Thymoquinone, a therapeutic phytochemical derived from Nigella sativa, has been shown to have a potent anticancer activity. However, it has been identified that the tumor microenvironment (TME) can attenuate the anticancer effects of thymoquinone (TQ) in ovarian cancer. Lysophosphatidic acid (LPA), a lipid growth factor present in high concentration in the TME of ovarian cancer, has been shown to regulate multiple oncogenic pathways in ovarian cancer. Taking account of the crucial role of LPA in the genesis and progression of ovarian cancer, the present study is focused on assessing the efficacy of TQ in inhibiting LPA-stimulated oncogenic pathways in ovarian cancer cells. Our results indicate that TQ is unable to attenuate LPA-stimulated proliferation or metabolic reprogramming in ovarian cancer cells. However, TQ potently inhibits the basal as well as LPA-stimulated migratory responses of the ovarian cancer cells. Furthermore, TQ abrogates the invasive migration of ovarian cancer cells induced by Gαi2, through which LPA stimulates cell migration. TQ also attenuates the activation of JNK, Src, and FAK, the downstream signaling nodes of LPA-LPAR-Gαi2 signaling pathway. In addition to establishing the differential effects of TQ in ovarian cancer cells, our results unravel the antitherapeutic role of LPA in the ovarian cancer TME could override the inhibitory effects of TQ on cell proliferation and metabolic reprogramming of ovarian cancer cells. More importantly, the concomitant finding that TQ could still sustain its inhibitory effect on LPA-stimulated invasive cell migration, points to its potential use as a response-specific therapeutic agent in ovarian cancer.
Abstract Introduction: Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related death worldwide. Chronic infections with hepatitis B or C viruses are the leading cause of HCC. The cancer stem cell (CSC) marker, doublecortin-like kinase (DCLK1), is believed to be a key factor for the development of multiple cancer types including HCC. However, its impact on signaling pathways that promote hepatocarcinogenesis is largely unknown. We have shown DCLK1's enhanced expression in HCC and its stimulatory role in hepatitis C virus (HCV) replication. Our published reports further suggest that DCLK1 positively affects tumor-related miRNAs and transcription factors that promote epithelial-mesenchymal transition (EMT). These observations prompted us to investigate DCLK1 signaling module in virus-induced liver pathogenesis and HCC. Methods: Genome-wide transcriptome analysis was carried out in total RNAs isolated from DCLK1-overexpressing and control hepatoma cells. One hundred five clinical cases representing various stages of liver diseases in patients with chronic hepatitis B or C were analyzed for an array of markers including DCLK1 by immunohistochemical staining and Western blot. Huh7 hepatoma cells were transplanted into the flanks of athymic nude mice to generate HCC-like tumor xenografts. The tumors were analyzed for proteins, mRNAs and miRNAs or treated with siRNA against DCLK1 and scrambled siRNA to monitor tumor growth arrest. Results: We found that the expression of 19 genes (14 upregulated, 5 downregulated) was specifically affected by DCLK1 overexpression in hepatoma cell lines. Among these, the level of kinase-suppressor of Ras 1 (KSR1) scaffold protein of Ras-MAPK pathway was increased significantly when recombinant DCLK1 was co-expressed with HCV replicon. Examination of liver tissues derived from patients with chronic hepatitis B/C and HCC suggests that a pro-inflammatory S100A9 protein tends to correlate with DCLK1 overexpression in cirrhosis with or without HCC. Both DCLK1 and S100A9 proteins were mainly localized in the regenerative nodules, fibrotic septa, mesenchymal cells, endothelium and areas with lymphocytes aggregates. Analysis of tumor xenografts developed by hepatoma cells suggest that overexpression of DCLK1 is accompanied by high-level expression of S100A9 and c-Myc as well as activation of NFκB. Conversely, siRNA-led inhibition of DCLK1 causes decrease in tumor volume considerably in a xenograft model. Conclusions: DCLK1 overexpression appears to be intimately related to the activation of pro-inflammatory and MAPK signaling pathways during the development of virus-induced pre-neoplastic conditions and initiation of tumors in liver. Thus, targeting DCLK1 at early stage of liver diseases may prevent virus-induced cirrhosis and HCC. Citation Format: Naushad Ali, Parthasarathy Chandrakesan, Mark Huycke, Sanam Husain, Allison F. Gillaspy, Randal May, William L. Berry, Sripathi Sureban, Dongfeng Qu, Nathaniel Weygant, Michael S. Bronze, Danny N. Dhanasekaran, Courtney W. Houchen. Overexpression of a cancer stem cell marker doublecortin-like kinase (DCLK1) leads to activation of inflammatory cascade during development of virus-induced hepatocellular carcinoma. [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 3171. doi:10.1158/1538-7445.AM2014-3171
The GTPase-deficient, activated mutant of Galpha12 (Galpha12Q229L, or Galpha12QL) induces neoplastic growth and oncogenic transformation of NIH 3T3 cells. Using microarray analysis, we have previously identified a role for platelet-derived growth factor receptor alpha (PDGFRalpha) in Galpha12-mediated cell growth (R. N. Kumar et al., Cell Biochem. Biophys. 41:63-73, 2004). In the present study, we report that Galpha12QL stimulates the functional expression of PDGFRalpha and demonstrate that the expression of PDGFRalpha by Galpha12QL is dependent on the small GTPase Rho. Our results indicate that it is cell type independent as the transient expression of Galpha12QL or the activation of Galpha12-coupled receptors stimulates the expression of PDGFRalpha in NIH 3T3 as well as in human astrocytoma 1321N1 cells. Furthermore, we demonstrate the presence of an autocrine loop involving PDGF-A and PDGFRalpha in Galpha12QL-transformed cells. Analysis of the functional consequences of the Galpha12-PDGFRalpha signaling axis indicates that Galpha12 stimulates the phosphatidylinositol 3-kinase (PI3K)-AKT signaling pathway through PDGFR. In addition, we show that Galpha12QL stimulates the phosphorylation of forkhead transcription factor FKHRL1 via AKT in a PDGFRalpha- and PI3K-dependent manner. Since AKT promotes cell growth by blocking the transcription of antiproliferative genes through the inhibitory phosphorylation of forkhead transcription factors, our results describe for the first time a PDGFRalpha-dependent signaling pathway involving PI3K-AKT-FKHRL1, regulated by Galpha12QL in promoting cell growth. Consistent with this view, we demonstrate that the expression of a dominant negative mutant of PDGFRalpha attenuated Galpha12-mediated neoplastic transformation of NIH 3T3 cells.
Abstract Ovarian cancer patients show increased levels of lysophosphatidic acid (LPA) in their serum and ascitic fluid. Our previous studies have established a critical role for LPA in inducing proliferation as well as migration of ovarian cancer cells. In the present study, we focused on identifying novel therapeutic targets in the oncogenic signaling pathway regulated by LPA. Using a commercial pathway reporter array of 45 transcription factors, we found that hypoxia-induced factor-1 alpha (HIF-1 alpha) was the most activated transcription factor induced by LPA. Both stable as well as transient silencing of Galphai2, a downstream signaling mediator for LPA, abrogates LPA induced increase in HIF-1 alpha expression. In addition, our results indicate that LPA stimulated an increase in the expression of Hexokinase-2 (HK2) and Glucose transporter 1 (GLUT1), which are targets of HIF-1 alpha, are also mediated via Galphai2 signaling pathway. To determine the outcome LPA induced increase in HK2 and GLUT1 on glycolytic rates, we measured the extracellular acidification rate (ECAR) in ovarian cancer cells following treatment with LPA, using an XFe analyzer. In both SKOV3-ip and OVCA429 cells, LPA increased ECAR in a dose-dependent manner. Silencing Galphai2 as well as HIF-1 alpha in ovarian cancer cells reduced ECAR indicative of reduced glycolytic rate. The current study, points to the identification of LPA(R)-Galphai2-HIF-1 alpha-GLUT1 signaling axis as potential metabolism-targets in ovarian cancer treatment. Citation Format: Ji Hee Ha, Rangasudhagar Radhakrishnan, Danny Dhanasekaran. LPA stimulates glycolytic shift of ovarian cancer cells via gip2 oncogenes. [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 1141. doi:10.1158/1538-7445.AM2015-1141
The cellular signaling network involves co-ordinated regulation of numerous signaling molecules that aid the maintenance of cellular as well as organismal homeostasis. Aberrant signaling plays a major role in the pathophysiology of many diseases. Recent studies have unraveled the superfamily of long non-coding RNAs (lncRNAs) as critical signaling nodes in diverse signaling networks. Defective signaling by lncRNAs is emerging as a causative factor underlying the pathophysiology of many diseases. LncRNAs have been shown to be involved in the multiplexed regulation of diverse pathways through both genetic and epigenetic mechanisms. They can serve as decoys, guides, scaffolds, and effector molecules to regulate cell signaling. In comparison with the other classes of RNAs, lncRNAs possess unique structural modifications that contribute to their diversity in modes of action within the nucleus and cytoplasm. In this review, we summarize the structure and function of lncRNAs as well as their vivid mechanisms of action. Further, we provide insights into the role of lncRNAs in the pathogenesis of four major disease paradigms, namely cardiovascular diseases, neurological disorders, cancers, and the metabolic disease, diabetes mellitus. This review serves as a succinct treatise that could open windows to investigate the role of lncRNAs as novel therapeutic targets.
Tumor dormancy is the extended period during which patients are asymptomatic before recurrence, and it represents a difficult phenomenon to target pharmacologically. The relapse of tumors, for instance arising from the interruption of dormant metastases, is frequently observed in ovarian cancer patients and determines poor survival. Inflammatory cytokines present in the tumor microenvironment likely contribute to such events. Cancer cell dormancy and autophagy are interconnected at the molecular level through ARH-I (DIRAS3) and BECLIN-1, two tumor suppressors often dysregulated in ovarian cancers. IL-6 disrupts autophagy in ovarian cancer cells via miRNAs downregulation of ARH-I, an effect contrasted by the nutraceutical protein restriction mimetic resveratrol (RV). By using three ovarian cancer cell lines with different genetic background in 2D and 3D models, the latter mimicking the growth of peritoneal metastases, we show that RV keeps the cancer cells in a dormant-like quiescent state contrasting the IL-6 growth-promoting activity. Mechanistically, this effect is mediated by BECLIN-1-dependent autophagy and relies on the availability of ARH-I. We also show that ARH-I (DIRAS3) is a bona fide target of miR-1305, a novel oncomiRNA upregulated by IL-6 and downregulated by RV. Clinically relevant, bioinformatic analysis of a transcriptomic database showed that the high expression of DIRAS3 and MAP1LC3B mRNAs together with that of CDKN1A, directing a cellular dormant phenotype, predicts better overall survival in ovarian cancer patients, and this correlates with MIR1305 downregulation. The possibility of maintaining a permanent cell dormancy in ovarian cancer by the chronic administration of RV should be considered as a therapeutic option to prevent the "awakening" of cancer cells in response to a permissive microenvironment, thus limiting the risk of tumor relapse and metastasis.
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