Yozo Suzuki

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.

Emerging Methods for Preparing iPS Cells

In 1998, human embryonic stem cells were first generated and were expected to contribute greatly to regenerative medicine. However, when medical treatments were performed using human embryonic stem cells, there were problems, such as transplant rejection, as well as bioethical issues. Induced pluripotent stem cells were generated from mouse and human fibroblasts in 2006 and 2007 by introducing four transcription factors (Oct3/4, Sox2, c-Myc and Klf4). This process was defined as direct reprogramming, and induced pluripotent stem cells were better tolerated. Although induced pluripotent stem cells have contributed greatly to biomedical research and regenerative medicine, high tumorigenic potential is still a critical problem due to the introduction of the oncogene c-Myc and reprogramming with a virus vector. To address this, we reprogrammed somatic cells by transfection with microribonucleic acids to avoid using virus vectors for genomic integration into the host genome. We found that it was possible to reprogram mouse and human cells to pluripotency by direct transfection of three mature microribonucleic acids (mir-200c, -302s and -369s) with increased expression levels in embryonic stem cells and induced pluripotent stem cells. The microribonucleic acid-induced pluripotent stem cells have a reduced risk of mutations and tumorigenesis. Our laboratory also introduced four transcription factors (Oct3/4, Sox2, c-Myc and Klf4) into cancer cells, generating induced pluripotent cancer cells that exhibited strikingly less malignant features, suggesting the possibility of a novel type of cancer therapy. However, the gene transduction method is not yet safe for clinical applications, due to a genomic integration that may cause tumor formation. We are currently investigating the reprogramming method using microribonucleic acids in cancer cells to develop a very safe, highly efficient and highly complete reprogramming for clinical applications.

Functional interactions of the cystine/glutamate antiporter, CD44v and MUC1-C oncoprotein in triple-negative breast cancer cells

The xCT light chain of the cystine/glutamate transporter (system XC−) is of importance for the survival of triple-negative breast cancer (TNBC) cells. The MUC1-C transmembrane oncoprotein is aberrantly overexpressed in TNBC and, like xCT, has been linked to maintaining glutathione (GSH) levels and redox balance. However, there is no known interaction between MUC1-C and xCT. Here we show that silencing MUC1-C is associated with decreases in xCT expression in TNBC cells. The results demonstrate that MUC1-C forms a complex with xCT and the CD44 variant (CD44v), which interacts with xCT and thereby controls GSH levels. MUC1-C binds directly with CD44v and in turn promotes stability of xCT in the cell membrane. The interaction between MUC1-C and xCT is further supported by the demonstration that targeting xCT with silencing or the inhibitor sulfasalazine suppresses MUC1 gene transcription by increasing histone and DNA methylation on the MUC1 promoter. In terms of the functional significance of the MUC1-C/xCT interaction, we show that MUC1-C protects against treatment with erastin, an inhibitor of XC− and inducer of ferroptosis, a form of non-apoptotic cell death. These findings indicate that targeting this novel MUC1-C/xCT pathway could represent a potential therapeutic approach for promoting TNBC cell death.

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 the 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.

MUC1-C activates BMI1 in human cancer cells

B-cell-specific Moloney murine leukemia virus integration site 1 (BMI1) is a component of the polycomb repressive complex 1 (PRC1) complex that is overexpressed in breast and other cancers, and promotes self-renewal of cancer stem-like cells. The oncogenic mucin 1 (MUC1) C-terminal (MUC1-C) subunit is similarly overexpressed in human carcinoma cells and has been linked to their self-renewal. There is no known relationship between MUC1-C and BMI1 in cancer. The present studies demonstrate that MUC1-C drives BMI1 transcription by a MYC-dependent mechanism in breast and other cancer cells. In addition, we show that MUC1-C blocks miR-200c-mediated downregulation of BMI1 expression. The functional significance of this MUC1-C→︀BMI1 pathway is supported by the demonstration that targeting MUC1-C suppresses BMI1-induced ubiquitylation of H2A and thereby derepresses homeobox HOXC5 and HOXC13 gene expression. Notably, our results further show that MUC1-C binds directly to BMI1 and promotes occupancy of BMI1 on the CDKN2A promoter. In concert with BMI1-induced repression of the p16INK4a tumor suppressor, we found that targeting MUC1-C is associated with induction of p16INK4a expression. In support of these results, analysis of three gene expresssion data sets demonstrated highly significant correlations between MUC1-C and BMI1 in breast cancers. These findings uncover a previously unrecognized role for MUC1-C in driving BMI1 expression and in directly interacting with this stem cell factor, linking MUC1-C with function of the PRC1 in epigenetic gene silencing.

SSEA-3 as a novel amplifying cancer cell surface marker in colorectal cancers

Findings from studies on stem cells have been applied to cancer stem cell (CSC) research, but little is known about the relationship between ES cell-related cell surface markers and CSCs. In this study, we focused on stage-specific embryonic antigen 3 (SSEA-3), a marker of mesenchymal stem cells and Muse cells in colorectal cancer (CRC). Expression of SSEA-3 in human CRC cell lines and clinical specimens, specifically the relationship of SSEA-3 expression and the representative CSC markers (CD44, CD166, ALDH, CD24 and CD26) as well as with mesenchymal stem cell/Muse cell marker (CD105) were assessed. To characterize SSEA-3-expressing cells, tumorigenicity, sphere formation ability, expression of iPS genes (Oct4, NANOG, SOX2 and c-Myc), cell proliferation and cell cycle status were assessed. SSEA-3 expression was identified in Caco-2, DLD-1, HT-29, SW480 and HCT116, but not in CaR-1 cells. No significant relationship between SSEA-3 and other stem cell markers was detected. SSEA-3+ cells showed increased tumorigenicity in vivo, but lower sphere formation ability in vitro than SSEA-3-. iPS gene expression was not correlated with SSEA-3 expression status. SSEA-3+ cells showed higher proliferative ability than SSEA-3- through enhanced cell cycles by decreased expression of p21Cip1/Waf1 and p27Kip1. Immunofluorescence analysis in clinical specimens indicated that expression of SSEA-3 is limited to stromal cells in normal mucosa but broad in poorly differentiated adenocarcinoma. These observations indicated that SSEA-3+ cells in CRC have immature phenotype but decreased self-renewal ability and may function as tumor transient amplifying cells or delayed contributing tumor-initiating cells.

MUC1-C Induces PD-L1 and Immune Evasion in Triple-Negative Breast Cancer

The immune checkpoint ligand PD-L1 and the transmembrane mucin MUC1 are upregulated in triple-negative breast cancer (TNBC), where they contribute to its aggressive pathogenesis. Here, we report that genetic or pharmacological targeting of the oncogenic MUC1 subunit MUC1-C is sufficient to suppress PD-L1 expression in TNBC cells. Mechanistic investigations showed that MUC1-C acted to elevate PD-L1 transcription by recruitment of MYC and NF-κB p65 to the PD-L1 promoter. In an immunocompetent model of TNBC in which Eo771/MUC1-C cells were engrafted into MUC1 transgenic mice, we showed that targeting MUC1-C associated with PD-L1 suppression, increases in tumor-infiltrating CD8+ T cells and tumor cell killing. MUC1 expression in TNBCs also correlated inversely with CD8, CD69, and GZMB, and downregulation of these markers associated with decreased survival. Taken together, our findings show how MUC1 contributes to immune escape in TNBC, and they offer a rationale to target MUC1-C as a novel immunotherapeutic approach for TNBC treatment.Significance: These findings show how upregulation of the transmembrane mucin MUC1 contributes to immune escape in an aggressive form of breast cancer, with potential implications for a novel immunotherapeutic approach. Cancer Res; 78(1); 205-15. ©2017 AACR.

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

Apical–basal polarity and epithelial integrity are maintained in part by the Crumbs (CRB) complex. The C-‐terminal subunit of MUC1 (MUC1-C) is a transmembrane protein that is expressed at the apical border of normal epithelial cells and aberrantly at high levels over the entire surface of their transformed counterparts. However, it is not known whether MUC1-C contributes to this loss of polarity that is characteristic of carcinoma cells. Here it is demonstrated that MUC1-C downregulates expression of the Crumbs complex CRB3 protein in triple-negative breast cancer (TNBC) cells. MUC1-C associates with ZEB1 on the CRB3 promoter and represses CRB3 transcription. Notably, CRB3 activates the core kinase cassette of the Hippo pathway, which includes LATS1 and LATS2. In this context, targeting MUC1-C was associated with increased phosphorylation of LATS1, consistent with activation of the Hippo pathway, which is critical for regulating cell contact, tissue repair, proliferation, and apoptosis. Also shown is that MUC1-C‐-mediated suppression of CRB3 and the Hippo pathway is associated with dephosphorylation and activation of the oncogenic YAP protein. In turn, MUC1-C interacts with YAP, promotes formation of YAP/β-catenin complexes, and induces the WNT target gene MYC. These data support a previously unrecognized pathway in which targeting MUC1-C in TNBC cells (i) induces CRB3 expression, (ii) activates the CRB3-driven Hippo pathway, (iii) inactivates YAP, and thereby (iv) suppresses YAP/β-catenin–mediated induction of MYC expression. Implications: 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.

Enhancement of perpendicular magnetic anisotropy and its electric field-induced change through interface engineering in Cr/Fe/MgO

Recently, perpendicular magnetic anisotropy (PMA) and its voltage control (VC) was demonstrated for Cr/Fe/MgO. In this study, we shed light on the origin of large voltage-induced anisotropy change in Cr/Fe/MgO. Analysis of the chemical structure of Cr/Fe/MgO revealed the existence of Cr atoms in the proximity of the Fe/MgO interface, which can affect both magnetic anisotropy (MA) and its VC. We showed that PMA and its VC can be enhanced by controlled Cr doping at the Fe/MgO interface. For Cr/Fe (5.9 Å)/Cr (0.7 Å)/MgO with an effective PMA of 0.8 MJ/m3, a maximum value of the voltage-controlled magnetic anisotropy (VCMA) effect of 370 fJ/Vm was demonstrated due to Cr insertion.