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Direct routes to liver-like cells Two groups report new approaches that could lead to the generation of hepatocyte-like cells for liver engineering and regenerative medicine. Lijian Hui and colleagues use a combination of Gata4, Hnf1a and Foxa3 overexpression and p19Arf inactivation to convert mouse fibroblasts directly into induced hepatic (iHep) cells that have gene-expression profiles close to that of mature hepatocytes. Sayaka Sekiya and Atsushi Suzuki identify three combinations of two transcription factors, comprising Hnf4a plus Foxa1, Foxa2 or Foxa3, that can convert mouse embryonic and adult fibroblasts directly into functional iHep cells. Both groups show that when iHep cells are transplanted into mice with a gene deficiency that models liver injury, the cells are able to repopulate the livers and restore their function. The location and timing of cellular differentiation must be stringently controlled for proper organ formation. Normally, hepatocytes differentiate from hepatic progenitor cells to form the liver during development 1 , 2 . However, previous studies have shown that the hepatic program can also be activated in non-hepatic lineage cells after exposure to particular stimuli or fusion with hepatocytes 3 , 4 , 5 , 6 , 7 , 8 , 9 . These unexpected findings suggest that factors critical to hepatocyte differentiation exist and become activated to induce hepatocyte-specific properties in different cell types. Here, by screening the effects of twelve candidate factors, we identify three specific combinations of two transcription factors, comprising Hnf4α plus Foxa1, Foxa2 or Foxa3, that can convert mouse embryonic and adult fibroblasts into cells that closely resemble hepatocytes in vitro . The induced hepatocyte-like (iHep) cells have multiple hepatocyte-specific features and reconstitute damaged hepatic tissues after transplantation. The generation of iHep cells may provide insights into the molecular nature of hepatocyte differentiation and potential therapies for liver diseases.

Abtract Forkhead box A1 (FOXA1) is a pioneer transcription factor that is essential for the normal development of several endoderm-derived organs, including the prostate gland 1 , 2 . FOXA1 is frequently mutated in hormone-receptor-driven prostate, breast, bladder and salivary-gland tumours 3 – 8 . However, it is unclear how FOXA1 alterations affect the development of cancer, and FOXA1 has previously been ascribed both tumour-suppressive 9 – 11 and oncogenic 12 – 14 roles. Here we assemble an aggregate cohort of 1,546 prostate cancers and show that FOXA1 alterations fall into three structural classes that diverge in clinical incidence and genetic co-alteration profiles, with a collective prevalence of 35%. Class-1 activating mutations originate in early prostate cancer without alterations in ETS or SPOP, selectively recur within the wing-2 region of the DNA-binding forkhead domain, enable enhanced chromatin mobility and binding frequency, and strongly transactivate a luminal androgen-receptor program of prostate oncogenesis. By contrast, class-2 activating mutations are acquired in metastatic prostate cancers, truncate the C-terminal domain of FOXA1, enable dominant chromatin binding by increasing DNA affinity and—through TLE3 inactivation—promote metastasis driven by the WNT pathway. Finally, class-3 genomic rearrangements are enriched in metastatic prostate cancers, consist of duplications and translocations within the FOXA1 locus, and structurally reposition a conserved regulatory element—herein denoted FOXA1 mastermind (FOXMIND)—to drive overexpression of FOXA1 or other oncogenes. Our study reaffirms the central role of FOXA1 in mediating oncogenesis driven by the androgen receptor, and provides mechanistic insights into how the classes of FOXA1 alteration promote the initiation and/or metastatic progression of prostate cancer. These results have direct implications for understanding the pathobiology of other hormone-receptor-driven cancers and rationalize the co-targeting of FOXA1 activity in therapeutic strategies. Comprehensive genomic analyses and mechanistic studies uncover three structural, functional and clinical classes of activating FOXA1 mutations and locus rearrangements in prostate cancer.

Genomic analysis of tumours has led to the identification of hundreds of cancer genes on the basis of the presence of mutations in protein-coding regions. By contrast, much less is known about cancer-causing mutations in non-coding regions. Here we perform deep sequencing in 360 primary breast cancers and develop computational methods to identify significantly mutated promoters. Clear signals are found in the promoters of three genes. FOXA1 , a known driver of hormone-receptor positive breast cancer, harbours a mutational hotspot in its promoter leading to overexpression through increased E2F binding. RMRP and NEAT1 , two non-coding RNA genes, carry mutations that affect protein binding to their promoters and alter expression levels. Our study shows that promoter regions harbour recurrent mutations in cancer with functional consequences and that the mutations occur at similar frequencies as in coding regions. Power analyses indicate that more such regions remain to be discovered through deep sequencing of adequately sized cohorts of patients. High-depth sequencing of targeted regions in primary breast cancer identifies mutated promoter elements with recurrent mutations at specific and/or nearby bases, suggesting selection of certain non-coding events. Non-coding driver mutations for breast cancer Many cancer genomic studies have characterized the landscape of driver mutations in protein-coding regions, but there has been limited exploration of non-coding regions. Gad Getz and colleagues searched for significantly mutated regulatory regions with high-depth sequencing of targeted regions in 360 primary breast cancers. They identified significantly mutated promoter elements associated with nine genes, and found recurrent mutations at specific and/or nearby bases, suggesting targeting of a specific element. For three of these genes, FOXA1 , RMRP and NEAT1 , they show that these recurrent promoter-proximal mutations influence gene expression and protein binding affinity. The authors suggest that further deep sequencing studies in larger cohorts could identify more as yet undiscovered promoter regions.

FoxA genomic surveillance mechanism Transcriptional enhancers orchestrate cell-type-specific gene expression programs, but how they convey signal-activated transcriptional responses remains poorly understood. Here, the cell-lineage-specific transcription factor FoxA1 is shown to both facilitate and restrict the androgen receptor to act on distinct classes of enhancers. Lowered levels of FoxA1, such as those found in prostate cancer, can reprogram the hormonal response by causing a switch in androgen receptor binding to a set of pre-established, functional enhancers that are marked by enhancer-derived non-coding RNAs (eRNAs). This work points to the existence of a large repository of active enhancers that can be tuned to allow alternative gene expression programs in normal cell development and in disease progression. Mammalian genomes are populated with thousands of transcriptional enhancers that orchestrate cell-type-specific gene expression programs 1 , 2 , 3 , 4 , but how those enhancers are exploited to institute alternative, signal-dependent transcriptional responses remains poorly understood. Here we present evidence that cell-lineage-specific factors, such as FoxA1, can simultaneously facilitate and restrict key regulated transcription factors, exemplified by the androgen receptor (AR), to act on structurally and functionally distinct classes of enhancer. Consequently, FoxA1 downregulation, an unfavourable prognostic sign in certain advanced prostate tumours, triggers dramatic reprogramming of the hormonal response by causing a massive switch in AR binding to a distinct cohort of pre-established enhancers. These enhancers are functional, as evidenced by the production of enhancer-templated non-coding RNA (eRNA 5 ) based on global nuclear run-on sequencing (GRO-seq) analysis 6 , with a unique class apparently requiring no nucleosome remodelling to induce specific enhancer–promoter looping and gene activation. GRO-seq data also suggest that liganded AR induces both transcription initiation and elongation. Together, these findings reveal a large repository of active enhancers that can be dynamically tuned to elicit alternative gene expression programs, which may underlie many sequential gene expression events in development, cell differentiation and disease progression.

Prostate cancer (PC) progressed to castration resistance (CRPC) is a fatal disease. CRPC tumors develop resistance to new-generation antiandrogen enzalutamide through lineage plasticity, characterized by epithelial-mesenchymal transition (EMT) and a basal-like phenotype. FOXA1 is a transcription factor essential for epithelial lineage differentiation. Here, we demonstrate that FOXA1 loss leads to remarkable upregulation of transforming growth factor beta 3 (TGFB3), which encodes a ligand of the TGF-β pathway. Mechanistically, this is due to genomic occupancy of FOXA1 on an upstream enhancer of the TGFB3 gene to directly inhibit its transcription. Functionally, FOXA1 downregulation induces TGF-β signaling, EMT, and cell motility, which is effectively blocked by the TGF-β receptor I inhibitor galunisertib (LY2157299). Tissue microarray analysis confirmed reduced levels of FOXA1 protein and a concordant increase in TGF-β signaling, indicated by SMAD2 phosphorylation, in CRPC as compared with primary tumors. Importantly, combinatorial LY2157299 treatment sensitized PC cells to enzalutamide, leading to synergistic effects in inhibiting cell invasion in vitro and xenograft CRPC tumor growth and metastasis in vivo. Therefore, our study establishes FOXA1 as an important regulator of lineage plasticity mediated in part by TGF-β signaling, and supports a novel therapeutic strategy to control lineage switching and potentially extend clinical response to antiandrogen therapies.

Lineage plasticity, the ability of a cell to alter its identity, is an increasingly common mechanism of adaptive resistance to targeted therapy in cancer. An archetypal example is the development of neuroendocrine prostate cancer (NEPC) after treatment of prostate adenocarcinoma (PRAD) with inhibitors of androgen signaling. NEPC is an aggressive variant of prostate cancer that aberrantly expresses genes characteristic of neuroendocrine (NE) tissues and no longer depends on androgens. Here, we investigate the epigenomic basis of this resistance mechanism by profiling histone modifications in NEPC and PRAD patient-derived xenografts (PDXs) using chromatin immunoprecipitation and sequencing (ChIP-seq). We identify a vast network of cis -regulatory elements ( N ~15,000) that are recurrently activated in NEPC. The FOXA1 transcription factor (TF), which pioneers androgen receptor (AR) chromatin binding in the prostate epithelium, is reprogrammed to NE-specific regulatory elements in NEPC. Despite loss of dependence upon AR, NEPC maintains FOXA1 expression and requires FOXA1 for proliferation and expression of NE lineage-defining genes. Ectopic expression of the NE lineage TFs ASCL1 and NKX2-1 in PRAD cells reprograms FOXA1 to bind to NE regulatory elements and induces enhancer activity as evidenced by histone modifications at these sites. Our data establish the importance of FOXA1 in NEPC and provide a principled approach to identifying cancer dependencies through epigenomic profiling. The molecular processes that lead to neuroendocrine prostate cancer after treating prostate adenocarcinoma (PRAD) are not well understood. Here the authors show that regulation by FOXA1 and changes in the epigenomic profile drive the transition from PRAD to a neuroendocrine phenotype.

Mutations in ARID1A , a subunit of the SWI/SNF chromatin remodeling complex, are the most common alterations of the SWI/SNF complex in estrogen-receptor-positive (ER + ) breast cancer. We identify that ARID1A inactivating mutations are present at a high frequency in advanced endocrine-resistant ER + breast cancer. An epigenome CRISPR–CAS9 knockout (KO) screen identifies ARID1A as the top candidate whose loss determines resistance to the ER degrader fulvestrant. ARID1A inactivation in cells and in patients leads to resistance to ER degraders by facilitating a switch from ER-dependent luminal cells to ER-independent basal-like cells. Cellular plasticity is mediated by loss of ARID1A-dependent SWI/SNF complex targeting to genomic sites of the luminal lineage-determining transcription factors including ER, forkhead box protein A1 (FOXA1) and GATA-binding factor 3 (GATA3). ARID1A also regulates genome-wide ER–FOXA1 chromatin interactions and ER-dependent transcription. Altogether, we uncover a critical role for ARID1A in maintaining luminal cell identity and endocrine therapeutic response in ER + breast cancer. A CRISPR–CAS9 screen, analysis of patient data, and functional in vivo and in vitro experiments identify a critical role for ARID1A in determining breast luminal cell identity and endocrine therapeutic response in estrogen-receptor-positive breast cancer.

Mutations in the transcription factor FOXA1 define a unique subset of prostate cancers but the functional consequences of these mutations and whether they confer gain or loss of function is unknown 1 – 9 . Here, by annotating the landscape of FOXA1 mutations from 3,086 human prostate cancers, we define two hotspots in the forkhead domain: Wing2 (around 50% of all mutations) and the highly conserved DNA-contact residue R219 (around 5% of all mutations). Wing2 mutations are detected in adenocarcinomas at all stages, whereas R219 mutations are enriched in metastatic tumours with neuroendocrine histology. Interrogation of the biological properties of wild-type FOXA1 and fourteen FOXA1 mutants reveals gain of function in mouse prostate organoid proliferation assays. Twelve of these mutants, as well as wild-type FOXA1, promoted an exaggerated pro-luminal differentiation program, whereas two different R219 mutants blocked luminal differentiation and activated a mesenchymal and neuroendocrine transcriptional program. Assay for transposase-accessible chromatin using sequencing (ATAC-seq) of wild-type FOXA1 and representative Wing2 and R219 mutants revealed marked, mutant-specific changes in open chromatin at thousands of genomic loci and exposed sites of FOXA1 binding and associated increases in gene expression. Of note, ATAC-seq peaks in cells expressing R219 mutants lacked the canonical core FOXA1-binding motifs (GTAAAC/T) but were enriched for a related, non-canonical motif (GTAAAG/A), which was preferentially activated by R219-mutant FOXA1 in reporter assays. Thus, FOXA1 mutations alter its pioneering function and perturb normal luminal epithelial differentiation programs, providing further support for the role of lineage plasticity in cancer progression. Mutations in the transcription factor FOXA1 that are common in prostate cancer result in gain-of-function effects that promote changes in the differentiation of tumour cells.

Matthew Freedman and colleagues show that androgen receptor (AR) binding sites undergo extensive reprogramming during prostate epithelial transformation. They further show that FOXA1 and HOXB13 colocalize at reprogrammed AR binding sites in human tumor tissue and are able to reprogram the AR cistrome of an immortalized prostate cell line to resemble that of prostate tumors. Master transcription factors interact with DNA to establish cell type identity and to regulate gene expression in mammalian cells 1 , 2 . The genome-wide map of these transcription factor binding sites has been termed the cistrome 3 . Here we show that the androgen receptor (AR) cistrome undergoes extensive reprogramming during prostate epithelial transformation in man. Using human prostate tissue, we observed a core set of AR binding sites that are consistently reprogrammed in tumors. FOXA1 and HOXB13 colocalized at the reprogrammed AR binding sites in human tumor tissue. Introduction of FOXA1 and HOXB13 into an immortalized prostate cell line reprogrammed the AR cistrome to resemble that of a prostate tumor, functionally linking these specific factors to AR cistrome reprogramming. These findings offer mechanistic insights into a key set of events that drive normal prostate epithelium toward transformation and establish the centrality of epigenetic reprogramming in human prostate tumorigenesis.

Background Hepatocellular carcinoma (HCC) is the most common malignant liver tumor with poor clinical outcomes. Increasing amount of long non-coding RNAs (lncRNAs) have been revealed to be implicated in the carcinogenesis and progression of HCC. However, the expressions, clinical significances, and roles of most lncRNAs in HCC are still unknown. Methods The expression of lncRNA MCM3AP antisense RNA 1 (MCM3AP-AS1) in HCC tissues and cell lines was detected by qRT-PCR and fluorescence in situ hybridization. Immunoblotting, CCK-8, EdU, colony formation and flow cytometry were performed to investigate the role of MCM3AP-AS1 in HCC cell proliferation, cell cycle and apoptosis in vitro. A subcutaneous tumor mouse model was constructed to analyze in vivo growth of HCC cells after MCM3AP-AS1 knockdown. The interactions among MCM3AP-AS1, miR-194-5p and FOXA1 were measured by RNA pull-down, RNA immunoprecipitation and luciferase reporter assay. Results We revealed a novel oncogenic lncRNA MCM3AP-AS1, which is overexpressed in HCC and positively correlated with large tumor size, high tumor grade, advanced tumor stage and poor prognosis of HCC patients. MCM3AP-AS1 knockdown suppressed HCC cell proliferation, colony formation and cell cycle progression, and induced apoptosis in vitro, and depletion of MCM3AP-AS1 inhibited tumor growth of HCC in vivo. Mechanistically, MCM3AP-AS1 directly bound to miR-194-5p and acted as competing endogenous RNA (ceRNA), and subsequently facilitated miR-194-5p’s target gene forkhead box A1 (FOXA1) expression in HCC cells. Interestingly, FOXA1 restoration rescued MCM3AP-AS1 knockdown induced proliferation inhibition, G1 arrest and apoptosis of HCC cells. Conclusions Our results recognized MCM3AP-AS1 as a novel oncogenic lncRNA, which indicated poor clinical outcomes in patients with HCC. MCM3AP-AS1 exerted an oncogenic role in HCC via targeting miR-194-5p and subsequently promoted FOXA1 expression. Our findings suggested that MCM3AP-AS1 could be a potential prognostic biomarker and therapeutic target for HCC.