Osteopontin Expression Correlates With Prognostic Variables and Survival in Clear Cell Renal Cell Carcinoma

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The role of OPN in tumor progression was first analyzed in experimental animal models, where it was identified as phosphoprotein secreted by transformed cells, and associated with increased metastatic potential in rodents [5,7]. Following this observation, OPN mRNA and protein were also analyzed in several types of human cancer. Firstly, OPN RNA was found to be produced primarily by tumor-associated macrophages rather than tumor cells themselves, while both tumor cells and macrophages immunohistochemically stained for OPN protein [10]. Authors suggested that OPN secreted by macrophages might bind to tumor cells, possibly through the RGD-binding domain in OPN. Later on, tumor cells were also identified as a source of OPN [12,13,21]. Until now, OPN RNA and protein have been found to be overexpressed in a number of human tumor types, relative to normal tissue, and the results of this investigations support the hypothesis that OPN detected within tumor cells has a potential utility as a prognostic marker [9,13,22-25]. However, there are limited data regarding the OPN expression in human renal cell carcinoma [10,14]. In order to better define the role of OPN in the progression of CRCC, we performed an immunohistochemical staining for OPN protein in large cohort of CRCC specimens, and assessed its potential prognostic significance.

We have found that 39% of CRCC stained positively by the goat mAb to human OPN. This percentage is largely in agreement with the overall staining levels of OPN reported previously [10]. The staining was also observed in some reactive stromal cells, mainly macrophages and plasma cells, and was particularly pronounced near areas of tumor necrosis, as described by Brown et al. [10]. In tubule-forming areas, the OPN staining was present on the apical surface in malignant glands, similar to the pattern of staining seen in the normal distal tubules.

Brown et al. analyzed distribution of OPN mRNA and protein in 14 renal cell carcinomas, and found strong expression of OPN mRNA in 13 cases, and strong and diffuse cytoplasmic staining for OPN protein in 7 cases [10]. In their study, all tumors were moderately differentiated clear cell renal cell carcinoma, except for one well-differentiated papillary carcinoma, which was also positive. In our study, the level of OPN expression strongly correlated with tumor variables reported previously to be associated with patient outcome: histological grade, pathological stage, tumor size, and Ki-67 proliferation index. While all of grade 1 tumors were negative for OPN protein, the positivity increased with transformation to higher nuclear grade. In the recent work of Coppola et al., osteopontin was also found to be significantly associated with tumor stage in 36 RCC, including tumors of bladder, colon, kidney, larynx, mouth, and salivary gland [14]. They used tissue arrays to assess OPN protein levels in 350 tumors from 23 body sites, compared with 113 normal tissues. In that study, OPN was found to be elevated in tumors, relative to normal tissues, and correlated significantly overall with tumor stage, when considering all tumor sites, as well as with tumor stage for several sites individually. This association between OPN and tumor stage most likely reflects the effect of OPN on cell migration. OPN contains an Arg-Gly-Asp (RGD) sequence that binds to avb1, a3, and a5 integrins, and is capable of promoting cell attachment, chemotaxis, and signal transduction in several different cell types [26]. Due to the presence of this sequence, it is probable that high expression of OPN by tumor cells may play a role in tumor cell invasion and metastasis, a process in which adhesive interactions between tumor cells and extracellular matrix are critical.

In our study, the level of OPN expression was higher in large tumors, as well as in tumors with high growth fraction, expressed as Ki-67 proliferation index. Interestingly, in this regard, recent experiments suggest that OPN acts in concert with several growth factors, including hepatocyte growth factor [27], and epidermal growth factor (EGF) [28], to induce malignant properties. Moch et al. described the relationship between EGF-receptor (EGFR) expression and Ki-67 index in RCC, and their association with poor prognosis [29]. In the light of these findings, it would be interesting to analyze the relationship between Ki-67 index, EGFR, and OPN protein in RCC. Also, several studies have demonstrated that OPN delivers a prosurvival, antiapoptotic signal to the cell [30,31]. Since tumor size is largely defined by the number of cells in proliferation and cells that undergo apoptosis, the mechanisms described previously could explain the association of OPN and tumor size observed in our study.

We have also found significant association of OPN expression and poor survival of CRCC patients. So far, there were no reports on the impact of the OPN expression to the clinical outcome in RCC. However, OPN overexpression has been significantly associated with patient survival in lung carcinoma [15,16,22], and breast carcinoma [9,13].

The mechanisms by which osteopontin could promote tumor progression are still unknown. Through its adhesive properties OPN can induce changes in tumor cell gene expression, including induction of proteolytic enzymes, particularly urokinase plasminogen activator (uPA) [32], and activation of growth factor kinases, which in turn may lead to increased cell motility and invasion. Also, the role of OPN protein in angiogenesis could be the one of proposed mechanisms through which OPN can mediate tumor progression and metastasis. OPN augments endothelial cell migration induced by vascular endothelial growth factor (VEGF) in an avb3 integrin-dependent manner [33], and enhances survival of endothelial cells [34]. In this regard, Shijuba et al. have shown the role of VEGF and OPN coexpression in clinical outcome of patients with stage I lung carcinoma [16].

In conclusion, in this study we have shown for the first time the upregulation of OPN protein in a large group of CRCC, and its association with the parameters of poor prognosis and with shorter survival. Significance of increased OPN expression in predicting the biological behavior of CRCC is unknown at present, and has to be more evaluated in future.