The role of CXC-chemokine receptor CXCR2 and suppressor of cytokine signaling-3 (SOCS-3) in renal cell carcinoma

Abstract

Background: Chemokine receptor signaling pathways are implicated in the pathobiology of renal cell carcinoma (RCC). However, the clinical relevance of CXCR2 receptor, mediating the effects of all angiogenic chemokines, remains unclear. SOCS (suppressor of cytokine signaling)-3 is a negative regulator of cytokine-driven responses, contributing to interferon-α resistance commonly used to treat advanced RCC with limited information regarding its expression in RCC.

Methods: In this study, CXCR2 and SOCS-3 were immunohistochemically investigated in 118 RCC cases in relation to interleukin (IL)-6 and (IL)-8, their downstream transducer phosphorylated (p-)STAT-3, and VEGF expression, being further correlated with microvascular characteristics, clinicopathological features and survival. In 30 cases relationships with hypoxia-inducible factors, i.e. HIF-1a, p53 and NF-κΒ (p65/RelA) were also examined. Validation of immunohistochemistry and further investigation of downstream transducers, p-JAK2 and p-c-Jun were evaluated by Western immunoblotting in 5 cases.

Results: Both CXCR2 and IL-8 were expressed by the neoplastic cells their levels being interrelated. CXCR2 strongly correlated with the levels of HIF-1a, p53 and p65/RelA in the neoplastic cells. Although SOCS-3 was simultaneously expressed with p-STAT-3, its levels tended to show an inverse relationship with p-JAK-2 and p-c-Jun in Western blots and were positively correlated with HIF-1a, p53 and p65/p65/RelA expression. Neither CXCR2 nor SOCS-3 correlated with the extent of microvascular network. IL-8 and CXCR2 expression was associated with high grade, advanced stage and the presence/number of metastases but only CXCR2 adversely affected survival in univariate analysis. Elevated SOCS-3 expression was associated with progression, the presence/number of metastasis and shortened survival in both univariate and multivariate analysis.

Conclusions: Our findings implicate SOCS-3 overexpression in RCC metastasis and biologic aggressiveness advocating its therapeutic targeting. IL-8/CXCR2 signaling also contributes to the metastatic phenotype of RCC cells but appears of lesser prognostic utility. Both CXCR2 and SOCS-3 appear to be related to transcription factors induced under hypoxia.

Figure 1

IL-6, IL-8, CXCR2, SOCS-3 and p-STAT-3 protein levels in fresh-frozen tissue specimens by Western blot and immunohistochemical expression of CXCR2, SOCS-3 and IL-8 in formalin-fixed tissue from 2 representative cases (#1, 4: Clear cell RCCs, #2: Papillary RCC, #3, 5: Chromophobe RCCs). Detection of IL-6 (A), IL-8 (B) CXCR2 (C) SOCS-3 (D) and p-STAT-3 (E) in the 5 cases. Western immunoblotting validated the results of immunohistochemistry.

Figure 2

Box plots illustrating the correlations between IL-8 (A), CXCR-2 (B), p-STAT-3 (C) H-score and minor axis length (D) with histological grade.

Figure 3

Box plots illustrating the correlations between IL-8 (A) and CXCR-2 (B) H-score with stage.

Figure 4

Box plots illustrating the correlations between IL-8 (A), CXCR-2 (B), SOCS-3 (C), p-STAT-3 (D) and VEGF (E) H-score with the presence of metastases.

Figure 5

Box plots illustrating the correlations between MVD (A), TVA (B), shape factor (C) and compactness (D) with histological type.

Figure 6

Immunohistochemical staining of a clear cell (A) and a chromophobe (B) RCC.(C, D) Same fields as in (A, B). The outline of each vessel is traced; the red layer represents the section area of each vessel. Clear cell RCC (A, C) displays clearly higher MVD and TVA when compared to chromophobe RCC (B, D).

Figure 7

Plots illustrating the correlations of SOCS-3 and CXCR2 with p65/RelA, HIF-1a and p53 in the 30 cases analyzed. (A, B, C) SOCS-3 with p65/RelA, HIF-1a and p53. (D, E, F) CXCR2 with p65/RelA, HIF-1a and p53.

Figure 8

Expression of CXCR2, SOCS-3, p-JAK-2 and p-c-Jun in 5 cases. Western immunoblotting of CXCR2, SOCS-3, p-JAK2 and p-c-Jun expression in 5 tumor cases (A). Densitometric analysis of relative protein amounts normalized to the corresponding Actin levels was performed using Image J software (B).

Figure 9

Kaplan-Meier curves for cancer specific survival according to CXCR-2 (A), SOCS-3 (B) and p-STAT-3 (C) immunoexpression.