Archana De

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.

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.

Emblica officinalis extract induces autophagy and inhibits human ovarian cancer cell proliferation, angiogenesis, growth of mouse xenograft tumors.

Patients with ovarian cancer (OC) may be treated with surgery, chemotherapy and/or radiation therapy, although none of these strategies are very effective. Several plant-based natural products/dietary supplements, including extracts from Emblica officinalis (Amla), have demonstrated potent anti-neoplastic properties. In this study we determined that Amla extract (AE) has anti-proliferative effects on OC cells under both in vitro and in vivo conditions. We also determined the anti-proliferative effects one of the components of AE, quercetin, on OC cells under in vitro conditions. AE did not induce apoptotic cell death, but did significantly increase the expression of the autophagic proteins beclin1 and LC3B-II under in vitro conditions. Quercetin also increased the expression of the autophagic proteins beclin1 and LC3B-II under in vitro conditions. AE also significantly reduced the expression of several angiogenic genes, including hypoxia-inducible factor 1α (HIF-1α) in OVCAR3 cells. AE acted synergistically with cisplatin to reduce cell proliferation and increase expression of the autophagic proteins beclin1 and LC3B-II under in vitro conditions. AE also had anti-proliferative effects and induced the expression of the autophagic proteins beclin1 and LC3B-II in mouse xenograft tumors. Additionally, AE reduced endothelial cell antigen – CD31 positive blood vessels and HIF-1α expression in mouse xenograft tumors. Together, these studies indicate that AE inhibits OC cell growth both in vitro and in vivo possibly via inhibition of angiogenesis and activation of autophagy in OC. Thus AE may prove useful as an alternative or adjunct therapeutic approach in helping to fight OC.

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.

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.

Emblica officinalis extract downregulates pro-angiogenic molecules via upregulation of cellular and exosomal miR-375 in human ovarian cancer cells.

Ovarian cancer (OC) is highly resistant to current treatment strategies based on a combination of surgery, chemotherapy and radiation therapy. We have recently demonstrated the anti-neoplastic effect of Amla extract (Emblica officinalis, AE) on OC cells in vitro and in vivo. We hypothesized that AE attenuates growth of OC through microRNA (miR)-regulated mechanism(s). The inhibitory effect of AE on proliferation, migration and invasiveness (P≤0.001) of SKOV3 cells and >90% attenuation of tumor growth in a xenograft mouse model suggested multiple targets. RT-qPCR analysis of microRNAs associated with OC showed a >2,000-fold increase in the expression of miR-375 in AE-treated SKOV3 cells that was blocked by an exogenous miR-375 inhibitor (P≤0.001). AE also decreased the gene and protein expression of IGF1R, a target of miR-375 (P≤0.001), and SNAIL1 (P≤0.002), an EMT-associated transcription factor that represses E-cadherin expression (P≤0.003). AE increased E-cadherin expression (P≤0.001). Treatment of SKOV3 cells with AE resulted in increased miR-375 in exosomes in the medium (P≤0.01). Finally, AE significantly decreased the expression of IGF1R and SNAIL1 proteins during attenuation of SKOV3-derived xenograft tumor. Together, these results show that AE modulates cancer cells and the tumor microenvironment via activation of miR-375 and by targeting IGF1R and SNAIL1 in OC cells.

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 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