Targeting MUC1-C Inhibits TWIST1 Signaling in Triple-Negative Breast

Published OnlineFirst July 15, 2019; DOI: 10.1158/1535-7163.MCT-19-0156

Patient Focused Therapies in Breast Cancer Molecular Cancer Therapeutics Targeting MUC1-C Inhibits TWIST1 Signaling in Triple-Negative Breast Cancer Tsuyoshi Hata, Hasan Rajabi, Masaaki Yamamoto, Caining Jin, Rehan Ahmad, Yan Zhang, Ling Kui, Wei Li, Yota Yasumizu, Deli Hong, Masaaki Miyo, Masayuki Hiraki,Takahiro Maeda,Yozo Suzuki, Hidekazu Takahashi, Mehmet Samur, and Donald Kufe

Abstract

The oncogenic MUC1-C protein and the TWIST1 epithelial– capacity for cell invasion. We also demonstrate that the mesenchymal transition transcription factor (EMT-TF) are MUC1-C/TWIST1 circuit drives (i) expression of the stem cell aberrantly expressed in triple-negative breast cancer (TNBC) markers SOX2, BMI1, ALDH1, and CD44, (ii) self-renewal cells. However, there is no known association between capacity, and (iii) tumorigenicity. In concert with these results, MUC1-C and TWIST1 in TNBC or other cancer cells. Here, we show that MUC1-C and TWIST1 also drive EMT and we show that MUC1-C activates STAT3, and that MUC1-C and stemness in association with acquired paclitaxel (PTX) resis- pSTAT3 drive induction of the TWIST1 gene. In turn, MUC1-C tance. Of potential therapeutic importance, targeting MUC1-C binds directly to TWIST1, and MUC1-C/TWIST1 complexes and thereby TWIST1 reverses the PTX refractory phenotype as activate MUC1-C expression in an autoinductive circuit. The evidenced by synergistic activity with PTX against drug- functional significance of the MUC1-C/TWIST1 circuit is sup- resistant cells. These findings uncover a master role for ported by the demonstration that this pathway is sufficient for MUC1-C in driving the induction of TWIST1, EMT, stemness, driving (i) the EMT-TFs, ZEB1 and SNAIL, (ii) multiple genes and drug resistance, and support MUC1-C as a highly attractive in the EMT program as determined by RNA-seq, and (iii) the target for inhibiting TNBC plasticity and progression.

Introduction EMT-TFs, such as TWIST1, ZEB1, and SNAIL, are expressed and activated by autocrine and paracrine signaling pathways (7–9). The epithelial–mesenchymal transition (EMT) is a program TWIST1 is a regulator of embryonic morphogenesis that, when in which epithelial cells undergo remodeling of cell–cell junc- expressed in mammary cells, induces loss of cell–cell adhesion tions, loss of apical-basal polarity and the acquisition of in association with EMT and a metastatic phenotype (10, 11). migratory capabilities (1, 2). EMT is essential for embryonic TWIST1 and other EMT-TFs regulate EMT, MET and interme- tissue development (2) and the reprogramming of pluripotent diate states harboring epithelial and mesenchymal features stem cells (3). EMT is also involved in pathologic processes, by orchestrating gene expression patterns in concert with effec- such as wound healing and cancer (2, 4). EMT has been linked tors of epigenetic reprogramming (5, 7). In this way, EMT-TFs to cancer progression, the cancer stem cell (CSC) state and have the capacity to control cellular plasticity by directing resistance to treatment (2, 5, 6). EMT and the reverse process of stable and reversible regulation of genes that dictate the epi- mesenchymal–epithelial transition (MET) are driven in part by thelial and mesenchymal programs. EMT-TF–induced plasticity EMT transcription factors (EMT-TFs), miRNAs and epigenetic has been linked to cancer progression and dedifferentiation by regulatory mechanisms in development and cancer (7, 8). promoting tumor heterogeneity, the CSC state and therapy resistance (5). Accumulating evidence has supported involvement of the Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts. oncogenic MUC1-C protein in the epigenetic reprogramming Note: Supplementary data for this article are available at Molecular Cancer of cancer cells (12). MUC1-C is positioned at the apical Therapeutics Online (http://mct.aacrjournals.org/). borders of normal epithelial cells, where it is poised to Current address for R. Ahmad: College of Medicine, King Saud University, Riyadh, respond to stress signals, such as those associated with Saudi Arabia; current address for M. Miyo, Y. Suzuki, and H. Takahashi: Depart- inﬂammation and damage (13). MUC1-C contributes to loss ment of Gastrointestinal Surgery, Graduate School of Medicine, Osaka Univer- of epithelial cell polarity by downregulating Crumbs complex sity, Suita, Osaka, Japan; and current address for T. Maeda: Department of components (CRB3, PATJ), HUGL2 (14) and the adherens Urology, Keio University School of Medicine, Shinjuku-ku, Tokyo, Japan. junction effector E-cadherin (15). In association with driving Corresponding Author: Donald Kufe, Dana-Farber Cancer Institute, Harvard loss of polarity, MUC1-C is expressed at increased levels over Medical School, 450 Brookline Avenue, DANA 830, Boston, MA 02215. the entire surface of carcinoma cells, where it interacts with Phone: 617-632-3141; Fax: 617-632-2934; E-mail: [email protected] receptor tyrosine kinases (RTK), such as EGFR and HER2, which are normally positioned at basolateral borders, and Mol Cancer Ther 2019;18:1744–54 promotes their activation (13, 16). MUC1-C is also targeted doi: 10.1158/1535-7163.MCT-19-0156 to the nucleus, where it directly interacts with proinﬂamma- 2019 American Association for Cancer Research. tory TFs, including NF-kB p65 and STAT3, and contributes to

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MUC1-C Drives a Program of EMT, Stemness, and Drug Resistance

the regulation of their target genes (12). A mechanistic role Real-time quantitative reverse-transcription PCR (qRT-PCR) for MUC1-C in gene regulation was advanced by the dem- Total RNA was isolated using TRizol reagent (Invitrogen). onstration that MUC1-C activates the Polycomb Repressive cDNAs were generated using the High Capacity cDNA Reverse Complex 1 (PRC1) and BMI1-directed ubiquitylation of H2A Transcription Kit (Applied Biosystems; ref. 18). Samples were with depression of homeobox genes (17). MUC1-C also amplified using the Power SYBR Green PCR Master Mix (Applied activates PRC2, interacts with EZH2 and drives increases in Biosystems) and the CFX96 Touch Real-Time PCR Detection H3K27 trimethylation (H3K27me3) on promoters of the System (Bio-Rad Laboratories). Primers used for qRT-PCR anal- CDH1 and BRCA1 TSGs (18). In addition, MUC1-C induces ysis are listed in Supplementary Table S1. the DNA methyltransferases (DNMT) 1 and 3b with increases in promoter-specific DNA methylation patterns that further Immunoblot analysis contribute to repression of TSGs (19). These findings have Whole-cell and nuclear lysates were prepared in RIPA buffer collectively supported a role for MUC1-C in inducing epige- containing protease inhibitor cocktail (Thermo Scientific). Immu- netic modifications that contribute to progression of the noblotting was performed with anti–MUC1-C (HM-1630- cancer cell (12). P1ABX; Thermo Fisher Scientific), anti-TWIST1 (ab50887), TWIST1 is expressed at low to undetectable levels in ER-positive anti–NF-kB p65 (ab16502), anti-ALDH1 (ab134188; Abcam), breast cancer cells and is upregulated in TNBC cells (20). In anti-pSTAT3 (#9145), anti-STAT3 (#9139), anti-ZEB1 (#3396), addition, TWIST1 expression is associated with decreases in TNBC anti-SNAIL (#3879), anti-SOX2 (#3579), anti-ABCB1 (#13342), disease-free and overall survival (21). MUC1 is also expressed in anti–N-cadherin (#13116S), anti-BMI1 (#6964P), anti-CD44 TNBC; however, there is no known relationship between MUC1 (#5640S), anti-GAPDH (#5174S; Cell Signaling Technology), and TWIST1. The present studies support an autocrine function and anti–b-actin (#A5441; Sigma). for MUC1-C in activating STAT3 and inducing expression of the TWIST1 EMT-TF in TNBC cells. We show that MUC1-C binds to Chromatin immunoprecipitation assays TWIST1, forms an autoregulatory loop with TWIST1 and func- Soluble chromatin was precipitated with anti–MUC1-C, anti- tions as a master regulator of EMT, stemness, and drug resistance. STAT3, anti-TWIST1 or a control non-immune IgG (Santa Cruz These findings highlight the importance of targeting MUC1-C as a Biotechnology). The precipitates were analyzed by qPCR using the potential therapeutic approach for suppressing phenotypic plas- Power SYBR Green PCR Master Mix and the ABI Prism 7300 ticity of TNBCs. sequence detector (Applied Biosystems). Data are reported as fold enrichment relative to the IgG control (19). Primers used for chromatin immunoprecipitation (ChIP) qPCR are listed in Sup- Materials and Methods plementary Table S2. Cell culture Human BT-549 basal B TNBC cells (ATCC) were cultured in RNA-seq and bioinformatics analysis RPMI-1640 medium (Corning) containing 10% FBS, 100 mg/mL Total RNA was isolated from cells cultured in triplicates using streptomycin, 100 U/mL penicillin and 10 mg/mL insulin. TRizol reagent (Invitrogen). TruSeq Stranded mRNA (Illumina) SUM149 basal B TNBC cells (22) were grown in Ham's F-12 was used for library preparation. Data were analyzed using the medium (Corning) supplemented with 10 mmol/L HEPES, 5% Hallmark Gene Set Molecular Signatures Database (24) and FBS, 100 mg/mL streptomycin, 100 U/mL penicillin, 5 mg/mL Metacore. The accession number for the RNA-seq data reported insulin and 1 mg/mL hydrocortisone. Cells were treated with the in this paper is GEO ACCESSION GSE134147. STAT3 inhibitor S3I-201 (Sigma), the MUC1-C inhibitor GO- 203, which has the D-amino acid sequence [R]9-CQCRRKN (23), Colony formation assays and paclitaxel (PTX; Selleckchem). PTX-resistant cells were gen- Cells were seeded at 3,000 per well in 6-well plates, treated with erated by continuous exposure to increasing PTX concentrations 500 ng/mL DOX for 7 days and then stained with 0.5% crystal over 5 to 6 months. Cell number and viability were assessed by violet in 25% methanol. Colonies >25 cells were counted in trypan blue exclusion. Authentication of the cells was performed triplicate wells. by short tandem repeat (STR) analysis. Cells were monitored for mycoplasma contamination using the MycoAlert Mycoplasma Invasion assays Detection Kit (Lonza). Cell invasion assays were performed in Transwell chambers precoated with Matrigel as described previously (15). Tetracycline-inducible and stable gene silencing and overexpression Mammosphere assays MUC1shRNA (MISSION shRNA TRCN0000122938; Sigma) or Single-cell suspensions were cultured in MammoCult Human a control scrambled shRNA (CshRNA; Sigma) was inserted into Medium Kit (Stemcell Technologies) at a density of 5,000 cells per the pLKO-tet-puro vector (Plasmid #21915; Addgene). MUC1-C well of a 6-well ultralow attachment culture plate (Corning) and (AQA) cDNA was inserted into the pinducer20 vector (Plasmid treated with DMSO or 500 ng/mL DOX (Sigma) for 7 days. #44012; Addgene). The CshRNA, MUC1shRNA, NF-kB Mammospheres with a diameter 50 mm were counted under p65shRNA (MISSION shRNA TRCN0000014685; Sigma), a microscope. STAT3shRNA (MISSION shRNA TRCN0000020543; Sigma) and TWIST1shRNA (MISSION shRNA TRCN0000329811; Sigma) Mouse tumor model studies vectors were produced in HEK293T cells as described previous- Six-week-old female nude mice (Taconic Farms) were injected ly (18). Cells transduced with the vectors were selected for growth subcutaneously in the flank with 3 106 SUM149/tet- in 1–3 mg/mL puromycin or 100 mg/mL geneticin. MUC1shRNA cells in 50% Matrigel. When tumor size reached

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approximately 125 mm3, the mice were pair-matched and fed findings, targeting STAT3 genetically (Supplementary Fig. S3A without or with DOX (625 ppm, daily). Tumor measurements and S3B) or with the S3I-201 inhibitor (Supplementary Fig. S3C) and body weights were recorded twice each week. Mice were decreased TWIST1 expression, suggesting that MUC1-C induces sacrificed when tumors reached >1,000 mm3 as calculated by the TWIST1 in TNBC cells by a STAT3-mediated mechanism. formula: (width)2 length/2. These experiments were conducted in accordance with and approved by the Dana-Farber Cancer MUC1-C is necessary for driving TWIST1 activation Institute Animal Care and Use Committee (IACUC) under pro- The MUC1-C cytoplasmic domain contains a CQC motif that is tocol 03-029. necessary for MUC1-C homodimerization, nuclear import and oncogenic function (Fig. 2A; ref. 16). Treatment of BT-549 and Immunofluorescence (IF) microscopy SUM149 cells with the cell-penetrating GO-203 peptide, which Cells were fixed in 4% paraformaldehyde, incubated with 0.3% targets the CQC motif, decreased pSTAT3 and TWIST1 levels Triton X-100 and blocked with 3% BSA. The fixed cells were (Fig. 2B). In addition, DOX-induced overexpression of tet- incubated with anti–MUC1-C or anti-TWIST1 at 4C and then, MUC1-C with a CQC!AQA mutation, which functions as a after washing, with goat anti-Armenian hamster IgG H&L labeled dominant-negative for MUC1-C function (29), was associated with Alexa Fluor 488 (Abcam) and anti-mouse IgG H&L labeled with suppression of pSTAT3 and downregulation of TWIST1 with Alexa Fluor 647 (Abcam) at room temperature. DAPI (Fig. 2C), further supporting the notion that MUC1-C activates (Thermo Fisher Scientific) was used for staining of nuclei. pSTAT3 and thereby TWIST1 expression. The TWIST1 promoter contains three potential STAT3 binding motifs in the region from Cell viability and combination index positions 60 to 162 upstream to the transcription start site Cells were seeded at a density of 1,000 to 2,000 cells per well in (ref. 27; Fig. 2D). In this respect, we found that MUC1-C forms a 96-well plates. After 24 hours, the cells were treated with different nuclear complex with STAT3 in BT-549 and SUM149 cells (Sup- concentrations of GO-203 and PTX alone and in combination. plementary Fig. S4A and S4B). ChIP studies further demonstrated Cell viability was assessed by Alamar blue staining (Invitrogen) that MUC1-C and STAT3 occupy this region (Fig. 2E) and, and the Cell Counting Kit (DOJINDO, Kumamoto, Japan) of 6 importantly, that silencing MUC1-C decreases STAT3 occupancy replicates. The IC50 value was determined by non-linear regres- (Fig. 2F). Targeting MUC1-C with DOX-induced silencing or GO- sion of the dose–response data using Prism 7.0 (GraphPad 203 treatment was also associated with downregulation of Software). Drug interaction and dose–effect relationships were TWIST1 promoter activation as assessed using a pTWIST1-Luc analyzed as described previously (25). The combination index reporter (Fig. 2G and H), demonstrating that MUC1-C is neces- (CI) was calculated to assess synergism (CI<1) or antagonism sary for driving TWIST1 activation. (CI>1). MUC1-C and TWIST1 form an autoregulatory circuit Statistical analysis The MUC1-C cytoplasmic domain is an intrinsically disordered Each experiment was repeated at least three times. Data are structure that has the capacity to interact with multiple TFs, expressed as the mean SD. The unpaired Student t test was used including NF-kB, ZEB1 and STAT3 (15, 28, 30, 31). Accordingly, to assess differences between means of two groups. A P value of coimmunoprecipitation studies were performed to determine <0.05 was considered a statistically significant difference. whether MUC1-C also associates with TWIST1. In this way, we found that MUC1-C/TWIST1 complexes are detectable in the nucleus (Fig. 3A). In assessing whether the interaction is direct, Results we found that GST–MUC1–CD binds to purified recombinant MUC1-C induces TWIST1 expression in TNBC cells TWIST1 in vitro and that the CQC motif is dispensable for binding MUC1-C and TWIST1, which are constitutively expressed in (Fig. 3B). Experiments with MUC1-CD deletion fragments further TNBC cells, have been linked to the induction of EMT; however, demonstrated that MUC1-CD(46-72), and not MUC1-CD(1-45), there is no reported relationship between these proteins (15, 26). confers the interaction (Fig. 3C). Of interest in this regard, we Accordingly, BT-549 cells expressing a tet-inducible control found that the MUC1 promoter contains two consensus TWIST1 CshRNA or MUC1shRNA were treated with DOX and analyzed binding motifs (Fig. 3D). ChIP assays performed to determine if for TWIST1 expression. We found that downregulation of MUC1 MUC1-C and TWIST1 form a complex on the MUC1 promoter (Fig. 1A) is significantly associated with decreases in TWIST1 demonstrated that MUC1-C and TWIST1 are both detectable on mRNA levels (Fig. 1B). Similar results were obtained in DOX- the region containing the TWIST1 motifs (Fig. 3E, left). Re-ChIP treated SUM149/tet-CshRNA and SUM149/tet-MUC1shRNA studies further demonstrated that MUC1-C is detectable in a cells (Fig. 1C and D). TWIST1 is activated by NF-kB, STAT3 and complex with TWIST1 (Fig. 3E, right). Notably, silencing other TFs (27). MUC1-C activates the ZEB1 EMT-TF by NF-kB MUC1-C decreased TWIST1 occupancy (Fig. 3F), indicating that p65–mediated signaling (15). However, silencing NF-kB p65 had MUC1-C directly contributes to activation of TWIST1 on the no apparent effect on TWIST1 expression, supporting a distinct MUC1 promoter. In concert with these results, we found that regulatory mechanism (Supplementary Fig. S1A and S1B). Stud- silencing TWIST1 is associated with suppression of MUC1-C ies in BC cells have shown that the MUC1-C cytoplasmic domain mRNA levels (Fig. 3G), supporting a model in which MUC1-C binds directly to JAK1 and STAT3, and in turn drives JAK1- and TWIST1 form an autoregulatory loop. mediated STAT3 phosphorylation (28). Silencing MUC1-C in the TNBC cells had no detectable effect on STAT3 mRNA levels MUC1-C/TWIST1 circuit promotes EMT and stemness (Supplementary Fig. S2A and S2B). Instead, we found that silenc- EMT-TFs can function cooperatively to induce one another ing MUC1-C in BT-549 (Fig. 1E) and SUM149 (Fig. 1F) cells and regulate common sets of target genes (4, 8). In this respect results in suppression of pSTAT3 and TWIST1. In support of these and in addition to TWIST1, silencing MUC1-C was associated

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MUC1-C Drives a Program of EMT, Stemness, and Drug Resistance

Figure 1. Targeting MUC1-C suppresses TWIST1 expression. A–F, BT-549 and SUM149 cells stably expressing a tet-CshRNA or tet- MUC1shRNA were left untreated or treated with 500 ng/mL DOX for 7 days. Cells were analyzed for MUC1-C (A and C) and TWIST1 (B and D) mRNA levels by qRT-PCR using primers listed in Supplementary Table S1. The results (mean SD) are expressed as relative mRNA levels compared with that obtained for control untreated cells (assigned a value of 1). , P <0.05. Lysates were immunoblotted with antibodies against the indicated proteins (E and F).

with suppression of ZEB1 and SNAIL expression (Fig. 4A, left consistent with suppression of the EMT phenotype. Targeting and right). RNA-seq analysis further demonstrated that MUC1- MUC1-C was also associated with (i) downregulation of the C is highly associated with driving the EMT program as breast cancer stem cell markers SOX2, BMI1, ALDH1 and supported by the most significant correlation observed among CD44 (refs. 32, 33; Fig. 4C). In support of the MUC1-C/ the different Hallmark gene sets (Fig. 4B; Supplementary TWIST1 autoinductive circuit, we found that silencing TWIST1 Fig. S5 and S6). In addition, Metacore pathway analysis docu- similarly downregulates ZEB1, SNAIL, and N-cadherin, as well mented significant correlations for MUC1-C with adherens as the breast cancer stem cell markers (Fig. 4D). In addition, junction disassembly and the EMT program (Supplementary silencing MUC1-C was associated with (i) inhibition of growth Fig. S7 and S8), demonstrating that MUC1-C regulates mul- (Supplementary Fig. S10A and S10B), (ii) decreases in colony tiple genes, including E-cadherin, vimentin, fibronectin and formation (Fig. 4E; Supplementary Fig. S11) and loss of self- PDGF, among others, and signaling pathways associated with renewal capacity as determined by decreased mammosphere the EMT state. In concert with those results, silencing MUC1-C formation (Fig. 4F). Moreover, DOX-induced suppression of was associated with decreases in N-cadherin (Fig. 4C) and the MUC1-C attenuated tumorigenicity (Fig. 4G) in association capacity for invasion (Supplementary Fig. S9A and S9B), with decreases in TWIST1, ZEB1, SNAIL and N-cadherin

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Figure 2. MUC1-C drives TWIST1 activation. A, Schema of MUC1-C with the extracellular domain (ED), transmembrane (TM) region, and amino acid sequence of the intrinsically disordered cytoplasmic domain (CD). Highlighted is the CQC motif, which is necessary for MUC1-C homodimerization and nuclear import. Mutation of

the CQC motif to AQA inactivates MUC1-C function. The CQC motif is the target of the cell-penetrating GO-203 inhibitor (R9-CQCRRKN). Also highlighted is the STAT3 binding site. B, BT-549 (left) and SUM149 (right) cells were left untreated or treated with 5 mmol/L GO-203 for 48 hours. Lysates were immunoblotted with antibodies against the indicated proteins. C, BT-549 cells expressing a tet-MUC1-C(AQA) vector were left untreated or treated with 500 ng/mL DOX for 7 days. Lysates were analyzed by immunoblotting with antibodies against the indicated proteins. D, Schema of the TWIST1 promoter with depiction of putative STAT3 binding motifs. E, Soluble chromatin from BT-549 cells was precipitated with anti–MUC1-C, anti-STAT3, or a control IgG. The DNA samples were ampliﬁed by qPCR with primers for the TWIST1 promoter (Supplementary Table S2). The results (mean SD of three determinations) are expressed as the relative fold enrichment compared with that obtained with the IgG control (assigned a value of 1). F, BT-549/tet-MUC1shRNA cells were left untreated or treated with 500 ng/mL DOX for 7 days. Soluble chromatin was precipitated with anti-STAT3 or a control IgG. The DNA samples were ampliﬁed by qPCR with primers for the TWIST1 promoter. The results (mean SD of three determinations) are expressed as the relative fold enrichment compared with that obtained with the IgG control (assigned a value of 1). G, BT-549/tet-MUC1shRNA cells were left untreated or treated with 500 ng/mL DOX for 5 days. H, BT-549 cells were left untreated or treated with 5 mmol/L GO-203 for 48 hours. The cells were transfected with a pTWIST1-Luc reporter for 48 hours and then analyzed for Luc activity. The results (mean SD) are expressed as relative Luc activity compared with that obtained for control untreated cells (assigned a value of 1).

(Fig. 4H; Supplementary Fig. S12). We also found that target- downregulation of TWIST1, ABCB1, SOX2, ALDH1, and ing MUC1-C in vivo suppresses expression of SOX2, BMI1, CD44 (Fig. 5E), and increased sensitivity to PTX treatment ALDH1, and CD44 (Fig. 4H), supporting a role for MUC1-C in (Fig. 5F), indicating that MUC1-C contributes to this program integrating EMT and stemness. of drug resistance. In extending these results, BT-549/PTX-R cells were treated MUC1-C/TWIST1 circuit promotes drug resistance with the GO-203 inhibitor, which directly blocks MUC1-C To investigate the potential role of MUC1-C/TWIST1 sig- function and is selective against MUC1-C expressing naling in drug resistance, we selected BT-549 cells for survival cells (35–37). GO-203 treatment was associated with suppres- in the presence of increasing concentrations of paclitaxel sion of MUC1-C, TWIST1, ABCB1, SOX2, BMI1, ALDH1 and (PTX; Supplementary Fig. S13A). As compared with drug CD44 expression (Fig. 6A), further indicating that MUC1-C na€ve BT-549 cells, we found partial decreases in MUC1-C contributes EMT and stemness in the setting of PTX resistance. levels and upregulation of TWIST1, the ABCB1 drug trans- In support of this notion, we found that combining GO-203 porter and SOX2, which also contributes to stemness and and PTX is synergistic against BT-549/PTX-R cells with CI values drug resistance (ref. 34; Fig. 5A). Immunoﬂuorescence less than 1.0 (Fig. 6B and C; Supplementary Fig. S13B and microscopy studies showed increased TWIST1 expression in S13C; Supplementary Table S3), demonstrating that (i) both the nucleus (Fig. 5B). In concert with these results, we also agents contribute to loss of viability, and (ii) targeting MUC1-C detected increases in the occupancy of TWIST1 complexes on reverses PTX resistance. Drug resistance is associated with the the MUC1 (Fig. 5C) and ABCB1 (Fig. 5D) promoters. Impor- CSC state (6, 38). In this respect and in concert with the above tantly, silencing MUC1-C in BT-549/PTX-R cells resulted in results, we found that the mammosphere forming capacity of

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Figure 3. MUC1-C forms a complex with TWIST1 on the MUC1 promoter. A, Nuclear lysates were incubated with anti–MUC1-C or a control IgG. The input and precipitates were analyzed by immunoblotting with antibodies against the indicated proteins. B and C, Purified recombinant TWIST1 was incubated with GST or the indicated GST–MUC1–CD fusion proteins. Precipitates obtained with glutathione beads were immunoblotted with anti-TWIST1. Loading of the GST proteins was analyzed by Coomassie Blue staining. D, Schema of the MUC1 promoter with highlighting of the putative TWIST1-binding motifs. E, Soluble chromatin was precipitated with anti–MUC1-C, anti-TWIST1, or a control IgG (left). Soluble chromatin was precipitated with anti–MUC1-C (ChIP) and then re-precipitated with anti-TWIST1 or a control IgG (re-ChIP; right). The DNA samples were amplified by qPCR with primers for the MUC1 promoter. The results (mean SD of three determinations) are expressed as the relative fold enrichment compared with that obtained with the IgG control (assigned a value of 1). F, BT-549/tet-MUC1shRNA cells were left untreated or treated with 500 ng/mL DOX for 7 days. Soluble chromatin was precipitated with anti-TWIST1 or a control IgG. The DNA samples were amplified by qPCR with primers for the MUC1 promoter. The results (mean SD of three determinations) are expressed as the relative fold enrichment compared with that obtained with the IgG control (assigned a value of 1). G, BT-549/CshRNA and BT-549/TWIST1shRNA cells were analyzed for TWIST1 and MUC1-C mRNA levels by qRT-PCR. The results (mean SD) are expressed as relative mRNA levels compared with that obtained for control cells (assigned a value of 1).

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Figure 4. MUC1-C promotes EMT and stemness. A and B, BT-549/tet-MUC1shRNA cells left untreated or treated with 500 ng/mL DOX for 7 days were analyzed for ZEB1 and SNAIL mRNA levels. The results (mean SD) are expressed as relative mRNA levels compared with that obtained for control cells (assigned a value of 1; left). Lysates were analyzed by immunoblotting (right). B, RNA-seq was performed in triplicate on (i) BT-549/CshRNA and BT-549/MUC1shRNA and (ii) SUM149/ CshRNA and SUM149/MUC1shRNA cells. The datasets were analyzed using the Hallmark gene signature collection. EMT was identified as the pathway with the highest association (Supplementary Fig. S5 and S6). C, Lysates from BT-549/tet-MUC1shRNA cells left untreated or treated with 500 ng/mL DOX were immunoblotted with antibodies against the indicated proteins. D, Lysates from BT-549/CshRNA and BT-549/TWIST1shRNA cells were immunoblotted with antibodies against the indicated proteins. E, BT-549/tet-MUC1shRNA cells left untreated (blue bars) or treated with 500 ng/mL DOX (red bars) for 7 days were analyzed for colony formation. The results (mean SD of at least three determinations) are expressed as the number of colonies/field. F, BT-549/tet- MUC1shRNA cells left untreated (blue bars) or treated with 500 ng/mL DOX (red bars) for 7 days were analyzed for mammosphere formation. The results (mean SD of at least three determinations) are expressed as the number of mammospheres/well. G, Six-week-old nude female mice were injected subcutaneously in the flank with 3 106 SUM149/tet-MUC1shRNA cells. Mice were pair-matched into two groups when tumor volumes reached 100–150 mm3 and were fed without (blue circles) and with DOX (red circles). Tumor volumes are expressed as the mean SD for 6 mice. H, Lysates from tumors harvested on day 21 were immunoblotted with antibodies against the indicated proteins.

BT-549/PTX-R cells is increased as compared with drug-na€ve Discussion cells (Fig. 6D). Moreover, targeting MUC1-C with silencing EMT has been linked to cancer cell progression, stemness, and (Fig. 6E) or treatment with GO-203 (Fig. 6F) signiﬁcantly drug resistance (2). The cellular plasticity among EMT, MET and decreased BT-549/PTX-R mammosphere formation. Notably, their intermediate states contributes to tumor heterogeneity and complete silencing of MUC1-C is not achievable with the tet- thereby emergence of more aggressive CSC populations (2). MUC1shRNA and, as a result, suppression of mammosphere Surprisingly, how these clinically important hallmarks of the formation is also not complete (Fig. 6E). In contrast, treatment cancer cell are interconnected has largely remained unclear (6). with GO-203, which suppresses MUC1-C expression in part The TWIST1 EMT-TF is an essential regulator of cell migration and and inhibits function of remaining MUC1-C by direct binding, morphogenesis during embryonic development (27). In contrast completely blocks mammosphere formation (Fig. 6F).

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MUC1-C Drives a Program of EMT, Stemness, and Drug Resistance

Figure 5. MUC1-C drives drug resistance. A, Lysates from drug-na€ve (WT) BT-549 and BT-549/PTX-R cells were immunoblotted with antibodies against the indicated proteins. B, BT-549 WT and PTX-R cells were analyzed for nuclear MUC1-C and TWIST1 localization by IF microscopy. C and D, Chromatin from BT-549 WT and PTX-R cells was precipitated with anti–MUC1-C, anti-TWIST1, or a control IgG. The DNA samples were ampliﬁed by qPCR with primers for the MUC1 (C)andABCB1 (D) promoters. The results (mean SD of three determinations) are expressed as the relative fold enrichment compared with that obtained with the IgG control (assigned a value of 1). E, Lysates from BT-549/PTX-R/tet-MUC1shRNA cells left untreated or treated with 500 ng/mL DOX for 7 days were analyzed by immunoblotting. F, BT-549/PTX-R/tet-MUC1shRNA cells were left untreated or treated with 500 ng/mL DOX for 7 days. The cells were then exposed to vehicle control (CTR) or 30 mmol/L PTX for 72 hours. The results (mean SD) are expressed as relative survival as compared with that obtained with DOX-untreated/CTR cells (assigned a value of 1).

with ER-positive breast cancer cells that have low to undetectable that, unlike ZEB1, MUC1-C induces TWIST1 expression by acti- levels of TWIST1 expression, TWIST1 is upregulated in TNBC cells vation of the proinﬂammatory STAT3 pathway. MUC1-C has a and promotes their invasion, genetic instability and CSC unique capacity to associate with TFs, such as NF-kB p65 and state (20, 21, 39). The oncogenic MUC1-C protein has been ZEB1, and to promote their occupancy on target genes (12). Along linked to the induction of EMT by activating the NF-kB p65 these lines, we found that MUC1-C induces pSTAT3 in TNBC cells pathway and in turn expression of the ZEB1 EMT-TF (15). By and, in turn, pSTAT3-mediated activation of TWIST1 expression. contrast and importantly, there has been no reported link Mechanistically, MUC1-C and STAT3 form a complex on the between MUC1-C and TWIST1. The present studies demonstrate TWIST1 promoter and activate TWIST1 transcription. Our results

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Hata et al.

Figure 6. Targeting MUC1-C reverses PTX resistance and decreases self-renewal capacity. A, Lysates from BT-549/ PTX-R cells left untreated or treated with 5 mmol/L GO-203 for 48 hours were analyzed by immunoblotting. B and C, BT-549/PTX-R cells were left untreated or treated with 0.1, 0.3, or 1.0 mmol/L GO-203 in the absence or presence of the indicated PTX concentrations. Cell viability (mean SD of at least three determinations) was determined by Alamar blue staining (B) and was used to calculate the combination index (CI) values (C), which at <1.0 denote synergism. D–F, BT-549 drug-na€ve (WT) and BT-549/PTX-R cells (D), BT-549/ PTX-R/tet-MUC1shRNA cells left untreated or treated with 500 ng/mL DOX for 7 days (E), and BT-549/PTX-R cells left untreated and treated with 5 mmol/L GO-203 for 7 days (F) were analyzed for mammosphere formation. The results (mean SD of at least three determinations) are expressed as the number of mammospheres/well. G, Proposed model for involvement of MUC1-C in integrating TWIST1, EMT, stemness, and drug resistance. MUC1-C activates the STAT3 pathway and is necessary for TWIST1 activation. In turn, MUC1-C binds directly to TWIST1 and forms an autoregulatory MUC1-C!TWIST1 circuit. Importantly, our results show that this circuit is sufﬁcient for driving (i) ZEB1 and SNAIL expression, (ii) multiple genes associated with the EMT program and invasion, (iii) BC stem cell markers, (iv) self-renewal and tumorigenicity, and (v) ABCB1 with the acquisition of drug resistance. , P <0.05.

further demonstrate that targeting MUC1-C decreases STAT3 way reciprocally contributing to the regulation of MUC1-C occupancy on the TWIST1 promoter and downregulates TWIST1 expression. Indeed, the identification of potential TWIST1 bind- mRNA and protein. These findings support a previously unrec- ing motifs in the MUC1 promoter and the demonstration that ognized model in which MUC1-C is necessary for pSTAT3-medi- MUC1-C/TWIST1 complexes occupy those sites, invoked the ated TWIST1 activation (Fig. 6G). possibility that MUC1-C and TWIST1 function in an autoinduc- The MUC1-C cytoplasmic domain is an intrinsically disordered tive manner. In support of a MUC1-C/TWIST1 circuit, (i) targeting protein with the plasticity to directly interact with TFs and MUC1-C suppressed TWIST1 occupancy on the MUC1 promoter epigenetic modulators of gene expression (12). In this way, and (ii) silencing TWIST1 downregulated MUC1-C expression MUC1-C induces ZEB1 expression and, in turn, binds to ZEB1 (Fig. 6G). These findings support an autoregulatory loop; how- with the formation of MUC1-C/ZEB1 complexes on the miR-200c ever, they do not exclude a role for TWIST1 in the fine-tuning of promoter and induction of the EMT program (15). In concert with MUC1 activation as may have been observed with upregulation those findings and the demonstration that MUC1-C drives of TWIST1 and partial suppression of MUC1-C in the setting of TWIST1 expression, we also found that MUC1-C directly associ- PTX resistance. Induction of the EMT program is associated with ates with TWIST1 and that MUC1-C/TWIST1 complexes are the activation of TWIST1, as well as other EMT-TFs (8, 40). constitutively detectable in the nucleus. During the course of Notably, disruption of the MUC1-C/TWIST1 circuit was also these experiments, it became apparent that TWIST1 was in some associated with suppression of ZEB1 and SNAIL, in concert

1752 Mol Cancer Ther; 18(10) October 2019 Molecular Cancer Therapeutics

MUC1-C Drives a Program of EMT, Stemness, and Drug Resistance

with a role for MUC1-C in driving expression of multiple EMT-TFs dules, which were cumbersome for patients. Accordingly, GO-203 (refs. 15, 41; Fig. 6G). These findings and the results of RNA-seq has been reformulated in nanoparticles for weekly administration analyses demonstrating highly significant correlations between and further clinical evaluation (52). Antibodies generated against MUC1-C and the EMT program provide evidence that MUC1-C the MUC1-C extracellular domain have also been developed to contributes to the regulation of multiple genes associated with the target MUC1-C–expressing tumors with antibody-drug conju- EMT state. Our findings that MUC1-C is sufficient to drive EMT gates and other immune-based therapies (53). When taken in and invasive capacity in TNBC cells further support an autocrine the context of a role for MUC1-C in driving stemness and process that may be independent of paracrine TGFb or inflam- resistance to treatment, the availability of these agents could matory cytokines. provide the basis for designing more effective therapeutic strat- In cancer cells, activation of the EMT program is linked to egies. In summary, our findings represent a previously unrecog- acquisition of the CSC state, a characteristic consistent with a nized paradigm for MUC1-C–induced integration of TWIST1, process of dedifferentiation (42–44). Relatively few insights are EMT, stemness and drug resistance, and support the targeting of available regarding the mechanistic basis for these highly MUC1-C for inhibiting cancer progression (Fig. 6G). interconnected programs. MUC1-C activates the BMI1 and ALDH1A1 effectors of BC stem cells (17, 45). However, it was Disclosure of Potential Conflicts of Interest not known if MUC1-C contributes to the interconnectivity D. Kufe is on the board of directors at Genus Oncology and Hillstream between EMT and stemness. The present results provide evi- BioPharma; a consultant at Genus Oncology, Reata Pharmaceuticals, CanBas dence that, in concert with induction of EMT, MUC1-C drives Co. Ltd., and Victa Biotherapeutics; is a scientificadvisoryboardmember self-renewal capacity. In this way, targeting MUC1-C genetically for Nanogen Therapeutics; and is on the board of directors and has own- or pharmacologically suppressed (i) the BC stem cell markers, ership interest (including patents) at Genus Oncology, Reata Pharmaceu- fl SOX2, BMI1, ALDH1, and CD44, (ii) mammosphere formation ticals, Victa Biotherapeutics, Nanogen Therapeutics. No potential con icts of interest were disclosed by the other authors. and tumorigenicity, indicating that MUC1-C also promotes stemness. Other evidence has linked MUC1-C to the epigenetic regulation of gene expression by interacting with pathways of Authors' Contributions effectors, such as DNMTs, BMI1/PRC1 and EZH2/PRC2 (18). Conception and design: T. Hata, M. Miyo, D. Kufe Further studies will now be needed to define how MUC1-C may Development of methodology: T. Hata, M. Miyo, Y. Suzuki, H. Takahashi integrate epigenetic modifications with the EMT program, Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): T. Hata, H. Rajabi, M. Yamamoto, C. Jin, Y. Zhang, stemness and process of dedifferentiation. EMT and the CSC W. Li, Y. Yasumizu, D. Hong, M. Miyo, T. Maeda, H. Takahashi state are intimately linked to the development of drug resis- Analysis and interpretation of data (e.g., statistical analysis, biostatistics, tance (6, 38). In cancer cells, MUC1-C localizes to the mito- computational analysis): T. Hata, H. Rajabi, M. Yamamoto, R. Ahmad, L. Kui, chondrial outer membrane, where it attenuates the intrinsic D. Hong, M. Miyo, M. Samur apoptotic response to cytotoxic anticancer agents (46–48). Writing, review, and/or revision of the manuscript: T. Hata, H. Rajabi, MUC1-C also confers resistance to targeted agents, such as M. Yamamoto, M. Miyo, H. Takahashi, M. Samur, D. Kufe Administrative, technical, or material support (i.e., reporting or organizing tamoxifen and trasutuzumab (49, 50), and contributes to the data, constructing databases): T. Hata, M. Miyo, M. Hiraki fi evasion of immune destruction (51). These ndings have Study supervision: T. Hata, H. Takahashi, D. Kufe collectively supported a potential role for MUC1-C in pleo- Other (bioinformatics analysis): L. Kui tropic states, such as heterogeneity and stemness, which can protect tumors against treatment. In support of this premise, we Acknowledgments found that MUC1-C plays a role in the acquisition of PTX Research reported in this publication was supported by the National Cancer resistance in association with driving expression of TWIST1, Institute of the National Institutes of Health under grant numbers CA97098, ABCB1 and SOX2, BMI1, ALDH1, and CD44. Importantly in CA166480, CA216553, and CA229716 (to D.W. Kufe). this respect, targeting MUC1-C with GO-203 (i) suppressed TWIST1, ABCB1 and the BC stem cell markers, (ii) reversed PTX The costs of publication of this article were defrayed in part by the resistance, and (iii) conferred synergistic activity with PTX payment of page charges. This article must therefore be hereby marked against these drug-resistant cells. advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate Regarding potential clinical relevance, a Phase I trial of GO-203 this fact. in patients with solid tumors demonstrated an acceptable safety profile and evidence of antitumor activity. However, a short Received February 14, 2019; revised May 21, 2019; accepted July 8, 2019; circulating GO-203 half-life necessitated frequent dosing sche- published first July 15, 2019.

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1754 Mol Cancer Ther; 18(10) October 2019 Molecular Cancer Therapeutics

Targeting MUC1-C Inhibits TWIST1 Signaling in Triple-Negative Breast Cancer

Tsuyoshi Hata, Hasan Rajabi, Masaaki Yamamoto, et al.

Mol Cancer Ther 2019;18:1744-1754. Published OnlineFirst July 15, 2019.

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