Jianlin Gong

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.

Fusion cell vaccination of patients with metastatic breast and renal cancer induces immunological and clinical responses.

Purpose: Dendritic cells (DCs) are potent antigen-presenting cells that are uniquely capable of inducing tumor-specific immune responses. We have conducted a Phase I trial in which patients with metastatic breast and renal cancer were treated with a vaccine prepared by fusing autologous tumor and DCs. Experimental Design: Accessible tumor tissue was disrupted into single cell suspensions. Autologous DCs were prepared from adherent peripheral blood mononuclear cells that were obtained by leukapheresis and cultured in granulocyte macrophage colony-stimulating factor, interleukin 4, and autologous plasma. Tumor cells and DCs were cocultured in the presence of polyethylene glycol to generate the fusions. Fusion cells were quantified by determining the percentage of cells that coexpress tumor and DC markers. Patients were vaccinated with fusion cells at 3-week intervals and assessed weekly for toxicity, and tumor response was assessed at 1, 3, and 6 months after completion of vaccination. Results: The vaccine was generated for 32 patients. Twenty-three patients were vaccinated with 1 × 105 to 4 × 106 fusion cells. Fusion cells coexpressed tumor and DC antigens and stimulated allogeneic T-cell proliferation. There was no significant treatment-related toxicity and no clinical evidence of autoimmunity. In a subset of patients, vaccination resulted in an increased percentage of CD4 and CD8+ T cells expressing intracellular IFN-γ in response to in vitro exposure to tumor lysate. Two patients with breast cancer exhibited disease regressions, including a near complete response of a large chest wall mass. Five patients with renal carcinoma and one patient with breast cancer had disease stabilization. Conclusions: Our findings demonstrate that fusion cell vaccination of patients with metastatic breast and renal cancer is a feasible, nontoxic approach associated with the induction of immunological and clinical antitumor responses.

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.

Message in a bottle: Role of the 70-kDa heat shock protein family in anti-tumor immunity

Extracellular heat shock protein 70 (HSP70) is a potent agent for tumor immunotherapy, which can break tolerance to tumor‐associated antigens and cause specific tumor cell killing by cytotoxic CD8+ T cells. The pro‐immune effects of extracellular HSP70 are, to some extent, extensions of its molecular properties as an intracellular stress protein. The HSP70 are characterized by massive inducibility after stress, preventing cell death by inhibiting aggregation of cell proteins and directly antagonizing multiple cell death pathways. HSP70 family members possess a domain in the C terminus that chaperones unfolded proteins and peptides, and a N‐terminal ATPase domain that controls the opening and closing of the peptide binding domain. These properties not only enable intracellular HSP70 to inhibit tumor apoptosis, but also promote formation of stable complexes with cytoplasmic tumor antigens that can then escape intact from dying cells to interact with antigen‐processing cells (APC) and stimulate anti‐tumor immunity. HSP70 may be released from tumors undergoing therapy at high local extracellular concentrations, and send a danger signal to the host leading to APC activation. Extracellular HSP70 bind to high‐affinity receptors on APC, leading to activation of maturation and re‐presentation of the peptide antigen cargo of HSP70 by the APC. The ability of HSP70‐peptide complexes (HSP70‐PC) to break tolerance and cause tumor regression employs these dual properties as signaling ligand and antigen transporter. HSP70‐PC thus coordinately activate innate immune responses and deliver antigens for re‐presentation by MHC class I and II molecules on the APC cell surface, leading to specific anti‐tumor immunity.

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.

Induction of heat shock proteins by heregulin β1 leads to protection from apoptosis and anchorage-independent growth

Elevation of heat shock protein (HSP) levels is widespread in cancer and predicts a poor prognosis and resistance to therapy. We show that HSP elevation in tumor cells can be induced by the highly malignant factor heregulin β1 (HRGβ1), which induces HSP expression through heat shock transcription factor 1 (HSF1). Inactivation of the hsf1 gene prevents HSP induction by HRGβ1. HSP expression is induced through a cascade response initiated by HRGβ1 binding to c-erbB receptors on the cell surface and which leads to the inhibition of intracellular HSF1 antagonist glycogen synthase kinase 3. HSF1 activated by this pathway plays a key role in the protection of cells from apoptosis and the mediation of anchorage independent growth by HRGβ1, indicating a role for HSF1 in this tumorigenic pathway.

mTOR is essential for the proteotoxic stress response, HSF1 activation and heat shock protein synthesis.

The target of rapamycin (TOR) is a high molecular weight protein kinase that regulates many processes in cells in response to mitogens and variations in nutrient availability. Here we have shown that mTOR in human tissue culture cells plays a key role in responses to proteotoxic stress and that reduction in mTOR levels by RNA interference leads to increase sensitivity to heat shock. This effect was accompanied by a drastic reduction in ability to synthesize heat shock proteins (HSP), including Hsp70, Hsp90 and Hsp110. As HSP transcription is regulated by heat shock transcription factor 1 (HSF1), we examined whether mTOR could directly phosphorylate this factor. Indeed, we determined that mTOR could directly phosphorylate HSF1 on serine 326, a key residue in transcriptional activation. HSF1 was phosphorylated on S326 immediately after heat shock and was triggered by other cell stressors including proteasome inhibitors and sodium arsenite. Null mutation of S326 to alanine led to loss of ability to activate an HSF1-regulated promoter-reporter construct, indicating a direct role for mTOR and S326 in transcriptional regulation of HSP genes during stress. As mTOR is known to exist in at least two intracellular complexes, mTORC1 and mTOR2 we examined which complex might interact with HSF1. Indeed mTORC1 inhibitor rapamycin prevented HSF1-S326 phosphorylation, suggesting that this complex is involved in HSF1 regulation in stress. Our experiments therefore suggest a key role for mTORC1 in transcriptional responses to proteotoxic stress.

Heat shock factor 1 represses estrogen-dependent transcription through association with MTA1

Heat shock factor 1 (HSF1), the transcriptional activator of the heat shock genes, is increasingly implicated in cancer. We have shown that HSF1 binds to the corepressor metastasis-associated protein 1 (MTA1) in vitro and in human breast carcinoma samples. HSF1–MTA1 complex formation was strongly induced by the transforming ligand heregulin and complexes incorporated a number of additional proteins including histone deacetylases (HDAC1 and 2) and Mi2α, all components of the NuRD corepressor complex. These complexes were induced to assemble on the chromatin of MCF7 breast carcinoma cells and associated with the promoters of estrogen-responsive genes. Such HSF1 complexes participate in repression of estrogen-dependent transcription in breast carcinoma cells treated with heregulin and this effect was inhibited by MTA1 knockdown. Repression of estrogen-dependent transcription may contribute to the role of HSF1 in cancer.

Dendritic Cells Fused with Allogeneic Colorectal Cancer Cell Line Present Multiple Colorectal Cancer–Specific Antigens and Induce Antitumor Immunity against Autologous Tumor Cells

The aim of antitumor immunotherapy is to induce CTL responses against autologous tumors. Previous work has shown that fusion of human dendritic cells and autologous tumor cells induce CTL responses against autologous tumor cells in vitro. However, in the clinical setting of patients with colorectal carcinoma, a major difficulty is the preparation of sufficient amounts of autologous tumor cells. In the present study, autologous dendritic cells from patients with colorectal carcinoma were fused to allogeneic colorectal tumor cell line, COLM-6 (HLA-A2−/HLA-24−), carcinoembryonic antigen (CEA)+, and MUC1+ as an alternative strategy to deliver shared colorectal carcinoma antigens to dendritic cells. Stimulation of autologous T cells by the fusion cells generated with autologous dendritic cells (HLA-A2+ and/or HLA-A24+) and allogeneic COLM-6 resulted in MHC class I– and MHC class II–restricted proliferation of CD4+ and CD8+ T cells, high levels of IFN-γ production in both CD4+ and CD8+ T cells, and the simultaneous induction of CEA- and MUC1-specific CTL responses restricted by HLA-A2 and/or HLA-A24. Finally, CTL induced by dendritic cell/allogeneic COLM-6 fusion cells were able to kill autologous colorectal carcinoma by HLA-A2- and/or HLA-A24-restricted mechanisms. The demonstration of CTL activity against shared tumor-associated antigens using an allogeneic tumor cell line, COLM-6, provides that the presence of alloantigens does not prevent the development of CTL with activity against autologous colorectal carcinoma cells. The fusion of allogeneic colorectal carcinoma cell line and autologous dendritic cells could have potential applicability to the field of antitumor immunotherapy through the cross-priming against shared tumor antigens and provides a platform for adoptive immunotherapy.