Osteopontin is a promoter for hepatocellular carcinoma metastasis: a summary of 10 years of studies

Abstract

In this review, we summarize the novel findings from our series of studies on the leading metastasis-related gene, osteopontin (OPN). In our previous gene expression profiling study, OPN was identified as one of the leading genes associated with the metastasis of hepatocellular carcinoma (HCC). We focused on OPN to evaluate its prognostic values and important roles in HCC metastasis. A retrospective study of large cohorts of HCC patients demonstrated that plasma OPN level was one of the leading independent prognostic factors for HCC patients, even in the early stage of HCC, and could serve as a surrogate serologic biomarker for monitoring the treatment response and tumor recurrence after HCC resection. Using both in vitro and in vivo investigations, we found that OPN has an important role in metastasis and tumor growth of HCC and is an attractive potential therapeutic target for combating HCC metastasis. We also found that OPN⁺ HCC cells have much more amplifications at chromosomal regions, and promoter polymorphisms are important in the regulation of OPN expression and tumor growth and lung metastasis of HCC.

Keywords

osteopontin (OPN) / hepatocellular carcinoma / metastasis / prognosis / therapeutic target / biomarker / genetic polymorphism

Introduction

Osteopontin (OPN or SPP1) is a secreted non-collagenous, sialic acid-rich, chemokine-like extracellular matrix (ECM) protein. OPN is a glycosylated phosphoprotein that acts as a cytokine and binds to αvβ integrins and receptors of the CD44 family to deliver signals to cells to promote cell adhesion, chemotaxis, ECM degradation, angiogenesis, apoptosis prevention, and indolent tumor growth [1,2]. Moreover, OPN has a crucial role in determining the oncogenic potential of various cancers, contributing to tumor invasion and metastasis [3-6]. This protein has also been regarded as a major transcription target induced by hepatocyte growth factor (HGF) and may contribute to HGF-mediated cell-cell dissociation, growth, and invasiveness [7-9]. OPN is overexpressed in highly metastatic tumor cell lines of breast cancer [10,11], and high OPN expression can confer a metastatic phenotype on benign tumor cells [12,13].

In our previous gene expression profiling study, OPN has been identified as one of the leading genes associated with the metastasis of hepatocellular carcinoma (HCC) [14]. We focused on OPN to evaluate its prognostic values and important roles in HCC metastasis. A retrospective study of large cohorts of HCC patients demonstrated that plasma OPN level was one of the leading independent prognostic factors for HCC patients, even in those with early stage of HCC [15-17] and can serve as a surrogate serologic biomarker for monitoring treatment response and tumor recurrence after HCC resection [18]. Using both in vitro and in vivo investigations, we found that OPN has an important role in HCC metastasis and tumor growth and is an attractive potential therapeutic target against HCC metastasis [19,20]. We also found that OPN⁺ HCC cells have much more amplifications at the chromosomal regions [21], and promoter polymorphisms are important in the regulation of OPN expression and HCC tumor growth and lung metastasis [22]. In this review, we summarized the novel findings of these series of studies.

OPN was identified as the leading genes associated with HCC metastasis

To identify the metastasis-related genes of HCC, we analyzed the expression profiles of 9180 genes in primary HCC tumors from 40 patients with or without intra-hepatic metastases. A supervised machine learning algorithm approach was used to classify patients based on their gene expression signatures for their potential to develop metastasis and for survival. We generated a molecular signature that correctly classified patients with or without intra-hepatic metastases [14]. Several genes with metastatic characteristics, including OPN, α9 integrin, H-cadherin, interleukin 2 receptor, serine proteinase inhibitor member 5, matrix metalloproteinase (MMP) 9, leukocyte immunoglobulin-like receptor subfamily A member 2, and CD37 antigen, were identified in our classifier. OPN exhibits the greatest increase in expression in primary HCC with accompanying metastasis (Fig. 1). OPN expression also correlates with invasiveness of HCC cells in tissue cultures, and neutralizing antibody to OPN can suppress the in vitro invasion and in vivo lung metastasis of highly metastatic HCC cells. These findings indicate that OPN may be needed for primary HCC to support metastasis [14].

Prognostic values of OPN for HCC

OPN overexpression has also been observed in various human tumors, including carcinomas of the stomach, lung, breast, colon, prostate, and pancreas [23-27]. The role of OPN in HCC has also generated significant interest, especially with regard to its roles as prognostic factor [28,29]. Based on these findings, we further evaluated the prognostic values of both tumor tissue and blood OPN levels for HCC patients.

Association of OPN levels in tumor tissue with HCC prognosis

First, we detected the OPN expression in tumor tissue from large cohorts of HCC patients and found that elevated OPN levels in HCC tissue were significantly associated with vascular or bile duct invasion, Edmondson’s grade, and intrahepatic spreading [18]. In addition, patients with high OPN expression had significantly poorer overall survival (OS) and shorter time to tumor recurrence (TTR) than the patients with low OPN expression (Fig. 2) [16]. These findings are consistent with the previous gene expression profiling study, suggesting that OPN level in tumor tissue is a dismal predictor for HCC patients.

Furthermore, we found that OPN, combined with caspase-3, acts as an even better independent indicator for HCC patients after curative resection [16]. Factors acting downstream of OPN interrupt the cell cycle, prevent apoptosis, and promote cell survival, all of which correlate with tumor progression [30]. Caspase-3 is a principal enzyme in the apoptotic cascade and frequently used to detect apoptotic activity [31]. Previous studies have found that OPN enhanced the chemoresistance in small-cell lung cancer and breast cancer by blocking caspase-9- and caspase-3-dependent cell apoptosis [32,33]. We examined OPN and caspase-3 expression in HCC tissue from 248 HCC patients to evaluate the predictive value of OPN and caspase-3 for HCC prognosis. OPN alone or combined with caspase-3 density was determined to be an independent prognosticator of both OS and TTR. Patients with high OPN density and low caspase-3 density had a higher probability of recurrence and death than the patients with low OPN and high caspase-3 levels. Moreover, according to the univariate and multivariate Cox proportional hazard regression analyses, the predictive power of the co-index was superior to that of OPN or caspase-3 levels alone as well as the other clinicopathological parameters, such as venous invasion, tumor size, and encapsulation. Moreover, patients with higher OPN levels and lower levels of caspase-3 had significantly poorer prognosis. These findings suggest that OPN/caspase-3 co-index can act as a more accurate predictor of early metastatic recurrence after HCC resection [16].

Another important finding is that thrombin is an independent prognostic indicator for HCCs with elevated OPN levels [20]. Thrombin, a serine protease that performs a multi-faceted role in coagulation, cleaves OPN at the cleavage site (RSK) into two fragments of approximately equivalent size, which changes the topological structure of OPN to display the integrin and CD44 binding domains [34]. This cleavage by thrombin improves the bioactivity of OPN and is necessary for efficient engagement with the integrin receptor [35,36]. Previous studies have demonstrated that thrombin-cleaved OPN is critically involved in the pathogenesis of various diseases including cancers [37,38]. Thrombin also contributes to tumor progression in coagulation-dependent and coagulation-independent manners [39,40]. In our study, thrombin expression was significantly associated with the metastatic potential of HCC, postoperative tumor recurrence, and poor prognosis of HCC patients. This finding is supported by the fact that high thrombin expression is significantly associated with the aggressive histopathological characteristics of HCC, such as big tumor size, vascular invasion, and high TNM staging. The prognostic value of thrombin was further confirmed to be independent of the other clinicopathological characteristics of HCC in multivariate analysis. This finding indicates that thrombin may serve as an independent predictor for tumor recurrence and prognosis of HCC patients. More interestingly, the correlations of thrombin to HCC prognosis differ in patients with varying OPN expression levels. Thrombin expression was closely associated with tumor recurrence and survival in HCC patients with higher OPN levels. However, this association was not significant in patients with lower OPN expression. HCC patients with thrombin⁺/OPN⁺ have the poorest prognosis. These findings show that thrombin makes a substantial contribution, together with OPN, to HCC malignancy [20].

Prognostic value of preoperative plasma/serum OPN level for HCC

OPN is an ideal diagnostic marker because it can be found in all bodily fluids and because elevated plasma OPN can be found in patients with malignant tumors [4-6,26,41]. We detected the preoperative plasma/serum OPN expression levels from large cohorts of HCC patients who underwent resection, and found that serum OPN levels of HCC patients were significantly higher than those of patients with chronic liver disease (CLD) and healthy individuals. Patients with early stage HCC (BCLC stage A) also had significantly elevated OPN levels compared with the CLD patients, although advanced HCC patients had even higher OPN levels. Moreover, OPN levels of both AFP^- and AFP⁺ HCC patients are similar and closely associated with tumor size, vascular invasion, liver function, and later disease stages. OPN is one of the leading independent prognostic factors for HCC patients, even in the early stage of HCC. This finding suggests that the preoperative plasma/serum OPN level can be used as a predictive marker for HCC recurrence and may be advantageous in assessing the prognosis of HCC patients after surgery [15,17,18].

This prognostic value is still significant even in the early stage of HCC. A higher plasma OPN level is associated with a larger tumor size and a higher probability of tumor recurrence after HCC resection. OPN is one leading independent prognostic factor for both OS and relapse-free survival in multivariate Cox models [17].

Postoperative serum OPN level is a novel monitor for the treatment response and tumor recurrence after HCC resection

We also investigated the dynamic changes in the serum OPN level after operation and its clinical significance in 110 patients with resectable HCCs. We found that serum OPN levels were transiently increased within the first week after HCC resection, and then dropped to a stable level 5 weeks after HCC resection (Fig. 3). Similar perioperative fluctuation was also observed in patients with non-small-cell lung cancer [42]. However, the possible reason for this phenomenon remains unclear, which is possibly associated with acute inflammatory immune response related to surgical resection because we found that it was closely correlated to serum CRP.

We further evaluated the monitoring values of serum OPN levels in the diagnosis and treatment response of recurrent HCCs. Among the 38 patients with tumor recurrences during the follow-up, we found that 11 patients (including 5 AFP^- ones) who have available serum samples when the tumor recurrences were diagnosed had accompanying increased OPN levels when the tumor relapses occurred. Moreover, second resections for the recurrent HCCs induced similar postoperative changes in serum OPN levels to the first surgical resections for the primary HCCs. By contrast, the serum OPN levels of the patients without tumor recurrence were relatively stable at a lower level during the follow-up period. Moreover, postoperative OPN in AFP^- and AFP⁺ HCC patients exhibited changing patterns. Postoperative serum OPN can serve as a surrogate serologic biomarker for monitoring treatment response and tumor recurrence after HCC resection, including those AFP^- ones (Fig. 4) [18], which deserves further validation in a larger population.

OPN is a prospective target for combating HCC metastasis

The abundance of clinical and experimental evidence regarding the link between OPN and HCC metastasis makes OPN an attractive potential therapeutic target against HCC metastasis. We have shown that neutralizing antibody to OPN can inhibit the in vitro invasion and in vivo lung metastasis of highly metastatic HCC cells [14]. To further analyze the roles of OPN in HCC metastasis, we constructed three lenti-viral vectors (which can induce a prolonged downregulation in gene expression) encoding microRNA against OPN, Lenti.OPNi-1, Lenti.OPNi-2, and Lenti.OPNi-3, which were found to downregulate the OPN level by 62%, 78%, and 95%, respectively, in HCC-LM3 cells with a constitutive OPN overexpression and higher metastatic potential. Both Lenti.OPNi-2 and Lenti.OPNi-3 induced a significant decrease in MMP-2 and urokinase plasminogen activator expression, and consequently led to an obvious inhibition of both in vitro invasion and in vivo lung metastasis of HCC-LM3 cells. This finding provides further evidence to support the fact that OPN has an important role and can be a therapeutic target for HCC metastasis [19]. Moreover, we reported, for the first time, that OPN has an important role in metastasis, as well as HCC tumor growth, in which different minimum threshold levels of OPN are needed. OPN may regulate tumor growth through the activation of the MAPK pathway. Furthermore, inducing the NF-κB translocation and the production/activation of MMP-2 may be an important mechanism by which OPN is involved in HCC metastasis [19]. Similarly, Zhao et al. found that OPN silencing resulted in the suppression of integrin expressions, blockade of NF-κB activation, and induction of a mitochondria-mediated apoptosis, and consequently suppressed the tumor growth and metastasis of HCC. Moreover, RNA interference-mediated depletion of OPN may be a promising strategy for the treatment of HCC by sensitizing the chemotherapeutic drugs [43]. These data suggest that OPN can be a hopeful target for the control of metastasis and HCC tumor growth.

Direct targeting of OPN is difficult because OPN-specific inhibitory compounds are not yet available; thus, we have to explore an alternative strategy that indirectly targets OPN by the signaling cascade components, leading to OPN-dependent HCC metastasis. Previous studies have shown that thrombin-induced modification can lead to changes in OPN activity [34,37,44-47]. Thus, investigating the role of thrombin in the OPN-mediated pathway is helpful in understanding the mechanisms by which OPN regulates the proliferation and metastasis of HCC, and in the development of a potential therapeutic target to block OPN function and control HCC metastasis. We first identified the important role of thrombin in OPN-dependent HCC metastasis. We investigated the correlation of thrombin levels to HCC prognosis in patients with various OPN levels and evaluated the effects of OPN fragments generated by thrombin cleavage on the proliferation and adhesion of HCC cells. We found that thrombin level was strongly associated with the metastatic potential of the HCC cell lines, and that thrombin was remarkably overexpressed in HCC tissue compared with the adjacent non-tumor tissue. In vitro and in vivo assays have demonstrated that thrombin can only promote proliferation and adhesion, as well as pulmonary metastases, of HCC cells with OPN overexpression. These effects were significantly suppressed by treatment with Argatroban, a thrombin inhibitor. These results suggest that OPN is necessary for the effects of thrombin on the proliferation and adhesion of HCC cells. In other words, thrombin affects HCC malignancy by the functional roles of the OPN pathway [20]. Furthermore, we found that thrombin collaborated with OPN to induce the increased integrin-β1 expression and activated the FAK pathway of the OPN⁺ HCC cells. These findings indicate that thrombin has an important role in OPN-mediated aggressive phenotype of HCC through activation of integrin β1-FAK signaling and may be a potential therapeutic target to inhibit HCC metastasis in OPN⁺ patients [20].

Exploration of the genetic regulations of OPN

OPN⁺ HCC cells have much more genomic aberrations

We also found significant intratumor genomic heterogeneity between the OPN⁺ and OPN^- HCC cells. OPN⁺ HCC cells have much more amplifications on 4q13.1-q13.3, 4q21.23-q22.1, and 13q32.1-q32.3 compared with the OPN^- ones. Some candidate tumor-related genes, such as SMR3B, MUC7, EPHA5, OPN, and CLDN10, were detected. OPN⁺ HCC cells have more important role in the development of HCC malignancy than their OPN^- counterparts [21].

Polymorphism at Locus -443 is more important in the regulation of OPN expression and metastasis of HCC

The human OPN gene is localized on chromosome 4ql3 [48]. The OPN promoter contains a number of potential regulatory elements for transcription factors [49]. More than 10 single nucleotide polymorphisms (SNPs) have been identified in the promoter. These polymorphisms may affect the transcriptional activity of OPN [50], and some are thought to be genetic risk factors for disease susceptibility [49,51-57]. These findings suggest that genetic variations in OPN may also induce differences in the incidence risks and outcomes of cancers. However, little is known about the impact of OPN polymorphisms on cancer progression.

To determine the effects of genetic variants of OPN on the progression and prognosis of HCC, we characterized the allelic architecture of the OPN promoter by direct sequencing and identified the SNPs at four loci -1748, -616, -443, and -155, and three most common haplotypes (Ht1, Ht2, and Ht3) of the OPN promoter region in HCCs. Interestingly, only one nucleotide different at locus -443 was found between the two major haplotypes (Ht2 and Ht3) [22]. Furthermore, -443TT and TC genotypes are more frequently found, consistent with the previous reports on Egyptian and Japanese chronic hepatitis C patients [56-58]. The -443TT genotype is a significant variable reflecting hepatitis activity in chronic hepatitis C patients [56,57]. These results suggest that SNP at locus -443 may be more important in the progression of liver diseases including HCC.

Another important finding is that only SNP at locus -443 and their related haplotypes (Ht2, Ht3) are closely associated with HCC prognosis. We evaluated the prognostic values of the selected ones in two large cohorts of 826 HCC patients who underwent radical liver resection. The -443TT/TC genotype and Ht2 significantly increased the probability of HCC recurrence and resulted in a poorer prognosis. By contrast, the patients with -443CC genotype or Ht3 had lower probability of tumor recurrence and longer survival (Fig. 5). Cox regression analyses indicated that the -443 genotypes and related haplotypes were independent prognostic factors. More importantly, this prognostic value of SNP at locus -443 still remains significant in patients with small HCC (≤5 cm). These findings suggest that, in addition to the OPN expression level, OPN promoter polymorphisms, particularly at -443, can also be also a part of the genetic variations that underlie the phenotypic variations seen in individual outcome to cancer and may serve as a powerful predictor for HCC prognosis [22].

To explore how OPN promoter polymorphisms affect HCC prognosis, we further analyzed the functional effects of SNP at locus -443 or the related haplotypes on the promoter activity and OPN expression and found that Ht2 (-443T allele) can increase transcriptional activity and expression level of OPN compared with the protective -443C allele [22]. Given that SNP at locus -443 is located just at the upstream of the cis-acting enhancing element of human OPN [59], such SNPs can affect the OPN expression. The presence of a strong transcription factor site, such as in the -443T/C allele, within the context of OPN promoter can probably alter the balance of the basic transcription complex toward the enhanced transcription and expression of OPN. Some transcription factors, such as MYT1 [50] and c-Myb [60], have been reported to bind OPN promoter at position -443. We also found that Ht2 (-443T allele) can lead to significant increases both in in vitro invasion and in vivo tumor growth and lung metastasis of HCC cells. Hence, we propose that the Ht2 of the OPN promoter in primary tumors may promote the progression and metastasis of HCC by regulating OPN expression. These findings provide an interesting possibility for developing a potential therapeutic strategy to block the OPN function and control HCC metastasis [22].

In summary, these findings indicate that the genetic variation at locus -443 of OPN promoter has important roles in the regulation of OPN expression and cancer progression of HCCs, which is a novel determinant and target for HCC metastasis and prognosis. These phenomena provide further evidence for the important role of OPN in HCC progression and new insight into the regulatory mechanism governing OPN expression, which may be a novel therapeutic target for HCC [22].

Conclusions

Based on these findings from our series of studies, we provide strong evidence that OPN overexpression is correlated with the metastatic potential of primary HCC. OPN can serve as a prognostic marker for HCC patients, even those with early stage HCC, and as a monitor for the treatment response and tumor recurrence after HCC resection. However, the diagnostic and prognostic roles in HCC need to be further validated before becoming a clinical routine because OPN was recently inferred to be a novel diagnostic marker for HCC [61]. Moreover, we propose that OPN is an attractive potential therapeutic target against HCC metastasis because OPN is an extracellular cytokine ligand, and this kind of receptor-ligand interaction is more readily reached and intervened by pharmaceuticals than intracellular drug targets. Thrombin may be an alternative target to inhibit HCC metastasis in OPN⁺ patients.

Compliance with ethics guidelines

Lun-Xiu Qin declares that he has no conflict of interest. This manuscript is a review article and does not involve a research protocol requiring approval by the relevant institutional review board or ethics committee.

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