Bioinformatics approach
Significant differential expression of proteins from tumor tissues in comparison to adjacent normal tissues was obtained of selected previous studies that include 1055, 589, and 983 proteins for ccRCC, pRCC, and chRCC, respectively (Supplementary Table 1). The results of the Venn diagram analysis illustrated 110 common differential expression proteins between subtypes of RCC (Fig. 1). PPI network analysis result explored 52 hub genes with the highest confidence > 0.9 (Fig. 2) that 24 key genes have reported for cancer on both Jensen and DisGeNET databases (Supplementary Table 1). Pathway analysis displays that most key genes contribute to metabolism pathways and several genes involved in FAK and degradation of extracellular matrix (ECM) pathways (Fig. 3). The major part of GO analysis results for 24 key genes indicates these genes are present in the microbody and play role in fatty acid catabolic process. Also, the basement membrane organization is a remarkable part of gene annotation results (Supplementary Fig. 1). According to these results, key genes involved in FAK and pathways related to ECM, as key drivers for both development and cancer progression (41), were highlighted for selection marker. Our key genes list, including COL4A1, LAMB2, NID1, and TLN1 (Talin-1) were evaluated among literature and led to the selection of Talin-1 as our final biomarker for investigation in RCC. Talin-1 as a master integrin-associated protein is a focal adhesion (FK) complex that mediates ECM proteolysis through different matrix metallopeptidases (MMPs) expression (42). FAK activation triggers the following processes, including survival, angiogenesis, cell growth, migration, and invasion. Since FAK functions potentially regulate gene expression to affect cancer progression (43), it seems that its signaling to be vital in the neoplastic cells invasion, and its activity inhibition decrease RCC metastasis both in vitro and in vivo (44–46). Although the FAK signaling pathway is deregulated in different cancer types and its importance approved in the regulation of cell adhesion and motility due to the tumor invasive behavior (47–49), its evidence in RCC is few and it is necessary to explore which is crucial (42). Further, UALCAN database revealed a higher expression of Talin-1 protein in ccRCC tissue samples compared to normal samples (Fig. 4).
Characteristics of RCC patients
Two hundred and ninety-five RCC patients were included in this study, while technical problems led to a loss of some cases, and finally, 269 cases remained for evaluating. These samples include 195 (73.58%) ccRCC, 20 (7.43%) pRCC type I, 20 (7.43%) pRCC type II, and 34 (12.63%) chRCCs. The patients’ clinicopathological characteristics were summarized in Table 1 based on whole RCC samples and histological subtypes of RCC.
Expression of Talin-1 protein in the subtypes of RCC and adjacent normal tissue samples
The expression level of Talin-1 protein was evaluated using the IHC method on TMA sections by three scoring methods, including the intensity of staining, percentage of positive tumor cells, and H-score in histological subtypes of RCC and adjacent normal tissue samples. Talin-1 protein was expressed at different intensities in the cell membrane, cytoplasm, and nucleus in the samples (Table 2) (Fig. 5), however, the expression of Talin-1 protein in adjacent normal tissue samples was less compared to tumor samples.
Comparison of Talin-1 protein expression based on histological subtypes of RCC
In this part of the study, to compare differences between the median expression levels of Talin-1 protein in the membrane, cytoplasm, and nucleus among the histological subtypes of RCC, the non-parametric Kruskal–Wallis and Mann–Whitney U tests were applied. The results of the Kruskal–Wallis test revealed a statistically significant difference between the membranous and cytoplasmic Talin-1 protein expression levels in different RCC subtypes (P < 0.001, P = 0.003, respectively). Also, Mann–Whitney U test showed a statistically significant difference in the median levels of membranous and cytoplasmic Talin-1 protein expression between ccRCC and pRCC (type I & II) (P < 0.001, P = 0.001, respectively) and also pRCC (type I & II) and chRCC (P = 0.006, P = 0.029, respectively) (Fig. 6A, B). In contrast, the Kruskal-Wallis test showed that there is no statistically significant difference between the median expression level of nuclear Talin-1 protein in different subtypes of RCC (P = 0.904) (Table 3). Moreover, we did not find a statistically significant difference in the median expression level of nuclear Talin-1 protein between the RCC subtypes (Fig. 6C).
Associations between membranous, cytoplasmic, and nuclear Talin-1 protein expression and clinicopathological parameters in histological subtypes of RCC
ccRCC:
In ccRCC, membranous and cytoplasmic expression of Talin-1 protein were observed in 195 (100.0%) patients and nuclear Talin-1 expression in 180 (92.3%) cases.
Pearson’s chi-square test was used to examine the association between Talin-1 protein expression and clinicopathological parameters in histological RCC subtypes. The results of Pearson's chi-square test showed a highly significant association between expression of membranous and cytoplasmic Talin-1 protein and increased nucleolar grades (I + II versus III + IV) (Pearson's chi-square, P < 0.001, all) (Table 4). Kruskal–Wallis and Mann–Whitney U tests also showed that statistically significant differences in the median levels of membranous and cytoplasmic Talin-1 protein expression in various nucleolar grades (P = 0.001, P < 0.001, respectively) (Fig. 7A, B). Moreover, Pearson's chi-square test revealed that significant differences between higher levels of expression of membranous and cytoplasmic Talin-1 protein and MVI (P = 0.007, P = 0.004, respectively), histological TN (P = 0.020, P = 0.018, respectively) as well as Gerota’s fascia invasion (P = 0.025, P = 0.050, respectively). Furthermore, there was a statistically significant difference between higher level of membranous Talin-1 protein expression and invasion to renal pelvis (P = 0.038). Kruskal–Wallis and Mann–Whitney U tests also indicated a statistically significant difference between the median expression levels of membranous and cytoplasmic Talin-1 protein and MVI (P = 0.022, P = 0.007, respectively) (Fig. 7C, D). Bivariate analysis (Spearman’s correlation) showed a significant direct correlation between membranous and cytoplasmic expression and advanced in nucleolar grades (Spearman’s rho, P < 0.001, P = 0.001, respectively), MVI (P = 0.007, P = 0.004, respectively), histological TN (P = 0.015, P = 0.014, respectively), and invasion to Gerota’s fascia (P = 0.025, P = 0.050, respectively). The results of Spearman’s correlation analysis also revealed that membranous Talin-1 protein expression and renal pelvis (P = 0.038) were directly correlated. In this study, we did not find any significant association and correlation between nuclear Talin-1 protein expression and clinicopathological features in ccRCC cases (Table 4).
pRCC (type I & II) and chRCC:
Membranous and cytoplasmic expression of Talin-1 protein was observed in all cases 20 (100.0%), 20 (100.0%), and nuclear Talin-1 expression in 11 (55.0%), 12 (60.0%) patients of pRCC type I and pRCC type II, respectively. In the cases of chRCC, membranous and cytoplasmic Talin-1 protein expression were found in 34 (100.0%) patients and nuclear expression in 25 (73.5%) cases. The results of Pearson's chi-square and Spearman’s correlation tests revealed that there are no significant association and correlation between membranous, cytoplasmic, and nuclear Talin-1 protein expression and clinicopathological parameters in pRCC (type I & II) (Tables 5, 6). In chRCC, we observed a statistically significant association between membranous expression of Talin-1 and tumor stage (P = 0.050) (Table 7). Moreover, Spearman’s correlation analysis showed a statistically significant positive correlation between increased Talin-1 protein expression and higher tumor stage (Spearman’s rho, P = 0.023).
Prognostic value of expression of Talin-1 protein for clinical outcomes in histological subtypes of RCC
In the current study, the mean and median duration of the follow-up time for DSS were 52 (SD = 27.5) months and 49 (Q1, Q3 = 34, 71) and for PFS were 49 (SD = 29.6) and 47 (Q1, Q3 = 31, 71) months, respectively. Also, the minimum and maximum of follow-up time were 1 and 117 months and the range was 116 months. The results of the analysis showed that distant metastasis and tumor recurrence occurred in 55 (20.4%) and 41 (15.2%) patients, respectively whereas, 214 (79.6%) and 228 (84.8%) were not positive for mentioned parameters. Also, 63 (23.4%) patients were positive for distant metastasis or tumor recurrence and 206 (76.6%) were not positive for these features. During the follow-up, cancer-related death occurred in 40 patients (14.9%) and disease-related death in 5 (1.9%). The main characteristics of patients enrolled for survival analysis according to the histological subtypes of RCC were summarized in (Table 8).
Survival outcomes based on the expression of Talin-1 protein in the histological subtypes of RCC
The association between expression of membranous, cytoplasmic, and nuclear Talin-1 protein and DSS or PFS were evaluated using Kaplan–Meier analysis curves and the log-rank test.
ccRCC:
Survival outcomes based on membranous Talin-1 protein expression
The results of the Kaplan–Meier curve demonstrated that significant differences between DSS and the patients with high and low membranous expression of Talin-1 protein (Log-rank test: P = 0.043) (Fig. 8A). The mean DSS time for patients with high and low membranous expression of Talin-1 protein was 91 (SD = 4.6) and 103 (SD = 3.5) months, respectively. Additionally, the 5-year survival rates for DSS in patients whose specimens expressed high and low membranous expression of Talin-1 protein were 78 and 88%, respectively (Log-rank test: P = 0.050). Moreover, the results of the Kaplan-Meier analysis for PFS showed that there are no significant differences between PFS and the patients with high and low expression of membranous Talin-1 protein (Log-rank test: P = 0.081) (Fig. 8B).
Survival outcomes based on cytoplasmic Talin-1 protein expression
Our finding of Kaplan–Meier curves analysis in the cytoplasmic expression of Talin-1 revealed that significant differences between DSS (Log-rank test: P = 0.024) or PFS (Log-rank test: P = 0.046) and the patients with high and low cytoplasmic Talin-1 protein expression, respectively (Fig. 8C, D). Further, the mean DSS or PFS time for patients with high and low cytoplasmic expression of Talin-1 protein were 90 (SD = 4.7), 87 (SD = 5.0) and 103 (SD = 3.4), 99 (SD = 3.9) months, respectively. The 5-year survival rates for DSS or PFS of patients with high cytoplasmic Talin-1 protein expression were 77 and 72% and with low expression were 89 and 85% (Log-rank test: P = 0.044, P = 0.050, respectively).
Survival outcomes based on nuclear Talin-1 protein expression
Kaplan Meier survival curves showed that there are no significant differences between DSS or PFS and the patients with high and low nuclear expression of Talin-1 protein in ccRCC cases (Log-rank test: P = 0.160, P = 0.219, respectively) (Fig. 9A, B).
Our finding from univariate analysis showed that the membranous and cytoplasmic expression of Talin-1 protein (P = 0.048, P = 0.028, respectively), tumor size (P = 0.009), nucleolar grade (P < 0.001), and tumor stage (P = 0.029) were significant risk factors affecting the DSS with hazard ratio (HR) more than 1 and cytoplasmic expression of Talin-1 protein (P = 0.050), tumor size (P = 0.004), nucleolar grade (P < 0.001), and tumor stage (P = 0.047) were significant risk factors affecting the PFS of patients with ccRCC (Tables 9, 10). As demonstrated in Tables 9 and 10, nucleolar grade was the only independent prognostic factor for DSS (membranous expression; HR: 2.563, 95% CI: 1.119–5.869; P = 0.026, cytoplasmic expression; HR: 2.483, 95% CI: 1.095–5.633; P = 0.029) or PFS (cytoplasmic expression; HR: 2.243, 95% CI: 1.061–4.739; P = 0.034) in the multivariate analysis. Expression of membranous and cytoplasmic Talin-1 protein for DSS (P = 0.356, P = 0.219, respectively) and cytoplasmic expression of Talin-1 for PFS (P = 0.312) were not significant risk factors for prognosis in the multivariate analysis (Tables 9, 10). Other clinicopathologic variables were not significant factors affecting the DSS or PFS in univariate and multivariate analyses of ccRCC patients.
pRCC (type I & II) and chRCC:
Kaplan–Meier survival analysis showed that there is no statistically significant association between membranous, cytoplasmic, and nuclear Talin-1 protein expression and patients’ survival outcomes in pRCC (type I & II) and chRCC patients. Additionally, univariate and multivariate analyses demonstrated that the listed clinicopathologic variables are not significant factors affecting the DSS or PFS of patients with papillary and chRCC.
Combined analysis of Talin-1/B7-H3 populations
In this part of the study, we first chose the samples which have stained in two markers, thus the number of patients was 138. The results showed that a statistically significant correlation between expression of cytoplasmic Talin-1 and cytoplasmic B7-H3 proteins (Spearman’s rho, 0.318; P < 0.001), however, in membranous expression, we did not find any significant correlation between two markers (Spearman’s rho, 0.026; P = 0.756). Therefore, we examined the association between co-expression of cytoplasmic Talin-1 and cytoplasmic B7-H3 proteins with clinicopathologic parameters. The expression levels of Talin-1 and B7-H3 were divided into two categories based on median expression and four phenotypes including Talin-1 High/B7-H3 High, Talin-1 High/B7-H3 Low, Talin-1 Low/B7-H3 High, and Talin-1 Low/B7-H3 Low (Table 11). The analysis showed that a highly significant association between cytoplasmic Talin-1 High/B7-H3 High phenotype and nucleolar grade (P = 0.002). The cytoplasmic Talin-1 High/B7-H3 High phenotype was more often found in patients with MVI present (P = 0.002), histological TN present (P = 0.026), Gerota’s fascia invasion (P = 0.043), and distant metastasis (P = 0.022) compared to patients without MVI and histological TN, as well as no invasion to Gerota’s fascia and no distant metastasis (Table 11).
Survival outcomes of Talin-1/B7-H3 expressions
The survival rate of patients with co-expression of cytoplasmic Talin-1 High/B7-H3 High phenotype was compared with other phenotypes. Higher cytoplasmic expression of Talin-1 High/B7-H3 High phenotype was associated with worse DSS and PFS than another phenotype (Log-rank test; P = 0.007, P = 0.021, respectively) (Fig. 10A, B). Further, the 5-year survival rates for DSS and PFS were 87.0% and 83.0% in cytoplasmic Talin-1 High/B7-H3 High phenotype expression, and 67.0% and 65.0% in another phenotype of cytoplasmic Talin-1/B7-H3 expression (Log-rank test; P = 0.010, P = 0.029, respectively). Moreover, the stratified analysis indicated that in the patients with distant metastasis, the differences in DSS between patients with cytoplasmic Talin-1 High/B7-H3 High phenotype and other phenotypes of Talin-1/B7-H3 expression is significant (P = 0.050) (Fig. 10C, D).
Tumor size (10.1 cm versus < 4 cm) and nucleolar grade were the significant risk factors affecting the DSS or PFS of patients with ccRCC in univariate analysis, with hazard ratios of 6.476, 4.338 and 6.737, 3.418 and P values of 0.016, 0.001, and 0.013, 0.001, respectively.