The immunoreactivity of GLI1 and VEGFA is a potential prognostic factor in kidney renal clear cell carcinoma

Abstract

Kidney renal clear cell carcinoma (KIRC) is the most common type of kidney cancer and its pathogenesis is strongly associated with VHL-HIF-VEGF signaling. SHH ligand is the upstream SHH pathway regulator, while GLI1 is its major effector that stimulates as a transcription factor, i.a. expression of VEGFA gene. The aim of present study was to assess the prognostic significance of SHH, GLI1 and VEGFA immunoreactivity in KIRC tissues. The analysis included paired tumor and normal samples from 34 patients with KIRC. The immunoreactivity of SHH, GLI1 and VEGFA proteins was determined by immunohistochemical (IHC) renal tissues staining. The IHC staining results were assessed using the immunoreactive score (IRS) method which takes into account the number of cells showing a positive reaction and the intensity of the reaction. Increased GLI1 protein immunoreactivity was observed in KIRC tissues, especially in early-stage tumors, according to the TNM classification. Elevated expression of the VEGFA protein was noted primarily in high-grade KIRC samples according to the Fuhrman/WHO/ISUP scale. Moreover, a directly proportional correlation was observed between SHH and VEGFA immunoreactivity in TNM 3 + 4 and Fuhrman/ISUP/WHO 3 + 4 tumor tissues as well as in samples of patients with shorter survival. We also observed an association between shorter patient survival as well as increased and decreased immunoreactivity, of the VEGFA and GLI1, respectively. The aforementioned findings suggest that the expression pattern of SHH, GLI1 and VEGFA demonstrates prognostic potential in KIRC.

Keywords: GLI1; IHC; KIRC; Prognostic factors; SHH; Sonic hedgehog pathway; VEGFA.

Fig. 1

Representative microphotographs of GLI1 immunohistochemical reactions in KIRC samples (A, C) and corresponding morphologically unchanged kidney tissues (B, D). A – KIRC stage 1 (based on TNM classification), Fuhrman/WHO grade 3, follow-up period: 36 months; C - KIRC stage 3 (based on TNM classification), Fuhrman/WHO grade 3, death at 30 months of follow-up. Immunohistochemistry was carried out as described in Methods, sections were counterstained by hematoxylin. Scale bars, 50 μm. GLI1 - glioma-associated oncogene family zinc finger 1, KIRC - kidney renal clear cell carcinoma

Fig. 2

Representative microphotographs of SHH (A, B, E, F) and VEGFA (C, D, G, H) immureactivity in KIRC samples (A, C, E, G) and morphologically unchanged kidney (B, D, F, H). Slides A, B, C, D derived from the patient diagnosed with KIRC stage 1 (based on TNM classification), Fuhrman/WHO grade 3, follow-up period: 30 months; slides E, F, G, H derived from the patient diagnosed with KIRC stage 3 (based on TNM classification), Fuhrman/WHO grade 4, death at 6 months of follow-up. Immunohistochemistry was carried out as described in Methods, sections were counterstained by hematoxylin. Scale bars, 50 μm. SHH - sonic hedgehog, VEGFA - vascular endothelial growth factor A, KIRC - kidney renal clear cell carcinoma

Fig. 3

Analysis of the SHH, GLI1 and VEGFA immunoreactivity in KIRC. IRS was assessed as described in the Methods. Comparison between tumor and normal kidney samples. (A) – SHH protein, (B) – GLI1 protein, (C) – VEGFA protein. Bars and whiskers represent mean ± standard deviation normalized to control kidney samples. ^*P < 0.05, **P < 0.01, Wilcoxon signed‑rank test. IRS – immunoreactivity score, SHH - sonic hedgehog, GLI1 - glioma-associated oncogene family zinc finger 1, VEGFA - vascular endothelial growth factor A, KIRC - kidney renal clear cell carcinoma

Fig. 4

Immunoexpression analysis of the SHH, GLI1 and VEGFA proteins in KIRC classified by clinical TNM staging. IRS scoring was assessed as described in the Methods. Comparison between tumor and normal kidney samples; (A) – SHH protein, (B) – GLI1 protein, (C) – VEGFA protein. Bars and whiskers represent mean ± standard deviation normalized to control kidney samples. *P < 0.05, ### P < 0.001 between KIRC subgroup and control samples (Kruskal Wallis test). SHH - sonic hedgehog, GLI1 - glioma-associated oncogene family zinc finger 1, VEGFA - vascular endothelial growth factor A, KIRC - kidney renal clear cell carcinoma

Fig. 5

Analysis of the SHH, GLI1 and VEGFA immunoreactivity in KIRC samples classified by histological ISUP grading. IRS was assessed as described in the Methods. Comparison between tumor and normal kidney samples. (A) – SHH protein, (B) – GLI1 protein, (C) – VEGFA protein. Bars and whiskers represent mean ± standard deviation normalized to control kidney samples. # P < 0.05 between KIRC subgroup and control samples (Kruskal Wallis test). SHH - sonic hedgehog, GLI1 - glioma-associated oncogene family zinc finger 1, VEGFA - vascular endothelial growth factor A, KIRC - kidney renal clear cell carcinoma

Fig. 6

Kaplan-Meier’s overall survival analysis for KIRC patients in relations to clinical data and immunoexpression of SHH, GLII and VEGFA proteins. (A) TNM classification. (B) ISUP grade. (C) SHH, (D) GLI1, (E) VEGFA protein immunoexpression, respectively. Cut-off values between increased and decreased immunoreactivity scores were arbitrarily classified using median expression values of each protein in control samples. Log-rank (Mantel-Cox) test was applied