Targeting MUC1-C Research Articles

MUC1-C is a target in lenalidomide resistant multiple myeloma.

Lenalidomide (LEN) acts directly on multiple myeloma (MM) cells by inducing cereblon-mediated degradation of interferon regulatory factor 4, Ikaros (IKZF)1 and IKZF3, transcription factors that are essential for MM cell survival. The mucin 1 (MUC1) C-terminal transmembrane subunit (MUC1-C) oncoprotein is aberrantly expressed by MM cells and protects against reactive oxygen species (ROS)-mediated MM cell death. The present studies demonstrate that targeting MUC1-C with GO-203, a cell-penetrating peptide inhibitor of MUC1-C homodimerization, is more than additive with LEN in downregulating the WNT/β-catenin pathway, suppressing MYC, and inducing late apoptosis/necrosis. We show that the GO-203/LEN combination acts by synergistically increasing ROS and, in turn, suppressing β-catenin. LEN resistance has been linked to activation of the WNT/β-catenin→CD44 pathway. In this regard, our results further demonstrate that targeting MUC1-C is effective against LEN-resistant MM cells. Moreover, GO-203 resensitized LEN-resistant MM cells to LEN treatment in association with suppression of β-catenin and CD44. Targeting MUC1-C also resulted in downregulation of CD44 on the surface of primary MM cells. These findings, and the demonstration that expression of MUC1 and CD44 significantly correlate in microarrays from primary MM cells, provide support for combining GO-203 with LEN in the treatment of MM and in LEN-resistance.

Intracellular targeting of the oncogenic MUC1-C protein with a GO-203 nanoparticle formulation overcomes MCL-1- and BFL-1-mediated resistance in human carcinoma cells.

e14053 Background: Aberrant expression of MCL-1 and BFL-1, the pro-survival members of the Bcl-2 family, is a major cause of drug resistance in human cancers. Mucin 1 (MUC1) is a heterodimeric oncoprotein that is aberrantly expressed in most human carcinomas. Notably, there is no known relationship between the oncogenic MUC1 C-terminal subunit (MUC1-C) and these anti-apoptotic proteins. Methods: MUC1-C was targeted in breast, lung and colon cancer cells by a stable shRNA, a tetracycline-inducible shRNA, or a pharmacologic peptide inhibitor GO-203. MCL-1 was inhibited by siRNA or the MS-1 peptide. In vitro and in vivo studies were conducted using our polymeric nanoparticles (NPs) for intracellular delivery of peptide cargos. Cells were selected for resistance to ABT-737 or ABT-263, which target BCL-2, BCL-XL and BCL-w, but not MCL-1 and BFL-1. Results: Targeting MCL-1 with MS-1/NPs inhibited the survival of parental cancer cells in vitro and in vivo, and was associated with upregulation of BFL-1 levels. In addition, MS-1/NPs treatment had limited effects on ABT-resistant cells because of increased BFL-1 expression. Importantly, we found that targeting MUC1-C is associated with suppression of both MCL-1 and BFL-1. Mechanistically, MUC1-C (i) stabilizes MCL-1 by activating the MEK→ERK and PI3K→AKT pathways, and (ii) induces BFL-1 through the NF-κB p65 pathway. Treatment with GO-203/NPs suppressed proliferation of parental and ABT-resistant cells. In addition, we show that combining GO-203 with ABT-737 is synergistic in inhibiting survival of parental and ABT-resistant cells. Conclusions: These findings demonstrate that targeting MUC1-C with GO-203/NPs is a potential strategy for abrogating MCL-1- and BFL-1-mediated resistance.

Targeting MUC1-C inhibits the AKT-S6K1-elF4A pathway regulating TIGAR translation in colorectal cancer

BackgroundColorectal cancer is third most common malignancy and is the second most common cause of cancer-related death. The MUC1 heterodimeric protein is aberrantly overexpressed in colorectal cancer and has been linked to poor outcomes in this disease. Here, we investigate the effects of the MUC1-C subunit inhibitor (GO-203), which disrupts MUC1-C homo-oligomerization, on human colorectal cancer cells.MethodsTIGAR mRNA level was determined using qRT-PCR. Western blotting was used to measure TIGAR protein level and AKT-mTOR-S6K1 pathways. Reactive oxygen species and apoptosis were measured by flow cytometry. Effect of MUC1-C peptide, GO-203 was studied on colorectal xenograft tumors. Immunohistochemistry was utilized for TIGAR staining.ResultsTreatment of MUC1-overexpressing SKCO-1 and Colo-205 colon cancer cells with GO-203 was associated with downregulation of the TP53-inducible glycolysis and apoptosis regulator (TIGAR) protein. TIGAR promotes the shunting of glycolytic intermediates into the pentose phosphate pathway and thus is of importance for maintaining redox balance. We show that GO-203-induced suppression of TIGAR is mediated by inhibition of AKT and the downstream mTOR pathway. The results also demonstrate that targeting MUC1-C blocks eIF4A cap-dependent translation of TIGAR. In concert with these results, GO-203-induced suppression of TIGAR was associated with decreases in GSH levels. GO-203 treatment also resulted in increases in reactive oxygen species (ROS) and loss of mitochondrial transmembrane potential. Consistent with these results, GO-203 inhibited the growth of colon cancer cells in vitro and as xenografts in nude mice. Inhibition of MUC1-C also downregulated TIGAR expression in xenograft tissues.ConclusionsThese findings indicate that MUC1-C is a potential target for the treatment of colorectal cancer. Colorectal cancer patients who overexpress MUC1-C may be candidates for treatment with the MUC1-C inhibitor alone or in combination therapy with other agents.

MUC1-C Represses the Crumbs Complex Polarity Factor CRB3 and Downregulates the Hippo Pathway.

These findings demonstrate a previously unrecognized role for the MUC1-C oncoprotein in the regulation of polarity and the Hippo pathway in breast cancer. Mol Cancer Res; 14(12); 1266-76. ©2016 AACR.

Abstract 3824: Targeting MUC1-C oncoprotein inhibits AKT-S6K1-elF4A pathway regulated TIGAR translation in colorectal cancer

Abstract Colorectal cancer is third most common malignancy and is the second most common cause of cancer-related death. The MUC1 multi-domain oncoprotein is aberrantly overexpressed in colorectal cancer and has been linked to poor outcomes in this disease. However, it is not known if MUC1 is of functional significance to colorectal tumors. Here, we investigate the effects of the MUC1-C inhibitor (GO-203), which disrupts MUC1-C homo-oligomerization, on AKT-mTORC-S6K translation pathway in human colorectal cancer cells. The present study demonstrates that GO-203 treatment was associated with inhibition of AKT-S6K1 signaling in colorectal cancer cells. Targeting MUC1-C with GO-203 downregulates TP53-inducible glycolysis and apoptosis regulator (TIGAR) protein. We show that GO-203-induced suppression of TIGAR is mediated by inhibition of AKT and the downstream mTOR pathway. The results also demonstrate that targeting MUC1-C blocks eIF4A cap-dependent translation of TIGAR. TIGAR promotes the shunting of glycolytic intermediates into the pentose phosphate pathway and thus is of importance for maintaining redox balance. In concert with these results, GO-203-induced suppression of TIGAR was associated with decreases glutathione levels. GO-203 treatment also resulted increases in reactive oxygen species (ROS) and loss of mitochondrial transmembrane potential. Consistent with these results, GO-203 inhibited the growth of colon cancer cells in vitro and xenografts in nude mice. These findings thus indicate that MUC1-C is a potential target for the treatment of colorectal cancer. Citation Format: Rehan Ahmad, Maroof Alam, Omar Al-Obaid, Masanori Hasegawa, Donald Kufe. Targeting MUC1-C oncoprotein inhibits AKT-S6K1-elF4A pathway regulated TIGAR translation in colorectal cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3824.

MUC1-C induces DNA methyltransferase 1 and represses tumor suppressor genes in acute myeloid leukemia.

Aberrant DNA methylation is a hallmark of acute myeloid leukemia (AML); however, the regulation of DNA methyltransferase 1 (DNMT1), which is responsible for maintenance of DNA methylation patterns, has largely remained elusive. MUC1-C is a transmembrane oncoprotein that is aberrantly expressed in AML stem-like cells. The present studies demonstrate that targeting MUC1-C with silencing or a pharmacologic inhibitor GO-203 suppresses DNMT1 expression. In addition, MUC1 expression positively correlates with that of DNMT1 in primary AML cells, particularly the CD34+/CD38− population. The mechanistic basis for this relationship is supported by the demonstration that MUC1-C activates the NF-κB p65 pathway, promotes occupancy of the MUC1-C/NF-κB complex on the DNMT1 promoter and drives DNMT1 transcription. We also show that targeting MUC1-C substantially reduces gene promoter-specific DNA methylation, and derepresses expression of tumor suppressor genes, including CDH1, PTEN and BRCA1. In support of these results, we demonstrate that combining GO-203 with the DNMT1 inhibitor decitabine is highly effective in reducing DNMT1 levels and decreasing AML cell survival. These findings indicate that (i) MUC1-C is an attractive target for the epigentic reprogramming of AML cells, and (ii) targeting MUC1-C in combination with decitabine is a potentially effective clinical approach for the treatment of AML.

MUC1-C drives MYC in multiple myeloma

AbstractMultiple 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 β-catenin/transcription factor 4 (TCF4)-mediated mechanism. In this way, MUC1-C (1) increases β-catenin occupancy on the MYC promoter, (2) forms a complex with β-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.

DNA methylation by DNMT1 and DNMT3b methyltransferases is driven by the MUC1-C oncoprotein in human carcinoma cells.

Aberrant expression of the DNA methyltransferases (DNMTs) and disruption of DNA methylation patterns are associated with carcinogenesis and cancer cell survival. The oncogenic MUC1-C protein is aberrantly overexpressed in diverse carcinomas; however, there is no known link between MUC1-C and DNA methylation. Our results demonstrate that MUC1-C induces expression of DNMT1 and DNMT3b, but not DNMT3a, in breast and other carcinoma cell types. We show that MUC1-C occupies the DNMT1 and DNMT3b promoters in complexes with NF-κB p65 and drives DNMT1 and DNMT3b transcription. In this way, MUC1-C controls global DNA methylation as determined by analysis of LINE-1 repeat elements. The results further demonstrate that targeting MUC1-C downregulates DNA methylation of the CDH1 tumor suppressor gene in association with induction of E-cadherin expression. These findings provide compelling evidence that MUC1-C is of functional importance to induction of DNMT1 and DNMT3b and, in turn, changes in DNA methylation patterns in cancer cells.

MUC1-C Stabilizes MCL-1 in the Oxidative Stress Response of Triple-Negative Breast Cancer Cells to BCL-2 Inhibitors.

Aberrant expression of myeloid cell leukemia-1 (MCL-1) is a major cause of drug resistance in triple-negative breast cancer (TNBC) cells. Mucin 1 (MUC1) is a heterodimeric oncoprotein that is aberrantly overexpressed in most TNBC. The present studies show that targeting the oncogenic MUC1 C-terminal subunit (MUC1-C) in TNBC cells with silencing or pharmacologic inhibition with GO-203 is associated with downregulation of MCL-1 levels. Targeting MUC1-C suppresses the MEK → ERK and PI3K → AKT pathways, and in turn destabilizes MCL-1. The small molecules ABT-737 and ABT-263 target BCL-2, BCL-XL and BCL-w, but not MCL-1. We show that treatment with ABT-737 increases reactive oxygen species and thereby MUC1-C expression. In this way, MUC1-C is upregulated in TNBC cells resistant to ABT-737 or ABT-263. We also demonstrate that MUC1-C is necessary for the resistance-associated increases in MCL-1 levels. Significantly, combining GO-203 with ABT-737 is synergistic in inhibiting survival of parental and drug resistant TNBC cells. These findings indicate that targeting MUC1-C is a potential strategy for reversing MCL-1-mediated resistance in TNBC.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

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.

Immunomodulatory Effect of MUC1-C in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is characterized by an immunosuppressive milieu in the bone marrow microenvironment that fosters tolerance, immune escape, and disease growth. Upregulation of PD-L1 expression by leukemia cells induces an exhausted phenotype in tumor reactive lymphocytes that blocks the activation and expansion of leukemia specific lymphocytes. Identifying pathways that regulate PD-L1 expression is vital for the development of novel therapeutic targets for immune based therapies. MUC1 is an oncoprotein that is aberrantly expressed on a majority of primary AML. MUC1 contains a transmembrane subunit (MUC1-C) that forms dimers and translocates to the nucleus where it mediates downstream signaling. MUC1-C expression on malignant cells promotes cell proliferation and resistance to apoptosis.We have demonstrated that MUC1-C is a critical regulator of PD-L1 expression on AML cells. Silencing of MUC1 expression was documented following lentiviral transduction with MUC1-C specific shRNA using western blotting. MUC1 silencing results in the near abrogation of MUC1-C expression by the human AML cell lines, MOLM-14 and THP1. Silencing of MUC1-C on MOLM-14 and THP1 AML cells resulted in a two-fold increase in susceptibility to T cell mediated lysis as determined by a flourochrome based CTL assay.To assess the effect of MUC1-C silencing on PD-L1 expression in-vivo, C57BL/6J mice were challenged with 100,000 GFP transfected TIB-49 murine AML cells in which MUC1-C was silenced using a lentiviral shRNA hairpin against MUC1-C. Following leukemia establishment, TIB-49 GFP+ cells were isolated from the bone marrows and spleens and PD-L1 expression on leukemic cells was measured. TIB-49 GFP+ cells of mice engrafted with MUC1 silenced AML cells demonstrated significantly lower PD-L1 expression compared to mice inoculated with TIB-49 cells transduced with a control vector (18% versus 3%; n=4). T cells isolated from bone marrow of mice inoculated with AML with silenced MUC1-C demonstrated a three-fold increase in INF-γ production when stimulated ex-vivo with autologous tumor lysate as compared to T cells from mice inoculated with control AML cells (n=4). Our group has developed a peptide inhibitor drug (G0-203), a cell-penetrating peptide that binds to the MUC1-C CQC motif, disrupting MUC1-C interaction with downstream effectors. As was observed following MUC1 silencing, bone marrow derived CD4+ and CD8+ T cells isolated from mice treated with G0-203 were shown to have two-fold increased intracellular IFN-γ production compared to control mice following ex-vivo exposure to AML lysate (n=4).To investigate the mechanism by which MUC1-C regulates PD-L1 expression we examined the role of noncoding RNAs that have been shown to be effectors of oncogenic modulation. MicroRNAs (miRNAs) are a conserved class of small RNAs that post-transcriptionally regulate gene expression by interacting with the 3′ untranslated region (3′ UTR) of target mRNAs. The 3'UTR of the PD-L1 gene contains putative binding sites for miR-200 family of micro-RNAs, supporting the hypothesis that miR-200 plays a role in regulating the expression of PD-L1.In the present study, we evaluated the effect of targeting MUC1-C mediated signaling, on miR200c levels and PD-L1 expression. MUC1-C silenced MOLM-14 cells demonstrated a two-fold increase in miR-200c expression, as demonstrated by qPCR and was associated with a reduction of PD-L1 expression from 77% to 13%. These data were confirmed in THP1 cells with PDL-1 expression decreasing from 95% to 40% following MUC1-C silencing. In support of the observation that miR200c is involved in the regulation of PD-L1 expression, lentiviral overexpression of miR200c in MOLM-14 cells led to a decrease in PD-L1 expression from 90% to 2% as demonstrated by flow cytometric analysis.In conclusion, these results demonstrate that MUC1-C mediated signaling regulates the expression of PD-L1 on AML cells. Silencing MUC1-C results in reduced expression of PD-L1, interfering with immune escape and enhancing the expansion of leukemia reactive T cells. We define a novel mechanism by which MUC1-C governs PD-L1 expression through its modulation of miR200c. As such, MUC1-C functions as a critical regulator of immune escape and represents a novel target for immunotherapy. DisclosuresKufe:Genus Oncology: Consultancy, Equity Ownership.

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

AbstractCutaneous 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 Induces the LIN28B→LET‐7→HMGA2 Axis to Regulate Self‐renewal in NSCLC

ObjectiveThe LIN28B→let‐7 pathway contributes to regulation of the epithelial‐mesenchymal transition (EMT) and stem cell self‐renewal. The oncogenic MUC1‐C transmembrane protein is aberrantly overexpressed in lung and other human carcinomas; however, there is no known association between MUC1‐C and the LIN28B→let‐7 pathway. 
 Methods: Stable silencing of MUC1‐C using MUC1 shRNA. Targeting MUC1‐C function with a dominant‐negative mutant or a peptide inhibitor was used to study the link between MUC1 expression and LIN28B→let‐7 signaling.
 Results: In non‐small cell lung cancer (NSCLC), silencing MUC1‐C downregulates the RNA binding protein LIN28B and coordinately increases the miRNA let‐7. Targeting MUC1‐C function with a dominant‐negative mutant or a peptide inhibitor provides confirming evidence that MUC1‐C induces LIN28B→let‐7 signaling. MUC1‐C promotes NF‐κB p65 chromatin occupancy of the LIN28B first intron and activates LIN28Btranscription which is associated with suppression of let‐7. Consistent with let‐7‐mediated inhibition of HMGA2 transcripts, targeting of MUC1‐C also decreases HMGA2 expression. HMGA2 has been linked to stemness, and functions as a competing endogenous RNA (ceRNA) of let‐7‐mediated regulation of the TGFβ co‐receptor TGFBR3. Accordingly, targeting MUC1‐C suppresses HMGA2 mRNA and protein which is associated with decreases in TGFBR3, reversal of the EMT phenotype and inhibition of self‐renewal capacity.
ConclusionThese findings support a model in which MUC1‐C activates the ⇑LIN28B→⇓let‐7→⇑HMGA2 axis in NSCLC and thereby promotes EMT traits and stemness.

MUC1-C Induces the LIN28B→LET-7→HMGA2 Axis to Regulate Self-Renewal in NSCLC.

The LIN28B→let-7 pathway contributes to regulation of the epithelial-mesenchymal transition (EMT) and stem cell self-renewal. The oncogenic MUC1-C transmembrane protein is aberrantly overexpressed in lung and other carcinomas; however, there is no known association between MUC1-C and the LIN28B→let-7 pathway. Here in non-small cell lung cancer (NSCLC), silencing MUC1-C downregulates the RNA-binding protein LIN28B and coordinately increases the miRNA let-7. Targeting MUC1-C function with a dominant-negative mutant or a peptide inhibitor provided confirming evidence that MUC1-C induces LIN28B→let-7 signaling. Mechanistically, MUC1-C promotes NF-κB p65 chromatin occupancy of the LIN28B first intron and activates LIN28B transcription, which is associated with suppression of let-7. Consistent with let-7-mediated inhibition of HMGA2 transcripts, targeting of MUC1-C also decreases HMGA2 expression. HMGA2 has been linked to stemness, and functions as a competing endogenous RNA (ceRNA) of let-7-mediated regulation of the TGFβ coreceptor TGFBR3. Accordingly, targeting MUC1-C suppresses HMGA2 mRNA and protein, which is associated with decreases in TGFBR3, reversal of the EMT phenotype, and inhibition of self-renewal capacity. These findings support a model in which MUC1-C activates the ⇑LIN28B→⇓let-7→⇑HMGA2 axis in NSCLC and thereby promotes EMT traits and stemness. A novel pathway is defined in which MUC1-C drives LIN28B→let-7→HMGA2 signaling, EMT, and self-renewal in NSCLC.

MUC1-C activates the TAK1 inflammatory pathway in colon cancer

The mucin 1 (MUC1) oncoprotein has been linked to the inflammatory response by promoting cytokine-mediated activation of the NF-κB pathway. The TGF-β-activated kinase 1 (TAK1) is an essential effector of proinflammatory NF-κB signaling that also regulates cancer cell survival. The present studies demonstrate that the MUC1-C transmembrane subunit induces TAK1 expression in colon cancer cells. MUC1 also induces TAK1 in a MUC1+/−/IL-10−/− mouse model of colitis and colon tumorigenesis. We show that MUC1-C promotes NF-κB-mediated activation of TAK1 transcription and, in a positive regulatory loop, MUC1-C contributes to TAK1-induced NF-κB signaling. In this way, MUC1-C binds directly to TAK1 and confers the association of TAK1 with TRAF6, which is necessary for TAK1-mediated activation of NF-κB. Targeting MUC1-C thus suppresses the TAK1→NF-κB pathway, downregulates BCL-XL, and in turn sensitizes colon cancer cells to MEK inhibition. Analysis of colon cancer databases further indicates that MUC1, TAK1 and TRAF6 are upregulated in tumors associated with decreased survival and that MUC1-C-induced gene expression patterns predict poor outcomes in patients. These results support a model in which MUC1-C-induced TAK1→NF-κB signaling contributes to intestinal inflammation and colon cancer progression.

MUC1 As a Potential Therapeutic Target in Cutaneous T-Cell Lymphoma

Introduction: MUC1 is an oncoprotein that is aberrantly expressed in many epithelial tumors and hematologic malignancies, conferring cell growth and survival. Expression and functional significance of MUC1 in cutaneous T-cell lymphoma (CTCL) has not been previously characterized. In the present study, we evaluated the potential role that MUC1 mediated signaling plays in the pathogenesis of Mycosis Fungoides (MF) and Sezary Syndrome cells (SS), the 2 common subtypes of CTCL.Methods and results: MUC1 was strongly expressed on the SS cell lines HuT-78 (60%), H9 (77%) and SeAx (59%) and the MF cell lines, Myla (94%) and HuT-102 (87%) when incubated with anti-MUC1 antibody and analyzed by flow cytometry (FCM) . In contrast, B-cell lymphoma (BCL) cell lines (Val, K422, Toledo, SU-DHL-2, OCI-Ly7, OCI-Ly8, SU-DHL-5, SU-DHL-8, OCI-Ly3, RCK8, Ramos, Daudi, JVM-2) expressed minimal levels of MUC1 (0-7%), as did circulating T-cell populations isolated from healthy controls (0-3%). The finding of high MUC1 expression on CTCL and minimal expression on BCL and normal circulating T cells was confirmed byimmunoblotting (IB) with another antibody directed against MUC1-C, the oncogenic subunit. The expression profile of MUC1-C was found to be similar at the mRNA level by RT-PCR analysis. We subsequently examined MUC1 as a therapeutic target in CTCL. We have developed an inhibitor of the MUC1-C receptor subunit, designated GO-203, which blocks MUC1-C dimerization necessary for nuclear translocation and downstream signaling. Growth inhibition was determined using CellTiter-Glo that measures ATP produced by live and proliferating cells and demonstrated that the concentration required to inhibit growth by 50% (IC50) following 72-hour exposure ranged from 3 uM to 3.5 uM in most cell lines (HuT-78, H9 and Myla). Culture of HuT-78, H9 and Myla cells in the presence of 5 uM GO-203 concentration for 72-hours resulted in 70, 80 and 90% cell death respectively as determined by Annexin V and propidium iodide staining by FCM. Cell proliferation, as assessed by incorporation of tritiated thymidine, was inhibited by 70% and 80% in 2 cell lines (HuT-78 and Myla respectively) when treated with GO-203 at concentration of 5 uM for 72-hours. To determine mechanism of action of GO-203 induced apoptosis in vitro, level of hydrogen peroxide (H2O2), a reactive oxygen species (ROS) was measured using FCM based conversion of carboxy-2′7′ dichlorodihydrofluorescein diacetate to 2′7′-dichlorodihydrofluorescein. Cell lysates were analyzed for expression of the protein TIGAR, a fructose-2-6-bisphosphatase p53-inducible regulator of glycolysis and apoptosis by IB. Exposure of Hut-78, Myla and H9 cells to GO-203 for 72-hours was associated with an increase in ROS and down regulation of TIGAR expression. To confirm ROS mediated apoptosis by GO-203, in vitro treatment with the antioxidant N-acetylcysteine (NAC) was performed and levels of H2O2 were measured including IB with anti-TIGAR antibody. Treatment with NAC blocked the decreases in TIGAR levels, supporting the hypothesis that this response was mediated by increases in ROS. The ability of GO-203 to eradicate disease was evaluated in vivo across a spectrum of tumor volumes in 2 xenograft mouse models. Five million HuT-78 and 2.5 million Myla cells were injected subcutaneously (SC) into NOD-SCID IL2Rgammanull (NSG) mice. Treatment with GO-203 was administered daily at the dose of 14mg/kg SC for 3 weeks. Tumor volumetric analysis by three-dimensional ultrasound (3D-US) in HuT-78 and Myla xenograft NSG mouse models demonstrated a statistically significant decrease in tumor volume in mice treated with GO-203 compared to control (In the HuT-78 xenograft, average tumor volume was 707 mm3 in the GO-203 treated arm versus 1171 mm3 in the control arm rendering a p-value of 0.0029; whereas in the Myla xenograft average tumor volume was 184 mm3 in the GO-203 treated arm versus 1080 mm3 in the control arm rendering a p-value of 0.0001).Conclusion: The data demonstratesthat MUC1-C is highly expressed in CTCL cell lines, and that blockade of the oncogenic MUC1-C subunit with the peptide inhibitor GO-203 induces apoptosis in vitro and in vivo. Targeting MUC1-C in in vitro is associated with down-regulation of TIGAR and increases in ROS, indicating that MUC1-C functions in maintaining redox balance. Our results highlight the potential of MUC1-C to be an exciting new therapeutic target in CTCL. DisclosuresKufe:Genus Oncology: Consultancy, Equity Ownership.

Targeting the oncogenic MUC1-C protein inhibits mutant EGFR-mediated signaling and survival in non-small cell lung cancer cells.

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. 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. 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. 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.

MUC1-C confers EMT and KRAS independence in mutant KRAS lung cancer cells.

Non-small cell lung cancers (NSCLCs) that harbor an oncogenic KRAS mutation are often associated with resistance to targeted therapies. The MUC1-C transmembrane protein is aberrantly overexpressed in NSCLCs and confers a poor outcome; however, the functional role for MUC1-C in mutant KRAS NSCLC cells has remained unclear. The present studies demonstrate that silencing MUC1-C in A549/KRAS(G12S) and H460/KRAS(Q61H) NSCLC cells is associated with downregulation of AKT signaling and inhibition of growth. Overexpression of a MUC1-C(CQC→AQA) mutant, which inhibits MUC1-C homodimerization and function, suppressed both AKT and MEK activation. Moreover, treatment with GO-203, an inhibitor of MUC1-C homodimerization, blocked AKT and MEK signaling and decreased cell survival. The results further demonstrate that targeting MUC1-C suppresses expression of the ZEB1 transcriptional repressor by an AKT-mediated mechanism, and in turn induces miR-200c. In concert with these effects on the ZEB1/miR-200c regulatory loop, targeting MUC1-C was associated with reversal of the epithelial-mesenchymal transition (EMT) and inhibition of self-renewal capacity. Loss of MUC1-C function also attenuated KRAS independence and inhibited growth of KRAS mutant NSCLC cells as tumors in mice. These findings support a model in which targeting MUC1-C inhibits mutant KRAS signaling in NSCLC cells and thereby reverses the EMT phenotype and decreases self-renewal.

Targeting MUC1-C is synergistic with bortezomib in downregulating TIGAR and inducing ROS-mediated myeloma cell death

AbstractThe proteosome inhibitor bortezomib (BTZ) induces endoplasmic reticulum and oxidative stress in multiple myeloma (MM) cells. The mucin 1 C-terminal subunit (MUC1-C) oncoprotein is aberrantly expressed in most MM cells, and targeting MUC1-C with GO-203, a cell-penetrating peptide inhibitor of MUC1-C homodimerization, is effective in inducing reactive oxygen species (ROS)-mediated MM cell death. The present results demonstrate that GO-203 and BTZ synergistically downregulate expression of the p53-inducible regulator of glycolysis and apoptosis (TIGAR), which promotes shunting of glucose-6-phosphate into the pentose phosphate pathway to generate reduced glutathione (GSH). In turn, GO-203 blocks BTZ-induced increases in GSH and results in synergistic increases in ROS and MM cell death. The results also demonstrate that GO-203 is effective against BTZ-resistant MM cells. We show that BTZ resistance is associated with BTZ-induced increases in TIGAR and GSH levels, and that GO-203 resensitizes BTZ-resistant cells to BTZ treatment by synergistically downregulating TIGAR and GSH. The GO-203/BTZ combination is thus highly effective in killing BTZ-resistant MM cells. These findings support a model in which targeting MUC1-C is synergistic with BTZ in suppressing TIGAR-mediated regulation of ROS levels and provide an experimental rationale for combining GO-203 with BTZ in certain settings of BTZ resistance.