Priyanka Grover

A novel association of neuropilin-1 and MUC1 in pancreatic ductal adenocarcinoma: role in induction of VEGF signaling and angiogenesis

We report that Mucin1 (MUC1), a transmembrane glycoprotein that is overexpressed in >80% of pancreatic ductal adenocarcinoma (PDA), induced a pro-angiogenic tumor microenvironment by increasing the levels of neuropilin-1 (NRP1, a co-receptor of vascular endothelial growth factor (VEGF)) and its ligand VEGF. Expression of tumor-associated MUC1 (tMUC1) positively correlated with NRP1 levels in human and mouse PDA. Further, tMUC1hi PDA cells secreted high levels of VEGF and expressed high levels of VEGF receptor 2 (VEGFR2) and its phosphorylated forms as compared with tMUC1low/null PDA. This enabled the tMUC1hi/NRP1hi PDA cells to (a) induce endothelial cell tube formation, (b) generate long ectopic blood vessels and (c) enhance distant metastasis in a zebrafish xenograft model. Concurrently, the proteins associated with epithelial-to-mesenchymal transition, N-cadherin and Vimentin, were highly induced in these tMUC1/NRP1hi PDA cells. Hence, blocking signaling via the NRP1–VEGF axis significantly reduced tube formation, new vessel generation and metastasis induced by tMUC1hi PDA cells. Finally, we show that blocking the interaction between VEGF165 and NRP1 with a NRP1 antagonist significantly reduced VEGFR signaling and PDA tumor growth in vivo. Taken together, our data suggest a novel molecular mechanism by which tMUC1 may modulate NRP1-dependent VEGFR signaling in PDA cells.

Mucin 1 Regulates Cox-2 Gene in Pancreatic Cancer

Objective Eighty percent of pancreatic ductal adenocarcinomas (PDAs) overexpress mucin 1 (MUC1), a transmembrane mucin glycoprotein. MUC1high PDA patients also express high levels of cyclooxygenase 2 (COX-2) and show poor prognosis. The cytoplasmic tail of MUC1 (MUC1-CT) partakes in oncogenic signaling, resulting in accelerated cancer progression. Our aim was to understand the regulation of Cox-2 expression by MUC1. Methods Levels of COX-2 and MUC1 were determined in MUC1−/−, MUC1low, and MUC1high PDA cells and tumors using reverse transcriptase–polymerase chain reaction, Western blot, and immunohistochemistry. Proliferative and invasive potential was assessed using MTT and Boyden chamber assays. Chromatin immunoprecipitation was performed to evaluate binding of MUC1-CT to the promoter of COX-2 gene. Results Significantly higher levels of COX-2 mRNA and protein were detected in MUC1high versus MUC1low/null cells, which were recapitulated in vivo. In addition, deletion of MUC1 gene and transient knockdown of MUC1 led to decreased COX-2 level. Also, MUC1-CT associated with the COX-2 promoter at ∼1000 base pairs upstream of the transcription start site, the same gene locus where nuclear factor &kgr;B p65 associates with the COX-2 promoter. Conclusions Data supports a novel regulation of COX-2 gene by MUC1 in PDA, the intervention of which may lead to a better therapeutic targeting in PDA patients.

Antibody-Guided In Vivo Imaging for Early Detection of Mammary Gland Tumors.

BACKGROUND: Earlier detection of transformed cells using target-specific imaging techniques holds great promise. We have developed TAB 004, a monoclonal antibody highly specific to a protein sequence accessible in the tumor form of MUC1 (tMUC1). We present data assessing both the specificity and sensitivity of TAB 004 in vitro and in genetically engineered mice in vivo. METHODS: Polyoma Middle T Antigen mice were crossed to the human MUC1.Tg mice to generate MMT mice. In MMT mice, mammary gland hyperplasia is observed between 6 and 10 weeks of age that progresses to ductal carcinoma in situ by 12 to 14 weeks and adenocarcinoma by 18 to 24 weeks. Approximately 40% of these mice develop metastasis to the lung and other organs with a tumor evolution that closely mimics human breast cancer progression. Tumor progression was monitored in MMT mice (from ages 8 to 22 weeks) by in vivo imaging following retro-orbital injections of the TAB 004 conjugated to indocyanine green (TAB-ICG). At euthanasia, mammary gland tumors and normal epithelial tissues were collected for further analyses. RESULTS: In vivo imaging following TAB-ICG injection permitted significantly earlier detection of tumors compared with physical examination. Furthermore, TAB-ICG administration in MMT mice enabled the detection of lung metastases while sparing recognition of normal epithelia. CONCLUSIONS: The data highlight the specificity and the sensitivity of the TAB 004 antibody in differentiating normal versus tumor form of MUC1 and its utility as a targeted imaging agent for early detection, tumor monitoring response, as well as potential clinical use for targeted drug delivery.

Abstract 475: Combinational treatment with MUC1 vaccine and Indomethacin reduces breast tumor burden via a COX-independent pathway.

Introduction: While much advancement has been made in breast cancer treatment, metastatic breast cancer remains an incurable disease. The glycoprotein MUC1 is over-expressed and underglycosylated in over 90% of human breast tumors and 100% of metastatic lesions, and thus was ranked the second most targetable tumor antigen (TA) by NCI. Vaccines against TAs have several benefits, including the chance to eliminate metastatic lesions that express the TA. To this end, we have proposed vaccinating with peptides from the MUC1 protein core, which is only visible to the immune system on the tumor-associated form of the protein. We have previously shown that vaccination does elicit a MUC1-specific immune response that can only be functional if the immunosuppressive tumor microenvironment is blocked to allow efficient tumor cell killing. We investigated the effectiveness of MUC1 vaccination in combination with drugs known to inhibit immunosuppression. Methods: Mice that are transgenic for human MUC1 were orthotopically injected with syngenic breast cancer cells expressing human MUC1. Mice were vaccinated after palpable tumor formation three times in 10 day intervals. Previous work has shown that blocking the cyclooxygenase pathway (COX) resulted in an inhibition of immunosuppression. Thus we treated daily with following drugs in combination with the MUC1-vaccine therapy: Indomethacine (COX1/2 inhibitor), Celecoxib (COX2 inhibitor), 1-methyl tryptophan (IDO inhibitor), and AH6809 (EP2 receptor antagonist). Tumor volume was monitored every day by caliper measurements. Immune activation was measured via an IFN-γ ELISPOT assay using T cells from tumor-draining lymph nodes as responders and dendritic cells pulsed with the vaccinating peptides as simulators. Results: MUC1 vaccine therapy alone causes a slight reduction in tumor burden, although not significant, but does induce IFN-γ production from T cells, indicating immune cell activation. Only the combinational therapy of Vaccine + Indomethacin results in a significant reduction in tumor size. All drugs cause significantly higher levels of IFN-γ production from T cells compared to vaccine alone but no reduction in tumor burden. The combinational treatments of Vaccine + Indomethacine and Vaccine + Celecoxib reduce intra-tumoral PGE2 levels compared to vaccine alone. In a repeat experiment, we found that vaccine alone and indomethacin alone caused a slight reduction in tumor burden, but tumor burden was only significantly reduced when the therapy was given in combination. Conclusion: In this model, MUC1 vaccine efficacy can be enhanced when given in combination with Indomethacin. Since Indomethacin but not Celecoxib reduced tumor burden when given in combination with the MUC1 vaccine, we suggest that Indomethacin enhanced vaccine efficacy via a COX-independent pathway. Future experiments will explore this mechanism. Citation Format: Jennifer M. Curry, Dahlia M. Besmer, Lopamudra Das Roy, Priyanka Grover, Sritama Nath, Shanti Rao, Pinku Mukherjee. Combinational treatment with MUC1 vaccine and Indomethacin reduces breast tumor burden via a COX-independent pathway. [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 475. doi:10.1158/1538-7445.AM2013-475

Early detection of pancreatic cancer in mouse models using a novel antibody, TAB004

Pancreatic ductal adenocarcinoma (PDA) is the fourth-leading cause of cancer death in the United States with a 5-year overall survival rate of 8% for all stages combined. But this decreases to 3% for the majority of patients that present with stage IV PDA at time of diagnosis. The lack of distinct early symptoms for PDA is one of the primary reasons for the late diagnosis. Common symptoms like weight loss, abdominal and back pains, and jaundice are often mistaken for symptoms of other issues and do not appear until the cancer has progressed to a late stage. Thus the development of novel imaging platforms for PDA is crucial for the early detection of the disease. MUC1 is a tumor-associated antigen (tMUC1) expressed on 80% of PDA. The goal of this study was to determine the targeting and detection capabilities of a tMUC1 specific antibody, TAB004. TAB004 antibody conjugated to a near infrared fluorescent probe was injected intraperitoneally into immune competent orthotopic and spontaneous models of PDA. Results show that fluorophore conjugated TAB004 specifically targets a) 1 week old small tumor in the pancreas in an orthotopic PDA model and b) very early pre-neoplastic lesions (PanIN lesions) that develop in the spontaneous PDA model before progression to adenocarcinoma. Thus, TAB004 is a promising antibody to deliver imaging agents directly to the pancreatic tumor microenvironment, significantly affecting early detection of PDA.

SMAD4-independent activation of TGF-β signaling by MUC1 in a human pancreatic cancer cell line

Pancreatic Ductal Adenocarcinoma (PDA) has a mortality rate that nearly matches its incidence rate. Transforming Growth Factor Beta (TGF-β) is a cytokine with a dual role in tumor development switching from a tumor suppressor to a tumor promoter. There is limited knowledge of how TGF-β function switches during tumorigenesis. Mucin 1 (MUC1) is an aberrantly glycosylated, membrane-bound, glycoprotein that is overexpressed in >80% of PDA cases and is associated with poor prognosis. In PDA, MUC1 promotes tumor progression and metastasis via signaling through its cytoplasmic tail (MUC1-CT) and interacting with other oncogenic signaling molecules. We hypothesize that high levels of MUC1 in PDA may be partly responsible for the TGF-β functional switch during oncogenesis. We report that overexpression of MUC1 in BxPC3 human PDA cells (BxPC3.MUC1) enhances the induction of epithelial to mesenchymal transition leading to increased invasiveness in response to exogenous TGF-β1. Simultaneously, these cells resist TGF-β induced apoptosis by downregulating levels of cleaved caspases. We show that mutating the tyrosines in MUC1-CT to phenylalanine reverses the TGF-β induced invasiveness. This suggests that the tyrosine residues in MUC1-CT are required for TGF-β induced invasion. Some of these tyrosines are phosphorylated by the tyrosine kinase c-Src. Thus, treatment of BxPC3.MUC1 cells with a c-Src inhibitor (PP2) significantly reduces TGF-β induced invasiveness. Similar observations were confirmed in the Chinese hamster ovarian (CHO) cell line. Data strongly suggests that MUC1 may regulate TGF-β function in PDA cells and thus have potential clinical relevance in the use of TGF-β inhibitors in clinical trials.

Abstract 4708: A novel anti-MUC1 CAR T cell drives immunity to pancreatic cancer

Background: Pancreatic cancer is the 4th leading cause of cancer deaths in the US with very poor prognosis. Treatment options are limited to surgery and chemo/radiation that often times do not increase survival and are associated with high toxicity. Targeted immune-based therapies have shown some promise but needs further exploration. Mucin 1 (MUC1), a glycoprotein expressed on the apical surface of epithelial cells of most epithelial organs, undergoes hypoglycozylation in tumors. This tumor-form of MUC1 (tMUC1) is over-expressed in 80% of pancreatic ductal adenocarcinomas (PDAC). tMUC1 therefore remains a promising target for therapeutic intervention. We have developed a patented antibody (TAB004) which specifically detects tMUC1 and spares normal MUC1. Using a novel technology, functional fragments of TAB004 antibody (scFv) were incorporated into the chimeric antigen receptor (CAR) construct and used to genetically modify primary human T cells. ScFv domain which recognizes tMUC1 is linked to the co-stimulatory molecules of T cells (CD28 and CD3ζ). When the engineered T cells come in contact with tMUC1 expressing tumor cells, multiple T cell signaling pathways are initiated leading to fully activated cytotoxic T cells that lyse the tumor cells. Methods: Retroviral based technique was used to deliver the CAR gene into human PBMC derived primary T cells. A fluorescent tag (mKate) was fused to the C-terminus of CAR molecules, in order to visualize CAR expression on T cell membrane by fluorescent microscopy and potentially for in vivo tracking. Cytotoxicity was evaluated using co-culture method with varying T cell to target cell ratios followed by MTT assay. Intracellular IFNγ was measured by flow cytometry. Results: tMUC1-CAR-T cells show increased activation and proliferation compared to normal T cells. These cells bind strongly to tMUC1 expressing human pancreatic cancer cells forming immunologic synapse. Minimal binding of the tMUC1-CAR T cells was observed to normal or low MUC1 expressing tumors cells suggesting high specificity of these CAR T cells to tMUC1. CAR expression was distributed evenly on the cell surface of the T cells. Engineered tMUC1-CAR T cells exhibit robust cytotoxicity against a panel of PDA cell lines, associated with high IFNγ release. Fortunately, the same CAR T cells display minimum toxicity against normal epithelial cells. CAR T cell function will be evaluated in the preclinical mouse model of PDA, as single treatment and also in combination with checkpoint inhibitors and chemotherapy drugs. Conclusion: Despite the remarkable successes reported using CAR T cells in clinic, particularly CD19 CAR T for leukemia; some adverse effects have been attributed to this treatment. This highlights the urgent need for developing tumor-specific CAR T cells. This study demonstrates the specificity and effectiveness of tMUC1-CAR T cells against pancreatic cancer cells. Thus, tMUC1 CAR T cells have the potential to be further developed for future clinical use. Citation Format: Mahboubeh Yazdanifar, Ru Zhou, Shu-ta Wu, Priyanka Grover, Pinku Mukherjee. A novel anti-MUC1 CAR T cell drives immunity to pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4708. doi:10.1158/1538-7445.AM2017-4708

Abstract 2054: In pancreatic cancer, MUC1 regulates function of TGF-β and thus enhances metastasis

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Background Pancreatic Cancer (PC) is the 4th leading cause of cancer-related deaths in the United States with 94% of PC patients dying within 5 years of diagnosis. Pancreatic Ductal Adenocarcinoma (PDA) characterizes 90% of PC. Over 80% of PDA overexpresses tumor associated Mucin-1 (MUC1), a membrane bound glycoprotein that is hypoglycosylated compared to normally expressed MUC1. Overexpression of MUC1 is associated with increased metastasis and poor prognosis. However the mechanism remains elusive. Transforming growth factor-β (TGF-β) is similarly overexpressed in most PDA. TGF-β is a cytokine with dual functionality. In normal cells, TGF-β functions as a tumor suppressor and induces apoptosis. This effect is mediated by activation of the canonical Smad pathway via engagement of TGF-β Receptor 1 (TGF-βRI). However, during cancer development, TGF-β becomes a tumor promoter and stimulates epithelial to mesenchymal transition, migration, and invasion of tumor cells thus enhancing metastasis. This effect of TGF-β is mediated by activation of the noncanonical Erk1/2 pathway through the engagement of TGF-β Receptor 2 (TGF-βRII). Therefore, we hypothesize that overexpression of MUC1 in PDA transforms the function of TGF-β from a tumor suppressor to a tumor promoter. Further, we postulate that signaling through the MUC1 cytoplasmic tail (CT) is necessary to activate the noncanonical Erk1/2 pathway via phosphorylation of the TGF-βRII, thus leading to enhanced metastasis. Methods We first assessed the ability of TGF-β to induce apoptosis versus invasiveness in PDA cell lines that express variable levels of MUC1 with gain of function (forced expression) and loss of function (knock down using specific MUC1 siRNA) studies. We determined the levels of TGF-βRI, RII, MUC1, and Smad 4 proteins in these PDA cell lines. We evaluated the activation status of the Erk1/2 and Smad pathways. Since c-Src is known to phosphorylate the 6th tyrosine residue of MUC1 CT and is essential for oncogenic signaling, we determined if c-Src is associated with MUC1 and is phosphorylated upon treatment with exogenous TGF-β in PDA cells. Results In MUC1-high expressing PDA cells, TGF-β acts as a tumor promoter while in MUC1-low expressing PDA cells, TGF-β induces apoptosis and acts as a tumor suppressor. We show a clear association between the expression of MUC1 and TGF-βRI and RII activation. In MUC1-high expressing cells, TGF-βRII is over expressed while in MUC1-low expressing cells, TGF-βRI is over expressed and this correlates with the activation status of Erk1/2 and Smad 4. Finally, we show that MUC1 specifically associates with c-Src and without this association, downstream oncogenic signaling is impeded. Conclusion MUC1 expression is directly correlated with TGF-β function and expression of TGF-βRII, while being negatively correlated with TGF-βRI expression. This has high clinical significance for patients with PDA. Citation Format: Priyanka Grover, Sritama Nath, Mohammad Ahmad, Pinku Mukherjee. In pancreatic cancer, MUC1 regulates function of TGF-β and thus enhances metastasis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2054. doi:10.1158/1538-7445.AM2015-2054

Abstract 26: MUC1 enhances neuropilin-1 signaling in pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDA) has the worst prognosis of all cancers and is the 4th leading cause of cancer-related deaths in the United States. Mucin1 (MUC1) is a transmembrane glycoprotein over-expressed in more than 60% of PDA and its expression correlates with high metastases and poor prognosis. In PDA, there is a correlation between blood vessel density, tumor levels of VEGF, and disease progression. We have recently discovered a novel association between MUC1 and neuropilin-1 (NRP-1) expression. Neuropilin-1 (NRP-1) is a co-receptor for VEGF165 and blockade of NRP-1-VEGF165 interaction has been shown to inhibit angiogenesis and tumor growth in several other cancer types. Thus, we hypothesize that MUC1 induces a pro-angiogenic tumor microenvironment by increasing levels of VEGF and NRP-1, thus enhancing disease progression and metastases. We have generated Muc1-expressing KC and Muc1-null KCKO mouse PDA cell lines for this study. Compared to KCKO, KC cells secrete more VEGF and express higher levels of NRP-1 as well as VEGF receptors (1/2/3). Although the cell culture supernatants from KC do not alter the NRP-1 level on the endothelial line 2H11 cells, they significantly promote the 2H11 cell tube formation. Consistent with the in vitro results, tumor lysates from KC-bearing mice have higher levels of NRP-1 and VEGFR2 and its phosphorylations at Tyr residues 951, 996, and 1175. In human PDA cell lines, inhibition of MUC1 expression by specific siRNA reduced NRP-1 level while overexpression of full-length MUC1 increases NRP-1 expression. This suggests a direct regulation of NRP-1 by MUC1. Furthermore, the over-expression of MUC1 in BxPC3 human PDA cell line induces strong angiogenesis and promotes tumor metastasis in a zebrafish tumor xenograft model. Finally, we report that inhibition of NRP-1 by its specific blocking peptide suppresses BxPC3.MUC1 tumor growth in vivo. Taking together, MUC1 in PDA cells can enhance their own NRP-1 levels and promote the VEGF-VEGFR-NRP-1 signaling, which may favor the creation of a pro-angiogenic microenvironment for tumor growth and metastases. Our findings highlight the potential for MUC1 targeted blockade of VEGF-NRP-1 interaction in PDA by conjugating the NRP-1 inhibitory agents to a MUC1 targeting antibody. Citation Format: Ru Zhou, Jennifer Curry, Priyanka Grover, Lopamudra Das Roy, TinChung Leung, Pinku Mukherjee. MUC1 enhances neuropilin-1 signaling in pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 26. doi:10.1158/1538-7445.AM2014-26

Abstract 3119: MUC1 transcriptionally regulates COX-2 gene in pancreatic cancer cells.

Introduction: Pancreatic Cancer (PC) is the fourth leading cause of cancer-related deaths in the United States. Over 80% of human pancreatic ductal adenocarcinomas (PDA) overexpress MUC1, a transmembrane mucin glycoprotein. MUC1high tumors are aggressive and patients with such tumors have poor prognosis. Interestingly, MUC1high PDA tumors overexpress cyclooxygenase-2 (COX-2), an inducible pro-inflammatory enzyme known to promote tumor progression and metastasis. Since the cytoplasmic domain of MUC1 (MUC1-C) is critically involved in the signal transduction cascade that promotes tumor progression and metastasis, we hypothesize that MUC1 induces its effect via regulating COX-2 expression and function. Thus, we aim to establish the relationship between MUC1 and COX-2 expression and determine the mechanism by which MUC1 regulates COX-2 expression. Cell lines: Human BxPC3 and mouse Panc02 PC cell lines that express low endogenous MUC1 were stably transfected with full length MUC1 (MUC1), empty vector (Neo), or full length MUC1 with all 7 tyrosine residues of MUC1 CT mutated to Phenylalanine (Y0). Other cell lines: human HPAFII and Capan-1 and mouse KCM cells that express high endogenous MUC1; and mouse KCKO cells that genetically lack Muc1. Method: We measured the level of COX-2 mRNA and protein by RT-PCR and Western blot respectively. MUC1 was transiently knocked down using a smart pool of MUC1 specific siRNA in MUC1high cells. Immunohistochemistry (IHC) was performed on tumor sections to determine the in situ expression of COX-2 and MUC1. Chromatin immunoprecipitation (ChIP) assay was conducted to evaluate binding of MUC1 CT to the promoter of COX-2 gene. Results: Significantly higher levels of COX-2 mRNA and protein were detected in PC cells expressing high MUC1 compared to cells with low MUC1. This was recapitulated in vivo with higher COX-2 expression in MUC1high versus MUC1low tumors. A subsequent decrease in COX-2 protein was observed upon MUC1 knock down in the MUC1high PC cells. Thus, a direct correlation exists between MUC1 and COX-2 expression in PC cells. Importantly, this correlation was lost in the Y0 cells in which COX-2 expression was significantly decreased even though there was high MUC1 expression. Data clearly suggests that signaling through the tyrosine residues of MUC1 CT is essential for MUC1 induced COX-2 gene expression. Further, in the MUC1high cells, MUC1 CT associates with the COX-2 promoter around 1000 bp upstream of the transcription start site. This association does not occur in the Y0 cells suggesting that an intact CT is necessary for this interaction. Interestingly, MUC1 CT interacts with the COX-2 promoter around the same gene locus where NF-kB associates with the COX-2 promoter. This raises the possibility that MUC1 CT associates with NF-kB via its tyrosine residues and the MUC1/NFkB complex acts as a transcriptional regulator of the COX-2 gene. Citation Format: Sritama Nath, Lopamudra Das Roy, Shanti Rao, Priyanka Grover, Pinku Mukherjee. MUC1 transcriptionally regulates COX-2 gene in pancreatic cancer cells. [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 3119. doi:10.1158/1538-7445.AM2013-3119