Akriti Kharbanda

MUC1-C oncoprotein activates the ZEB1/miR-200c regulatory loop and epithelial-mesenchymal transition.

The epithelial–mesenchymal transition (EMT) is activated in cancer cells by ZEB1, a member of the zinc finger/homeodomain family of transcriptional repressors. The mucin 1 (MUC1) heterodimeric protein is aberrantly overexpressed in human carcinoma cells. The present studies in breast cancer cells demonstrate that the oncogenic MUC1-C subunit induces expression of ZEB1 by a NF-κB (nuclear factor kappa B) p65-dependent mechanism. MUC1-C occupies the ZEB1 promoter with NF-κB p65 and thereby promotes ZEB1 transcription. In turn, ZEB1 associates with MUC1-C and the ZEB1/MUC1-C complex contributes to the transcriptional suppression of miR-200c, an inducer of epithelial differentiation. The co-ordinate upregulation of ZEB1 and suppression of miR-200c has been linked to the induction of EMT. In concert with the effects of MUC1-C on ZEB1 and miR-200c, we show that MUC1-C induces EMT and cellular invasion by a ZEB1-mediated mechanism. These findings indicate that (i) MUC1-C activates ZEB1 and suppresses miR-200c with the induction of EMT and (ii) targeting MUC1-C could be an effective approach for the treatment of breast and possibly other types of cancers that develop EMT properties.

TARGETING THE MUC1-C ONCOPROTEIN DOWNREGULATES HER2 ACTIVATION AND ABROGATES TRASTUZUMAB RESISTANCE IN BREAST CANCER CELLS

Patients with HER2-positive breast cancer often exhibit intrinsic or acquired resistance to trastuzumab treatment. The transmembrane mucin 1 (MUC1) oncoprotein is aberrantly overexpressed in breast cancer cells and associates with HER2. The present studies demonstrate that silencing MUC1 C-terminal subunit (MUC1-C) in HER2-overexpressing SKBR3 and BT474 breast cancer cells results in the downregulation of constitutive HER2 activation. Moreover, treatment with the MUC1-C inhibitor, GO-203, was associated with disruption of MUC1–C/HER2 complexes and decreases in tyrosine-phosphorylated HER2 (p-HER2) levels. In studies of trastuzumab-resistant SKBR3R and BT474R cells, we found that the association between MUC1-C and HER2 is markedly increased (∼20-fold) as compared with that in sensitive cells. In addition, silencing MUC1-C in the trastuzumab-resistant cells or treatment with GO-203 decreased p-HER2 and AKT activation. Moreover, targeting MUC1-C was associated with the downregulation of phospho-p27 and cyclin E, which confer trastuzumab resistance. Consistent with these results, targeting MUC1-C inhibited the growth and clonogenic survival of both trastuzumab-resistant cells. Our results further demonstrate that silencing MUC1-C reverses resistance to trastuzumab and that the combination of GO-203 and trastuzumab is highly synergistic. These findings indicate that MUC1-C contributes to constitutive activation of the HER2 pathway and that targeting MUC1-C represents a potential approach to abrogate trastuzumab resistance.

Targeting the Oncogenic MUC1-C Protein Inhibits Mutant EGFR-Mediated Signaling and Survival in Non–Small Cell Lung Cancer Cells

Purpose: Non–small cell lung cancers (NSCLC) that express EGF receptor with activating mutations frequently develop resistance to EGFR kinase inhibitors. The mucin 1 (MUC1) heterodimeric protein is aberrantly overexpressed in NSCLC cells and confers a poor prognosis; however, the functional involvement of MUC1 in mutant EGFR signaling is not known. Experimental Design: Targeting the oncogenic MUC1 C-terminal subunit (MUC1-C) in NSCLC cells harboring mutant EGFR was studied for effects on signaling, growth, clonogenic survival, and tumorigenicity. Results: Stable silencing of MUC1-C in H1975/EGFR(L858R/T790M) cells resulted in downregulation of AKT signaling and inhibition of growth, colony formation, and tumorigenicity. Similar findings were obtained when MUC1-C was silenced in gefitinib-resistant PC9GR cells expressing EGFR(delE746_A750/T790M). The results further show that expression of a MUC1-C(CQC→AQA) mutant, which blocks MUC1-C homodimerization, suppresses EGFR(T790M), AKT and MEK→ERK activation, colony formation, and tumorigenicity. In concert with these results, treatment of H1975 and PC9GR cells with GO-203, a cell-penetrating peptide that blocks MUC1-C homodimerization, resulted in inhibition of EGFR, AKT, and MEK→ERK signaling and in loss of survival. Combination studies of GO-203 and afatinib, an irreversible inhibitor of EGFR, further demonstrate that these agents are synergistic in inhibiting growth of NSCLC cells harboring the activating EGFR(T790M) or EGFR(delE746-A750) mutants. Conclusions: These findings indicate that targeting MUC1-C inhibits mutant EGFR signaling and survival, and thus represents a potential approach alone and in combination for the treatment of NSCLCs resistant to EGFR kinase inhibitors. Clin Cancer Res; 20(21); 5423–34. ©2014 AACR.

MUC1-C Oncoprotein Activates ERK→C/EBPβ Signaling and Induction of Aldehyde Dehydrogenase 1A1 in Breast Cancer Cells

Background: Aldehyde dehydrogenase 1A1 (ALDH1A1) activity is up-regulated in breast cancer cells by mechanisms that are unclear. Results: The MUC1-C oncoprotein induces ERK-mediated activation of the C/EBPβ transcription factor and, thereby, ALDH1A1 expression. Conclusion: MUC1-C activates a novel ERK→C/EBPβ→ALDH1A1 pathway that up-regulates ALDH activity. Significance: Overexpression of MUC1-C in breast cancer cells contributes to an ALDH+ phenotype that is linked to stemness. 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 β (C/EBPβ) transcription factor. The results further show that MUC1-C and C/EBPβ 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/EBPβ→ALDH1A1 pathway, and (ii) promotes the induction of ALDH activity in breast cancer cells.

INHIBITION OF MUC1-C SUPPRESSES MYC EXPRESSION AND ATTENUATES MALIGNANT GROWTH IN KRAS MUTANT LUNG ADENOCARCINOMAS

Dysregulation of MYC expression is a hallmark of cancer, but the development of agents that target MYC has remained challenging. The oncogenic MUC1-C transmembrane protein is, like MYC, aberrantly expressed in diverse human cancers. The present studies demonstrate that MUC1-C induces MYC expression in KRAS mutant non-small cell lung cancer (NSCLC) cells, an effect that can be suppressed by targeting MUC1-C via shRNA silencing, CRISPR editing, or pharmacologic inhibition with GO-203. MUC1-C activated the WNT/β-catenin (CTNNB1) pathway and promoted occupancy of MUC1-C/β-catenin/TCF4 complexes on the MYC promoter. MUC1-C also promoted the recruitment of the p300 histone acetylase (EP300) and, in turn, induced histone H3 acetylation and activation of MYC gene transcription. We also show that targeting MUC1-C decreased the expression of key MYC target genes essential for the growth and survival of NSCLC cells, such as TERT and CDK4. Based on these results, we found that the combination of GO-203 and the BET bromodomain inhibitor JQ1, which targets MYC transcription, synergistically suppressed MYC expression and cell survival in vitro as well as tumor xenograft growth. Furthermore, MUC1 expression significantly correlated with that of MYC and its target genes in human KRAS mutant NSCLC tumors. Taken together, these findings suggest a therapeutic approach for targeting MYC-dependent cancers and provide the framework for the ongoing clinical studies addressing the efficacy of MUC1-C inhibition in solid tumors.

Oncogenic MUC1-C Promotes Tamoxifen Resistance in Human Breast Cancer

Tamoxifen resistance of estrogen receptor-positive (ER+) breast cancer cells has been linked in part to activation of receptor tyrosine kinases, such as HER2, and the PI3K-AKT pathway. Mucin 1 (MUC1) is aberrantly overexpressed in about 90% of human breast cancers, and the oncogenic MUC1-C subunit is associated with ERα. The present studies using HER2 overexpressing BT-474 breast cancer cells, which are constitutively resistant to tamoxifen, demonstrate that silencing MUC1-C is associated with (i) downregulation of p-HER2 and (ii) sensitivity to tamoxifen-induced growth inhibition and loss of clonogenic survival. In contrast, overexpression of MUC1-C in tamoxifen-sensitive MCF-7 breast cancer cells resulted in upregulation of p-AKT and tamoxifen resistance. We show that MUC1-C forms complexes with ERα on the estrogen-responsive promoter of Rab31 and that MUC1-C blocks tamoxifen-induced decreases in ERα occupancy. MUC1-C also attenuated tamoxifen-induced decreases in (i) recruitment of the coactivator CREB binding protein, (ii) Rab31 promoter activation, and (iii) Rab31 mRNA and protein levels. The importance of MUC1-C is further supported by the demonstration that targeting MUC1-C with the cell-penetrating peptide inhibitor, GO-203, sensitized tamoxifen-resistant cells to tamoxifen treatment. Moreover, we show that targeting MUC1-C in combination with tamoxifen is highly synergistic in the treatment of tamoxifen-resistant breast cancer cells. Combined, these findings indicate that MUC1-C contributes to tamoxifen resistance. Mol Cancer Res; 11(7); 714–23. ©2013 AACR.

Cooperative Interaction between the MUC1-C Oncoprotein and the Rab31 GTPase in Estrogen Receptor-Positive Breast Cancer Cells

Rab31 is a member of the Ras superfamily of small GTPases that has been linked to poor outcomes in patients with breast cancer. The MUC1-C oncoprotein is aberrantly overexpressed in most human breast cancers and also confers a poor prognosis. The present results demonstrate that MUC1-C induces Rab31 expression in estrogen receptor positive (ER+) breast cancer cells. We show that MUC1-C forms a complex with estrogen receptor α (ERα) on the Rab31 promoter and activates Rab31 gene transcription in an estrogen-dependent manner. In turn, Rab31 contributes to the upregulation of MUC1-C abundance in breast cancer cells by attenuating degradation of MUC1-C in lysosomes. Expression of an inactive Rab31(S20N) mutant in nonmalignant breast epithelial cells confirmed that Rab31 regulates MUC1-C expression. The functional significance of the MUC1-C/Rab31 interaction is supported by the demonstration that Rab31 confers the formation of mammospheres by a MUC1-C-dependent mechanism. Analysis of microarray databases further showed that (i) Rab31 is expressed at higher levels in breast cancers as compared to that in normal breast tissues, (ii) MUC1+ and ER+ breast cancers have increased levels of Rab31 expression, and (iii) patients with Rab31-positive breast tumors have a significantly decreased ten-year overall survival as compared to those with Rab31-negative tumors. These findings indicate that MUC1-C and Rab31 function in an autoinductive loop that contributes to overexpression of MUC1-C in breast cancer cells.

Intracellular Targeting of the Oncogenic MUC1-C Protein with a Novel GO-203 Nanoparticle Formulation

Purpose: The MUC1-C oncoprotein is an intracellular target that is druggable with cell-penetrating peptide inhibitors. However, development of peptidyl drugs for treating cancer has been a challenge because of unfavorable pharmacokinetic parameters and limited cell-penetrating capabilities. Experimental Design: Encapsulation of the MUC1-C inhibitor GO-203 in novel polymeric nanoparticles was studied for effects on intracellular targeting of MUC1-C signaling and function. Results: Our results show that loading GO-203 into tetrablock polylactic acid (PLA)-polyethylene glycol (PEG)-polypropylene glycol (PPG)-PEG copolymers is achievable and, notably, is enhanced by increasing PEG chain length. In addition, we found that release of GO-203 from these nanoparticles is controllable over at least 7 days. GO-203/nanoparticle treatment of MUC1-C–positive breast and lung cancer cells in vitro was more active with less frequent dosing than that achieved with nonencapsulated GO-203. Moreover, treatment with GO-203/nanoparticles blocked MUC1-C homodimerization, consistent with on-target effects. GO-203/nanoparticle treatment was also effective in downregulating TIGAR, disrupting redox balance, and inhibiting the self-renewal capacity of cancer cells. Significantly, weekly administration of GO-203/nanoparticles to mice bearing syngeneic or xenograft tumors was associated with regressions that were comparable with those found when dosing on a daily basis with GO-203. Conclusions: These findings thus define an effective approach for (i) sustained administration of GO-203 in polymeric PLA-(PEG-PPG-PEG) nanoparticles to target MUC1-C in cancer cells and (ii) the potential delivery of other anticancer peptide drugs. Clin Cancer Res; 21(10); 2338–47. ©2015 AACR.

A Monoclonal Antibody Against the Oncogenic Mucin 1 Cytoplasmic Domain

Mucin 1 (MUC1) is a heterodimeric protein that is aberrantly overexpressed in diverse human carcinomas and certain hematologic malignancies. The transmembrane MUC1-C subunit confers tumorigenicity and is a target for anti-cancer drug development. In this regard, the MUC1-C cytoplasmic domain interacts with multiple effectors that have been linked to transformation. Here we report on the generation of a mouse monoclonal antibody (MAb) against the human MUC1-C cytoplasmic domain (MUC1-CD). This IgG1 MAb, designated anti-MUC1-CD, reacts with the NYGQLDIFP epitope. We show that anti-MUC1-CD is useful in immunoblotting and immunoprecipitation experiments. In addition, anti-MUC1-CD can be used to detect expression of the MUC1-C subunit in formalin-fixed, paraffin-embedded tissues. The MUC1-C inhibitor has entered Phase I evaluation for patients with refractory solid tumors. The present results indicate that the anti-MUC1-CD antibody could be useful as a biomarker to identify patients with tumors that may be responsive to MUC1-C inhibitors.

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 β‐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.