Although several reports have demonstrated the important role of full-length OPN in modulating tumor progression , only a few studies have identified its gene expression-related profiling [5, 9]. This is the first report regarding gene expression data related to a specific OPN splice variant and correlation with its functional roles in tumor progression. Although other reports have pointed to individual pathways modulating the actions of some OPN splice variants in different tumor models , none of them has investigated putative interacting signaling networks and their relationships to functional data. The main finding of the current study is that OPNc splice variant overexpression can modulate key cancer pathways and gene transcriptional patterns associated with the progression of ovarian and prostate tumors. In total, changes in the expression of 34 genes in OvCar-3 and of 16 genes in PC-3 OPNc-overexpression cells were observed, with an overlap of 7 genes (8.3%) in the two cell lines (Figure 1A). Gene ontology and interactome analysis led to a categorization of our data set into functional categories and networks (Additional files 2 and 3, and Figure 2). The array data revealed that OPNc overexpression modulates the expression of genes related to cell cycle control, apoptosis, signal transduction molecules and transcription factors, adhesion, angiogenesis, invasion and metastasis in both cell-line tumor models. These data were correlated with previously described roles of OPNc in activating tumor progression. Among the significantly up-regulated genes identified were Vegf and other pro-angiogenic gene products. Based on these data, we could partially validate the results obtained here, using gene expression profiling to demonstrate that OPNc-overexpressing cells secrete factors that are able to activate early angiogenic processes.
Transcriptional patterns associated with cell cycle control, proliferation and apoptosis
Previous studies from our group demonstrated that OPNc favors the proliferation of ovarian and prostate carcinoma cells [6, 8]. Here, we identified several differentially expressed cell proliferation-related genes in both tumor cell line models as a result of OPNc overexpression. In OvCar-3 OPNc-overexpressing cells, Atm was significantly down-regulated, and 6 other genes were up-regulated (Rb1, Cdk2, Cdkn1a, Ccne1, S100a4 and Cdc25a). The deregulated patterns of these genes may be related to the progression of ovarian and prostate cancer, according to the known roles for these genes, as presented in Additional files 2 and 3 [15–25].
We also previously showed that OvCar-3 and PC-3 OPNc-overexpressing cells that were treated with the anti-OPNc antibody proliferated more slowly and were induced to die, further evidencing a survival role for OPNc [6, 8]. In this study we found that Bcl2l1 and Bad were up-regulated in OvCar-3 and PC-3 OPNc-overexpressing cells compared to control cells. Consistently with our findings, these gene products have been reported as involved in the survival and chemosensitivity of prostate and ovarian carcinoma cells [15–30]. We also observed a significant upregulation of Casp8 and Apaf1, which have been implicated in death-receptor-mediated apoptosis and chemoresistance [24, 25]. In PC-3 cells, we also identified Htatip2 and Tert deregulated expression in response to OPNc overexpression, which are also gene products that mediate cell survival, metastasis and cancer recurrence [28, 30].
Transcriptional patterns associated with signal transduction and transcription factors
The results of this study are also in accordance with previous data demonstrating pathways related to signal transduction and transcription factors that are typically activated in ovarian and prostate tumor progression [31, 32]. We have shown that PI3K/Akt has an important pro-survival role and mediates several pro-tumorigenic features evoked by OPNc overexpression in OvCar-3 and PC-3 cells [6, 8]. Our current data provide evidence of the existence of additional deregulated signal transduction pathways and transcription factors in OvCar-3 and PC-3 cells as a result of OPNc overexpression. The Fos gene was found to be down-regulated in response to OPNc overexpression in OvCar-3 and PC-3 cells. Specifically in OvCar-3 OPNc-overexpressing cells, down-regulation of Myc and up-regulation of Pik3r1, Raf1, Erbb2 and Akt1 genes were observed. It has been reported that the tumor environment down-regulates c-MYC protein levels, which might be a strategy for cancer cells to survive under conditions of limited energy resources . However, down-regulation of c-MYC has not been described previously in ovarian carcinoma cells. Additional clinical implications of differentially expressed genes able to modulate signaling pathways and transcription are listed in Additional files 2 and 3 [34–40].
Transcriptional patterns associated with cell adhesion and angiogenesis
Regarding gene expression patterns related to cancer-associated adhesion molecules, in OvCar-3 cells, in addition to up-regulation of integrins in response to OPNc overexpression, we found an up-regulation of Pinin (Pnn). The up-regulation of a number of integrin heterodimers and adhesion molecules in cells that constitutively overexpress OPNc may therefore represent additional mechanisms by which cells acquire a general ability to adhere, promoting ovarian and prostate tumor progression, consistent with the well-known integrin-mediated role of total OPN, especially in cancer cells .
Previously, we have also shown that OPNc significantly increases Vegfa mRNA in ovarian carcinoma and prostate cancer xenograft tumors [6, 8]. Here, we also observed Vegfa up-regulation in response to OPNc-overexpression in OvCar-3 and PC-3 cells. Published reports have also indicated that VEGF-A is overexpressed in ovarian carcinoma and prostate cancer, and has been associated with tumor growth and recurrence [42, 43]. In addition to Vegfa overexpression, we found that OvCar-3 OPNc-overexpressing cells up-regulate Epdr1, Pdgfa, Tgfbr1, Tnf and Fgfr2, all of which are able to directly or indirectly modulate different aspects of angiogenesis, such as vascular permeability, lymphatic metastasis and tumor-stroma interactions, endothelial cell survival and stable vasculature [42–52].
Based on the significant up-regulation of several pro-angiogenic transcripts in response to OPNc overexpression, we attempted to validate part of the data obtained here by investigating the effect of OPNc-CM on activating angiogenic properties. Our data clearly demonstrated that OPNc-CM activated different steps of early angiogenesis, such as endothelial cell proliferation, adhesion and migration. In the light of data previously published by our group and those presented here, we partially validated that OvCar-3 and PC-3 OPNc-overexpressing cells secrete specific proteins that create a permissive environment, favoring induction of their own angiogenesis. However, the specific factors or proteins mediating these pro-angiogenic features contained in this CM should be further validated and characterized. OPN has been broadly characterized as an inducer of tumor angiogenesis, with a particular correlation with VEGF expression [53, 54]. The specific actions of OPN splice variants regarding non-small-cell lung cancer angiogenesis and VEGF have been investigated . These authors showed that OPNa overexpression was associated with increased bovine capillary endothelial tubule length and VEGF secretion, whereas OPNc was associated with decreases in both. OPNc in this tumor model has opposite roles to the OPNc-induced angiogenic features that we observed in ovarian and prostate carcinoma cells. Considering the tumor-specific roles of the OPN splice variants, the means by which different splicing isoforms specifically modulate tumor angiogenesis should be further investigated.
Previously, we have also shown that OPNc overexpression stimulates OvCar-3 and PC-3 migration, invasion and the formation of colonies in semisolid medium. OvCar-3 and PC-3 cells overexpressing OPNc resulted in extremely rapid tumor growth in vivo. In these tumors, well-known markers of tumor progression able to modulate tumor invasion and metastatic potential, such as Mmp2 and Mmp9, were consistently up-regulated [6, 8]. Transcriptional levels of pro-metastatic genes such as Mmp1 and Serpine1 in both OvCar-3 and PC-3 cells; Mta1, Mta2 and Mmp2 in OvCar-3; and Plau and Mmp9 in PC-3 cells, were significantly modulated in OPNc-overexpressing cells. However, our present study provides the first indication that OPNc might down-regulate Mmp1 in OC. The literature has shown the involvement of these transcripts in the steps modulating tumor invasion and metastasis in both ovarian and prostate tumors [56–63]. Here, also consistent with our previous data [6, 8], we report that OPNc-overexpressing cells induce Mmp2 and Mmp9 overexpression in ovarian carcinoma and prostate cancer cells, respectively, further corroborating a role of these gene products in activating cell invasion in both tumor models [60, 62].
Interacting networks and associated transcriptional patterns
The existence of an interactome network indicates that these differentially expressed genes, besides inducing specific cancer-associated pathways, potentially interact with each other, further indicating that the roles of OPNc in activating ovarian and prostate cancer progression require multiple and crosstalk signaling. The different network patterns observed for each tumor model investigated here concord with previously discussed tissue- and tumor-specific roles for OPNc and other OPN splice variants . Further studies aimed at exploring the mechanisms by which OPNc modulates these target and interacting gene products will elucidate how it controls the proliferation of ovarian and prostate cancer cells.