atac-seq-optimization-for-cancer-epigenetics-research

ATAC-Seq Optimization for Cancer Epigenetics Research

The assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) probes deoxyribonucleic acid (DNA) accessibility using the ...

Dana-Farber Cancer Institute

The Brg/Brahma-associated factor (BAF, mSWI/SNF) chromatin remodeling complex is of importance in development and has been linked to prostate oncogenesis. The oncogenic MUC1-C protein promotes lineage plasticity in the progression of neuroendocrine prostate cancer (NEPC), however, there is no known association between MUC1-C and BAF. We report here that MUC1-C binds directly to the E2F1 transcription factor and that the MUC1-C→E2F1 pathway induces expression of embryonic stem cell-specific BAF (esBAF) components BRG1, ARID1A, BAF60a, BAF155, and BAF170 in castrate-resistant prostate cancer (CRPC) and NEPC cells. In concert with this previously unrecognized pathway, MUC1 was associated with increased expression of E2F1 and esBAF components in NEPC tumors as compared with CRPC, supporting involvement of MUC1-C in activating the E2F1→esBAF pathway with progression to NEPC. MUC1-C formed a nuclear complex with BAF and activated cancer stem cell (CSC) gene signatures and the core pluripotency factor gene network. The MUC1-C→E2F1→BAF pathway was necessary for induction of both the NOTCH1 effector of CSC function and the NANOG pluripotency factor, and collectively, this network drove CSC self-renewal. These findings indicate that MUC1-C promotes NEPC progression by integrating activation of E2F1 and esBAF with induction of NOTCH1, NANOG, and stemness. SIGNIFICANCE: These findings show that MUC1-C, which promotes prostate cancer progression, activates a novel pathway that drives the BAF remodeling complex, induces NOTCH1 and NANOG, and promotes self-renewal of prostate cancer stem cells.

MUC1-C Activates the BAF (mSWI/SNF) Complex in Prostate Cancer Stem Cells.

Immune checkpoint inhibitors (ICIs) have had a profound impact on the treatment of many tumors; however, their effectiveness against triple-negative breast cancers (TNBCs) has been limited. One factor limiting responsiveness of TNBCs to ICIs is a lack of functional tumor-infiltrating lymphocytes (TILs) in 'non-inflamed' or 'cold' tumor immune microenvironments (TIMEs), although by unknown mechanisms. Targeting MUC1-C in a mouse transgenic TNBC tumor model increases cytotoxic tumor-infiltrating CD8+ T cells (CTLs), supporting a role for MUC1-C in immune evasion. The basis for these findings and whether they extend to human TNBCs are not known.

MUC1-C integrates activation of the IFN-γ pathway with suppression of the tumor immune microenvironment in triple-negative breast cancer.

The polybromo-associated PBAF (SWI/SNF) chromatin remodeling complex, which includes PBRM1, ARID2, and BRD7, regulates cell differentiation and genomic integrity. MUC1-C is an oncogenic protein that drives lineage plasticity in prostate cancer (PC) progression. The present work demonstrates that MUC1-C induces PBRM1, ARID2, and BRD7 expression by the previously unrecognized E2F1-mediated activation of their respective promoters. The functional significance of the MUC1-C→PBAF pathway is supported by demonstrating involvement of MUC1-C in associating with nuclear PBAF and driving the NRF2 antioxidant gene transcriptome in PC cells. Mechanistically, MUC1-C forms a complex with NRF2 and PBRM1 on the NRF2 target SLC7A11 gene that encodes the xCT cystine-glutamate antiporter, increases chromatin accessibility and induces SLC7A11/xCT expression. We also show that MUC1-C and PBRM1 are necessary for induction of other NRF2 target genes, including G6PD and PGD that regulate the pentose phosphate pathway. Our results further demonstrate that MUC1-C integrates activation of PBRM1 with the regulation of antioxidant genes, ROS levels, pluripotency factor expression and the cancer stem cell (CSC) state. These findings reveal a role for MUC1-C in regulating PBAF, redox balance and lineage plasticity of PC CSC progression. Our findings also uncover involvement of MUC1-C in integrating the PBAF and BAF pathways in cancer.

MUC1-C activates the PBAF chromatin remodeling complex in integrating redox balance with progression of human prostate cancer stem cells.

Pancreatic ductal adenocarcinomas (PDAC) and poorly differentiated pancreatic neuroendocrine (NE) carcinomas are KRAS mutant malignancies with a potential common cell of origin. PDAC ductal, but not NE, lineage traits have been associated with cell-intrinsic activation of interferon (IFN) pathways. The present studies demonstrate that the MUC1 C-terminal subunit (MUC1-C), which evolved to protect mammalian epithelia from loss of homeostasis, is aberrantly overexpressed in KRAS mutant PDAC tumors and cell lines. We show that MUC1-C is necessary for activation of the type I and II IFN pathways and for expression of the Yamanaka OCT4, SOX2, KLF4 and MYC (OSKM) pluripotency factors. Our results demonstrate that MUC1-C integrates IFN signaling and pluripotency with NE dedifferentiation by forming a complex with MYC and driving the (i) achaete-scute homolog 1 and BRN2/POU3F2 neural, and (ii) NOTCH1/2 stemness transcription factors. Of translational relevance, targeting MUC1-C genetically and pharmacologically in PDAC cells (i) suppresses OSKM, NE dedifferentiation and NOTCH1/2, and (ii) inhibits self-renewal capacity and tumorigenicity. In PDAC tumors, we show that MUC1 significantly associates with activation of IFN signaling, MYC and NOTCH, and that upregulation of the MUC1-C → MYC pathway confers a poor prognosis. These findings indicate that MUC1-C dictates PDAC NE lineage specification and is a potential target for the treatment of recalcitrant pancreatic carcinomas with NE dedifferentiation.

MUC1-C dictates neuroendocrine lineage specification in pancreatic ductal adenocarcinomas.

Epigenetic regulation involves reversible changes in histones and DNA modifications that can be inherited without any changes in the DNA sequence. Dysregulation of normal epigenetic processes can lead to aberrant gene expression as observed in many diseases, notably cancer. Recent insights into the mechanisms of DNA methylation, histone modifications, and non-coding RNAs involved in altered gene expression profiles of tumor cells have caused a paradigm shift in the diagnostic and therapeutic approaches towards cancer. There has been a surge in search for compounds that could modulate the altered epigenetic landscape of tumor cells, and to exploit their therapeutic potential against cancers. Flavonoids are naturally occurring phenol compounds which are abundantly found among phytochemicals and have potentials to modulate epigenetic processes. Knowledge of the precise flavonoid-mediated epigenetic alterations is needed for the development of epigenetics drugs and combinatorial therapeutic approaches against cancers. This review is aimed to comprehensively explore the epigenetic modulations of flavonoids and their anti-tumor activities.

Role of Flavonoids as Epigenetic Modulators in Cancer Prevention and Therapy.

The oncogenic MUC1-C protein promotes dedifferentiation of castrate-resistant prostate cancer (CRPC) and triple-negative breast cancer (TNBC) cells. Chromatin remodeling is critical for the cancer stem cell (CSC) state; however, there is no definitive evidence that MUC1-C regulates chromatin accessibility and thereby expression of stemness-associated genes. We demonstrate that MUC1-C drives global changes in chromatin architecture in the dedifferentiation of CRPC and TNBC cells. Our results show that MUC1-C induces differentially accessible regions (DAR) across their genomes, which are significantly associated with differentially expressed genes (DEG). Motif and cistrome analysis further demonstrated MUC1-C-induced DARs align with genes regulated by the JUN/AP-1 family of transcription factors. MUC1-C activates the BAF chromatin remodeling complex, which is recruited by JUN in enhancer selection. In studies of the NOTCH1 gene, which is required for CRPC and TNBC cell self-renewal, we demonstrate that MUC1-C is necessary for (i) occupancy of JUN and ARID1A/BAF, (ii) increases in H3K27ac and H3K4me3 signals, and (iii) opening of chromatin accessibility on a proximal enhancer-like signature. Studies of the EGR1 and LY6E stemness-associated genes further demonstrate that MUC1-C-induced JUN/ARID1A complexes regulate chromatin accessibility on proximal and distal enhancer-like signatures. These findings uncover a role for MUC1-C in chromatin remodeling that is mediated at least in part by JUN/AP-1 and ARID1A/BAF in association with driving the CSC state.

MUC1-C Dictates JUN and BAF-Mediated Chromatin Remodeling at Enhancer Signatures in Cancer Stem Cells.

The oncogenic MUC1-C protein drives dedifferentiation of castrate resistant prostate cancer (CRPC) cells in association with chromatin remodeling. The present work demonstrates that MUC1-C is necessary for expression of IFNGR1 and activation of the type II interferon-gamma (IFN-γ) pathway. We show that MUC1-C→ARID1A/BAF signaling induces  transcription and that MUC1-C-induced activation of the NuRD complex suppresses FBXW7 in stabilizing the IFNGR1 protein. MUC1-C and NuRD were also necessary for expression of the downstream STAT1 and IRF1 transcription factors. We further demonstrate that MUC1-C and PBRM1/PBAF are necessary for IRF1-induced expression of (i) IDO1, WARS and PTGES, which metabolically suppress the immune tumor microenvironment (TME), and (ii) the ISG15 and SERPINB9 inhibitors of T cell function. Of translational relevance, we show that MUC1 associates with expression of IFNGR1, STAT1 and IRF1, as well as the downstream IDO1, WARS, PTGES, ISG15 and SERPINB9 immunosuppressive effectors in CRPC tumors. Analyses of scRNA-seq data further demonstrate that MUC1 correlates with cancer stem cell (CSC) and IFN gene signatures across CRPC cells. Consistent with these results, MUC1 associates with immune cell-depleted "cold" CRPC TMEs. These findings demonstrate that MUC1-C integrates chronic activation of the type II IFN-γ pathway and induction of chromatin remodeling complexes in linking the CSC state with immune evasion.

MUC1-C integrates type II interferon and chromatin remodeling pathways in immunosuppression of prostate cancer.

Small cell lung cancer (SCLC) is a recalcitrant malignancy defined by subtypes based on differential expression of the ASCL1, NEUROD1 and POU2F3 transcription factors. The MUC1-C protein is activated in pulmonary epithelial cells by exposure to environmental carcinogens and promotes oncogenesis; however, there is no known association between MUC1-C and SCLC. We report that MUC1-C is expressed in classic neuroendocrine (NE) SCLC-A, variant NE SCLC-N and non-NE SCLC-P cells and activates the MYC pathway in these subtypes. In SCLC cells characterized by NE differentiation and DNA replication stress, we show that MUC1-C activates the MYC pathway in association with induction of E2F target genes and dysregulation of mitotic progression. Our studies further demonstrate that the MUC1-C->MYC pathway is necessary for induction of (i) NOTCH2, a marker of pulmonary NE stem cells that are the proposed cell of SCLC origin, and (ii) ASCL1 and NEUROD1. We also show that the MUC1-C->MYC->NOTCH2 network is necessary for self-renewal capacity and tumorigenicity of NE and non-NE SCLC cells. Analyses of datasets from SCLC tumors confirmed that MUC1 expression in single SCLC cells significantly associates with activation of the MYC pathway. These findings demonstrate that SCLC cells are addicted to MUC1-C and identify a potential new target for SCLC treatment. Implications: The present work uncovers addiction of SCLC cells to MUC1-C, which is a druggable target that could provide new opportunities for advancing SCLC treatment.

DEPENDENCE ON THE MUC1-C ONCOPROTEIN IN CLASSIC, VARIANT AND NON-NEUROENDOCRINE SMALL CELL LUNG CANCER.

The MUC1-C apical transmembrane protein is activated in the acute response of epithelial cells to inflammation. However, chronic MUC1-C activation promotes cancer progression, emphasizing the importance of MUC1-C as a target for treatment. We report here that MUC1-C is necessary for intrinsic expression of the RIG-I, MDA5 and cGAS cytosolic nucleotide pattern recognition receptors (PRRs) and the cGAS-stimulator of IFN genes (STING) in triple-negative breast cancer (TNBC) cells. Consistent with inducing the PRR/STING axis, MUC1-C drives chronic IFN-β production and activation of the type I interferon (IFN) pathway. MUC1-C thereby induces the IFN-related DNA damage resistance gene signature (IRDS), which includes ISG15, in linking chronic inflammation with DNA damage resistance. Targeting MUC1-C in TNBC cells treated with carboplatin or the PARP inhibitor olaparib further demonstrated that MUC1-C is necessary for expression of PRRs, STING and ISG15 and for intrinsic DNA damage resistance. Of translational relevance, MUC1 significantly associates with upregulation of STING and ISG15 in TNBC tumors and is a target for treatment with CAR T cells, antibody-drug conjugates (ADCs) and direct inhibitors that are under preclinical and clinical development.

Targeting MUC1-C Suppresses Chronic Activation of Cytosolic Nucleotide Receptors and STING in Triple-Negative Breast Cancer.

Merkel cell carcinoma (MCC) is an aggressive malignancy with neuroendocrine (NE) features, limited treatment options, and a lack of druggable targets. There is no reported involvement of the MUC1-C oncogenic protein in MCC progression. We show here that MUC1-C is broadly expressed in MCCs and at higher levels in Merkel cell polyomavirus (MCPyV)-positive (MCCP) relative to MCPyV-negative (MCCN) tumors. Our results further demonstrate that MUC1-C is expressed in MCCP, as well as MCCN, cell lines and regulates common sets of signaling pathways related to RNA synthesis, processing, and transport in both subtypes. Mechanistically, MUC1-C (i) interacts with MYCL, which drives MCC progression, (ii) is necessary for expression of the OCT4, SOX2, KLF4, MYC, and NANOG pluripotency factors, and (iii) induces the NEUROD1, BRN2 and ATOH1 NE lineage dictating transcription factors. We show that MUC1-C is also necessary for MCCP and MCCN cell survival by suppressing DNA replication stress, the p53 pathway, and apoptosis. In concert with these results, targeting MUC1-C genetically and pharmacologically inhibits MCC self-renewal capacity and tumorigenicity. These findings demonstrate that MCCP and MCCN cells are addicted to MUC1-C and identify MUC1-C as a potential target for MCC treatment.

Atrayee Bhattacharya

Dana-Farber Cancer Institute

ATAC-Seq Optimization for Cancer Epigenetics Research