Sandipto Sarkar

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.

Abstract 4915: Alcohol-induced reprogramming of tumor plasticity is mediated via suppression of CCN5 signaling in breast cancer cells

Background and Objective: The epidemiological and experimental studies suggest that alcohol consumption is associated with increased risk for breast cancer development and metastasis. However, the mechanisms of alcohol-induced breast cancer progression and metastasis remain unknown. In this study, we examined the roles of CCN5, a tumor suppressor gene in alcohol induced breast cancer cell migration/invasion, epithelial to mesenchymal transition (EMT) and stemness. Methods: Estrogen receptor-positive (ER+) human breast cancer cell line MCF-7 and immortalized normal human epithelial breast cell line MCF-12A were treated with ethanol at various concentrations in the presence and absence of human recombinant human CCN5 protein (hrCCN5) . Invasive/migratory ability of treated and untreated cells was measured by Boyden chamber assays. The mRNA and protein expression level of CCN5 was determined by real-time qRT-PCR and Western blot. EMT and stemness markers were evaluated by Western blot. Single-cell suspensions from pre-treated cells were re-suspended at a density of 500 cells/ml mammocult media in ultralow attachment dishes. Number as well as the size of the mammosphere in specified experimental set-up was monitored and recorded alternate day for 8-10 days. Results: The studies demonstrated that alcohol promotes significantly the invasive/migratory ability and EMT phenotypes of breast cancer cells through the suppression of CCN5. Interestingly, hrCCN5 protein treatment suppresses the effect of alcohol and reprograms MET (mesenchymal to epithelial transition). In addition, alcohol-induced mammosphere formation efficiency is also suppressed by hrCCN5 protein-treatment. Conclusions: Our findings suggest that the CCN5 signaling plays a preventive role in alcohol-induced reprogramming of tumor plasticity in breast cancer cells. Citation Format: Inamul Haque, Arvind Subramanian, Vijayalaxmi Gupta, Sandipto Sarkar, Snigdha Banerjee, Sushanta K. Banerjee. Alcohol-induced reprogramming of tumor plasticity is mediated via suppression of CCN5 signaling 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 4915. doi:10.1158/1538-7445.AM2017-4915

Abstract 134: CCN5 down regulates HER2/ neu expression in HER2 positive breast cancer cells

Breast cancers are broadly classified into four different subtypes which are Luminal A, Luminal B, Basal type and Her2 positive cancers. The cells in Her2 positive tumor lesions overexpress the tyrosine kinase receptor ErbB2 or Her2 which serve as the major oncoprotein and drives the unrestrained proliferation of the cells which is one of the major hallmarks of cancer. About 25% of the breast cancer overexpress Her2 (ErbB2) proto-oncogene resulting in aggressive tumor phenotype and is associated in poor prognosis in patients. Though Her2 targeted therapies are being used hugely in clinical practice, a significant fraction of the Her2 positive tumors develop mechanisms to evade the targeted therapies of Her2 inhibition. CCN5 or Wisp2 is a matricellular protein which is now considered as an ‘anti-invasive gene’. It has been already that established from multiple evidences that CCN5 protein inhibits progression of triple negative breast cancer. The goal of the study is to investigate if CCN5 can regulate Her2 in Her2 positive breast cancer. We have found that CCN5 treatment also negatively regulates expression and activity of Her2 receptor in Her2 overexpressed breast cancer cells. It has been indicated from Her2 promoter assays that Wisp2/CCN5 negatively regulates the expression of Her2 in a transcriptional level. Wisp2 /CCN5 treatment of Her2 overexpressed breast cancer cell lines, SKBR3 and Bt474, exerts a negative impact on the migration and proliferative properties of the cancer cells. Thus it can be concluded that our initial studies on effect of CCN5 on Her2 positive cells can pave the way for a novel approach of therapy to control Her2 overexpressed tumor cell growth. Citation Format: Sandipto Sarkar, Gargi Maity, Amlan Das, Monami Majumder, Snigdha Banerjee, Sushanta Banerjee. CCN5 down regulates HER2/ neu expression in HER2 positive breast cancer cells. [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 134. doi:10.1158/1538-7445.AM2015-134

Abstract 5410: Aspirin attenuates tumor initiating cell growth and induces reprogramming factors of mesenchymal to epithelial transition in breast cancer cell

Acetyl Salicylic Acid (ASA) also known as aspirin, a classic, non-steroidal, anti-inflammatory drug (NSAIDs) 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 chemo-preventive 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. Our studies, using in vitro and in vivo tumor xenograft model, show a strong beneficial effect of ASA in the prevention of breast carcinogenesis. We find ASA not only prevents breast tumor cell growth in vitro and tumor growth in nude mice xenograft model through the induction of apoptosis, it also significantly reduces the growth of tumor initiating cells (TICs)/ cancer stem cells (CSCs) and delayed 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 as well as reprogramming roles of ASA could be mediated through inhibition of TGF-β-signaling pathway, which is associated with growth, motility, invasion and metastasis in advanced breast cancers. Collectively, ASA has a therapeutic or preventive potential by attacking possible target such as TGF-β- SMAD4 signaling in breast carcinogenesis. [This work is funded by VA Merit Awarded Grant (SB and SKB)] Note: This abstract was not presented at the meeting. Citation Format: Gargi Maity, Archana De, Snigdha Banerjee, Amlan Das, Sandipto Sarkar, Sushanta K. Banerjee. Aspirin attenuates tumor initiating cell growth and induces reprogramming factors of mesenchymal to epithelial transition in breast cancer cell. [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 5410. doi:10.1158/1538-7445.AM2015-5410

Abstract 1552: CCN5/wisp-2 induced growth arrest of aggressive triple negative breast cancer cells is promoted through accumulation and trafficking of p27kip1

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA CCN5, a matricellular protein, represents promising treatment target in triple negative breast cancer (TNBC) as treatment or induced activation of CCN5 in TNBC cells promotes cell growth arrest at G0/G1 phase, reduces cell proliferation and delays tumor growth in xenograft model. Our studies found that p27Kip1 tumor suppressor protein is upregulated and relocalized to the nucleus from cytoplasm by CCN5 in these cells and that these two events (i.e., upregulation and relocalization) of p27Kip1 is required for CCN5-induced growth inhibition of TNBC cells. In the absence of CCN5, p27Kip1 remains mostly in the cytoplasm, a state of aggressive nature of cancer cells. Mechanistically, CCN5 inhibits Skp2 expression, which seems to stabilize p27Kip1 protein in these cells. On the other hand, CCN5 also recruits FOXO3a to mediate transcriptional regulation of p27Kip1. The recruitment of FOXO3a is achieved by the induction of its expression and activity through shifting from cytoplasm to the nucleus. Our data indicate that CCN5 blocks PI3K/AKT signaling to dephosphorylate at S318, S253 and Thr32 in FOXO3a for nuclear relocalization and activation of FOXO3a. Inhibition of α6β1 diminishes CCN5 action on p27Kip1 in TNBC cells. Collectively, CCN5 may have therapeutic potential for TNBC. Citation Format: Inamul Haque, Snigdha Banerjee, Archana De, Gargi Maity, Sandipto Sarkar, Douglas McGragor, Sushanta K. Banerjee. CCN5/wisp-2 induced growth arrest of aggressive triple negative breast cancer cells is promoted through accumulation and trafficking of p27kip1 . [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 1552. doi:10.1158/1538-7445.AM2014-1552

Abstract 2330: Transcription factor MAZ promotes cell growth and aggressive behavior of human pancreatic cancer cells

Pancreatic ductal adenocarcinoma (PDAC) is the fourth most common cancer in the United States and the eighth worldwide. PDAC is most aggressive type of cancer that spreads rapidly and is seldom detected in its early stages as signs and symptoms may not appear until pancreatic cancer is quite advanced. MAZ (Myc-associated zinc-finger protein) or SAF1 (Serum amyloid A activating factor 1) gene is a member of multiple Cys2-His2-type zinc finger proteins that are activated in response to various inflammatory signals and may act as a transcription factor with dual roles in transcription initiation and termination. Deregulation of MAZ expression has been shown to be related to the development and progression of various cancers like prostate cancer, breast cancer and pancreatic cancer. However, mode of action of MAZ in carcinogenesis is unclear. We found that MAZ mRNA and protein levels were markedly elevated in pancreatic adenocarcinoma tissue samples as compared to adjacent normal tissues and the expression profile was increased as the disease progressed. Similarly, MAZ mRNA and protein levels were increased several folds in aggressive pancreatic cancer cell lines as compared to non-transformed pancreatic cancer cell lines. Functional analysis demonstrates that siRNA-based depletion of MAZ significantly inhibits in vitro cellular growth, colony formation, anchorage independent growth and invasion in pancreatic cancer cells. Mechanistically, MAZ regulates BIRC5, a pancreatic cancer promoting gene, to possibly exert its pathobiological function. Moreover, we found that CCN1/Cyr61 is an upstream regulator of MAZ. Collectively, these findings indicate that MAZ is a downstream target of CCN1 and plays an important role in pancreatic cancer progression and differentiation. Thus, MAZ could be an ideal target for PDAC management. [This work is supported by VA Merit Award Grant (SB/SKB)] Citation Format: Gargi Maity, Sandipto Sarkar, Kakali Dhar, Gopal Dhar, Inamul Haque, Sushanta K. Banerjee, Snigdha Banerjee. Transcription factor MAZ promotes cell growth and aggressive behavior of human pancreatic cancer cells. [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 2330. doi:10.1158/1538-7445.AM2014-2330

Abstract 224: Targeting reprogramming the stemness in breast carcinogenesis by 2-Methoxyestradiol.

2-Methoxyestradiol (2ME2), a natural metabolite of estradiol is a potent antitumor and antiangiogenic agent. 2ME2 inhibits the proliferation of a wide variety of cancer cell and primary cultures and blocks tumor growth in the tumor xenograft model. 2-ME 2 is currently undergoing Phase I and II clinical trial under the commercial name Panzem. Although various molecular targets have been proposed for this compound, the impact of 2ME2 on reprogramming of stemness is unknown. Thus, the purpose of this was to determine whether 2ME2 can modulate pathophysiological events like epithelial to mesenchymal transition (EMT), cell migration and stemness in breast cancer cell. Our study indicated that 2ME2 inhibits EMT by down regulating mesenchymal markers like vimentin, Snail, Twist and upregulating epithelial markers like keratin-19, E-cadherin in both low metastatic (MCF-7) and high metastatic (MDA-MB-231) breast cancer cells. We found that 2ME2 significantly reduced in vitro breast cancer cell proliferation and migration. We also showed that 2ME2 not only decreased expression of stem cell markers CD44 and oct4 and increased CD24 expression in breast cancer cells it also inhibits stemness or self -renewal property of breast cancer stem cell as observed from mammosphere assay. Overall our results may help to understand the role of 2ME2 in EMT process followed by stemness, which may facilitate to develop new therapeutic targets in breast cancer management. Citation Format: Gargi Maity, Snigdha Banerjee, Archana De, Sandipto Sarkar, Amlan Das, Sushanta Banerjee. Targeting reprogramming the stemness in breast carcinogenesis by 2-Methoxyestradiol. [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 224. doi:10.1158/1538-7445.AM2013-224 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.