Amritha Kidiyoor

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.

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.

Soluble Tumor Necrosis Factor Receptors Shed by Breast Tumor Cells Inhibit Macrophage Chemotaxis

Breast tumor cells alter their microenvironment in part through the expression of protumor molecules that influence macrophages during tumor progression and metastasis. Macrophage recruitment is stimulated by chemotactic factors, including tumor necrosis factor alpha (TNF-α), which also stimulates the cytotoxic/tumor cell killing macrophage phenotype. Through TNF-α converting enzyme (TACE/ADAM17) activities, breast tumor cells shed membrane-bound proteins, including their TNF receptors (sTNFR1/2), which serve as decoys sequestering TNF-α and preventing TNF-α-driven apoptosis of tumor cells, thereby decreasing TNF-α bioavailability. Here we investigated the levels of sTNFRs shed by breast tumor cells and determined the effects of shed sTNFRs on macrophage migration toward TNF-α. TNF-α and sTNFRs concentrations were measured in murine normal epithelial, stromal, and mammary tumor cells. The migration of murine macrophages towards TNF-α in the presence of tumor derived soluble factors (TDSFs) shed by TACE was determined. TNF-α concentrations secreted by tumor and normal epithelial cells were below the detection limit contrasting with stromal cells, especially macrophages, which expressed higher levels of TNF-α (P<0.001). Regardless of the cell tested, treatment with the TACE inhibitor TAPI-0 led to a significant decrease in sTNFR2 shed (P<0.05). The dose-dependent macrophage migration toward TNF-α prevented by incubation with TDSFs was not observed with TDSFs collected following TAPI-0 treatment (P<0.05). Furthermore, the TNF-α-driven increased pAkt expression in macrophage was inhibited by TACE shed TDSFs (P<0.05). These results highlight the role of tumor-shed sTNFRs in TNF-α -driven macrophage chemotaxis.

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.

Abstract 1389: Arthritis augments breast cancer metastasis: Role of mast cells and SCF/c-Kit signaling

Background Breast Cancer remains the second leading cause of cancer-related deaths in the United States. In 2011, an estimated 458,000 women are expected to die from the disease due to metastasis. Metastasis is regulated not only by intrinsic genetic changes in malignant cells, but also by the microenvironment, especially those associated with chronic inflammation. We have reported that mice that suffer from 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 contributing to the increased metastasis. Our preliminary studies show that the metastatic niches (bone and lung) in the arthritic mice express significantly higher levels of mast cells than their non-arthritic counterparts. It is known the SCF/c-kit signaling within the metastatic niche triggers the activation of mast cells which are known to aid the metastatic process via up-regulation of various pro-inflammatory factors. Thus, we hypothesize that increase in mast cells triggered by SCF/cKit signaling may be the underlying cause for increased metastasis and that targeting the SCF-cKit interaction may prevent metastasis. Methods We used two mouse models; one that develops spontaneous autoimmune arthritis (SKG mice) injected with metastatic breast cancer cells (4T1), and second, that develops spontaneous breast cancer (MMTV-PyV MT mice) injected with type II collagen to induce autoimmune arthritis. Mice were left untreated or treated with anti cKit receptor antibody ± celecoxib (a known drug used to treat autoimmune arthritis). Mast cell levels and metastasis were monitored along with the pro-inflammatory factors associated with mast cell activation. Results i) Differentiation of mast cells from bone marrow derived stem cells was significantly higher in the arthritic versus the non-arthritic tumor-bearing mice; ii) Mast cell population within the bone and lung lesions were significantly higher in the arthritic versus non-arthritic tumor-bearing mice; iii) The SCF/cKit signaling was significantly up regulated within the metastatic lesions of the arthritic versus the non-arthritic tumor-bearing mice; and iv) Treatment with the anti-cKit receptor antibody + celecoxib significantly reduced the differentiation of mast cells and consequently reduced breast cancer-associated metastasis. Conclusion: Mast cell levels and SCF/cKit signaling are significantly higher in breast cancer mice that suffer from autoimmune arthritis versus their non-arthritic counterparts. Mice treated with anti-cKit receptor + celecoxib shows decreased metastasis and mast cells. Thus, mast cells play a critical role in not only remodeling the tumor microenvironment but also the metastatic niche to facilitate efficient metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1389. doi:1538-7445.AM2012-1389

Identification of the Most Commonly Used Laboratory Techniques in Regenerative Medicine: A Roadmap for Developing a Competency Based Education Curriculum

Here, we are proposing and testing the use of literature reviews as a method to identify essential competencies for specific fields. This has implications in how educators develop and structure both traditional and competency based curricula. Our focus will be on utilizing this method to identify the most relevant and commonly used techniques in the field of regenerative medicine. This publication review method may be used to develop competency based education (CBE) programs that focus on commonly utilized skills. CBE is an emerging trend in higher education that will greatly enhance student learning experiences. CBE works by providing students with field specific skills and knowledge; thus, it is imperative for educators to identify the most essential competencies in a given field. Therefore, we reason that a literature review of the techniques performed in studies published in prevalent peer reviewed journals for a given field offers an ideal method to identify and rank competencies that should be delivered to students by a respective curriculum. Here, we reviewed recent articles published on topics in the field of regenerative medicine as a proof of concept for the use of literature reviews as a guide for the development of a regenerative medicine CBE curriculum.

Abstract 1540: TNFRs shed by tumor cells inhibit the migration of macrophages.

Breast tumor cells transform their microenvironment through the expression of pro-tumor molecules critical to their progression and metastasis. Among the targets of tumor cell secretions are macrophages, which play an essential role in tumor progression. Although macrophages possess tumor cell killing abilities, the tumor-associated macrophages (TAMs) display primarily pro-tumor functions including increasing tumor angiogenesis and promoting tumor cell invasion. Breast tumor cells shed multiple membrane-bound proteins including their tumor necrosis factor (TNF) receptors through TACE/ADAM17 activities. The release of sTNFR1/2 serves a decoy sequestering TNF and preventing TNF-driven apoptosis of tumor cells. Additionally, tumor derived sTNFR1/2 also modulate TNF effects on macrophages including their migration. Here, we investigated whether sTNFRs were shed by cells present within the breast tumor microenvironment and determined their effects on macrophage migration in response to TNF. TNF and sTNFRs concentrations were measured in murine mammary tumor and normal cells including monocytes/macrophages. In addition, the migration of murine monocytes toward TNF in the presence tumor derived soluble factors (TDSFs) was determined by transwell assays. The effects of TDSFs obtained with and without the sheddase inhibitor TAPI-0 treatment on monocyte/macrophage migration were also assessed. No TNF was detected in the TDSFs of tumor cells or secretions from normal epithelial cells. In contrast, stroma cells especially macrophages expressed high levels of TNF (p [This works is supported by grants from the Department of Defense Era of Hope research program (BC044778) and the National Science Foundation EFRI program (CBE0736007)]. Citation Format: Stephen Rego, Rachel Helms, Alexander De Piante, Amritha Kidiyoor, Amanda Lance, Pinku Mukherjee, Didier Dreau. TNFRs shed by tumor cells inhibit the migration of macrophages. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1540. doi:10.1158/1538-7445.AM2013-1540

Abstract 5400: Investigating the role of IDO in MUC1 expressing breast cancers

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Breast cancer is the second most common cancer in the U.S. and contributes to 40,000 deaths a year. Breast cancer vaccines are currently being considered as a clinical intervention that may reduce the chance of metastasis and recurrence, and perhaps even function to be effective in cancer prevention. MUC1, a membrane tethered mucin glycoprotein, is over expressed in >90% of breast cancers, and therefore has been recently described as the second most targetable tumor antigen by the National Cancer Institute. There are currently ongoing trials using the MUC1 vaccine in patients with metastatic breast cancer. However, immunotherapy has had limited success because tumors have the ability to undergo immune evasion tactics. This includes expression of Indoleamine 2,3-dioxygenase (IDO) immunosuppressive enzymes. IDO was first identified in maintaining maternal tolerance towards the antigenically foreign fetus during pregnancy. Its activity is increased under pathological conditions, including during tumor development. IDO is emerging as a key player in T cell suppression and in the induction of immune tolerance to tumors. The present study is focused on understanding the role of IDO enzymatic activity on tumor development and immune function. In this study, we injected two cell lines that express IDO, and one cell line that is IDO null (MTAG, MTAG.MUC1 and IKOM, respectively). These cell lines were injected into either IDO null mice, or control c57/bk6 mice (n=3 each). We hypothesized that IDO expression in the tumor microenvironment of mice creates a pathological state of immune suppression resulting in altered tumor progression and immune function. We show that mice (whether they be IDO null or blk6) injected with IKOM cells have significantly slower tumor progression and rates of secondary metastasis as compared to those injected with IDO expressing tumor cells (p<0.05). In vitro, IKOM cells had significantly lower proliferative rates than the IDO expressing cells. Furthermore, mice injected with IKOM cells had a greater percentage of CD4+ and CD8+ T cells as compared to those mice injected with IDO expressing tumor cells. Mice injected with IDO expressing cells had a greater percentage of MDSCs characterized by the expression of gr1 and cd11b. Future studies would investigate the role of MUC1 based vaccines in combination with an IDO inhibitor, with the goal of reducing metastasis and increasing survival in patients with breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5400. doi:1538-7445.AM2012-5400

Abstract 3644: Pancreatic tumor cells that develop within Muc1 knock-out mice generate less immunosuppressive MDSCs in vitro

Epithelial Mucin 1 (MUC1) is a well characterized tumor associated antigen found to be over expressed and aberrantly glycosylated in human pancreatic adenocarcinomas. In recent years, Myeloid- derived suppressor cells (MDSCs), major regulators of the immune response, have been implicated in the progression of cancer. We have recently shown that Muc1 null mice have a higher propensity of MDSCs and are unable to reject allogeneic tumors as effectively as their wild-type counterparts. Here, we studied the effects of tumor derived soluble factors (TDSFs) from pancreatic cancer cell lines that over expressed human MUC1 (KCM) or were null for Muc1 (KCKO) on the generation of MDSCs from bone marrow (BM) cells in vitro. The two cell lines were derived from a spontaneous mouse model of pancreatic ductal adenocarcinoma (PDA) that was bred to the Muc1-null (KCKO) or the human MUC1 transgenic (KCM) mice. BM cells were cultured in complete DMEM supplemented with 10ng/ml GM-CSF and 10ng/ml IL-4 + 30% v/v conditioned media (TCCM) from KCKO or KCM cell lines for 5 days. On day 3, floating cells were removed and fresh media with cytokines and TCCM was added. On day 5, cells were stained for MDSCs and analyzed by flow cytometry. Statistical analysis was performed using GraphPad software and p values were calculated using One-way ANOVA. We observed a significant increase in the monocytic (CD11b+Ly6C+Ly6G-) and the granulocytic (CD11b+Ly6C+LyG+) MDSC subpopulations when naive BM cells were cultured in the presence of TCCM from KCKO versus KCM cells (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3644. doi:10.1158/1538-7445.AM2011-3644