Gargi Maity

Aspirin blocks growth of breast tumor cells and tumor-initiating cells and induces reprogramming factors of mesenchymal to epithelial transition.

Acetylsalicylic acid (ASA), also known as aspirin, a classic, nonsteroidal, anti-inflammatory drug (NSAID), is widely used to relieve minor aches and pains and to reduce fever. Epidemiological studies and other experimental studies suggest that ASA use reduces the risk of different cancers including breast cancer (BC) and may be used as a chemopreventive agent against BC and other cancers. These studies have raised the tempting possibility that ASA could serve as a preventive medicine for BC. However, lack of in-depth knowledge of the mechanism of action of ASA reshapes the debate of risk and benefit of using ASA in prevention of BC. Our studies, using in vitro and in vivo tumor xenograft models, show a strong beneficial effect of ASA in the prevention of breast carcinogenesis. We find that ASA not only prevents breast tumor cell growth in vitro and tumor growth in nude mice xenograft model through the induction of apoptosis, but also significantly reduces the self-renewal capacity and growth of breast tumor-initiating cells (BTICs)/breast cancer stem cells (BCSCs) and delays the formation of a palpable tumor. Moreover, ASA regulates other pathophysiological events in breast carcinogenesis, such as reprogramming the mesenchymal to epithelial transition (MET) and delaying in vitro migration in BC cells. The tumor growth-inhibitory and reprogramming roles of ASA could be mediated through inhibition of TGF-β/SMAD4 signaling pathway that is associated with growth, motility, invasion, and metastasis in advanced BCs. Collectively, ASA has a therapeutic or preventive potential by attacking possible target such as TGF-β in breast carcinogenesis.

Pancreatic Tumor Cell Secreted CCN1/Cyr61 Promotes Endothelial cell migration and Aberrant Neovascularization

The complex signaling networks between cancer cells and adjacent endothelial cells make it challenging to unravel how cancer cells send extracellular messages to promote aberrant vascularization or tumor angiogenesis. Here, in vitro and in vivo models show that pancreatic cancer cell generated unique microenvironments can underlie endothelial cell migration and tumor angiogenesis. Mechanistically, we find that pancreatic cancer cell secreted CCN1/Cyr61 matricellular protein rewires the microenvironment to promote endothelial cell migration and tumor angiogenesis. This event can be overcome by Sonic Hedgehog (SHh) antibody treatment. Collectively, these studies identify a novel CCN1 signaling program in pancreatic cancer cells which activates SHh through autocrine-paracrine circuits to promote endothelial cell migration and tumor angiogenesis and suggests that CCN1 signaling of pancreatic cancer cells is vital for the regulation of tumor angiogenesis. Thus CCN1 signaling could be an ideal target for tumor vascular disruption in pancreatic cancer.

Deficiency of CCN5/WISP-2-Driven Program in breast cancer Promotes Cancer Epithelial cells to mesenchymal stem cells and Breast Cancer growth

Breast cancer progression and relapse is conceivably due to tumor initiating cells (TICs)/cancer stem cells. EMT (epithelial-mesenchymal-transition)-signaling regulates TICs’ turnover. However, the mechanisms associated with this episode are unclear. We show that, in triple-negative-breast cancer (TNBC) cells enriched with TICs, CCN5 significantly blocks cellular growth via apoptosis, reversing EMT-signaling and impairing mammosphere formation, thereby blocking the tumor-forming ability and invasive capacity of these cells. To corroborate these findings, we isolated tumor-initiating side populations (SP) and non-side population (NSP or main population) from MCF-7 cell line, and evaluated the impact of CCN5 on these subpopulations. CCN5 was overexpressed in the NSP but downregulated in the SP. Characteristically, NSP cells are ER-α positive and epithelial type with little tumorigenic potency, while SP cells are very similar to triple-negative ones that do not express ER-α- and Her-2 and are highly tumorigenic in xenograft models. The overexpression of CCN5 in SP results in EMT reversion, ER-α upregulation and delays in tumor growth in xenograft models. We reasoned that CCN5 distinguishes SP and NSP and could reprogram SP to NSP transition, thereby delaying tumor growth in the xenograft model. Collectively, we reveal how CCN5-signaling underlies the driving force to prevent TNBC growth and progression.

Human pancreatic cancer progression: an anarchy among CCN-siblings

Decades of basic and translational studies have identified the mechanisms by which pancreatic cancer cells use molecular pathways to hijack the normal homeostasis of the pancreas, promoting pancreatic cancer initiation, progression, and metastasis, as well as drug resistance. These molecular pathways were explored to develop targeted therapies to prevent or cure this fatal disease. Regrettably, the studies found that majority of the molecular events that dictate carcinogenic growth in the pancreas are non-actionable (potential non-responder groups of targeted therapy). In this review we discuss exciting discoveries on CCN-siblings that reveal how CCN-family members contribute to the different aspects of the development of pancreatic cancer with special emphasis on therapy.

Detection of CCN1 and CCN5 mRNA in Human Cancer Samples Using a Modified In Situ Hybridization Technique

In situ hybridization is an ideal tool for the detection and localization of mRNA expression of specific gene(s) in tissue sections and cell lines for prognosis, predictive markers, and highlighted potential therapeutic targets. Given the importance of CCN1 and CCN5 in breast and pancreatic cancer progression, these two secretory proteins could be novel therapeutic targets. Thus, evaluating the distribution of mRNA of these targets using in situ hybridization could be important preclinical tools. This chapter describes a detailed in situ hybridization technique for the detection of CCN1 and CCN5 in formalin-fixed, paraffin-embedded patient samples of breast and pancreatic cancers.

Abstract 3572: Metformin inhibits the oncogenic potential and invasiveness of pancreatic cancer cells targeting CCN1-CXCR4 axis : A new perspective for an old antidiabetic drug

Pancreatic Ductal Adenocarcinoma (PDAC) is among the most aggressive of solid malignancies responsible for around 330,000 deaths globally and accounts for the fourth most common cause of deaths due to cancer in USA. There has been a little advancement in the treatment of PDAC patients and the mortality rate has remained unchanged and may even be climbing up. Metformin, an oral biguanide medication used to treat type-2 diabetes mellitus, has demonstrated potential therapeutic effect against PDAC. Recent meta-analysis and epidemiologic studies indicate that diabetic patients treated with metformin were less likely to develop pancreatic cancer exhibit longer overall survival than those using other oral antidiabetic medications. Although there are several reports of the possible mode of action of Metformin against PDAC, no direct cellular/ molecular target is till reported. The present study aims to identify the direct molecular target of metformin in pancreatic cancer cells. Previously, we have demonstrated that matricellular protein CCN1/ Cyr61 plays pivotal role in pancreatic cancer development, maintenance of stemness, and induction of tumor angiogenesis. Hence the goal of this study is to investigate whether CCN1/ Cyr61 signaling cascade acts as a potential target for metformin. The encouraging results we have obtained so far reveal that metformin, which reduces CCN1 expression, significantly inhibits SDF-1 induced invasion, formation of tumor spheres (pancospheres) and also results in the down regulation of CXCR4 receptor in PC cell lines (Panc-1 and AsPC-1). Interestingly we observed that CXCR4 expression is drastically down regulated in genetically engineered CCN1-knock out Panc-1 (Panc-1 KO CCN1 ) cells, compared to the scrambled shRNA transfected Panc-1 cells. Further, it is also observed that Panc-1 K. CCN1 cells are found to be more susceptible to metformin treatment, whereas extracellular supplementation of recombinant CCN1 protein significantly abrogates the effect of metformin on PC cells. Moreover, tumor progression was found to be drastically inhibited in metformin treated Panc-1 (CCN1+ve) tumor xenografts in nude mice model, while the anti-tumorigenic effect of metformin was found to be more drastic in Panc-1 KO CCN1 tumor xenografts. In this scenario, we conclude that CCN1 acts as a direct target for metformin in PC cells, and targeted knock down of CCN1 increases the effectiveness of metformin. [This project is funded by VA Merit Award grants (SB & SKB)] Citation Format: Amlan Das, Archana De, Inamul Haque, Gargi Maity, Sushanta Banerjee, Snigdha Banerjee. Metformin inhibits the oncogenic potential and invasiveness of pancreatic cancer cells targeting CCN1-CXCR4 axis : A new perspective for an old antidiabetic drug. [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 3572. doi:10.1158/1538-7445.AM2015-3572

Abstract 4385: The green tea polyphenol EGCG induces mesenchymal to epithelial transition (MET) and tumor regression in triple negative breast cancer (TNBC) cells and mouse xenograft model: involvement of CCN5.

Background: Epithelial to Mesenchymal transition (EMT) is an important and coordinated series of events associated with tumor metastasis and invasion. Recent studies had shown the importance of CCN5 (also known as WISP-2,Wnt-1-induced signaling protein-2) in the regulation of various carcinomas including the breast cancer. Recent studies had showed that ectopic expression of CCN5 can reverse Epithelial-Mesenchymal transition (EMT) and inhibit cancer metastasis. Epigallocatechin-3-gallate (EGCG), a major polyphenol in green tea, has been extensively studied as a bioactive dietary component against various types of carcinomas through multiple mechanisms such as anti-oxidation, induction of apoptosis, inhibition of angiogenesis and metastasis. However, the mechanism of action of EGCG in breast carcinoma is uncertain. Objective: The objective of the present study is to determine whether CCN5 plays any significant role in EGCG-mediated cytotoxicity in triple negative breast cancer cells. Results: Exposure of triple negative human breast cancer (TNBC) cells, MDA-MB-231 and HCC70 to EGCG resulted in a dose-dependent inhibition of proliferation after 72h and the IC50 was observed around 75μM for MDA-MB-231 cells and 50μM for HCC70 respectively. We found EGCG-treatment effectively induces MET and inhibits the in vitro migration parallel with the induction of CCN5 expression in TNBC cells in a dose-dependent fashion. Furthermore, consistent with in vitro findings, tumor progression was drastically inhibited in EGCG-treated MDA-MB-231-tumor xenograft in nude mouse model. Conclusion: EGCG imparts its anti-cancer activity in both TNBC cells as well as MB-231-tumor xenografts via induction of CCN5. Citation Format: Amlan Das, Snigdha Banerjee, Archana De, Inamul Haque, Gargi Maity, Matt McEwen, Sushanta K. Banerjee. The green tea polyphenol EGCG induces mesenchymal to epithelial transition (MET) and tumor regression in triple negative breast cancer (TNBC) cells and mouse xenograft model: involvement of CCN5. [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 4385. doi:10.1158/1538-7445.AM2013-4385

Abstract 84: CCN1/Cyr61 regulation of gemcitabine-resistant phenotype in pancreatic cancer: involvement of CTGF and dCK

With an estimated half a million new cases and similar mortality rates for 2016, pancreatic ductal adenocarcinoma (PDAC) remains a life-threatening and challenging disease to diagnose and treat. As per American Cancer Society (ACS), varying efficacy in different patients has led to an increase in the mortality rate of PDAC. Gemcitabine (GEM) remains the drug of choice either alone or in combination, but is unsuccessful in reducing or curing PDAC in most patients. The limited efficacies of these drugs are due to the acquisition of chemo-resistant characteristics of PDAC. Although several molecular and physiological factors have been shown to correlate with the GEM-resistance, defined molecular mechanism(s) of GEM-resistance remains a mystery. Previous studies have shown that CCN1, which is overexpressed in PDAC and known to associate with PDAC progression, is critical for drug resistance. Here, we found that while the pancreatic cancer cell lines (i.e., Panc-1 and AsPC-1) in which CCN1 is overexpressed are typically GEM-resistant, the knocking down of CCN1 makes them sensitive to GEM. Mechanistic studies revealed that CCN1 regulates two important genes that are directly involved in regulation GEM-sensitivity in PDAC. These include cancer cell-secreted connective tissue growth factor (CTGF), a regulator of desmoplasia, and Deoxycytidine kinase (dCK), an enzyme that enhances gemcitabine sensitivity and efficacy in cancer cells. The deletion of CCN1 in PDAC cells blocks CTGF expression and under co-cultured conditions prevent the growth of alpha-smooth muscle actin (α-SMA)-positive stellate cells, which are required for desmoplastic growth in pancreatic cancers. CCN1 ablation upregulates dCK expression in PDAC cell lines as compared to CCN1 expressed PDAC cells. These two events enhance the anti-proliferative effect of GEM and can be rescued by CTGF-treatment or blocking dCK. In conclusion, CCN1 promotes GEM-resistance in PDAC cell through the regulation of CTGF and dCK and the mechanistic insights provided by these studies may help in designing future therapeutic strategies to combat PDAC. Citation Format: Vijayalaxmi G. Gupta, Gargi Maity, Inamul Haque, Sushanta K. Banerjee, Snigdha Banerjee. CCN1/Cyr61 regulation of gemcitabine-resistant phenotype in pancreatic cancer: involvement of CTGF and dCK [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 84. doi:10.1158/1538-7445.AM2017-84

Abstract 2118: The food additive agent potassium bromate prevents growth and aggressive phenotypes by targeting multiple molecular signatures in breast cancer cells

Potassium bromate (KBrO3) is by-product of ozone that has found multifunctional purpose in modern society. It is used as disinfectant in drinking water, a bleaching agent to improve flour, a component of cold-wave hair lotion and an ingredient in the production of fish paste and fermented beverages. Potassium bromide has also been used in America for bread-baking as a safe food additive since 1914. Despite the commercial value of this chemical, some studies suggest that KBrO3 could be a carcinogen. During the bread-baking process, Potassium bromate is normally converted into a stable and inert compound, potassium bromide (KBr). However, due to incomplete reduction, the residual KBrO3 remains in the bread, which eventually acts as a potential carcinogen to humans. Interestingly, our studies, in breast cancer cells, found contrasting results. We found that KBrO3 delays growth of ER-α positive luminal type breast cancer cells and triple-negative breast cancer cells (TNBC) via inducing apoptosis in a dose-dependent manner. KBrO3-induced apoptosis is mediated via targeting BCL-2/Bax and Caspase-3 signaling pathway. Moreover, aggressive phenotypes such as migration, invasion and sphere-forming ability of breast cancer cells are significantly impaired by KBrO3-treatment via targeting multiple molecular signatures in breast cancer cells. The growth inhibition effect of KBrO3 is also documented in a tumor xenograft model. Collectively, our findings provide a rationale for the basic and pre-clinical evaluation of the role of KBrO3 in breast cancer progression and therapy. Citation Format: Priyanka Ghosh, Gargi Maity, Snigdha Banerjee, Sushanta Banerjee. The food additive agent potassium bromate prevents growth and aggressive phenotypes by targeting multiple molecular signatures in breast cancer cells [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 2118. doi:10.1158/1538-7445.AM2017-2118

Abstract 790: Regulation of tumor angiogenesis by low dose Aspirin

Background and Objective: Tumor angiogenesis is a pathophysiological process in which new blood vessels are formed in the primary tumor site or distant organs for the nourishment of cancer cells and metastatic growth. Thereby, targeting tumor angiogenesis is an important area of research for cancer therapy. Abnormal structure of blood vessels (i.e. leakiness due to abnormal lining of pericytes on the microvessels) is one of the critical features of tumor angiogenesis that sensitizes vascular cells to cytokines and helps circulating tumor cells to metastasize to distant organs. Our long term goal is to repurpose the drugs that may prevent tumor angiogenesis or normalize the vessels by repairing leakiness via recruiting pericytes or both. Our recent studies found that Aspirin (ASA) has the potency to inhibit breast cancer growth and metastasis, as well as reprogram the mesenchymal to epithelial transition (MET). Given the importance of ASA, we tested whether ASA may be able to regulate tumor angiogenesis. Methods: To do so, we determined the effect of low dose ASA (1mM, which is equivalent to 80mg human dose), ASA-treated (2.5mM) conditioned media (231-CMASA) or vehicle-treated conditioned media (231-CMVT) of MDA-MB-231 cells on different endothelial cell physiology. These include endothelial cells’ migration towards serum using 3D modified Boyden chamber assay, in vitro capillary-like structure formation on Matrigel, cell permeability using in vitro endothelial permeability assay and interactions of pericytes-endothelial cells. We also determined the effect of ASA on various angiogenic factors associated with tumor angiogenesis. Finally, we determined the effect of ASA on in vivo tumor angiogenesis using in vivo Angiogenesis Assay (Trevigen) Results and Conclusions: We found that 231-CMASA significantly blocks in vitro migration, the formation of in vitro capillary-like structures parallel with leakiness via incomplete interaction of pericytes and endothelial cells as compared to 231-CMASA. The antiangiogenic effect of ASA was also documented in in vivo assays. Mechanistically, ASA treatment blocks several angiogenic factors including VEGF-A that are associated with these three events, implicating a low dose of Aspirin is potentially therapeutic for breast cancer via blocking and normalizing tumor angiogenesis. Citation Format: Jinia Chakraborty, Gargi Maity, Snigdha Banerjee, Sushanta K. Banerjee. Regulation of tumor angiogenesis by low dose Aspirin [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 790. doi:10.1158/1538-7445.AM2017-790