Tatiana M. Tilli

Osteopontin-c Splicing Isoform Contributes to Ovarian Cancer Progression

Ovarian carcinoma is one of the most aggressive gynecological diseases and generally diagnosed at advanced stages. Osteopontin (OPN) is one of the proteins overexpressed in ovarian cancer and is involved in tumorigenesis and metastasis. Alternative splicing of OPN leads to 3 isoforms, OPNa, OPNb, and OPNc. However, the expression pattern and the roles of each of these isoforms have not been previously characterized in ovarian cancer. Herein, we have evaluated the expression profiling of OPN isoforms in ovarian tumor and nontumor samples and their putative roles in ovarian cancer biology using in vitro and in vivo functional assays. OPNa and OPNb were expressed both in tumor and nontumor ovarian samples, whereas OPNc was specifically expressed in ovarian tumor samples. The isoform OPNc significantly activated OvCar-3 cell proliferation, migration, invasion, anchorage-independent growth and tumor formation in vivo. Additionally, we have also shown that some of the OPNc-dependent protumorigenic roles are mediated by PI3K/Akt signaling pathway. OPNc stimulated immortalized ovarian epithelial IOSE cell proliferation, indicating a role for this isoform in ovarian cancer tumorigenesis. Functional assays using OPNc conditioned medium and an anti-OPNc antibody have shown that most cellular effects observed herein were promoted by the secreted OPNc. According to our data, OPNc-specific expression in ovarian tumor samples and its role on favoring different aspects of ovarian cancer progression suggest that secreted OPNc contributes to the physiopathology of ovarian cancer progression and tumorigenesis. Altogether, the data open possibilities of new therapeutic approaches for ovarian cancer that selectively down regulate OPNc, altering its properties favoring ovarian tumor progression. Mol Cancer Res; 9(3); 280–93. ©2011 AACR.

Both osteopontin-c and osteopontin-b splicing isoforms exert pro-tumorigenic roles in prostate cancer cells.

Alternative splicing of the osteopontin (opn, spp1) gene generates three protein splicing isoforms (OPN‐SI), designated as OPNa, OPNb, and OPNc, which have demonstrated specific roles in different tumor models. This work aims to investigate the roles of each OPN‐SI in prostate cancer (PCa) progression by using in vivo and in vitro functional assays.

Changes in the transcriptional profile in response to overexpression of the osteopontin-c splice isoform in ovarian (OvCar-3) and prostate (PC-3) cancer cell lines

BackgroundEspecially in human tumor cells, the osteopontin (OPN) primary transcript is subject to alternative splicing, generating three isoforms termed OPNa, OPNb and OPNc. We previously demonstrated that the OPNc splice variant activates several aspects of the progression of ovarian and prostate cancers. The goal of the present study was to develop cell line models to determine the impact of OPNc overexpression on main cancer signaling pathways and thus obtain insights into the mechanisms of OPNc pro-tumorigenic roles.MethodsHuman ovarian and prostate cancer cell lines, OvCar-3 and PC-3 cells, respectively, were stably transfected to overexpress OPNc. Transcriptomic profiling was performed on these cells and compared to controls, to identify OPNc overexpression-dependent changes in gene expression levels and pathways by qRT-PCR analyses.ResultsAmong 84 genes tested by using a multiplex real-time PCR Cancer Pathway Array approach, 34 and 16, respectively, were differentially expressed between OvCar-3 and PC-3 OPNc-overexpressing cells in relation to control clones. Differentially expressed genes are included in all main hallmarks of cancer, and several interacting proteins have been identified using an interactome network analysis. Based on marked up-regulation of Vegfa transcript in response to OPNc overexpression, we partially validated the array data by demonstrating that conditioned medium (CM) secreted from OvCar-3 and PC-3 OPNc-overexpressing cells significantly induced endothelial cell adhesion, proliferation and migration, compared to CM secreted from control cells.ConclusionsOverall, the present study elucidated transcriptional changes of OvCar-3 and PC-3 cancer cell lines in response to OPNc overexpression, which provides an assessment for predicting the molecular mechanisms by which this splice variant promotes tumor progression features.

Abstract 89: Osteopontin-c and osteopontin-b splicing isoforms activate prostate cancer progression features

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Osteopontin (OPN) is one of the proteins overexpressed in prostate cancer (PCa) and is involved in tumorigenesis and metastasis. Alternative splicing of the OPN gene generates three protein splicing isoforms (OPN-SI), designated as OPNa, OPNb and OPNc, which have demonstrated specific roles in different tumor models. However, the roles of each of these isoforms have not been previously characterized in PCa. Our previous study reported that OPN-SI were overexpressed in PCa tissues, strongly associated with PCa occurrence and with tumor cell differentiation. Besides, OPNc was the most upregulated variant. We have also demonstrated that OPNc splicing isoform can contribute for PCa diagnosis and prognosis. Based on these results, the present study aimed to evaluate the putative roles of OPN-SI in PCa biology using in vitro and in vivo functional assays. PC-3 was stably transfected with expression vectors containing OPNa, OPNb and OPNc, as well as empty vector control. PC-3 cells overexpressing each construct were analyzed for in vivo tumor growth and in relation to different aspects mimicking tumor progression, such as cell proliferation, migration, invasion and soft agar colony formation. OPNc and OPNb overexpressing cells significantly activated enhanced xenograft tumor growth and PC-3 proliferation, migration, invasion, and soft agar colony formation, as well as the expression of several cancer related genes. Additionally, we have also shown that some of the OPNc and OPNb-dependent pro-tumorigenic roles are mediated by PI3K/Akt signaling pathway. Inhibition of this pathway by using LY294002 specifically inhibited tumor progression features evoked by OPNc and OPNb overexpression. According to our data, OPNc and OPNb roles on favoring different aspects of PCa progression suggest that these isoforms contribute to the physiopathology of prostate cancer progression and tumorigenesis. Altogether, the data open possibilities of new therapeutic approaches for prostate cancer that selectively downregulate OPNc and OPNb, altering its properties favoring prostate tumor progression. 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 89. doi:1538-7445.AM2012-89

Abstract 1346: Osteopontin-b and Osteopontin-c splicing isofoms activate prostate cancer cells prosurvival features

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Alternative splicing of the osteopontin (OPN) gene generates three protein splicing isoforms (OPN-SI), designated as OPNa, OPNb and OPNc. We previously demonstrated that both OPNb e OPNc are able to activate typical features of prostate cancer (PCa) progression. These data suggested that some of these tumorigenic roles are mediated by the activation of pro-survival pathways in PCa cells. The aim of this study was to delineate molecular and signaling pathways by which these two isoforms are able to mediate PCa cell survival and growth. PC3 cells overexpressing the three OPN-SI were treated with 1x10-6μg/mL of docetaxel (DXT), which was used as an in vitro model to induce cell death. Cell survival was analyzed by phase-contrast microscopy, crystal violet staining and MTT assays, in the presence or not of PI3K/Akt specific inhibitor (LY294002). Cell death has been investigated by immunoblot and flow citometry. Quantitative real time PCR assays (qRT-PCR) and immunofluorescence evaluated the involvement of epithelial mesenchymal transition (EMT) on PC3 cell survival in response to DXT treatment. PC3 cells overexpressing OPNb or OPNc treated with DXT have a higher cell density and phosphorylated AKT (Ser473) expression level compared to control cells. An additional increase in Akt (Ser473) phosphorylation was also observed after DXT treatment. PC3 cells overexpressing OPNb or OPNc isoforms also promoted additional resistance to cell death induced by DXT treatment, as compared to cells overexpressing OPNa. Combined treatment with LY294002 and DXT significantly inhibited PC3 cell survival mediated by the overexpression of OPNb and OPNc isoforms. Cells overexpressing the 3 OPN-SI similarly express Bcl-2, activated caspase 3 and 7 and also similar annexin V staining index. However, PC3 overexpressing OPNb or OPNc treated with DXT differentially express Mcl-1 and Bim. Immunofluorescence and qRT-PCR assays demonstrated that PC3 cells overexpressing these two splice variants significantly down-regulate the epithelial and upregulate the mesenchymal EMT markers, indicating that activation of this pathway is one of the mechanisms modulating PC3 cell survival in response to DXT treatment. As a whole, our data indicate important roles for PI3K/Akt and EMT signaling, besides BL2-family members differential expression on modulating PC3 pro-survival features in response to OPNb e OPNc overexpression. Citation Format: Kivvi Duarte Mello, Tatiana Martins Tilli, Ana Carolina S. Ferreira, Claudete Esteves Klumb, Luiz Eurico Nasciutti, Etel Rodrigues Pereira Gimba. Osteopontin-b and Osteopontin-c splicing isofoms activate prostate cancer cells prosurvival features. [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 1346. doi:10.1158/1538-7445.AM2014-1346

Abstract B78: Osteopontin-c splicing isoform is a key molecule in ovarian cancer progression

Introduction: Osteopontin (OPN) is a glyphosphoprotein overexpressed in ovarian carcinoma (OC) and is involved in tumorigenesis and metastasis. Alternative splicing of OPN primary transcript generates 3 isoforms, named as OPNa, OPNb, and OPNc. Objective: This study aimed to characterize the expression profile and functional role of each OPN splicing isoform (OPN-SI) in OC. Methodology: The expression patterns of OPN-SI and a series of genes involved in key cancer pathways were analyzed by real-time PCR. In vitro and in vivo functional assays were performed using the ovarian cancer cell line OvCar-3, stably overexpressing the three OPN-SI and conditoned medium secreted by these cells. Results: OPNc, but not OPNa and OPNb, was specifically expressed on OC samples. OPNc splice variant significantly activated OvCar-3 cell proliferation, migration, invasion and anchorage independent cells growth, besides inducing tumor formation in nude mice. We also found that these features are mainly mediated by PI3K/Akt signaling pathway. By using a real time PCR Cancer Gene Array, we found that OvCar-3 OPNc-overexpressing cells are also able to modulate the expression of 34 genes involved in key cancer pathways. Notably, tumors formed by OPNc-overexpressing cells present high expression levels of typical angiogenic markers, such as VEGF-A, VEGFR-2 and CD34. Based on these data, we also investigated the molecular mechanisms by which OPNc stimulates angiogenic processes. Our data showed that OPNc overexpression activates VEGF-A expression and secretion, besides upregulating the expression of c-Fos, c-Jun and phospho-c-Jun. OPNc role on activating the phosphorylation of c-Jun is mediated by the integrin receptor (αvβ3), in an RGD-dependent manner. In addition, OPNc-conditioned medium is able to induce HUVEC endothelial cell proliferation, migration and adhesion. Conclusion: In summary, our data demonstrate that OPNc is able to activate sereral aspects of OC progression, including angiogenesis, mainly by the PI3K/Akt pathway, indicating that this splice variant could be a putative target for further studies aiming to investigate this molecule for new therapeutic approaches for OC. Citation Format: Tatiana M. Tilli, Kivvi D. Mello, Vanessa Franco, Bruno Robbs, Joao Luiz Wanderley, Fabricio Rass, Joao P B Viola, Georg Weber, Vincent Castronovo, Akeila Bellahcene, Etel R P Gimba, Etel Gimba. Osteopontin-c splicing isoform is a key molecule in ovarian cancer progression. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr B78.

Abstract 396: Osteopontin-c splicing isoform contributes to ovarian cancer progression.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Introduction: Ovarian carcinoma (OC) is one of the most aggressive gynecological diseases and generally diagnosed at advanced stages. Osteopontin (OPN) is overexpressed in OC and is involved in tumorigenesis and metastasis. Alternative splicing of OPN generates 3 isoforms, OPNa, OPNb, and OPNc. Objective: This study aims to characterize the expression profile and functional role of OPN splicing isoforms (OPN-SI) in OC. Methodology: The expression patterns of OPN-SI and a series of genes involved in cancer pathways were analyzed by real-time PCR. In vitro and in vivo functional assays were performed using the ovarian cancer cell line OvCar-3, stably overexpressing the three OPN-SI. Results: OPNc, but not OPNa and OPNb, was specifically expressed on OC samples. We then demonstrated that OPNc isoform has stimulatory effects on OvCar-3 cell proliferation, migration, invasion and anchorage independent cells growth, besides activating tumor formation in nude mice. We also found that these features are mainly mediated by PI3K/Akt signaling pathway. Moreover, OvCar-3 OPNc-overexpressing cells are able to modulate the expression of 34 genes involved in key cancer pathways. Notably, tumors formed by OPNc-overexpressing cells present high expression levels of typical angiogenic markers, such as VEGF-A, VEGFR-2 and CD34. Based on this observation, we also investigated the molecular mechanisms by which OPNc stimulates angiogenic processes. Our data showed that OPNc overexpression activates VEGF-A expression and secretion, also through the PI3K/Akt pathway. This splicing isoform is also able to activate the expression of c-Fos, c-Jun and phospho-c-Jun. OPNc role on activating the phosphorylation of c-Jun is mediated by integrin receptor, in an RGD-dependent manner. In addition, OPNc-conditioned medium is able to induce HUVEC endothelial cell proliferation, migration and adhesion. Conclusion: Thus, the specific targeting of OPNc and its regulated signaling network could be a putative novel strategy to inhibit key steps involved in OC progression and may represent a putative strategy for developing new therapeutic approaches using OPNc as interesting target for OC treatment strategies. Citation Format: Tatiana M. Tilli, Kivvi D. Mello, Vanessa Franco, Bruno Robbs, Joao Luiz Wanderley, Fabricio R A Silva, Joao P B Viola, Georg Weber, Vincent Castronovo, Akeila Bellahcene, Etel R P Gimba. Osteopontin-c splicing isoform contributes to ovarian cancer progression. [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 396. doi:10.1158/1538-7445.AM2013-396

Abstract B58: OPNc and OPNb osteopontin splicing isofoms activate prostate cancer prosurvival features through PI3K/AKT/BIM signaling pathway

Abstract Alternative splicing of the osteopontin (OPN) gene generates three protein splicing isoforms (OPN-SI), designated as OPNa, OPNb and OPNc. We previously demonstrated that both OPNb e OPNc are able to activate typical features of prostate cancer (PCa) progression. These data suggested that some of these pro-tumorigenic roles are mediated by the activation of pro-survival pathways in PCa cells. The aim of this study was to delineate molecular and signaling pathways by which these two isoforms are able to mediate PCa cell survival and growth. PC3 cells treated with docetaxel were used as an in vitro cell model to induce cell death. We then investigated the involvement of PI3K/Akt signaling and anti e pro-apoptotic proteins on inducing PC3 cell survival on cells overexpressing each OPN-SI. PC3 cells overexpressing the three OPN-SI were treated with 1×10-6μg/mL of docetaxel and then analysed for cell proliferation and viability using crystal violet and 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide) MMT assays, respectively. PC3 whole protein cell extracts were prepared to evaluate protein expression of members of PI3K/Akt and cell death signaling. PI3K/Akt signaling was analysed by immunoblot tests using specific antibodies against total and phosphorylated (ser473) Akt. The expression of apoptotic proteins was investigated by using Bcl-2, Mcl-1 e Bim specific antibodies. LY294002 was used as an specific inhibitor of PI3K signaling. PC3 cells were incubated with LY294002 in combination or not with docetaxel treatment in order to evaluate cell survival pathways. Cells overexpressing OPNb and OPNc isoforms presented a significant increase of Akt (Ser473) phosphorylation, as compared to cells overexpressing OPNa and empty vector control. An additional increase in Akt (Ser473) phosphorylation was also observed after docetaxel treatment. PC3 cells overexpressing OPNb and OPNc isoforms also promoted additional resistance to cell death induced by docetaxel treatment, as compared to cells overexpressing OPNa. Combined treatment with LY294002 and docetaxel significantly inhibited PC3 cell survival mediated by the overexpression of OPNb and OPNc isoforms. The expression of Bcl-2 protein was similar among cells overexpressing the three OPN-SI, before and after docetaxel treatment. On the other hand, cells overexpressing OPNb e OPNc presented significant reduction on the expression of Mcl-1. Cells overexpressing OPNc, but not OPNb, presented a significant inhibition of pro-apoptotic protein Bim. As a whole, our data indicate an important role of PI3K/Akt signaling activation and inhibition of pro-apoptotic proteins on the survival of PC3 cells as a result of OPNb e OPNc overexpression. Citation Format: Kivvi Duarte Mello, Tatiana Martins Tilli, Ana Carolina S. Ferreira, Claudete Esteves Klumb, Etel Rodrigues Pereira Gimba. OPNc and OPNb osteopontin splicing isofoms activate prostate cancer prosurvival features through PI3K/AKT/BIM signaling pathway [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr B58.

Abstract 5256: Osteopontin-c and Osteopontin-b splicing isoforms activate important hallmarks of cancer contributing to prostate carcinoma progression

Introduction: Osteopontin (OPN) is a secreted glycophosphoprotein and is involved in tumorigenesis and metastasis in many solid tumors, including prostate cancer (PCa). Alternative splicing of OPN mRNA leads to the expression of 3 isoforms, named OPNa, OPNb and OPNc. However, the expression pattern and the roles of these isoforms have not been previously characterized in PCa. Objective and Methodology: Herein we evaluated the expression pattern of these OPN isofoms in PCa and BPH (benign prostate hyperplasia) tissue samples by using qRT-PCR. ROC curve analysis evaluated the performance of OPN isoforms tissue levels as compared to PSA serum levels for the diagnosis of PCa. Additionally, we investigated the roles of these isoforms on PCa progression using PC3 cells ectopically overexpressing each OPN isoform and in vitro and in vivo assays. Results: Our data demonstrate that these 3 isoforms are overexpressed in PCa in relation to BPH tissue samples and that OPNc presents the highest expression level. ROC curve analysis demonstrate that OPNa, OPNb and OPNc expression levels perform better (AUC 0,9; 0,81 and 0,88, respectively, p 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 5256. doi:10.1158/1538-7445.AM2011-5256

Abstract 1194: The splicing isoform osteopontin-c contributes to ovarian cancer progression

Introduction: Osteopontin (OPN) is a secreted glycophosphoprotein that is involved in tumorigenesis and metastasis in many solid tumors. Alternative splicing of OPN mRNA generates 3 known species and proteins, OPNa, OPNb and OPNc. Objective and Methodology: The aim of the present work was to investigate the expression profiling and the functional roles of OPN isoforms in ovarian carcinoma. Here we investigate the expression of each OPN isoform in ovarian cell lines and ovarian tissues, by quantitative real-time PCR using OPN splice variants specific primer pairs. We then investigated the roles of each one of these isoforms by using in vitro and in vivo models. Cell proliferation, migration and anchorage independent cell growth analysis were performed using OvCAr-3 cells overexpressing OPNa, OPNb or OPNc isoforms. Tumor formation was evaluated in athymic nude mice inoculated with OvCar3 cells overexpressing each oisoform as compared to empty vector controls. Results: We show that OPNa and OPNb are expressed in all ovarian tissues analyzed. However, the isoform OPNc is selectively present in malignant and borderline ovarian tumors and malignant tumor cell lines, but not in benign and normal ovarian tissues. Cell proliferation, migration, anchorage-independent growth and tumor growth in vivo are dramatically increased in OvCar-3 cells overexpressing OPNc. In contrast, OPNa and OPNb do not present these same pro-tumorigenic properties. Conclusions: These results together provide compelling evidence that OPNc plays important roles in ovarian cancer progression. Firstly, OPNc is specifically expressed in borderline and malignant ovarian tumors. Secondly, OPNc expression facilitates tumor progression towards an aggressive phenotype. Targeting OPNc in ovarian cancer has potential as a novel therapeutic strategy. Financial Support: CNPq, CAPES, FAPERJ, Swiss Bridge Foundation, FAF/MS Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1194.