MUC1-C Oncoprotein Integrates a Program of EMT, Epigenetic Reprogramming and Immune Evasion in Human Carcinomas.

The MUC1 gene evolved in mammalian species to provide protection of epithelia. The transmembrane MUC1 C-terminal subunit (MUC1-C) signals stress to the interior of the epithelial cell and, when overexpressed as in most carcinomas, functions as an oncoprotein. MUC1-C induces the epithelial-mesenchymal transition (EMT) by activating the inflammatory NF-κB p65 pathway and, in turn, the EMT-transcriptional repressor ZEB1. Emerging evidence has indicated that MUC1-C drives a program integrating the induction of EMT with activation of stem cell traits, epigenetic reprogramming and immune evasion. This mini-review focuses on the potential importance of this MUC1-C program in cancer progression.

MUC1-mediated induction of myeloid-derived suppressor cells in patients with acute myeloid leukemia.

Myeloid-derived suppressor cells (MDSCs) play a critical role in promoting immune tolerance and disease growth. The mechanism by which tumor cells evoke the expansion of MDSCs in acute myeloid leukemia (AML) has not been well described. We have demonstrated that patients with AML exhibit increased presence of MDSCs in their peripheral blood, in comparison with normal controls. Cytogenetic studies demonstrated that MDSCs in patients with AML may be derived from leukemic or apparently normal progenitors. Engraftment of C57BL/6 mice with TIB-49 AML led to an expansion of CD11b+ Gr1+ MDSCs in bone marrow and spleen. Coculture of the AML cell lines MOLM-4, THP-1 or primary AML cells with donor peripheral blood mononuclear cells elicited a cell contact-dependent expansion of MDSCs. MDSCs were suppressive of autologous T-cell responses as evidenced by reduced T-cell proliferation and a switch from a Th1 to a Th2 phenotype. We hypothesized that the expansion of MDSCs in AML is accomplished by tumor-derived extracellular vesicles (EVs). Using tracking studies, we demonstrated that AML EVs are taken-up myeloid progenitor cells, resulting in the selective proliferation of MDSCs in comparison with functionally competent antigen-presenting cells. The MUC1 oncoprotein was subsequently identified as the critical driver of EV-mediated MDSC expansion. MUC1 induces increased expression of c-myc in EVs that induces proliferation in the target MDSC population via downstream effects on cell cycle proteins. Moreover, we demonstrate that the microRNA miR34a acts as the regulatory mechanism by which MUC1 drives c-myc expression in AML cells and EVs.

MUC1-C drives myeloid leukaemogenesis and resistance to treatment by a survivin-mediated mechanism.

Acute myeloid leukaemia (AML) is an aggressive haematological malignancy with an unmet need for improved therapies. Responses to standard cytotoxic therapy in AML are often transient because of the emergence of chemotherapy-resistant disease. The MUC1-C oncoprotein governs critical pathways of tumorigenesis, including self-renewal and survival, and is aberrantly expressed in AML blasts and leukaemia stem cells (LSCs). However, a role for MUC1-C in linking leukaemogenesis and resistance to treatment has not been described. In this study, we demonstrate that MUC1-C overexpression is associated with increased leukaemia initiating capacity in an NSG mouse model. In concert with those results, MUC1-C silencing in multiple AML cell lines significantly reduced the establishment of AML in vivo. In addition, targeting MUC1-C with silencing or pharmacologic inhibition with GO-203 led to a decrease in active ß-catenin levels and, in-turn, down-regulation of survivin, a critical mediator of leukaemia cell survival. Targeting MUC1-C was also associated with increased sensitivity of AML cells to Cytarabine (Ara-C) treatment by a survivin-dependent mechanism. Notably, low MUC1 and survivin gene expression were associated with better clinical outcomes in patients with AML. These findings emphasize the importance of MUC1-C to myeloid leukaemogenesis and resistance to treatment by driving survivin expression. Our findings also highlight the potential translational relevance of combining GO-203 with Ara-C for the treatment of patients with AML.

MUC1-C drives MYC in multiple myeloma.

Multiple myeloma (MM) cell lines and primary tumor cells are addicted to the MYC oncoprotein for survival. Little is known, however, about how MYC expression is upregulated in MM cells. The mucin 1 C-terminal subunit (MUC1-C) is an oncogenic transmembrane protein that is aberrantly expressed in MM cell lines and primary tumor samples. The present studies demonstrate that targeting MUC1-C with silencing by clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 editing or with the GO-203 inhibitor is associated with downregulation of MYC messenger RNA and protein. The results show that MUC1-C occupies the MYC promoter and thereby activates the MYC gene by a ß-catenin/transcription factor 4 (TCF4)-mediated mechanism. In this way, MUC1-C (1) increases ß-catenin occupancy on the MYC promoter, (2) forms a complex with ß-catenin and TCF4, and, in turn, (3) drives MYC transcription. Analysis of MM cells using quantitative real-time reverse transcription polymerase chain reaction arrays further demonstrated that silencing MUC1-C is associated with downregulation of MYC target genes, including CCND2, hTERT, and GCLC Analysis of microarray data sets further demonstrated that MUC1 levels positively correlate with MYC expression in MM progression and in primary cells from over 800 MM patients. These findings collectively provide convincing evidence that MUC1-C drives MYC expression in MM.

Bone marrow stroma protects myeloma cells from cytotoxic damage via induction of the oncoprotein MUC1.

Multiple myeloma (MM) is a lethal haematological malignancy that arises in the context of a tumour microenvironment that promotes resistance to apoptosis and immune escape. In the present study, we demonstrate that co-culture of MM cells with stromal cells results in increased resistance to cytotoxic and biological agents as manifested by decreased rates of cell death following exposure to alkylating agents and the proteosome inhibitor, bortezomib. To identify the mechanism of increased resistance, we examined the effect of the co-culture of MM cells with stroma cells, on expression of the MUC1 oncogene, known to confer tumour cells with resistance to apoptosis and necrosis. Co-culture of stroma with MM cells resulted in increased MUC1 expression by tumour cells. The effect of stromal cell co-culture on MUC1 expression was not dependent on cell contact and was therefore thought to be due to soluble factors secreted by the stromal cells into the microenvironment. We demonstrated that MUC1 expression was mediated by interleukin-6 and subsequent up-regulation of the JAK-STAT pathway. Interestingly, the effect of stromal cell co-culture on tumour resistance was partially reversed by silencing of MUC1 in MM cells, consistent with the potential role of MUC1 in mediating resistance to cytotoxic-based therapies.

Mucin 1 is a potential therapeutic target in cutaneous T-cell lymphoma.

Cutaneous T-cell lymphoma (CTCL) is an aggressive neoplasm with limited treatments for patients with advanced disease. The mucin 1 C-terminal subunit (MUC1-C) oncoprotein plays a critical role in regulating cell proliferation, apoptosis, and protection from cytotoxic injury mediated by reactive oxygen species (ROS). Although CTCL cells exhibit resistance to ROS-induced apoptosis, the expression and functional significance of MUC1 in CTCL have not been previously investigated. Present studies demonstrate that MUC1-C is overexpressed in CTCL cell lines and primary CTCL cells but is absent in resting T cells from healthy donors and B-cell lymphoma cells. We have developed a cell-penetrating peptide that disrupts homodimerization of the MUC1-C subunit necessary for its nuclear translocation and downstream signaling. We show that treatment of CTCL cells with the MUC1-C inhibitor is associated with downregulation of the p53-inducible regulator of glycolysis and apoptosis and decreases in reduced NAD phosphate and glutathione levels. In concert with these results, targeting MUC1-C in CTCL cells increased ROS and, in turn, induced ROS-mediated late apoptosis/necrosis. Targeting MUC1-C in CTCL tumor xenograft models demonstrated significant decreases in disease burden. These findings indicate that MUC1-C maintains redox balance in CTCL cells and is thereby a novel target for the treatment of patients with CTCL.

MUC1-C oncoprotein promotes FLT3 receptor activation in acute myeloid leukemia cells.

Blasts from approximately one-third of patients with acute myeloid leukemia (AML) harbor activating mutations in the FMS-like tyrosine kinase 3 (FLT3) receptor tyrosine kinase that confer a poor prognosis. The Mucin 1-C-terminal subunit (MUC1-C) oncoprotein is aberrantly expressed in AML blasts and stem cells; however, there is no known interaction between MUC1-C and FLT3. The present studies demonstrate that MUC1-C associates with wild-type and mutant FLT3 in AML cells. Targeting MUC1-C with the cell-penetrating peptide inhibitor GO-203 disrupts MUC1-C/FLT3 complexes and downregulates FLT3 activation. GO-203 treatment of AML cells was also associated with inhibition of the FLT3 downstream effectors AKT, extracellular signal-regulated kinase, and STAT5. The results further show that AML cells with FLT3-activating mutations and resistant to the FLT3 inhibitor midostaurin/PKC412 are sensitive to GO-203-induced growth arrest and death. Moreover, GO-203 increases sensitivity of mutant FLT3 AML cells to FLT3 inhibitor treatment. These results indicate that MUC1-C contributes to FLT3 activation in AML cells and that targeting MUC1-C inhibits the FLT3 signaling pathway. Our findings support the development of MUC1-C inhibitors alone and in combination with agents that target FLT3 for the treatment of wild-type and mutant FLT3 AML.

Retinoblastoma protein and MyoD function together to effect the repression of Fra-1 and in turn cyclin D1 during terminal cell cycle arrest associated with myogenesis.

The acquisition of skeletal muscle-specific function and terminal cell cycle arrest represent two important features of the myogenic differentiation program. These cellular processes are distinct and can be separated genetically. The lineage-specific transcription factor MyoD and the retinoblastoma protein pRb participate in both of these cellular events. Whether and how MyoD and pRb work together to effect terminal cell cycle arrest is uncertain. To address this question, we focused on cyclin D1, whose stable repression is required for terminal cell cycle arrest and execution of myogenesis. MyoD and pRb are both required for the repression of cyclin D1; their actions, however, were found not to be direct. Rather, they operate to regulate the immediate early gene Fra-1, a critical player in mitogen-dependent induction of cyclin D1. Two conserved MyoD-binding sites were identified in an intronic enhancer of Fra-1 and shown to be required for the stable repression of Fra-1 and, in turn, cyclin D1. Localization of MyoD alone to the intronic enhancer of Fra-1 in the absence of pRb was not sufficient to elicit a block to Fra-1 induction; pRb was also recruited to the intronic enhancer in a MyoD-dependent manner. These observations suggest that MyoD and pRb work together cooperatively at the level of the intronic enhancer of Fra-1 during terminal cell cycle arrest. This work reveals a previously unappreciated link between a lineage-specific transcription factor, a tumor suppressor, and a proto-oncogene in the control of an important facet of myogenic differentiation.

Identification of a novel mechanism of action of fingolimod (FTY720) on human effector T cell function through TCF-1 upregulation.

BACKGROUND: Fingolimod (FTY720), the first oral treatment for multiple sclerosis (MS), blocks immune cell trafficking and prevents disease relapses by downregulation of sphingosine-1-phosphate receptor. We determined the effect of FTY720 on human T cell activation and effector function. METHODS: T cells from MS patients and healthy controls were isolated to measure gene expression profiles in the presence or absence of FTY720 using nanostring and quantitative real-time polymerase chain reaction (qPCR). Cytokine protein expression was measured using luminex assay and flow cytometry analysis. Lentivirus vector carrying short hairpin RNA (shRNA) was used to knock down the expression of specific genes in CD4+ T cells. Chromatin immunoprecipitation was performed to assess T cell factor 1 (TCF-1) binding to promoter regions. Luciferase assays were performed to test the direct regulation of interferon gamma (IFN-γ) and granzyme B (GZMB) by TCF-1. Western blot analysis was used to assess the phosphorylation status of Akt and GSK3ß. RESULTS: We showed that FTY720 treatment not only affects T cell trafficking but also T cell activation. Patients treated with FTY720 showed a significant reduction in circulating CD4 T cells. Activation of T cells in presence of FTY720 showed a less inflammatory phenotype with reduced production of IFN-γ and GZMB. This decreased effector phenotype of FTY720-treated T cells was dependent on the upregulation of TCF-1. FTY720-induced TCF-1 downregulated the pathogenic cytokines IFN-γ and GZMB by binding to their promoter/enhancer regions and mediating epigenetic modifications. Furthermore, we observed that TCF-1 expression was lower in T cells from multiple sclerosis patients than in those from healthy individuals, and FTY720 treatment increased TCF-1 expression in multiple sclerosis patients. CONCLUSIONS: These results reveal a previously unknown mechanism of the effect of FTY720 on human CD4+ T cell modulation in multiple sclerosis and demonstrate the role of TCF-1 in human T cell activation and effector function.

MUC1-C oncoprotein activates ERK→C/EBPß signaling and induction of aldehyde dehydrogenase 1A1 in breast cancer cells.

Aldehyde dehydrogenase 1A1 (ALDH1A1) activity is used as a marker of breast cancer stem cells; however, little is known about the regulation of ALDH1A1 expression. Mucin 1 (MUC1) is a heterodimeric protein that is aberrantly overexpressed in most human breast cancers. In studies of breast cancer cells stably silenced for MUC1 or overexpressing the oncogenic MUC1-C subunit, we demonstrate that MUC1-C is sufficient for induction of MEK → ERK signaling and that treatment with a MUC1-C inhibitor suppresses ERK activation. In turn, MUC1-C induces ERK-mediated phosphorylation and activation of the CCAAT/enhancer-binding protein ß (C/EBPß) transcription factor. The results further show that MUC1-C and C/EBPß form a complex on the ALDH1A1 gene promoter and activate ALDH1A1 gene transcription. MUC1-C-induced up-regulation of ALDH1A1 expression is associated with increases in ALDH activity and is detectable in stem-like cells when expanded as mammospheres. These findings demonstrate that MUC1-C (i) activates a previously unrecognized ERK→C/EBPß→ALDH1A1 pathway, and (ii) promotes the induction of ALDH activity in breast cancer cells.

MUC1 oncoprotein activates the IkappaB kinase beta complex and constitutive NF-kappaB signalling.

Nuclear factor-kappaB (NF-kappaB) is constitutively activated in diverse human malignancies by mechanisms that are not understood. The MUC1 oncoprotein is aberrantly overexpressed by most human carcinomas and, similarly to NF-kappaB, blocks apoptosis and induces transformation. This study demonstrates that overexpression of MUC1 in human carcinoma cells is associated with constitutive activation of NF-kappaB p65. We show that MUC1 interacts with the high-molecular-weight IkappaB kinase (IKK) complex in vivo and that the MUC1 cytoplasmic domain binds directly to IKKbeta and IKKgamma. Interaction of MUC1 with both IKKbeta and IKKgamma is necessary for IKKbeta activation, resulting in phosphorylation and degradation of IkappaBalpha. Studies in non-malignant epithelial cells show that MUC1 is recruited to the TNF-R1 complex and interacts with IKKbeta-IKKgamma in response to TNFalpha stimulation. TNFalpha-induced recruitment of MUC1 is dependent on TRADD and TRAF2, but not the death-domain kinase RIP1. In addition, MUC1-mediated activation of IKKbeta is dependent on TAK1 and TAB2. These findings indicate that MUC1 is important for physiological activation of IKKbeta and that overexpression of MUC1, as found in human cancers, confers sustained induction of the IKKbeta-NF-kappaB p65 pathway.

The MUC1-C oncoprotein binds to the BH3 domain of the pro-apoptotic BAX protein and blocks BAX function.

The pro-apoptotic BAX protein contains a BH3 domain that is necessary for its dimerization and for activation of the intrinsic apoptotic pathway. The MUC1 (mucin 1) heterodimeric protein is overexpressed in diverse human carcinomas and blocks apoptosis in the response to stress. In this study, we demonstrate that the oncogenic MUC1-C subunit associates with BAX in human cancer cells. MUC1-C·BAX complexes are detectable in the cytoplasm and mitochondria and are induced by genotoxic and oxidative stress. The association between MUC1-C and BAX is supported by the demonstration that the MUC1-C cytoplasmic domain is sufficient for the interaction with BAX. The results further show that the MUC1-C cytoplasmic domain CQC motif binds directly to the BAX BH3 domain at Cys-62. Consistent with binding to the BAX BH3 domain, MUC1-C blocked BAX dimerization in response to (i) truncated BID in vitro and (ii) treatment of cancer cells with DNA-damaging agents. In concert with these results, MUC1-C attenuated localization of BAX to mitochondria and the release of cytochrome c. These findings indicate that the MUC1-C oncoprotein binds directly to the BAX BH3 domain and thereby blocks BAX function in activating the mitochondrial death pathway.

MUC1-C oncoprotein induces TCF7L2 transcription factor activation and promotes cyclin D1 expression in human breast cancer cells.

MUC1 is a heterodimeric glycoprotein that is overexpressed in breast cancers. The present studies demonstrate that the oncogenic MUC1 C-terminal subunit (MUC1-C) associates with the TCF7L2 transcription factor. The MUC1-C cytoplasmic domain (MUC1-CD) binds directly to the TCF7L2 C-terminal region. MUC1-C blocks the interaction between TCF7L2 and the C-terminal-binding protein (CtBP), a suppressor of TCF7L2-mediated transcription. TCF7L2 and MUC1-C form a complex on the cyclin D1 gene promoter and MUC1-C promotes TCF7L2-mediated transcription by the recruitment of ß-catenin and p300. Silencing MUC1-C in human breast cancer cells down-regulated activation of the cyclin D1 promoter and decreased cyclin D1 expression. In addition, a MUC1-C inhibitor blocked the interaction with TCF7L2 and suppressed cyclin D1 levels. These findings indicate that the MUC1-C oncoprotein contributes to TCF7L2 activation and thereby promotes cyclin D1 expression in breast cancer cells.

MUC1-C oncoprotein confers androgen-independent growth of human prostate cancer cells.

BACKGROUND: The mucin 1 (MUC1) heterodimeric oncoprotein is overexpressed in human prostate cancers with aggressive pathologic and clinical features. However, few insights are available regarding the functional role of MUC1 in prostate cancer. METHODS: Effects of MUC1-C on androgen receptor (AR) expression were determined by RT-PCR, immunoblotting and AR promoter activation. Coimmunoprecipitations, direct binding assays, and chromatin immunoprecipitation (ChIP) studies were performed to assess the interaction between MUC1-C and AR. Cells were analyzed for invasion, growth in androgen-depleted medium, and sensitivity to MUC1-C inhibitors. RESULTS: The present studies in androgen-dependent LNCaP and LAPC4 prostate cancer cells demonstrate that the oncogenic MUC1-C subunit suppresses AR expression. The results show that MUC1-C activates a posttranscriptional mechanism involving miR-135b-mediated downregulation of AR mRNA levels. The results further demonstrate that MUC1-C forms a complex with AR through a direct interaction between the MUC1-C cytoplasmic domain and the AR DNA-binding domain (DBD). In addition, MUC1-C associates with AR in a complex that occupies the PSA promoter. The interaction between MUC1-C and AR is associated with induction of the epithelial-mesenchymal transition (EMT) and increased invasion. MUC1-C also conferred growth in androgen-depleted medium and resistance to bicalutamide treatment. Moreover, expression of MUC1-C resulted in sensitivity to the MUC1-C inhibitor GO-203 with inhibition of growth in vitro. GO-203 treatment also inhibited growth of established tumor xenografts in nude mice. CONCLUSIONS: These findings indicate that MUC1-C suppresses AR expression in prostate cancer cells and confers a more aggressive androgen-independent phenotype that is sensitive to MUC1-C inhibition.

Dependence on the MUC1-C Oncoprotein in Classic, Variant, and Non-neuroendocrine Small Cell Lung Cancer.

Small cell lung cancer (SCLC) is a recalcitrant malignancy defined by subtypes on the basis of 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: This work uncovers addiction of SCLC cells to MUC1-C, which is a druggable target that could provide new opportunities for advancing SCLC treatment.

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

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. IMPLICATIONS: These findings show that MUC1-C, which is necessary for the CRPC and TNBC CSC state, activates a novel pathway involving JUN/AP-1 and ARID1A/BAF that regulates chromatin accessibility of stemness-associated gene enhancers.

Mucin 1 C-terminal subunit oncoprotein is a target for small-molecule inhibitors.

Mucin 1 (MUC1) is a heterodimeric protein that is overexpressed in diverse human carcinomas. The oncogenic function of the MUC1 C-terminal subunit (MUC1-C) subunit is dependent on the formation of dimers through its cytoplasmic domain; however, it is not known whether MUC1-C can be targeted with small-molecule inhibitors. In the present work, an assay using the MUC1-C cytoplasmic domain (MUC1-CD) was established to screen small-molecule libraries for compounds that block its dimerization. Using this approach, the flavone apigenin was identified as an inhibitor of MUC1-CD dimerization in vitro and in cells. By contrast, the structurally related flavone baicalein was ineffective in blocking the formation of MUC1-CD dimers. In concert with these results, apigenin, and not baicalein, blocked the localization of MUC1-C to the nucleus. MUC1-C activates MUC1 gene expression in an autoinductive loop, and apigenin, but not baicalein, treatment was associated with down-regulation of MUC1 mRNA levels and MUC1-C protein. The results also demonstrate that apigenin-induced suppression of MUC1-C expression is associated with apoptotic cell death and loss of clonogenic survival. These findings represent the first demonstration that the MUC1-C cytoplasmic domain is a target for the development of small-molecule inhibitors.

Androgen receptor regulates expression of the MUC1-C oncoprotein in human prostate cancer cells.

BACKGROUND: The MUC1 heterodimeric oncoprotein is aberrantly overexpressed in human prostate cancers with more aggressive pathologic and clinical features. However, the signals that regulate MUC1 expression in prostate cancer cells are not well understood. METHODS: MUC1 expression was studied in androgen-dependent and -independent prostate cancer cell lines by quantitative RT-PCR, immunoblotting and assessment of MUC1 promoter activation. Chromatin immunoprecipitation (ChIP) studies were performed to assess androgen receptor (AR) occupancy on the MUC1 promoter. Post-transcriptional regulation of MUC1 expression was assessed by miR-125b-mediated effects on activity of the MUC1 3' untranslated region (3'UTR). RESULTS: The present studies demonstrate that AR occupies a consensus AR element on the MUC1 promoter in androgen-dependent LNCaP, but not in androgen-independent DU145 and PC3, prostate cancer cells. The results further show that AR downregulates MUC1 gene transcription. Stable introduction of exogenous AR in PC3 (PC3/AR) cells and then silencing of AR confirmed AR-mediated repression of the MUC1 promoter. AR signaling has also been shown to drive miR-125b expression. The present studies further demonstrate that miR-125b suppresses MUC1 translation in LNCaP cells and that an anti-sense miR-125b upregulates expression of MUC1 protein. In addition, stable expression of miR-125b in DU145 cells resulted in decreases in MUC1 levels. CONCLUSIONS: These findings demonstrate that AR signaling regulates MUC1 expression by transcriptional and posttranscriptional mechanisms in prostate cancer cells.

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

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 BAF (mSWI/SNF) Complex in Prostate Cancer Stem Cells.

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.