Dongshu Chen

Human MUC1 carcinoma-associated protein confers resistance to genotoxic anticancer agents

The MUC1 transforming protein is overexpressed by most human carcinomas. The present studies demonstrate that the MUC1 C-terminal subunit (MUC1 C-ter) localizes to mitochondria in HCT116/MUC1 colon carcinoma cells and that heregulin stimulates mitochondrial targeting of MUC1 C-ter. We also show that MUC1 attenuates cisplatin-induced (1) release of mitochondrial apoptogenic factors, (2) activation of caspase-3, and (3) induction of apoptosis. Moreover, knockdown of MUC1 expression in A549 lung and ZR-75-1 breast carcinoma cells by MUC1siRNA was associated with increased sensitivity to genotoxic drugs in vitro and in vivo. These findings indicate that MUC1 attenuates the apoptotic response to DNA damage and that this oncoprotein confers resistance to genotoxic anticancer agents.

Interaction of Glycogen Synthase Kinase 3β with the DF3/MUC1 Carcinoma-Associated Antigen and β-Catenin

ABSTRACT The DF3/MUC1 mucin-like glycoprotein is highly overexpressed in human carcinomas. Recent studies have demonstrated that the cytoplasmic domain of MUC1 interacts with β-catenin. Here we show that MUC1 associates with glycogen synthase kinase 3β (GSK3β). GSK3β binds directly to an STDRSPYE site in MUC1 and phosphorylates the serine adjacent to proline. Phosphorylation of MUC1 by GSK3β decreases binding of MUC1 to β-catenin in vitro and in vivo. GSK3β-mediated phosphorylation of MUC1 had no apparent effect on β-catenin levels or the transcriptional coactivation function of β-catenin. The results, however, demonstrate that MUC1 expression decreases binding of β-catenin to the E-cadherin cell adhesion molecule. Negative regulation of the β-catenin–MUC1 interaction by GSK3β is associated with restoration of the complex between β-catenin and E-cadherin. These findings indicate that GSK3β decreases the interaction of MUC1 with β-catenin and that overexpression of MUC1 in the absence of GSK3β activity inhibits formation of the E-cadherin–β-catenin complex.

Fusions of Human Ovarian Carcinoma Cells with Autologous or Allogeneic Dendritic Cells Induce Antitumor Immunity

Human ovarian carcinomas express the CA-125, HER2/neu, and MUC1 tumor-associated Ags as potential targets for the induction of active specific immunotherapy. In the present studies, human ovarian cancer cells were fused to human dendritic cells (DC) as an alternative strategy to induce immunity against known and unidentified tumor Ags. Fusions of ovarian cancer cells to autologous DC resulted in the formation of heterokaryons that express the CA-125 Ag and DC-derived costimulatory and adhesion molecules. Similar findings were obtained with ovarian cancer cells fused to allogeneic DC. The fusion cells were functional in stimulating the proliferation of autologous T cells. The results also demonstrate that fusions of ovarian cancer cells to autologous or allogeneic DC induce cytolytic T cell activity and lysis of autologous tumor cells by a MHC class I-restricted mechanism. These findings demonstrate that fusions of ovarian carcinoma cells and DC activate T cell responses against autologous tumor and that the fusions are functional when generated with either autologous or allogeneic DC.

Human DF3/MUC1 carcinoma-associated protein functions as an oncogene.

The human DF3/MUC1 mucin-like glycoprotein is aberrantly overexpressed by most carcinomas of the breast and other epithelia. The contribution of MUC1 overexpression to the malignant phenotype is, however, not known. In the present studies, we have stably expressed MUC1 in rat 3Y1 fibroblasts. MUC1-positive cells were selected from independent transfections. The results demonstrate that, as found in human carcinomas, MUC1 is expressed on the cell surface and as a complex with β-catenin in the nucleus of the transfectants. Colony formation in soft agar demonstrates that cells expressing MUC1, but not the empty vector, exhibit anchorage-independent growth. The results also show that MUC1 expression confers tumor formation in nude mice. These findings provide the first evidence that MUC1 induces cellular transformation.

Induction of Antitumor Immunity by Vaccination of Dendritic Cells Transfected with MUC1 RNA

Dendritic cells (DC) are potent APCs. In this study, murine bone marrow-derived DC were transfected with RNA encoding the MUC1 Ag that is aberrantly overexpressed in human breast and other carcinomas. The MUC1 RNA-transfected DC exhibited cell surface expression of MUC1 and costimulatory molecules. After injection at the base of the tail, the transfected DC were detectable in inguinal lymph nodes by dual immunochemical staining. Vaccination of wild-type mice with MUC1 RNA-transfected DC induced anti-MUC1 immune responses against MUC1-positive MC38/MUC1, but not MUC1-negative, tumor cells. Mice immunized with the transfected DC were protected against challenge with MC38/MUC1 tumor cells. Furthermore, mice with established MC38/MUC1 tumors were eliminated after receiving the vaccination. CTLs isolated from mice immunized with the transfected DC exhibited specific cytolytic activity against MC38/MUC1 tumor cells. In contrast to these findings, there was little if any anti-MUC1 immunity induced with the transfected DC in MUC1 transgenic (MUC1.Tg) mice. However, coadministration of the transfected DC and IL-12 reversed the unresponsiveness to MUC1 Ag in MUC1.Tg mice and induced MUC1-specific immune responses. These findings demonstrate that vaccination of DC transfected with MUC1 RNA and IL-12 reverses tolerance to MUC1 and induces immunity against MUC1-positive tumors.

MUC1 Oncoprotein Blocks Glycogen Synthase Kinase 3β–Mediated Phosphorylation and Degradation of β-Catenin

Dysregulation of beta-catenin is of importance to the development of diverse human malignancies. The MUC1 oncoprotein is aberrantly overexpressed by most human carcinomas and associates with beta-catenin. However, the functional significance of the MUC1-beta-catenin interaction is not known. Here, we show that MUC1 increases beta-catenin levels in the cytoplasm and nucleus of carcinoma cells. Previous studies have shown that glycogen synthase kinase 3beta (GSK3beta) phosphorylates beta-catenin and thereby targets it for proteosomal degradation. Consistent with the up-regulation of beta-catenin levels, our results show that MUC1 blocks GSK3beta-mediated phosphorylation and degradation of beta-catenin. To further define the interaction between MUC1 and beta-catenin, we identified a serine-rich motif (SRM) in the MUC1 cytoplasmic domain that binds directly to beta-catenin Armadillo repeats. Mutation of the SRM attenuated binding of MUC1 to beta-catenin and MUC1-mediated inhibition of beta-catenin degradation. Importantly, disruption of the MUC1-beta-catenin interaction with the SRM mutant also attenuated MUC1-induced anchorage-dependent and -independent growth and delayed MUC1-mediated tumorigenicity. These findings indicate that MUC1 promotes transformation, at least in part, by blocking GSK3beta-mediated phosphorylation and thereby degradation of beta-catenin.

Breast cancer selective gene expression and therapy mediated by recombinant adenoviruses containing the DF3/MUC1 promoter.

The high molecular weight mucin-like glycoprotein, DF3 (MUC1), is overexpressed in the majority of human breast cancers. Here we demonstrate that replication defective recombinant adenoviral vectors, containing the DF3 promoter (bp -725 to +31), can be used to express beta-galactosidase (Ad.DF3-betagal) and the herpes simplex virus thymidine kinase (HSV-tk) gene (Ad.Df3-tk) in DF3 positive breast carcinoma cell lines. In vivo experiments using breast tumor implants in nude mice injected with Ad.DF3-betagal demonstrated that expression of the beta-galactosidase gene is limited to DF3-positive breast cancer xenografts. Moreover, in an intraperitoneal breast cancer metastases model, we show that i.p. injection of Ad.DF3-tk followed by GCV treatment results in inhibition of tumor growth. These results demonstrate that utilization of the DF3 promoter in an adenoviral vector can confer selective expression of heterologous genes in breast cancer cells in vitro and in vivo.

Induction of antigen-specific antitumor immunity with adenovirus-transduced dendritic cells.

Transduction of dendritic cells (DC) can result in presentation of tumor-associated antigens and induction of immunity against undefined epitopes. The present studies demonstrate adenovirus (Ad)-mediated transduction of the β-galactosidase gene in mouse DC. Similar transductions have been obtained with the gene encoding the DF3/MUC1 tumor-associated antigen. We show that the Ad-transduced DC are functional in primary allogeneic mixed lymphocyte reactions. Mice immunized with Ad-transduced DC develop cytotoxic T lymphocytes that are specific for the β-galactosidase or DF3/MUC1 antigens. The results also demonstrate that Ad.MUC1-transduced DC induce a specific response which inhibits the growth of DF3/MUC1- positive tumors. These findings support the usefulness of Ad-transduced DC for in vivo immunization against tumor-associated antigens.

Nuclear Import of the MUC1-C Oncoprotein Is Mediated by Nucleoporin Nup62

The MUC1 heterodimeric transmembrane protein is aberrantly overexpressed by most human carcinomas. The MUC1 C-terminal subunit (MUC1-C) is devoid of a classical nuclear localization signal and is targeted to the nucleus by an unknown mechanism. The present results demonstrate that MUC1-C associates with importin β and not importin α. The results also show that, like importin β, MUC1-C binds to Nup62 (nucleoporin p62). MUC1-C binds directly to the Nup62 central domain and indirectly to the Nup62 C-terminal α-helical coiled-coil domain. We demonstrate that MUC1-C forms oligomers and that oligomerization is necessary for binding to Nup62. The MUC1-C cytoplasmic domain contains a CQC motif that when mutated to AQA abrogates oligomerization and binding to Nup62. Stable expression of MUC1 with the CQC → AQA mutations was associated with targeting to the cell membrane and cytosol and attenuation of nuclear localization. The results further show that expression of MUC1(CQC-AQA) attenuates MUC1-induced (i) transcriptional coactivation, (ii) anchorage-independent growth, and (iii) tumorigenicity. These findings indicate that the MUC1-C oncoprotein is imported to the nucleus by a pathway involving Nup62.

MUC1 cytoplasmic domain coactivates Wnt target gene transcription and confers transformation.

The DF3/MUC1 mucin-like transmembrane oncoprotein is overexpressed by most human carcinomas. The MUC1 cytoplasmic domain (CD) binds directly to the Wnt effector, beta-catenin, and colocalizes with beta-catenin in the nucleus; however, the nuclear function of MUC1 is unknown. The present results demonstrate that MUC1 coactivates transcription of beta-catenin-Tcf-binding sites in the pTOPFLASH reporter. Activation of transcription was abrogated by expression of MUC1 with a Y-46->F mutation in the CD that attenuates binding of MUC1 and beta-catenin. We also show that transcription of the Wnt responsive cyclin D1 promoter is activated by MUC1, but not MUC1(Y46F), and that the cyclin D1 gene is upregulated in MUC1-positive cells. In concert with these results, MUC1-induced anchorage-independent growth and tumorigenicity were also abrogated by mutating MUC1 at the Y-46 site. These findings support a model in which the MUC1 functions as a transforming protein by coactivating transcription of Wnt target genes.