Dahlia M. Besmer

MUC1 enhances invasiveness of pancreatic cancer cells by inducing epithelial to mesenchymal transition

Increased motility and invasiveness of pancreatic cancer cells are associated with epithelial to mesenchymal transition (EMT). Snai1 and Slug are zinc-finger transcription factors that trigger this process by repressing E-cadherin and enhancing vimentin and N-cadherin protein expression. However, the mechanisms that regulate this activation in pancreatic tumors remain elusive. MUC1, a transmembrane mucin glycoprotein, is associated with the most invasive forms of pancreatic ductal adenocarcinomas (PDA). In this study, we show that over expression of MUC1 in pancreatic cancer cells triggers the molecular process of EMT, which translates to increased invasiveness and metastasis. EMT was significantly reduced when MUC1 was genetically deleted in a mouse model of PDA or when all seven tyrosines in the cytoplasmic tail of MUC1 were mutated to phenylalanine (mutated MUC1 CT). Using proteomics, RT–PCR and western blotting, we revealed a significant increase in vimentin, Slug and Snail expression with repression of E-Cadherin in MUC1-expressing cells compared with cells expressing the mutated MUC1 CT. In the cells that carried the mutated MUC1 CT, MUC1 failed to co-immunoprecipitate with β-catenin and translocate to the nucleus, thereby blocking transcription of the genes associated with EMT and metastasis. Thus, functional tyrosines are critical in stimulating the interactions between MUC1 and β-catenin and their nuclear translocation to initiate the process of EMT. This study signifies the oncogenic role of MUC1 CT and is the first to identify a direct role of the MUC1 in initiating EMT during pancreatic cancer. The data may have implications in future design of MUC1-targeted therapies for pancreatic cancer.

Pancreatic Ductal Adenocarcinoma Mice Lacking Mucin 1 Have a Profound Defect in Tumor Growth and Metastasis

MUC1 is overexpressed and aberrantly glycosylated in more than 60% of pancreatic ductal adenocarcinomas. The functional role of MUC1 in pancreatic cancer has yet to be fully elucidated due to a dearth of appropriate models. In this study, we have generated mouse models that spontaneously develop pancreatic ductal adenocarcinoma (KC), which are either Muc1-null (KCKO) or express human MUC1 (KCM). We show that KCKO mice have significantly slower tumor progression and rates of secondary metastasis, compared with both KC and KCM. Cell lines derived from KCKO tumors have significantly less tumorigenic capacity compared with cells from KCM tumors. Therefore, mice with KCKO tumors had a significant survival benefit compared with mice with KCM tumors. In vitro, KCKO cells have reduced proliferation and invasion and failed to respond to epidermal growth factor, platelet-derived growth factor, or matrix metalloproteinase 9. Further, significantly less KCKO cells entered the G(2)-M phase of the cell cycle compared with the KCM cells. Proteomics and Western blotting analysis revealed a complete loss of cdc-25c expression, phosphorylation of mitogen-activated protein kinase (MAPK), as well as a significant decrease in nestin and tubulin-α2 chain expression in KCKO cells. Treatment with a MEK1/2 inhibitor, U0126, abrogated the enhanced proliferation of the KCM cells but had minimal effect on KCKO cells, suggesting that MUC1 is necessary for MAPK activity and oncogenic signaling. This is the first study to utilize a Muc1-null PDA mouse to fully elucidate the oncogenic role of MUC1, both in vivo and in vitro.

Vesicular Stomatitis Virus as an Oncolytic Agent against Pancreatic Ductal Adenocarcinoma

ABSTRACT Vesicular stomatitis virus (VSV) is a promising oncolytic agent against a variety of cancers. However, it has never been tested in any pancreatic cancer model. Pancreatic ductal adenocarcinoma (PDA) is the most common and aggressive form of pancreatic cancer. In this study, the oncolytic potentials of several VSV variants were analyzed in a panel of 13 clinically relevant human PDA cell lines and compared to conditionally replicative adenoviruses (CRAds), Sendai virus and respiratory syncytial virus. VSV variants showed oncolytic abilities superior to those of other viruses, and some cell lines that exhibited resistance to other viruses were successfully killed by VSV. However, PDA cells were highly heterogeneous in their susceptibility to virus-induced oncolysis, and several cell lines were resistant to all tested viruses. Resistant cells showed low levels of very early VSV RNA synthesis, indicating possible defects at initial stages of infection. In addition, unlike permissive PDA cell lines, most of the resistant cell lines were able to both produce and respond to interferon, suggesting that intact type I interferon responses contributed to their resistance phenotype. Four cell lines that varied in their permissiveness to VSV-ΔM51 and CRAd dl1520 were tested in mice, and the in vivo results closely mimicked those in vitro. While our results demonstrate that VSV is a promising oncolytic agent against PDA, further studies are needed to better understand the molecular mechanisms of resistance of some PDAs to oncolytic virotherapy.

MUC1 regulates PDGFA expression during pancreatic cancer progression

Pancreatic ductal adenocarcinoma (PDA) has one of the worst prognoses of all cancers. Mucin 1 (MUC1), a transmembrane mucin glycoprotein, is a key modulator of several signaling pathways that affect oncogenesis, motility and metastasis. Its expression is known to be associated with poor prognosis in patients. However, the precise mechanism remains elusive. We report a novel association of MUC1 with platelet-derived growth factor-A (PDGFA). PDGFA is one of the many drivers of tumor growth, angiogenesis and metastasis in PDA. Using mouse PDA models as well as human samples, we show clear evidence that MUC1 regulates the expression and secretion of PDGFA. This, in turn, influences proliferation and invasion of pancreatic cancer cells leading to higher tumor burden in vivo. In addition, we reveal that MUC1 overexpressing cells are heavily dependent on PDGFA both for proliferation and invasion, whereas MUC1-null cells are not. Moreover, PDGFA and MUC1 are critical for translocation of β catenin to the nucleus for oncogenesis to ensue. Finally, we elucidate the underlying mechanism by which MUC1 regulates PDGFA expression and secretion in pancreatic cancer cells. We show that MUC1 associates with Hif1-α, a known transcription factor involved in controlling PDGFA expression. Furthermore, MUC1 facilitates Hif1-α translocation to the nucleus. In summary, we have demonstrated that MUC1-induced invasion and proliferation occurs via increased exogenous production of PDGFA. Thus, impeding MUC1 regulation of PDGFA signaling may be therapeutically beneficial for patients with PDA.

The use of a novel MUC1 antibody to identify cancer stem cells and circulating MUC1 in mice and patients with pancreatic cancer.

MUC1 is over‐expressed and aberrantly glycosylated in >60% of human pancreatic cancer (PC). Development of novel approaches for detection and/or targeting of MUC1 are critically needed and should be able to detect MUC1 on PC cells (including cancer stem cells) and in serum.

Systemic neutralization of IL-17A significantly reduces breast cancer associated metastasis in arthritic mice by reducing CXCL12/SDF-1 expression in the metastatic niches

BackgroundIL-17A is a pro-inflammatory cytokine that is normally associated with autoimmune arthritis and other pro-inflammatory conditions. Recently, IL-17A has emerged as a critical factor in enhancing breast cancer (BC)-associated metastases. We generated immune competent arthritic mouse models that develop spontaneous BC-associated bone and lung metastasis. Using these models, we have previously shown that neutralization of IL-17A resulted in significant reduction in metastasis. However, the underlying mechanism/s remains unknown.MethodsWe have utilized two previously published mouse models for this study: 1) the pro-arthritic mouse model (designated SKG) injected with metastatic BC cell line (4T1) in the mammary fat pad, and 2) the PyV MT mice that develop spontaneous mammary gland tumors injected with type II collagen to induce autoimmune arthritis. Mice were treated with anti-IL-17A neutralizing antibody and monitored for metastasis and assessed for pro-inflammatory cytokines and chemokines associated with BC-associated metastasis.ResultsWe first corroborate our previous finding that in vivo neutralization of IL-17A significantly reduced metastasis to the bones and lungs in both models. Next, we report that treatment with anti-IL17A antibody significantly reduced the expression of a key chemokine, CXCL12 (also known as stromal derived factor-1 (SDF - 1)) in the bones and lungs of treated mice. CXCL12 is a ligand for CXCR4 (expressed on BC cells) and their interaction is known to be critical for metastasis. Interestingly, levels of CXCR4 in the tumor remained unchanged with treatment. Consequently, protein lysates derived from the bones and lungs of treated mice were significantly less chemotactic for the BC cells than lysates from untreated mice; and addition of exogenous SDF-1 to the lysates from treated mice completely restored BC cell migration. In addition, cytokines such as IL-6 and M-CSF were significantly reduced in the lung and bone lysates following treatment. The data presented suggests that systemic neutralization of IL-17A can block the CXCR4/SDF-1 signaling pathway by reducing the expression of SDF-1 in the metastatic niches and significantly reducing metastasis in both mouse models.ConclusionIn our model, neutralization of IL-17A regulates SDF-1 expression in the metastatic niches either directly or indirectly via reducing levels of IL-6 and M-CSF.

Arthritis augments breast cancer metastasis: role of mast cells and SCF/c-Kit signaling

IntroductionBreast cancer remains the second leading cause of cancer-related deaths for women in the United States. Metastasis is regulated not only by intrinsic genetic changes in malignant cells, but also by the microenvironment, especially those associated with chronic inflammation. We recently reported that mice with autoimmune arthritis have significantly increased incidence of bone and lung metastasis and decreased survival associated with breast cancer. In this study, we evaluated the mechanism underlying the increased metastasis.MethodsWe used two mouse models; one that develops spontaneous autoimmune arthritis (SKG mice) injected with metastatic breast cancer cells (4T1), and another that develops spontaneous breast cancer (MMTV-PyV MT mice) injected with type II collagen to induce autoimmune arthritis. Mast cell levels and metastasis were monitored.ResultsFirst, we confirmed that breast tumor-bearing arthritic mice have a significantly higher incidence of bone and lung metastasis than do their nonarthritic counterparts. Next, we showed increased recruitment of mast cells within the primary tumor of arthritic mice, which facilitates metastasis. Next, we report that arthritic mice without any tumors have higher numbers of mast cells in the bones and lungs, which may be the underlying cause for the enhanced lung and bone metastases observed in the arthritic mice. Next, we showed that once the tumor cells populate the metastatic niches (bones and lungs), they further increase the mast cell population within the niche and assist in enhancing metastasis. This may primarily be due to the interaction of c-Kit receptor present on mast cells and stem cell factor (SCF, the ligand for ckit) expressed on tumor cells. Finally, we showed that targeting the SCF/cKit interaction with an anti-ckit antibody reduces the differentiation of mast cells and consequently reduces metastasis.ConclusionThis is the first report to show that mast cells may play a critical role in remodeling not only the tumor microenvironment but also the metastatic niche to facilitate efficient metastasis through SCF/cKit interaction in breast cancer with arthritis.

Intratumoral delivery of CpG-conjugated anti-MUC1 antibody enhances NK cell anti-tumor activity.

Monoclonal antibodies (mAbs) against tumor-associated antigens are useful anticancer agents. Antibody-dependent cellular cytotoxicity (ADCC) is one of the major mechanisms responsible for initiating natural killer cell (NK)-mediated killing of tumors. However, the regulation of ADCC via NK cells is poorly understood. We have investigated the cytolytic activity of NK cells against pancreatic cancer cells that were coated with an antibody directed against the human tumor antigen, Mucin-1 designated HMFG-2, either alone or conjugated to CpG oligodeoxynucleotide (CpG ODN). Conjugated antibodies were tested for their ability to elicit ADCC in vitro and in vivo against pancreatic cancer cells. NK cells cultured in the presence of immobilized CpG ODN, HMFG-2 Ab, or CpG ODN-conjugated HMFG-2 Ab were able to up-regulate perforin similarly. Interestingly, a significant higher ADCC was observed when CpG ODN-conjugated HMFG-2-coated tumor cells were co-cultured with NK cells compared to unconjugated HMFG-2 Ab or CpG ODN alone. Moreover, MyD88-deficient NK cells can perform ADCC in vitro. Furthermore, intratumoral injections of CpG ODN-conjugated HMFG-2 induced a significant reduction in tumor burden in vivo in an established model of pancreatic tumor in nude mice compared to CpG ODN or the HMFG-2 alone. Depletion of macrophages or NK cells before treatment confirmed that both cells were required for the anti-tumor response in vivo. Results also suggest that CpG ODN and HMFG-2 Ab could be sensed by NK cells on the mAb-coated tumor cells triggering enhanced ADCC in vitro and in vivo.

Oncolytic Vesicular Stomatitis Virus in an Immunocompetent Model of MUC1-Positive or MUC1-Null Pancreatic Ductal Adenocarcinoma

ABSTRACT Vesicular stomatitis virus (VSV) is a promising oncolytic agent against various malignancies. Here, for the first time, we tested VSV in vitro and in vivo in a clinically relevant, immunocompetent mouse model of pancreatic ductal adenocarcinoma (PDA). Our system allows the study of virotherapy against PDA in the context of overexpression (80% of PDA patients) or no expression of human mucin 1 (MUC1), a major marker for poor prognosis in patients. In vitro, we tested three VSV recombinants, wild-type VSV, VSV-green fluorescent protein (VSV-GFP), and a safe oncolytic VSV-ΔM51-GFP, against five mouse PDA cell lines that either expressed human MUC1 or were MUC1 null. All viruses demonstrated significant oncolytic abilities independent of MUC1 expression, although VSV-ΔM51-GFP was somewhat less effective in two PDA cell lines. In vivo administration of VSV-ΔM51-GFP resulted in significant reduction of tumor growth for tested mouse PDA xenografts (+MUC1 or MUC1 null), and antitumor efficacy was further improved when the virus was combined with the chemotherapeutic drug gemcitabine. The antitumor effect was transient in all tested groups. The developed system can be used to study therapies involving various oncolytic viruses and chemotherapeutics, with the goal of inducing tumor-specific immunity while preventing premature virus clearance.

Pancreatic Cancer Cells Isolated from Muc1-Null Tumors Favor the Generation of a Mature Less Suppressive MDSC Population

Mucin 1 (MUC1) is a transmembrane mucin glycoprotein that is over-expressed and aberrantly glycosylated in >80% of human pancreatic ductal adenocarcinoma (PDA) and is associated with poor prognosis. To understand the role of MUC1 in PDA, we have recently developed two mouse models of spontaneous PDA, one that expresses full-length human MUC1 transgene (KCM mice) and one that is null for MUC1 (KCKO mice). We have previously reported that KCM mice express high levels of myeloid derived suppressor cells (MDSCs) in their tumors and develop highly aggressive PDA. To further understand the underlying mechanism for high MDSC levels in KCM-tumors, we generated primary cell lines from KCM and KCKO-tumors. In this study, we report that MDSCs derived using KCM cells express significantly higher levels of arginase 1 and inducible nitric oxide synthase (markers associated with immune suppression) and lower levels of CD115 (a marker associated with maturation of myeloid cells) as compared to KCKO-derived MDSCs. Functionally, KCM-derived MDSCs secrete significantly higher levels of urea and nitric oxide (NO) when co-cultured with normal splenic cells as compared to KCKO-derived MDSCs. Data indicates that KCM-derived MDSCs remain immature and are more suppressive as compared to KCKO-derived MDSCs. This was further corroborated in vivo where MDSCs isolated from KCM-tumor-bearing mice retained their immature state and were highly suppressive as compared to MDSCs derived from KCKO-tumor-bearing mice. Finally, we show that KCM cells secrete significantly higher levels of prostaglandin E2 (PGE2), a COX-2 metabolite and a known driver of suppressive MDSCs as compared to KCKO cells. Thus, inhibiting PGE2 with a specific COX-2 inhibitor reverses the immunosuppressive and immature phenotype of KCM-derived MDSCs. This is the first report that clearly suggests a functional role of pancreatic tumor-associated MUC1 in the development of functional MDSCs.