermethylated endometrial cancer cells with epigenetic inhibitors reactivated miR-137. Moreover, genetic overexpression of miR-137 suppressed cancer cell proliferation and colony formation in vitro. When transfected cancer cells were implanted into nude mice, the cells that overexpressed miR-137 grew more slowly and formed smaller tumors (P < 0.05) than vector transfectants. Histologically, xenograft tumors from cancer cells expressing miR-137 were less proliferative (P < 0.05), partly due to inhibition of EZH2 and LSD1 expression (P < 0.01) in both the transfected cancer cells and tumors. Reporter assays indicated that miR-137 targets EZH2 and LSD1. These results suggest that miR-137 is a tumor suppressor that is repressed in endometrial cancer because the promoter of its gene becomes hypermethylated.

Reprimo, a Potential p53-Dependent Tumor Suppressor Gene, Is Frequently Hypermethylated in Estrogen Receptor α-Positive Breast Cancer.

Aberrant DNA methylation is a hallmark of many cancers. Currently, there are four intrinsic molecular subtypes in breast cancer (BC): Luminal A, B, Her2-positive, and triple negative (TNBC). Recently, The Cancer Genome Atlas (TCGA) project has revealed that Luminal subtypes have higher levels of genome-wide methylation that may be a result of Estrogen/Estrogen receptor α (E2/ERα) signaling pathway activation. In this study, we analyze promoter CpG-island (CGIs) of the Reprimo (RPRM) gene in breast cancers (n = 77), cell lines (n = 38), and normal breast tissue (n = 10) using a MBDCap-seq database. Then, a validation cohort (n = 26) was used to confirm the results found in the MBDCap-seq platform. A differential methylation pattern was found between BC and cell lines compared to normal breast tissue. In BC, a higher DNA methylation was observed in tissues that were ERα-positive than in ERα-negative ones; more precisely, subtypes Luminal A compared to TNBC. Also, significant reverse correlation was observed between DNA methylation and RPRM mRNA expression in BC. Our data suggest that ERα expression in BC may affect the DNA methylation of CGIs in the RPRM gene. This approach suggests that DNA methylation status in CGIs of some tumor suppressor genes could be driven by E2 availability, subsequently inducing the activation of the ERα pathway.

Reprimo as a modulator of cell migration and invasion in the MDA-MB-231 breast cancer cell line.

BACKGROUND: Reprimo (RPRM), a highly glycosylated protein, is a new downstream effector of p53-induced cell cycle arrest at the G2/M checkpoint, and a putative tumor suppressor gene frequently silenced via methylation of its promoter region in several malignances. The aim of this study was to characterize the epigenetic inactivation and its biological function in BC cell lines. METHODS: The correlation between RPRM methylation and loss of mRNA expression was assessed in six breast cancer cell lines by methylation specific PCR (MSP), 5'-Aza-2'-deoxycytidine treatment and RT-PCR assays. MDA-MB-231 cells were chosen to investigate the phenotypic effect of RPRM in cell proliferation, cell cycle, cell death, cell migration and invasion. RESULTS: In the cancer methylome system (CMS) (web-based system for visualizing and analyzing genome-wide methylation data of human cancers), the CpG island region of RPRM (1.1 kb) was hypermethylated in breast cancer compared to normal breast tissue; more interesting still was that ERα(+) tumors showed higher methylation intensity than ERα(-). Downregulation of RPRM mRNA by methylation was confirmed in MDA-MB-231 and BT-20 cell lines. In addition, overexpression of RPRM in MDA-MB-231 cells resulted in decreased rates of cell migration, wound healing and invasion in vitro. However, RPRM overexpression did not alter cell viability, phosphatidylserine (PS) translocation or G2/M cell cycle transition. CONCLUSION: Taken together, these data suggest that RPRM is involved in decreased cell migration and invasion in vitro, acting as a potential tumor suppressor gene in the MDA-MB-231 cell line.

DNA methylation screening of primary prostate tumors identifies SRD5A2 and CYP11A1 as candidate markers for assessing risk of biochemical recurrence.

BACKGROUND: Altered DNA methylation in CpG islands of gene promoters has been implicated in prostate cancer (PCa) progression and can be used to predict disease outcome. In this study, we determine whether methylation changes of androgen biosynthesis pathway (ABP)-related genes in patients' plasma cell-free DNA (cfDNA) can serve as prognostic markers for biochemical recurrence (BCR). METHODS: Methyl-binding domain capture sequencing (MBDCap-seq) was used to identify differentially methylated regions (DMRs) in primary tumors of patients who subsequently developed BCR or not, respectively. Methylation pyrosequencing of candidate loci was validated in cfDNA samples of 86 PCa patients taken at and/or post-radical prostatectomy (RP) using univariate and multivariate prediction analyses. RESULTS: Putative DMRs in 13 of 30 ABP-related genes were found between tumors of BCR (n = 12) versus no evidence of disease (NED) (n = 15). In silico analysis of The Cancer Genome Atlas data confirmed increased DNA methylation of two loci-SRD5A2 and CYP11A1, which also correlated with their decreased expression, in tumors with subsequent BCR development. Their aberrant cfDNA methylation was also associated with detectable levels of PSA taken after patients' post-RP. Multivariate analysis of the change in cfDNA methylation at all of CpG sites measured along with patient's treatment history predicted if a patient will develop BCR with 77.5% overall accuracy. CONCLUSIONS: Overall, increased DNA methylation of SRD5A2 and CYP11A1 related to androgen biosynthesis functions may play a role in BCR after patients' RP. The correlation between aberrant cfDNA methylation and detectable PSA in post-RP further suggests their utility as predictive markers for PCa recurrence. .

Chloroquine eliminates cancer stem cells through deregulation of Jak2 and DNMT1.

Triple negative breast cancer (TNBC) is known to contain a high percentage of CD44(+) /CD24(-/low) cancer stem cells (CSCs), corresponding with a poor prognosis despite systemic chemotherapy. Chloroquine (CQ), an antimalarial drug, is a lysotropic reagent which inhibits autophagy. CQ was identified as a potential CSC inhibitor through in silico gene expression signature analysis of the CD44(+) /CD24(-/low) CSC population. Autophagy plays a critical role in adaptation to stress conditions in cancer cells, and is related with drug resistance and CSC maintenance. Thus, the objectives of this study were to examine the potential enhanced efficacy arising from addition of CQ to standard chemotherapy (paclitaxel) in TNBC and to identify the mechanism by which CQ eliminates CSCs in TNBCs. Herein, we report that CQ sensitizes TNBC cells to paclitaxel through inhibition of autophagy and reduces the CD44(+) /CD24(-/low) CSC population in both preclinical and clinical settings. Also, we are the first to report a mechanism by which CQ regulates the CSCs in TNBC through inhibition of the Janus-activated kinase 2 (Jak2)-signal transducer and activator of transcription 3 signaling pathway by reducing the expression of Jak2 and DNA methyltransferase 1.

Integrated analysis identifies a class of androgen-responsive genes regulated by short combinatorial long-range mechanism facilitated by CTCF.

Recently, much attention has been given to elucidate how long-range gene regulation comes into play and how histone modifications and distal transcription factor binding contribute toward this mechanism. Androgen receptor (AR), a key regulator of prostate cancer, has been shown to regulate its target genes via distal enhancers, leading to the hypothesis of global long-range gene regulation. However, despite numerous flows of newly generated data, the precise mechanism with respect to AR-mediated long-range gene regulation is still largely unknown. In this study, we carried out an integrated analysis combining several types of high-throughput data, including genome-wide distribution data of H3K4 di-methylation (H3K4me2), CCCTC binding factor (CTCF), AR and FoxA1 cistrome data as well as androgen-regulated gene expression data. We found that a subset of androgen-responsive genes was significantly enriched near AR/H3K4me2 overlapping regions and FoxA1 binding sites within the same CTCF block. Importantly, genes in this class were enriched in cancer-related pathways and were downregulated in clinical metastatic versus localized prostate cancer. Our results suggest a relatively short combinatorial long-range regulation mechanism facilitated by CTCF blocking. Under such a mechanism, H3K4me2, AR and FoxA1 within the same CTCF block combinatorially regulate a subset of distally located androgen-responsive genes involved in prostate carcinogenesis.

Epigenetic deregulation of the anaplastic lymphoma kinase gene modulates mesenchymal characteristics of oral squamous cell carcinomas.

DNA hypermethylation of promoter CpG islands is associated with epigenetic silencing of tumor suppressor genes in oral squamous cell carcinomas (OSCCs). We used a methyl-CpG-binding domain protein capture method coupled with next-generation sequencing (MBDCap-seq) to survey global DNA methylation patterns in OSCCs with and without nodal metastasis and normal mucosa (total n = 58). Of 1462 differentially methylated CpG islands identified in OSCCs relative to normal controls, MBDCap-seq profiling uncovered 359 loci linked to lymph node metastasis. Interactive network analysis revealed a subset of these loci (n = 23), including the anaplastic lymphoma kinase (ALK) gene, are potential regulators and effectors of invasiveness and metastatic progression. Promoter methylation of ALK was preferentially observed in OSCCs without node metastasis, whereas relatively lower methylation levels were present in metastatic tumors, implicating an active state of ALK transcription in the latter group. The OSCC cell line, SCC4, displayed reduced ALK expression that corresponded to extensive promoter CpG island methylation. SCC4 treatment with demethylating agents induced ALK expression and increased invasion and migration characteristics. Inhibition of ALK activity in OSCC cells with high ALK expression (CAL27, HSC3 and SCC25), decreased cell growth and resulted in changes in invasive potential and mesenchymal marker expression that were cell-line dependent. Although ALK is susceptible to epigenetic silencing during oral tumorigenesis, overwriting this default state may be necessary for modulating invasive processes involved in nodal metastases. Given the complex response of OSCC cells to ALK inhibition, future studies are required to assess the feasibility of targeting ALK to treat invasive OSCCs.

Single-cell analysis of circulating tumor cells identifies cumulative expression patterns of EMT-related genes in metastatic prostate cancer.

BACKGROUND: Prostate tumors shed circulating tumor cells (CTCs) into the blood stream. Increased evidence shows that CTCs are often present in metastatic prostate cancer and can be alternative sources for disease profiling and prognostication. Here we postulate that CTCs expressing genes related to epithelial-mesenchymal transition (EMT) are strong predictors of metastatic prostate cancer. METHODS: A microfiltration system was used to trap CTCs from peripheral blood based on size selection of large epithelial-like cells without CD45 leukocyte marker. These cells individually retrieved with a micromanipulator device were assessed for cell membrane physical properties using atomic force microscopy. Additionally, 38 CTCs from eight prostate cancer patients were used to determine expression profiles of 84 EMT-related and reference genes using a microfluidics-based PCR system. RESULTS: Increased cell elasticity and membrane smoothness were found in CTCs compared to noncancerous cells, highlighting their potential invasiveness and mobility in the peripheral circulation. Despite heterogeneous expression patterns of individual CTCs, genes that promote mesenchymal transitioning into a more malignant state, including IGF1, IGF2, EGFR, FOXP3, and TGFB3, were commonly observed in these cells. An additional subset of EMT-related genes (e.g., PTPRN2, ALDH1, ESR2, and WNT5A) were expressed in CTCs of castration-resistant cancer, but less frequently in castration-sensitive cancer. CONCLUSIONS: The study suggests that an incremental expression of EMT-related genes in CTCs is associated with metastatic castration-resistant cancer. Although CTCs represent a group of highly heterogeneous cells, their unique EMT-related gene signatures provide a new opportunity for personalized treatments with targeted inhibitors in advanced prostate cancer patients.

Phagocytosis-initiated tumor hybrid cells acquire a c-Myc-mediated quasi-polarization state for immunoevasion and distant dissemination.

While macrophage phagocytosis is an immune defense mechanism against invading cellular organisms, cancer cells expressing the CD47 ligand send forward signals to repel this engulfment. Here we report that the reverse signaling using CD47 as a receptor additionally enhances a pro-survival function of prostate cancer cells under phagocytic attack. Although low CD47-expressing cancer cells still allow phagocytosis, the reverse signaling delays the process, leading to incomplete digestion of the entrapped cells and subsequent tumor hybrid cell (THC) formation. Viable THCs acquire c-Myc from parental cancer cells to upregulate both M1- and M2-like macrophage polarization genes. Consequently, THCs imitating dual macrophage features can confound immunosurveillance, gaining survival advantage in the host. Furthermore, these cells intrinsically express low levels of androgen receptor and its targets, resembling an adenocarcinoma-immune subtype of metastatic castration-resistant prostate cancer. Therefore, phagocytosis-generated THCs may represent a potential target for treating the disease.

Integrative genome-wide chromatin signature analysis using finite mixture models.

Regulation of gene expression has been shown to involve not only the binding of transcription factor at target gene promoters but also the characterization of histone around which DNA is wrapped around. Some histone modification, for example di-methylated histone H3 at lysine 4 (H3K4me2), has been shown to bind to promoters and activate target genes. However, no clear pattern has been shown to predict human promoters. This paper proposed a novel quantitative approach to characterize patterns of promoter regions and predict novel and alternative promoters. We utilized high-throughput data generated using chromatin immunoprecipitation methods followed by massively parallel sequencing (ChIP-seq) technology on RNA Polymerase II (Pol-II) and H3K4me2. Common patterns of promoter regions are modeled using a mixture model involving double-exponential and uniform distributions. The fitted model obtained were then used to search for regions displaying similar patterns over the entire genome to find novel and alternative promoters. Regions with high correlations with the common patterns are identified as putative novel promoters. We used this proposed algorithm, RNA-seq data and several transcripts databases to find alternative promoters in MCF7 (normal breast cancer) cell line. We found 7,235 high-confidence regions that display the identified promoter patterns. Of these, 4,167 regions (58%) can be mapped to RefSeq regions. 2,444 regions are in a gene body or overlap with transcripts (non-coding RNAs, ESTs, and transcripts that are predicted by RNA-seq data). Some of these maybe potential alternative promoters. We also found 193 regions that map to enhancer regions (represented by androgen and estrogen receptor binding sites) and other regulatory regions such as CTCF (CCCTC binding factor) and CpG island. Around 5% (431 regions) of these correlated regions do not overlap with any transcripts or regulatory regions suggesting that these might be potential new promoters or markers for other annotation which are currently undiscovered.

Downregulation of the tumor-suppressor miR-16 via progestin-mediated oncogenic signaling contributes to breast cancer development.

INTRODUCTION: Experimental and clinical evidence points to a critical role of progesterone and the nuclear progesterone receptor (PR) in controlling mammary gland tumorigenesis. However, the molecular mechanisms of progesterone action in breast cancer still remain elusive. On the other hand, micro RNAs (miRNAs) are short ribonucleic acids which have also been found to play a pivotal role in cancer pathogenesis. The role of miRNA in progestin-induced breast cancer is poorly explored. In this study we explored progestin modulation of miRNA expression in mammary tumorigenesis. METHODS: We performed a genome-wide study to explore progestin-mediated regulation of miRNA expression in breast cancer. miR-16 expression was studied by RT-qPCR in cancer cell lines with silenced PR, signal transducer and activator of transcription 3 (Stat3) or c-Myc, treated or not with progestins. Breast cancer cells were transfected with the precursor of miR-16 and proliferation assays, Western blots or in vivo experiments were performed. Target genes of miR-16 were searched through a bioinformatical approach, and the study was focused on cyclin E. Reporter gene assays were performed to confirm that cyclin E 3'UTR is a direct target of miR-16. RESULTS: We found that nine miRNAs were upregulated and seven were downregulated by progestin in mammary tumor cells. miR-16, whose function as a tumor suppressor in leukemia has already been shown, was identified as one of the downregulated miRNAs in murine and human breast cancer cells. Progestin induced a decrease in miR-16 levels via the classical PR and through a hierarchical interplay between Stat3 and the oncogenic transcription factor c-Myc. A search for miR-16 targets showed that the CCNE1 gene, encoding the cell cycle regulator cyclin E, contains conserved putative miR-16 target sites in its mRNA 3' UTR region. We found that, similar to the molecular mechanism underlying progestin-modulated miR-16 expression, Stat3 and c-Myc participated in the induction of cyclin E expression by progestin. Moreover, overexpression of miR-16 abrogated the ability of progestin to induce cyclin E upregulation, revealing that cyclin E is a novel target of miR-16 in breast cancer. Overexpression of miR-16 also inhibited progestin-induced breast tumor growth in vitro and in vivo, demonstrating for the first time, a role for miR-16 as a tumor suppressor in mammary tumorigenesis. We also found that the ErbB ligand heregulin (HRG) downregulated the expression of miR-16, which then participates in the proliferative activity of HRG in breast tumor cells. CONCLUSIONS: In this study, we reveal the first progestin-regulated miRNA expression profile and identify a novel role for miR-16 as a tumor suppressor in progestin- and growth factor-induced growth in breast cancer.

GLI3 Is Stabilized by SPOP Mutations and Promotes Castration Resistance via Functional Cooperation with Androgen Receptor in Prostate Cancer.

Although the Sonic hedgehog (SHH) signaling pathway has been implicated in promoting malignant phenotypes of prostate cancer, details on how it is activated and exerts its oncogenic role during prostate cancer development and progression is less clear. Here, we show that GLI3, a key SHH pathway effector, is transcriptionally upregulated during androgen deprivation and posttranslationally stabilized in prostate cancer cells by mutation of speckle-type POZ protein (SPOP). GLI3 is a substrate of SPOP-mediated proteasomal degradation in prostate cancer cells and prostate cancer driver mutations in SPOP abrogate GLI3 degradation. Functionally, GLI3 is necessary and sufficient for the growth and migration of androgen receptor (AR)-positive prostate cancer cells, particularly under androgen-depleted conditions. Importantly, we demonstrate that GLI3 physically interacts and functionally cooperates with AR to enrich an AR-dependent gene expression program leading to castration-resistant growth of xenografted prostate tumors. Finally, we identify an AR/GLI3 coregulated gene signature that is highly correlated with castration-resistant metastatic prostate cancer and predictive of disease recurrence. Together, these findings reveal that hyperactivated GLI3 promotes castration-resistant growth of prostate cancer and provide a rationale for therapeutic targeting of GLI3 in patients with castration-resistant prostate cancer (CRPC). IMPLICATIONS: We describe two clinically relevant mechanisms leading to hyperactivated GLI3 signaling and enhanced AR/GLI3 cross-talk, suggesting that GLI3-specific inhibitors might prove effective to block prostate cancer development or delay CRPC.

Cancer epigenetics: a perspective on the role of DNA methylation in acquired endocrine resistance.

Epigenetic mechanisms, including DNA methylation, are responsible for determining and maintaining cell fate, stably differentiating the various tissues in our bodies. Increasing evidence shows that DNA methylation plays a significant role in cancer, from the silencing of tumor suppressors to the activation of oncogenes and the promotion of metastasis. Recent studies also suggest a role for DNA methylation in drug resistance. This perspective article discusses how DNA methylation may contribute to the development of acquired endocrine resistance, with a focus on breast cancer. In addition, we discuss DNA methylome profiling and how recent developments in this field are shedding new light on the role of epigenetics in endocrine resistance. Hormone ablation is the therapy of choice for hormone-sensitive breast tumors, yet as many as 40% of patients inevitably relapse, and these hormone refractory tumors often have a poor prognosis. Epigenetic studies could provide DNA methylation biomarkers to predict and diagnose acquired resistance in response to treatment. Elucidation of epigenetic mechanisms may also lead to the development of new treatments that specifically target epigenetic abnormalities or vulnerabilities in cancer cells. Expectations must be tempered by the fact that epigenetic mechanisms of endocrine resistance remain poorly understood, and further study is required to better understand how altering epigenetic pathways with therapeutics can promote or inhibit endocrine resistance in different contexts. Going forward, DNA methylome profiling will become increasingly central to epigenetic research, heralding a network-based approach to epigenetics that promises to advance our understanding of the etiology of cancer in ways not previously possible.

Aberrant DNA methylation profile and frequent methylation of KLK10 and OXGR1 genes in hepatocellular carcinoma.

Investigating aberrant DNA methylation in the cancer genome may identify genes that play an important role in tumor progression. In this study, we combined differential methylation hybridization and a CpG microarray platform to characterize methylation profiles and identify novel candidate genes associated with hepatocellular carcinoma (HCC). The genomic DNA of 21 paired adjacent normal and HCC samples was used, and results were analyzed by hierarchical clustering. Twenty-seven hypermethylated candidates and 38 hypomethylated candidates were obtained. Six candidate genes from the hypermethylated group were validated by combined bisulfite restriction analysis; two genes, human kallikrein 10 gene (KLK10) and oxoglutarate (alpha-ketoglutarate) receptor 1 gene (OXGR1), were further analyzed by bisulfite sequencing. The DNA hypermethylation status of KLK10 and OXGR1 were subsequently examined in HCC cell lines and clinical samples using methylation-specific PCR. In 49 HCC samples, 46 (94%) showed that at least one of these two genes was highly methylated. Moreover, KLK10 and OXGR1 mRNA levels were inversely correlated (r = -0.435 and -0.497, P < 0.05) with DNA methylation as examined in paired adjacent normal and tumor samples. Statistical analyses further indicated that KLK10 hypermethylation was significantly associated with cirrhosis (P = 0.042) and HCV infection (P = 0.017) as well as inversely associated with HBV infection (P = 0.023). Furthermore, restoration of KLK10 and OXGR1 expression reduced the ability of anchorage-independent growth, and sensitized HCC cells to doxorubicin- or 5-fluorouracil-induced cytotoxicity. Our results suggest that the hypermethylated KLK10 and OXGR1 are frequent in HCC and may be useful as markers for clinical application.

Menin and Menin-Associated Proteins Coregulate Cancer Energy Metabolism.

The interplay between glycolysis and mitochondrial oxidative phosphorylation (OXPHOS) is central to maintain energy homeostasis. It remains to be determined whether there is a mechanism governing metabolic fluxes based on substrate availability in microenvironments. Here we show that menin is a key transcription factor regulating the expression of OXPHOS and glycolytic genes in cancer cells and primary tumors with poor prognosis. A group of menin-associated proteins (MAPs), including KMT2A, MED12, WAPL, and GATA3, is found to restrain menin's full function in this transcription regulation. shRNA knockdowns of menin and MAPs result in reduced ATP production with proportional alterations of cellular energy generated through glycolysis and OXPHOS. When shRNA knockdown cells are exposed to metabolic stress, the dual functionality can clearly be distinguished among these metabolic regulators. A MAP can negatively counteract the regulatory mode of menin for OXPHOS while the same protein positively influences glycolysis. A close-proximity interaction between menin and MAPs allows transcriptional regulation for metabolic adjustment. This coordinate regulation by menin and MAPs is necessary for cells to rapidly adapt to fluctuating microenvironments and to maintain essential metabolic functions.

Enhancer reprogramming driven by high-order assemblies of transcription factors promotes phenotypic plasticity and breast cancer endocrine resistance.

Acquired therapy resistance is a major problem for anticancer treatment, yet the underlying molecular mechanisms remain unclear. Using an established breast cancer cellular model, we show that endocrine resistance is associated with enhanced phenotypic plasticity, indicated by a general downregulation of luminal/epithelial differentiation markers and upregulation of basal/mesenchymal invasive markers. Consistently, similar gene expression changes are found in clinical breast tumours and patient-derived xenograft samples that are resistant to endocrine therapies. Mechanistically, the differential interactions between oestrogen receptor α and other oncogenic transcription factors, exemplified by GATA3 and AP1, drive global enhancer gain/loss reprogramming, profoundly altering breast cancer transcriptional programs. Our functional studies in multiple culture and xenograft models reveal a coordinated role of GATA3 and AP1 in re-organizing enhancer landscapes and regulating cancer phenotypes. Collectively, our study suggests that differential high-order assemblies of transcription factors on enhancers trigger genome-wide enhancer reprogramming, resulting in transcriptional transitions that promote tumour phenotypic plasticity and therapy resistance.

Differential methylation hybridization array of endometrial cancers reveals two novel cancer-specific methylation markers.

PURPOSE: To identify novel endometrial cancer-specific methylation markers and to determine their association with clinicopathologic variables and survival outcomes. EXPERIMENTAL DESIGN: Differential methylation hybridization analysis (DMH) was done for 20 endometrioid endometrial cancers using normal endometrial DNA as a reference control. Combined bisulfite restriction analysis (COBRA) was used to verify methylation of sequences identified by DMH. Bisulfite sequencing was undertaken to further define CpG island methylation and to confirm the reliability of the COBRA. The methylation status of newly identified markers and the MLH1 promoter was evaluated by COBRA in a large series of endometrioid (n=361) and non-endometrioid uterine cancers (n=23). RESULTS: DMH and COBRA identified two CpG islands methylated in tumors but not in normal DNAs: SESN3 (PY2B4) and TITF1 (SC77F6/154). Bisulfite sequencing showed dense methylation of the CpG islands and confirmed the COBRA assays. SESN3 and TITF1 were methylated in 20% and 70% of endometrioid tumors, respectively. MLH1 methylation was seen in 28% of the tumors. TITF1 and SESN3 methylation was highly associated with MLH1 methylation (P<0.0001). SESN3 and TITF1 were methylated in endometrioid and non-endometrioid tumors, whereas MLH1 methylation was restricted to endometrioid tumors. Methylation at these markers was not associated with survival outcomes. CONCLUSIONS: The 5' CpG islands for SESN3 and TITF1 are novel cancer-specific methylation markers. Methylation at these loci is strongly associated with aberrant MLH1 methylation in endometrial cancers. SESN3, TITF1 and MLH1 methylation did not predict overall survival or disease-free survival in this large cohort of patients with endometrioid endometrial cancer.

Cross-talk among writers, readers, and erasers of m6A regulates cancer growth and progression.

The importance of RNA methylation in biological processes is an emerging focus of investigation. We report that altering m6A levels by silencing either N 6-adenosine methyltransferase METTL14 (methyltransferase-like 14) or demethylase ALKBH5 (ALKB homolog 5) inhibits cancer growth and invasion. METTL14/ALKBH5 mediate their protumorigenic function by regulating m6A levels of key epithelial-mesenchymal transition and angiogenesis-associated transcripts, including transforming growth factor-ß signaling pathway genes. Using MeRIP-seq (methylated RNA immunoprecipitation sequencing) analysis and functional studies, we find that these target genes are particularly sensitive to changes in m6A modifications, as altered m6A status leads to aberrant expression of these genes, resulting in inappropriate cell cycle progression and evasion of apoptosis. Our results reveal that METTL14 and ALKBH5 determine the m6A status of target genes by controlling each other's expression and by inhibiting m6A reader YTHDF3 (YTH N 6-methyladenosine RNA binding protein 3), which blocks RNA demethylase activity. Furthermore, we show that ALKBH5/METTL14 constitute a positive feedback loop with RNA stability factor HuR to regulate the stability of target transcripts. We discover that hypoxia alters the level/activity of writers, erasers, and readers, leading to decreased m6A and consequently increased expression of target transcripts in cancer cells. This study unveils a previously undefined role for m6A in cancer and shows that the collaboration among writers-erasers-readers sets up the m6A threshold to ensure the stability of progrowth/proliferation-specific genes, and protumorigenic stimulus, such as hypoxia, perturbs that m6A threshold, leading to uncontrolled expression/activity of those genes, resulting in tumor growth, angiogenesis, and progression.