SPARC inhibits LPA-mediated mesothelial-ovarian cancer cell crosstalk.
ABSTRACT: The interplay between peritoneal mesothelial cells and ovarian cancer cells is critical for the initiation and peritoneal dissemination of, and ascites formation in, ovarian cancer. The production of lysophosphatidic acid (LPA) by both peritoneal mesothelial cells and ovarian cancer cells has been shown to promote metastatic phenotype in ovarian cancer. Herein, we report that exogenous addition or ectopic overexpression of the matricellular protein SPARC (secreted protein acidic and rich in cysteine) significantly attenuated LPA-induced proliferation, chemotaxis, and invasion in both highly metastatic SKOV3 and less metastatic OVCAR3 ovarian cancer cell lines. SPARC appears to modulate these functions, at least in part, through the regulation of LPA receptor levels and the attenuation of extracellular signal-regulated kinase (ERK) 1/2 and protein kinase B/AKT signaling. Moreover, our results show that SPARC not only significantly inhibited both basal and LPA-induced interleukin (IL) 6 production in both cell lines but also attenuated IL-6-induced mitogenic, chemotactic, and proinvasive effects, in part, through significant suppression of ERK1/2 and, to a lesser extent, of signal transducers and activators of transcription 3 signaling pathways. Our results strongly suggest that SPARC exerts a dual inhibitory effect on LPA-induced mesothelial-ovarian cancer cell crosstalk through the regulation of both LPA-induced IL-6 production and function. Taken together, our findings underscore the use of SPARC as a potential therapeutic candidate in peritoneal ovarian carcinomatosis.
Project description:Epithelial ovarian cancer (EOC) metastasis occurs by exfoliation of cells and multicellular aggregates (MCAs) from the tumor into the peritoneal cavity, adhesion to and retraction of peritoneal mesothelial cells and subsequent anchoring. Elevated levels of lysophosphatidic acid (LPA) have been linked to aberrant cell proliferation, oncogenesis, and metastasis. LPA disrupts junctional integrity and epithelial cohesion in vitro however, the fate of free-floating cells/MCAs and the response of host peritoneal tissues to LPA remain unclear. EOC MCAs displayed significant LPA-induced changes in surface ultrastructure with the loss of cell surface protrusions and poor aggregation, resulting in increased dissemination of small clusters compared to untreated control MCAs. LPA also diminished the adhesive capacity of EOC single cells and MCAs to murine peritoneal explants and impaired MCA survival and mesothelial clearance competence. Peritoneal tissues from healthy mice injected with LPA exhibited enhanced mesothelial surface microvilli. Ultrastructural alterations were associated with restricted peritoneal susceptibility to metastatic colonization by single cells as well as epithelial-type MCAs. The functional consequence is an LPA-induced dissemination of small mesenchymal-type clusters, promoting a miliary mode of peritoneal seeding that complicates surgical removal and is associated with worse prognosis.
Project description:The matricellular glycoprotein SPARC (secreted protein acidic and rich in cysteine) has been accorded major roles in regulation of cell adhesion and proliferation, as well as tumorigenesis and metastasis. We have recently reported that in addition to its potent antiproliferative and proapoptotic functions, SPARC also abrogates ovarian carcinoma cell adhesion, a key step in peritoneal implantation. However, the underlying molecular mechanism through which SPARC ameliorates peritoneal ovarian carcinomatosis seems to be multifaceted and has yet to be delineated. Herein, we show that SPARC significantly inhibited integrin-mediated ovarian cancer cell adhesion to extracellular matrix proteins, as well as to peritoneal mesothelial cells. This counteradhesive effect of SPARC was shown to be mediated in part through significant attenuation of cell surface expression and clustering of alpha(v)-integrin subunit, alpha(v)beta(3)- and alpha(v)beta(5)-heterodimers, and beta(1)-subunit, albeit to a lesser extent, in ovarian cancer cells. Moreover, SPARC significantly suppressed both anchorage-dependent and -independent activation of AKT and mitogen-acti-vated protein kinase survival signaling pathways in ovarian cancer cells in response to serum and epidermal growth factor stimulation. In summary, we have identified a novel role of SPARC as a negative regulator of both integrin-mediated adhesion and growth factor-stimulated survival signaling pathways in ovarian cancer.
Project description:Ovarian cancer is mainly confined in peritoneal cavity and its metastasis is often associated with the formation of malignant ascites. As lysophosphatidic acid (LPA) is present at high levels in ascites of ovarian cancer patients and potently stimulates cell migration, we reason that LPA-stimulated cell migration may play an important role in ovarian cancer metastasis. Here, we show that only ovarian cancer cell lines with LPA migratory response undergo peritoneal metastatic colonization. LPA-stimulated cell migration is required for metastatic colonization because knockdown of LPA receptor subtype 1 (LPAR(1)) abolishes this event. However, the difference in metastatic potentials is not caused by the absence of LPAR(1) because both metastatic and nonmetastatic lines express similar levels of LPAR(1). Instead, we find that LPA can activate Rac only in metastatic cells and that metastatic colonization of ovarian cancer cells necessitates Rac activity. These results thus suggest that LPA-induced Rac activation is a prerequisite for ovarian cancer metastasis. In metastatic cells, Rac activation is facilitated by SOS1/EPS8/ABI1 tri-complex and the integrity of this tri-complex is essential for LPA-stimulated cell migration and metastatic colonization. We show that at least 1 member of SOS1/EPS8/ABI1 tri-complex is absent in nonmetastatic ovarian cancer cells and reexpressing the missing one conferred them with metastatic capability. Importantly, coexpression of SOS1, EPS8, and ABI1, but not of any individual member of SOS1/EPS8/ABI1 tri-complex, correlates with advanced stages and shorter survival of ovarian cancer patients. Our study implicates that the integrity of SOS1/EPS8/ABI1 tri-complex is a determinant of ovarian cancer metastasis.
Project description:Advanced ovarian cancers are highly metastatic due to frequent peritoneal dissemination, resulting in dismal prognosis. Here we report the functions of cancer-derived extracellular vesicles (EVs), which are emerging as important mediators of tumour metastasis. The EVs from highly metastatic cells strongly induce metastatic behaviour in moderately metastatic tumours. Notably, the cancer EVs efficiently induce apoptotic cell death in human mesothelial cells in vitro and in vivo, thus resulting in the destruction of the peritoneal mesothelium barrier. Whole transcriptome analysis shows that MMP1 is significantly elevated in mesothelial cells treated with highly metastatic cancer EVs and intact MMP1 mRNAs are selectively packaged in the EVs. Importantly, MMP1 expression in ovarian cancer is tightly correlated with a poor prognosis. Moreover, MMP1 mRNA-carrying EVs exist in the ascites of cancer patients and these EVs also induce apoptosis in mesothelial cells. Our findings elucidate a previously unknown mechanism of peritoneal dissemination via EVs.
Project description:Metastatic dissemination of ovarian tumors involves the invasion of tumor cell clusters into the mesothelial cell lining of peritoneal cavity organs; however, the tumor-specific factors that allow ovarian cancer cells to spread are unclear. We used an in vitro assay that models the initial step of ovarian cancer metastasis, clearance of the mesothelial cell layer, to examine the clearance ability of a large panel of both established and primary ovarian tumor cells. Comparison of the gene and protein expression profiles of clearance-competent and clearance-incompetent cells revealed that mesenchymal genes are enriched in tumor populations that display strong clearance activity, while epithelial genes are enriched in those with weak or undetectable activity. Overexpression of transcription factors SNAI1, TWIST1, and ZEB1, which regulate the epithelial-to-mesenchymal transition (EMT), promoted mesothelial clearance in cell lines with weak activity, while knockdown of the EMT-regulatory transcription factors TWIST1 and ZEB1 attenuated mesothelial clearance in ovarian cancer cell lines with strong activity. These findings provide important insights into the mechanisms associated with metastatic progression of ovarian cancer and suggest that inhibiting pathways that drive mesenchymal programs may suppress tumor cell invasion of peritoneal tissues.
Project description:Missense mutations in the TP53 gene resulting in the accumulation of mutant proteins are extremely common in advanced ovarian cancer, which is characterised by peritoneal metastasis. Attachment of cancer cells to the peritoneal mesothelium is regarded as an initial, key step for the metastatic spread of ovarian cancer. In the present study, we investigated the possible role of a p53 mutant in the mesothelial adhesion of ovarian cancer cells. We found that OVCAR-3 cells with the R248 TP53 mutation (p53(R248)) were more adhesive to mesothelial Met5A cells than were A2780 cells expressing wild-type p53. In addition, ectopic expression of p53(R248) in p53-null SKOV-3 cells significantly increased adhesion to Met5A cells. Knockdown of mutant p53 significantly compromised p53(R248)-induced cell adhesion to Met5A cells. Microarray analysis revealed that several adhesion-related genes, including integrin β4, were markedly up-regulated, and certain signalling pathways, including PI3K/Akt, were activated in p53(R248) transfectants of SKOV-3 cells. Inhibition of integrin β4 and Akt signalling using blocking antibody and the inhibitor LY294002, respectively, significantly attenuated p53(R248)-mediated ovarian cancer-mesothelial adhesion. These data suggest that the p53(R248) mutant endows ovarian cancer cells with increased adhesiveness and that integrin β4 and Akt signalling are associated with the mutation-enhanced ovarian cancer-mesothelial cell adhesion.
Project description:TGFBI has been shown to sensitize ovarian cancer cells to the cytotoxic effects of paclitaxel via an integrin receptor-mediated mechanism that modulates microtubule stability. Herein, we determine that TGFBI localizes within organized fibrillar structures in mesothelial-derived ECM. We determined that suppression of SPARC expression by shRNA decreased the deposition of TGFBI in mesothelial-derived ECM, without affecting its overall protein expression or secretion. Conversely, overexpression of SPARC increased TGFBI deposition. A SPARC-YFP fusion construct expressed by the Met5a cell line co-localized with TGFBI in the cell-derived ECM. Interestingly, in vitro produced SPARC was capable of precipitating TGFBI from cell lysates dependent on an intact SPARC carboxy-terminus with in vitro binding assays verifying a direct interaction. The last 37 amino acids of SPARC were shown to be required for the TGFBI interaction while expression of a SPARC-YFP construct lacking this region (aa 1-256) did not interact and co-localize with TGFBI in the ECM. Furthermore, ovarian cancer cells have a reduced motility and decreased response to the chemotherapeutic agent paclitaxel when plated on ECM derived from mesothelial cells lacking SPARC compared to control mesothelial-derived ECM. In conclusion, SPARC regulates the fibrillar ECM deposition of TGFBI through a novel interaction, subsequently influencing cancer cell behavior.
Project description:Malignant mesothelioma (MM) is an aggressive cancer of mesothelial cells of pleural and peritoneal cavities. In 85% of cases both pleural and peritoneal MM is caused by asbestos exposure. Although both are asbestos-induced cancers, the incidence of pleural MM is significantly higher (85%) than peritoneal MM (15%). It has been proposed that carcinogenesis is a result of asbestos-induced inflammation but it is not clear what contributes to the differences observed between incidences of these two cancers. We hypothesize that the observed differences in incidences of pleural and peritoneal MM are the result of differences in the direct response of these cell types to asbestos rather than to differences mediated by the in vivo microenvironment. To test this hypothesis we characterized cellular responses to asbestos in a controlled environment. We found significantly greater changes in genome-wide expression in response to asbestos exposure in pleural mesothelial cells as compared to peritoneal mesothelial cells. In particular, a greater response in many common genes (IL-8, ATF3, CXCL2, CXCL3, IL-6, GOS2) was seen in pleural mesothelial cells as compared to peritoneal mesothelial cells. Unique genes expressed in pleural mesothelial cells were mainly pro-inflammatory (G-CSF, IL-1?, IL-1?, GREM1) and have previously been shown to be involved in development of MM. Our results are consistent with the hypothesis that differences in incidences of pleural and peritoneal MM upon exposure to asbestos are the result of differences in mesothelial cell physiology that lead to differences in the inflammatory response, which leads to cancer.
Project description:<h4>Background</h4>Lysophosphatidic acid (LPA) acts through the cell surface G protein-coupled receptors, LPA1, LPA2, or LPA3, to elicit a wide range of cellular responses. It is present at high levels in intraperitoneal effusions of human ovarian cancer increasing cell survival, proliferation, and motility as well as stimulating production of neovascularizing factors. LPA2 and LPA3 and enzymes regulating the production and degradation of LPA are aberrantly expressed by ovarian cancer cells, but the consequences of these expression changes in ovarian cancer cells were unknown.<h4>Methods</h4>Expression of LPA1, LPA2, or LPA3 was inhibited or increased in ovarian cancer cells using small interfering RNAs (siRNAs) and lentivirus constructs, respectively. We measured the effects of changes in LPA receptor expression on cell proliferation (by crystal violet staining), cell motility and invasion (using Boyden chambers), and cytokines (interleukin 6 [IL-6], interleukin 8 [IL-8], and vascular endothelial growth factor [VEGF]) production by enzyme-linked immunosorbent assay. The role of LPA receptors in tumor growth, ascites formation, and cytokine production was assessed in a mouse xenograft model. All statistical tests were two-sided.<h4>Results</h4>SKOV-3 cells with increased expression of LPA receptors showed increased invasiveness, whereas siRNA knockdown inhibited both migration (P < .001, Student t test) and invasion. Knockdown of the LPA2 or LPA3 receptors inhibited the production of IL-6, IL-8, and VEGF in SKOV-3 and OVCAR-3 cells. SKOV-3 xenografts expressing LPA receptors formed primary tumors of increased size and increased ascites volume. Invasive tumors in the peritoneal cavity occurred in 75% (n = 4) of mice injected with LPA1 expressing SKOV-3 and 80% (n = 5) of mice injected with LPA2 or LPA3 expressing SKOV-3 cells. Metastatic tumors expressing LPA1, LPA2, and LPA3 were identified in the liver, kidney, and pancreas; tumors expressing LPA2 and LPA3 were detected in skeletal muscle; and tumors expressing LPA2 were also found in the cervical lymph node and heart. The percent survival of mice with tumors expressing LPA2 or LPA3 was reduced in comparison with animals with tumors expressing beta-galactosidase.<h4>Conclusions</h4>Expression of LPA2 or LPA3 during ovarian carcinogenesis contributes to ovarian cancer aggressiveness, suggesting that the targeting of LPA production and action may have potential for the treatment of ovarian cancer.