Osteopontin-c gene expression and subcellular localization in ovarian cancer cells: Implications for prognosis and therapeutic responses

OPN gene structure and known roles

Osteopontin (OPN) is a highly phosphorylated glycoprotein, encoded by the secreted phosphoprotein 1 (SPP1) gene, also known as early T-lymphocyte activation 1 protein (ETA1).^1,2 The SPP1 gene is located on chromosome 4q1322.1, containing seven exons. While the exon 1 is untranslated, exons 2–7 contain the coding sequences.^3,4 OPN modulates interactions with the extracellular matrix through integrins and CD44 receptors.^5,6 OPN has also been proposed as a biomarker for human malignancies, including breast, ⁷ ovarian, ⁸ and gastric cancers. ⁹ Differential expression of OPN has also been observed during viral infections, such as influenza and HIV. ¹⁰

OPN isoforms

OPN presents several isoforms, generated by post-translational modifications (PTMs), alternative translation initiation, and alternative splicing. The sum of all these isoforms constitutes total OPN (tOPN), generally referred as OPN. The OPN protein undergoes extensive PTMs, such as phosphorylation, ¹¹ O-linked glycosylation, ¹² tyrosine sulfation, ¹³ transglutamination, ¹⁴ and sialylation. ¹⁵ These modifications are cell-type specific and depend on pathophysiological mechanisms to modulate OPN functions. Alternative translation of full-length OPN transcript can generate secreted OPN (sOPN) and/or intracellular OPN (iOPN) isoforms. Although most studies focus on sOPN, intracellular roles have been also described for iOPN. ¹⁶ Depending on its intracellular or extracellular localization, these two isoforms exhibit different levels in various cell types, leading to distinct biological effects. The translation of sOPN is initiated from a canonical AUG start codon, while the iOPN begins translation from a non-AUG alternative start codon located downstream of the canonical AUG, preventing this isoform from being directed to secretory vesicles.^16,17 In cancer cells, tOPN aberrantly activates several signaling pathways, such as phosphoinositide 3-kinase (PI3K)/protein kinase B (PKB/AKT), mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK), and focal adhesion kinase (FAK).^18–20 These signaling pathways, in turn, can activate both intracellular and secreted OPN expression in tumor cells, promoting tumor progression and resistance to therapies. ¹⁹ The hyperactivation of PI3K/AKT signaling pathway has been shown to be a critical mechanism of drug resistance induced by OPN in non-small-cell lung cancer cells. ²⁰ Additionally, overexpression of OPN and integrin αVβ3 has been demonstrated to contribute to acquired resistance to gefitinib and cisplatin via activation of the PI3K/AKT/FAK signaling pathway.^18–20

The alternative splicing of the OPN human primary transcript results in at least five isoforms (OPN-SI), named as OPN-a (the full length gene product), OPN-b (exon 5 is deleted), OPN-c (exon 4 is deleted), OPN-4 (both exons 4 and 5 are deleted), and OPN-5 (arises from an inclusion of intron 3).^21–24 OPN-SI have been described as key modulators of cancer hallmarks, due to their differential aberrant expression in tumor cells. ²⁵ The expression of these isoforms has been correlated with tumor development and progression,^26,27 metastasis formation,^28,29 and response to treatment. ³⁰ The OPN-SI expression is tumor and cell-type specific and may influence tOPN roles in malignancy, besides being widely reported as cancer biomarkers.^31–33 Since OPN-SI presents differential levels in tumor in relation to non-tumor tissues, it is necessary to investigate the mechanisms by which OPN-SI may contribute to tumor progression.^34–38

Ovarian cancer and total osteopontin

Ovarian carcinoma (OC) is a highly heterogeneous and aggressive disease. It is the most frequent among gynecological tumors and exhibits high mortality rates, mainly due to late diagnosis. ³⁹ Histologically, the most frequent epithelial ovarian carcinomas are serous, clear-cell, endometrioid, and mucinous tumors, although they are treated as a single disease. ⁴⁰ Despite significant advances in the treatment of OC and initially high responsiveness to platinum-based chemotherapy, therapeutic failures are still observed, contributing to tumor progression and high mortality rates. ⁴¹ Therefore, a better comprehension of the expression patterns and functional roles of OPN gene products aberrantly expressed in OC, including those mediating drug resistance, is key to better treatment options for this tumor type.

Total OPN is interestingly found overexpressed in OC and is currently one of the most studied serum biomarkers in this neoplasia. OPN levels in plasma are higher in patients with OC than those presenting ovarian benign tumors or samples of healthy ovarian epithelium. Moreover, OPN may be clinically useful when used complementary to CA125 in detecting and predicting OC, being proposed as a useful tumor biomarker for OC screening tests and as an improvement factor for diagnostic performance of a multimarker OC diagnostic test.^42–44 Moreover, OPN levels have been associated with OC progression and metastasis. ⁴⁵ In the context of OC microenvironment, an RNA single-cell analysis has revealed that OPN levels are found high in exhausted T cell and associated with worse prognosis, providing new clues for OPN as an immunotherapeutic target in OC. ⁴⁶

OPN-c in ovarian cancer and related signaling pathways

The specific OPN splicing patterns in OC were largely unknown until the studies conducted by our group. Among the OPN-SI, the OPN-c variant has been the most extensively studied across several tumor contexts. ⁴⁷ In 2011, we were the first to show that OPN-c splicing isoform is found highly expressed in aggressive serous ovarian adenocarcinoma patients, compared to non-neoplastic ovarian tissues. ⁴⁸ Therefore, in this review, we will focus on discussing this splice variant in more detail in OC.

Total OPN has been widely implicated in the modulation of multiple oncogenic signaling pathways in cancer proliferation and drug resistance. ²² In OC, tOPN has been shown to promote tumor growth in an AKT/HIF1α-dependent manner. ⁴⁹ Notably, we have previously demonstrated that OC cells overexpressing OPN-c exhibit enhanced proliferative capacity, as shown by functional assays in vitro and in vivo. Interestingly, when OC cells overexpressing OPN-c were exposed to a neutralizing polyclonal anti-OPN-c antibody, their proliferation rates were suppressed, and apoptosis was induced, suggesting that these effects depend on the secreted form of OPN-c. ⁴⁸ Considering the impact of OPN-c on tumor proliferation, we investigated the phosphorylation levels of the AKT, ERK, and FAK oncogenic kinases in our OC cell model stably expressing OPN-c. Remarkably, we found that phosphorylated ERK and AKT levels are reduced upon OPN-c overexpression (Figure 1(a) and 1(b)), while FAK phosphorylation is increased (Figure 1(c)). This finding initially appears unexpected and may suggest that these parallel pathways could compensate for each other depending on the cellular context. Adding complexity to the interplay between signaling pathways in cancer, a recent study showed that AKT and FAK kinase are differentially expressed depending on cell contractility, extracellular matrix stiffness, and the epithelial–mesenchymal transition (EMT) phenotype. ⁵⁰ Although these findings were obtained in a model of oral squamous cell carcinoma, they suggest that AKT and FAK requirements and concomitant activation may be context-dependent.OPEN IN VIEWER

Consistent with the differential modulation of signaling pathways in OPN-c overexpressing cancer cells, OPN-c has been implicated in additional features of OC progression. Using in vitro and in vivo approaches, we found that OPN-c promoted migratory and invasive phenotypes in OC cell models. OC cell lines overexpressing OPN-c presented higher VEGF and MMP-2 and MMP-9 transcript levels, when compared to cells overexpressing OPN-a and OPN-b. ⁴⁸ Later, OPN-c overexpression was reported to induce gene expression patterns involved not only in migration but also in angiogenesis, again including changes in the VEGF gene. Moreover, we functionally demonstrated that conditioned media secreted by OC overexpressing OPN-c stimulated proliferation, migration, and adhesion of human endothelial cells, further evidencing OPN-c roles on promoting angiogenesis. ⁵¹ Notably, cells overexpressing OPN-c resulted in extremely rapid tumor growth and formation of larger tumors, when compared to those tumors formed by cells overexpressing OPN-a and OPN-b. We also found that OPN-c overexpression in OC cells increased the number of soft agar colonies formed, whereas OPN-b inhibited it, evidencing a higher metastatic potential of OPN-c in OC cells. Otherwise, OPN-a promoted no significant effect on the number of colonies formed. ⁴⁸ While our studies do not directly demonstrate how each OPN splice variant influences tumor metastasis, the observed effects for OPN-c strongly suggest that this splice variant likely plays a role in stimulating ovarian cancer metastasis. To definitively clarify the specific contributions of OPN-c to OC metastatic potential, future research using appropriate animal models will be necessary.

Consistently, we found that OPN-c was expressed in higher levels in an OC cell line model resistant to cisplatin, compared to OPN-a and OPN-b isoforms. Silencing OPN-c in these cells affected the gene expression of epithelial–mesenchymal transition (EMT) markers and related cytokines. According to these findings, OPN-c may be involved in the modulation of EMT that contributes to cisplatin resistance in these cells. These transcriptional alterations were directly linked to decreased cell viability in response to cisplatin and doxorubicin chemotherapeutic agents, further highlighting OPN-c as an additional factor in the complex response of cancer cells to drugs. ⁵² Otherwise, OPN-c-overexpressing cells had higher cell viability but were interestingly more sensitive to cisplatin than control cells. This suggested that OPN-c could be a preferential target for cisplatin’s cytotoxic effects in OC cells, leading to reduced cell survival and viability. These data are in alignment with previous information showing that OPN-c supports prostate cancer cell survival by mediating resistance to docetaxel. ⁵³ In gastric cancer, low OPN-c expression is correlated with poor prognosis. In vitro studies showed that OPN-c overexpression suppressed cell proliferation, adhesion, migration, and invasiveness of gastric cancer cells and increased reactive oxygen species (ROS) production after 5-fluorouracil treatment. ⁵⁴

Altogether, these data implicate OPN-c in a wide range of ovarian tumor hallmarks, including proliferation, migration, invasion, angiogenesis, metastatic potential, and several biological processes linked to drug resistance, such as cell survival, the EMT phenotype, and the production of EMT-related cytokines. These initial insights into the role of OPN-c in OC cancer offer a basis for exploring this variant and its related signaling pathways as new molecular targets to control OC progression.

OPN-c expression in ovarian cancer: Biological and clinical implications

Reports on OPN-c transcript expression patterns in OC are quite scarce to date. Our analysis of OPN-a, OPN-b, and OPN-c RNA levels in ovarian cancer cell lines and human ovarian tissues (both cancerous and non-cancerous) revealed that OPN-c was uniquely expressed in tumor samples, while OPN-a and OPN-b are expressed in both normal and tumor tissues. Within ovarian tumor cell lines, OPN-a and OPN-b showed higher expression levels compared to an immortalized non-tumoral ovarian cell line. Importantly, OPN-c expression was absent in benign and non-tumoral ovarian tissues, evidencing the diagnostic potential of OPN-c levels in this tumor type. ⁴⁸ The mechanisms underlying the deregulated expression of OPN-SI in OC remain poorly understood, but some evidence points to aberrant expression of genes encoding splicing factors, such as serine/arginine-rich (SR) and heterogeneous ribonucleoproteins (hnRNsP). ⁵⁵ It was found that high OPN-c expression levels are associated with a higher hnRNP to SR transcriptional ratio and that OPN exon 4 (which is deleted in OPN-c) and adjacent introns have predicted binding sites for some of these splicing factors. Thus, differential expression of these splicing factors in OC cells might be a mechanism for aberrant OPN-c expression in OC cells. This hypothesis is further reinforced by findings showing that silencing of the splicing regulator SRSF7 impacts the OPN-SI levels and leads to a reduction in the proliferation of kidney cancer cells. ⁵⁶ Future work aiming at preventing OC progression by downregulating OPN-c might include strategies regulating these splicing factors.

Moreover, we have extracted data for endometrioid OC patient samples from the GEPIA2 public data bank, which revealed that OPN-c isoform is also found to be overexpressed compared to non-tumoral tissues (Figure 2(a)). This data further suggests that OPN-c could be a useful diagnostic biomarker in OC, irrespective of the molecular subtype. Interestingly, early diagnosis blood tests including tOPN and additional serological markers have been increasingly proposed for OC, indicating that OPN seems to be robust and useful in distinguishing OC. ⁵⁷ Other studies have also reported OPN-c expression and their roles in different cancer types, such as melanoma, ²⁹ prostate, ⁵⁸ leukemia, ³⁰ breast, ⁵⁹ and lung cancer ⁶⁰ (Table 1).OPEN IN VIEWER

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Table 1.

Roles and clinical impacts of OPN-c splice variant expression in ovarian carcinoma and other tumor types.

Cancer type	Biological roles and/or expression patterns	Clinical impacts
AML 61	Higher levels in bone marrow samples in response to therapy	Cancer relapse and response to therapy
BC 62	Higher levels in the blood than carcinomas in situ	Cancer screening and diagnosis
BC 63	High tissue levels correlated with lymph node metastasis, advanced TNM stage, histologic grade, tumor recurrence, and triple-negative subtype	Prognosis
BC 64	High levels associated with worse prognosis, aggressive subtypes HER-2, and triple-negative/basal-like, and higher risk of recurrence	Cancer relapse and prognosis
BC 65	Fibrous nature; druggable pockets in the CD44 interaction region	Therapeutic target
BC 66	Nuclear osteopontin-c is strongly associated with mortality in patients with early breast cancer	Prognosis in early breast cancer
BC 67	Variable response to specific treatment	Response to therapy
CRC 68	Overexpressed in CCR samples and associated with worse overall survival	Diagnosis and prognosis
B-ALL 69	Cell migration: Overexpression in KMT2A-AFF1 samples and associated with CNS infiltration	Prognosis
B-ALL 70	Cell adhesion to matrix, viability and response to vincristine and etoposide	Response to therapy
ESCC 71	High levels in ESCC tissues	Diagnosis and prognosis
OC 48	Cell proliferation, migration and invasion, and colony and tumor formation	Diagnosis
OC 52	Cell proliferation and viability, colony formation, and chemoresistance	Response to therapy
OC 51	Endothelial cell adhesion, proliferation, and migration	Tumor progression
OC 55	High levels associated with higher hnRNP to SR splicing factor ratios	N/A
OC 72	High nuclear OPN-c in patients responsive to PLD treatment; association with longer OS and PFS	Response to therapy
PDA 73	Activates MMP-9 and VEGF; metastatic potential in response to nicotine	Therapeutic target
PDA 74	Associated with nicotine response and alpha-7-nicotine acetylcholine receptor; cell migration	Therapeutic target
PCA 58	High tissue levels	Diagnosis and prognosis
PCA 75	Cell proliferation, migration, invasion, and colony formation	Tumor progression
PCA 51	Endothelial cell adhesion, proliferation, and migration	Tumor progression
PCA 76	Activation of androgen responsive genes	Tumor progression
PCC 53	Cell survival, EMT, and response to DXT	Response to therapy
PDA 62	High blood levels	Cancer screening and diagnosis

We have found OPN-c was the most upregulated OPN-SI in prostate cancer tissue samples. In this tumor model, high OPN-c levels were strongly associated with prostate cancer occurrence and high Gleason score, providing evidence that OPN-c can improve prostate cancer diagnosis and prognosis. ⁵⁸ In human esophageal squamous cell carcinomas (ESCCs), OPN-c is significantly upregulated and could also serve as a diagnostic marker, given its association with disease staging and patient survival. ⁷¹ Not only does OPN-c expression provide important diagnostic information, but it can also predict clinical evolution in OC. In the same cohort of patients with endometrioid OC, we found that high OPN-c transcriptional levels were associated with decreased overall survival (Figure 2(b)). When it comes to disease-free survival, we have found no significant differences among patients with high and low OPN-c transcript levels (Figure 2(c)). Similar findings have been observed in serous ovarian cancer subtype, in which higher OPNc expression levels were also associated with worse overall survival in patients treated or not with platinum chemotherapies (Figure 2(d) and 2(g)), although with no evident impact to worse progression-free survival (Figure 2(e) and 2(h)) and disease-free survival (Figure 2(f) and 2(i)).

As shown in our studies in OC, the OPN-c isoform has also been reported as a prognostic indicator for invasive breast cancer following papillomatous growth. ⁷⁷ In human breast tumors, OPN-c expression correlates with lymph node metastasis, ⁷⁸ advanced TNM stage, histologic grade, and survival rates. ⁶³ OPN-c in breast tumors may be critical for metastasis. ⁷⁸ Studies on metastatic melanoma samples also show that high OPN-c expression levels are associated with advanced tumor progression and poor prognosis. ²⁶ Our group also evaluated OPN-c expression levels in B-cell acute lymphoblastic leukemia (B-ALL) cases presenting KMT2F-AFF1 and ETV6-RUNX1 gene fusions, where OPN-c expression contributed to poor prognostic features in B-ALL patient subgroups, suggesting it could be a potential biomarker in B-ALL. ⁶⁹ In prostate cancer, OPN-c expression supports sustained proliferative survival, primarily mediated through PI3K signaling. ⁷⁵ OPN-c overexpression in the PC-3 prostate cancer cell line enhanced xenograft tumor growth, proliferation, migration, invasion, and soft agar colony formation, as well as MMP2, MMP-9, and vascular endothelial growth factor (VEGF) expression, as has been found in OC cells.^48,75 OPN-c also appears to be a biomarker for PCa and modulates androgen receptor (AR) signaling, contributing to PCa progression. ⁷⁶ Conditioned medium secreted by prostate cells overexpressing OPN-c activated the expression levels of AR-responsive genes through the PI3K pathway. ⁷⁶ In colorectal cancer, high levels of OPN-c correlate with poor clinicopathological features and shorter progression-free survival (PFS), and they are associated with the BRAFV600E activating mutation, which promotes tumor progression. ⁶⁸ Moreover, in colon cancer cell lines (HT115 and HCT-8) undergoing 5-fluorouracil (5-FU) treatment, stress signals could be transduced by secreting OPN-c, which promotes cell survival in the tumor microenvironment. Secreted OPN-c isoform activates nuclear calcium signaling and modulates epigenetic markers such as methyl-CpG binding protein 2 (MeCP2), demonstrating the role of OPN-c in adapting tumor cells within a pro-inflammatory microenvironment. ⁷⁹

Subcellular localization of OPN-c in OC and other tumor types

Total OPN has been widely reported as a matricellular and secreted protein. Indeed, most data concerning its physiological and pathological roles in tumor cells are mainly related to total secreted OPN. Typically, the immunohistochemical (IHC) detection of tOPN reveals a strong signal in the perinuclear area, indicating its passage through the Golgi apparatus and endoplasmic reticulum for secretion. ⁸⁰

In distinct cell compartments and specific tumor models, its diagnostic or prognostic implications have been explored.^71,72,81 Recent IHC analysis of OC tissues found that high nuclear OPN-c expression in patients exposed to PEGylated liposomal doxorubicin (PDL) treatment was associated with longer OS and longer PFS. Thus, nuclear OPN-c expression appears to be a promising marker of therapy response (PLD or other chemotherapy) in OC patients. ⁷² Additionally, using immunofluorescence assays, we identified predominant perinuclear OPN-c in OC cells overexpressing this OPN splice variant (Figure 3). In these cells, OPN-c is also found in the cytoplasm and faintly labeled in the nucleus. Nuclear staining for OPN-c has been detected for the first time in breast cancer, although it has not been quantified. Later, high nuclear OPN-c staining patterns were strongly associated with mortality in patients with early breast cancer, serving as a poor prognostic indicator for patient survival. It has also been suggested that incorporating OPN-c IHC into standard pathology evaluations could offer prognostic advantages in the early diagnosis of breast cancer. ⁸¹ OPEN IN VIEWER

In parallel, cytoplasmic OPN-c has been detected by IHC analysis in esophageal squamous cell carcinoma (ESSC) and found significantly upregulated compared to normal tissues, supporting its value as a potential diagnostic marker. It was also found that high cytoplasmic OPN-c expression in ESCC samples was significantly associated with pathological T stage and overall tumor stage, but not with histological grade or lymph node metastasis. ⁷¹ These findings suggest that the impact of assessing the subcellular localization of OPNc in cancer tissues might be tumor-specific and should be further explored in additional cancer types.