Monami Majumder

Cyr61/CCN1 signaling is critical for epithelial-mesenchymal transition and stemness and promotes pancreatic carcinogenesis

BackgroundDespite recent advances in outlining the mechanisms involved in pancreatic carcinogenesis, precise molecular pathways and cellular lineage specification remains incompletely understood.ResultsWe show here that Cyr61/CCN1 play a critical role in pancreatic carcinogenesis through the induction of EMT and stemness. Cyr61 mRNA and protein were detected in the early precursor lesions and their expression intensified with disease progression. Cyr61/CCN1 expression was also detected in different pancreatic cancer cell lines. The aggressive cell lines, in which the expressions of mesenchymal/stem cell molecular markers are predominant; exhibit more Cyr61/CCN1 expression. Cyr61 expression is exorbitantly higher in cancer stem/tumor initiating Panc-1-side-population (SP) cells. Upon Cyr61/CCN1 silencing, the aggressive behaviors are reduced by obliterating interlinking pathobiological events such as reversing the EMT, blocking the expression of stem-cell-like traits and inhibiting migration. In contrast, addition of Cyr61 protein in culture medium augments EMT and stemness features in relatively less aggressive BxPC3 pancreatic cancer cells. Using a xenograft model we demonstrated that cyr61/CCN1 silencing in Panc-1-SP cells reverses the stemness features and tumor initiating potency of these cells. Moreover, our results imply a miRNA-based mechanism for the regulation of aggressive behaviors of pancreatic cancer cells by Cyr61/CCN1.ConclusionsIn conclusion, the discovery of the involvement of Cyr61/CCN1 in pancreatic carcinogenesis may represent an important marker for PDAC and suggests Cyr61/CCN1 can be a potential cancer therapeutic target.

Tumor cell-derived PDGF-B potentiates mouse mesenchymal stem cells-pericytes transition and recruitment through an interaction with NRP-1

BackgroundNew blood vessel formation, or angiogenic switch, is an essential event in the development of solid tumors and their metastatic growth. Tumor blood vessel formation and remodeling is a complex and multi-step processes. The differentiation and recruitment of mural cells including vascular smooth muscle cells and pericytes are essential steps in tumor angiogenesis. However, the role of tumor cells in differentiation and recruitment of mural cells has not yet been fully elucidated. This study focuses on the role of human tumor cells in governing the differentiation of mouse mesenchymal stem cells (MSCs) to pericytes and their recruitment in the tumor angiogenesis process.ResultsWe show that C3H/10T1/2 mouse embryonic mesenchymal stem cells, under the influence of different tumor cell-derived conditioned media, differentiate into mature pericytes. These differentiated pericytes, in turn, are recruited to bind with capillary-like networks formed by endothelial cells on the matrigel under in vitro conditions and recruited to bind with blood vessels on gel-foam under in vivo conditions. The degree of recruitment of pericytes into in vitro neo-angiogenesis is tumor cell phenotype specific. Interestingly, invasive cells recruit less pericytes as compared to non-invasive cells. We identified tumor cell-secreted platelet-derived growth factor-B (PDGF-B) as a crucial factor controlling the differentiation and recruitment processes through an interaction with neuropilin-1 (NRP-1) in mesenchymal stem cells.ConclusionThese new insights into the roles of tumor cell-secreted PDGF-B-NRP-1 signaling in MSCs-fate determination may help to develop new antiangiogenic strategies to prevent the tumor growth and metastasis and result in more effective cancer therapies.

Cysteine-rich 61-connective tissue growth factor-nephroblastoma-overexpressed 5 (CCN5)/Wnt-1-induced signaling protein-2 (WISP-2) regulates microRNA-10b via hypoxia-inducible factor-1α-TWIST signaling networks in human breast cancer cells.

Background: Because CCN5 is an anti-invasive gene, present studies were designed to determine whether CCN5 exerts its anti-invasive function through controlling microRNA-10b expression. Results: Up-regulation of TWIST1, a miR-10b activator, can be achieved by CCN5 silencing through the activation of HIF-1α JNK signaling. Conclusion: CCN5 is a negative regulator of miR-10b in breast cancer cells. Significance: The reactivation of CCN5 could be a unique therapeutic strategy for Triple negative breast cancer. MicroRNAs (miRNAs) are naturally occurring single-stranded RNA molecules that post-transcriptionally regulate the expression of target mRNA transcripts. Many of these target mRNA transcripts are involved in regulating processes commonly altered during tumorigenesis and metastatic growth. These include cell proliferation, differentiation, apoptosis, migration, and invasion. Among the several miRNAs, miRNA-10b (miR-10b) expression is increased in metastatic breast cancer cells and positively regulates cell migration and invasion through the suppression of the homeobox D10 (HOXD10) tumor suppressor signaling pathway. In breast metastatic cells, miR-10b expression is enhanced by a transcription factor TWIST1. We find that miR-10b expression in breast cancer cells can be suppressed by CCN5, and this CCN5 effect is mediated through the inhibition of TWIST1 expression. Moreover, CCN5-induced inhibition of TWIST1 expression is mediated through the translational inhibition/modification of hypoxia-inducible factor-1α via impeding JNK signaling pathway. Collectively, these studies suggest a novel regulatory pathway exists through which CCN5 exerts its anti-invasive function. On the basis of these findings, it is plausible that reactivation of CCN5 in miR-10b-positive invasive/metastatic breast cancers alone or in combination with current therapeutic regimens could provide a unique, alternative strategy to existing breast cancer therapy.

The Matricellular Protein CCN1/Cyr61 is a Critical regulator of Sonic Hedgehog in Pancreatic Carcinogenesis

Background: CCN1 plays a vital role in pancreatic carcinogenesis with an unknown mechanism. Results: CCN1 regulates Sonic-Hedgehog in pancreatic cancer cells via integrin-Notch-signaling pathway to promote in vitro motility and in vivo tumorigenic growth. Conclusion: CCN1 is a critical regulator of Sonic-Hedgehog signaling in pancreatic cancer cells. Significance: Studies suggest a mechanism whereby CCN1 regulates carcinogenic events in the pancreas. CCN1 is a matricellular protein and a member of the CCN family of growth factors. CCN1 is associated with the development of various cancers including pancreatic ductal adenocarcinoma (PDAC). Our recent studies found that CCN1 plays a critical role in pancreatic carcinogenesis through the induction of EMT and stemness. CCN1 mRNA and protein were detected in the early precursor lesions, and their expression intensified with disease progression. However, biochemical activity and the molecular targets of CCN1 in pancreatic cancer cells are unknown. Here we show that CCN1 regulates the Sonic Hedgehog (SHh) signaling pathway, which is associated with the PDAC progression and poor prognosis. SHh regulation by CCN1 in pancreatic cancer cells is mediated through the active Notch-1. Notably, active Notch-1is recruited by CCN1 in these cells via the inhibition of proteasomal degradation results in stabilization of the receptor. We find that CCN1-induced activation of SHh signaling might be necessary for CCN1-dependent in vitro pancreatic cancer cell migration and tumorigenicity of the side population of pancreatic cancer cells (cancer stem cells) in a xenograft in nude mice. Moreover, the functional role of CCN1 could be mediated through the interaction with the αvβ3 integrin receptor. These extensive studies propose that targeting CCN1 can provide a new treatment option for patients with pancreatic cancer since blocking CCN1 simultaneously blocks two critical pathways (i.e. SHh and Notch1) associated with the development of the disease as well as drug resistance.

2-Methoxyestradiol Inhibits Barrett's Esophageal Adenocarcinoma Growth and Differentiation through Differential Regulation of the β-Catenin–E-Cadherin Axis

The purpose of this study was to evaluate whether 2-methoxyestradiol (2-ME2), a promising anticancer agent, modulates Barretts esophageal adenocarcinoma (BEAC) cell growth and behavior through a cellular pathway involving β-catenin in partnership with E-cadherin, which seems to play a critical role in the induction of antitumor responses in cancer cells. We found that 2-ME2 markedly reduced the BEAC cell proliferation through regulating apoptotic machinery such as Bcl-2 and Bax. It may nullify the aggressive behavior of the cells by reducing the migratory behavior. Expressions of β-catenin and E-cadherin and binding of these two proteins is activated in a 2-ME2–dependent fashion in Bic-1 cells. Moreover, overexpressions of these two proteins may be due to the stabilization of these proteins by 2-ME2. We found that 2-ME2–induced antimigratory effects are mediated through the β-catenin–E-cadherin signaling pathways. In view of these results, we determined whether 2-ME2 reduces BEAC tumor growth. Administration of 2-ME2 significantly decreased the growth of BEAC cells xenografted on the flank of nude mice. The evidence presented points out that the effect of 2-ME2 on β-catenin–orchestrated signal transduction plausibly plays a multifaceted functional role to inhibit the proliferation and cell migration of 2-ME2–treated malignant cells and it could be a potential candidate in novel treatment strategies for Barretts esophageal adenocarcinoma. Mol Cancer Ther; 9(3); 523–34

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 3037: Cyr61/CCN1 regulates SHh through Notch signaling in pancreatic cancer cells

Cyr61 (cysteine-rich 61)/CCN1, a member of the CCN family of growth factors that includes CTGF, NOV, WISP-1, WISP-2 and WISP-3, is associated with the development of various cancers. Our recent studies found Cyr61/CCN1 play a critical role in pancreatic carcinogenesis through the induction of EMT and stemness. Cyr61 mRNA and protein were detected in the early precursor lesions and their expression intensified with disease progression. However, the mode of action of CCN1 in the pancreatic carcinogenesis is unknown. In this study, we have shown that CCN1 regulates the expression of Sonic hedgehog (SHh), a lipid-modified secreted signaling protein, plays a critical role in pancreatic cancer development from Pan1N lesions to invasive growth of the disease. Moreover, we also found that SHh regulation by CCN1in pancreatic cancer cells is mediated through the regulation Notch1 signaling pathway. These extensive studies proposed that targeting Cyr61/CCN1 can provide a new treatment option for patients with pancreatic cancer as blocking CCN1 simultaneously two critical pathways (i.e., SHh and Notch1) associated with the development of the disease as well as drug resistence. (THIS WORK IS SUPPORTED BY VA MERIT GRANT TO SB). 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 3037. doi:1538-7445.AM2012-3037

Abstract 1314: Estrogen receptor-α is activated in breast ductal epithelial cells by CCN5 in CCN5-conditional tri-transgenic mice

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Estrogen receptor-alpha (ER-α) is localized in the nuclei of approximately 3-7% of normal resting breast epithelial cells with a higher proportion in lobular cells than in ductal. This number is significantly increased in atypical ductal hyperplasia, ductal carcinoma in situ (DCIS), pure intraductal carcinoma, and intraductal carcinoma with invasive duct carcinoma. Despite having this information for last 15 years, it is still uncertain how ER-α is activated in the nuclei of breast epithelial cells. CCN5/WISP-2, a member of the CCN (Cysteine-rich61/Connective Tissue Growth Factor/Nephroblastoma overexpressed) family of growth factors, is becoming an increasingly important focus in breast cancer research. CCN5/WISP-2 is a two-faced signaling molecule and plays differently in breast carcinogenesis under different micro-environmental setups. Our previous studies have shown that the transcriptional and translational activation of the WISP-2/CCN5 gene is a characteristic of estrogen receptor positive non-invasive breast tumor cell lines. Expression of the WISP-2/CCN5 gene was virtually undetected in non-transformed mammary epithelial cells. Moreover, multiple studies suggest a fine tune between CCN5 signaling and ER-α pathways, but this has not yet been fully elucidated. The main objective of this work was to establish the effect of excessive activation of CCN5 on the ER-α signaling. To do so, we generated a tri-transgenic inducible mouse model that overexpresses CCN5 under the Doxycycline (dox) environment. After the confirmation of the conditional expression of CCN5 in a mammary-specific and dox-dependent manner, morphology as well as the status of ER-α was evaluated. We found that induced expression of CCN5 by Dox in breast epithelial cells significantly increased ER-α expression and activity along with no morphological changes. Collectively, these studies indentify CCN5 as a regulator of ER-α in breast epithelial cells in a tri-transgenic mouse model. 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 1314. doi:10.1158/1538-7445.AM2011-1314

Abstract 1684: 2-Methoxyestradiol reverses the epithelial-mesenchymal transition in Barrett's adenocarcinoma cells

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL 2-Methoxyestradiol (2-ME2) is a novel anti-cancer agent because of its ability to potentiate apoptotic cell death and inhibit cancer cell growth and angiogenesis. In our study, we have investigated whether 2-ME2 is able to modulate epithelial-mesenchymal transition (EMT) in Barretts esophageal adenocarcinoma cell (OE33), which has been postulated as an absolute requirement for tumor invasion and metastasis. Expression of epithelial (E-cadherin, β-catenin, Keratin-19) and mesenchymal markers (Oct-4, Vimentin) and stem cell markers (CD44, CD24) were studied at mRNA and protein levels in OE33 cells that were treated with 5µmolar/L of 2-ME2 for 24 h and 48 h. Our results indicate expression of β-catenin and E-cadherin is activated in a 2-ME2 dependent fashion in OE33 cells. Expression of Vimentin and Oct-4 is significantly down-regulated in 2-ME2 treated OE33 cells. Altogether, these results provide new insights into the role of 2-ME2 in EMT modulation, which may facilitate development of new therapeutic strategies to prevent the tumor invasion and metastasis. 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 1684. doi:10.1158/1538-7445.AM2011-1684

Abstract 2007: Epigenetic reactivation of CCN5/WISP2 by combined effect of green tea polyphenol and epigenetic modulators in highly aggressive breast cancer cells

CCN5/WISP-2 has been considered as an antiinvasive gene because CCN5/WISP-2 silencing augments the invasive phenotypes in vitro. One of the major functions of the CCN5 protein is to block aggressive behavior, such as the mesenchymal-to-epithelial transition (MET) of cancer cells. CCN5/WISP-2 is constitutively expressed in less aggressive human breast cancer cells, whereas its expression is undetected in the highly aggressive breast cancer cells. Dietary green tea polyphenol, (−)-epigallocatechin-3-gallate (EGCG), is believed to be an anticancer agent in part through its regulation of epigenetic processes. Our studies were aimed to address the epigenetic mechanisms of CCN5 reactivation by EGCG in highly aggressive breast cancer cells. In our current studies, we found that EGCG can reactivate CCN5 expression in highly aggressive breast cancer cell line (MDA-MB-231) and augments mesenchymal-epithelial transition process by modulating multiple EMT regulators. Combination studies using EGCG with the epigenetic modulators including the histone deacetylase (HDAC) inhibitor, trichostatin (TSA) and the DNA methyltransferase (DNMT) inhibitor such as 5-aza-2’-deoxycytidine (5- aza), revealed a synergistic effect of reactivation of CCN5 expression in highly aggressive breast cancer cells. Our findings help to assess the key mechanisms of EGCG chemoprevention and therapy by impacting epigenetic pathways toward aggressive breast tumors. 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 2007. doi:10.1158/1538-7445.AM2011-2007