Although much progress has been made big changes in the treatment of RCC, drug resistance to targeted therapies brings more failures in advanced RCC patients' treatment 7. Thus, it is urgently needed to explore novel and practical clinical prognostic markers of RCC in the patient's early diagnosis in future renal cancer therapy.
The bioinformatics analysis would be conducive to molecular markers research in clinical RCC. This study set out with the aim to identify the novel biomarker that was potentially involved in the various RCC subtypes. In this regard, we focused on PPI network analysis for proteomics results of other studies related to RCC subtypes and used bioinformatics software investigations to identify hub genes. The hub genes were generated based on the highest degree of connectivity and were screened on Enricher for cancer disease. Besides, enrichment analysis determined the involved pathways and molecular function of hub genes. These results suggested several proteins as hub genes related to cancer, which among them we selected Talin-1 and warranted further investigation. Therefore, to validate the Talin-1 protein as a prognostic marker for RCC, for the first time, expression levels of Talin-1 protein was investigated in a well-characterized series of 269 tissues specimen from patients treated with radical nephrectomy in three main subtypes of RCC including 195 (73.58%) ccRCC, 20 (7.43%) pRCC type I, 20 (7.43%) pRCC type II, and 34 (12.63%) chRCC tissue samples through IHC on TMA slides and evaluation of the association between its expression and clinicopathological characteristics as well as patient survival outcomes. The pattern of Talin-1 protein expression in RCCs also was classified into membranous, cytoplasmic, and nuclear expression.
It is important to investigate histological subtypes of specific cancer, giving consideration to various subtypes which may be associated with different biologic behavior, pattern, and prognosis, hence each of them needs different treatment. The evaluation of the staining pattern in each subtype of RCC displayed differential expression of Talin-1 protein in membrane, cytoplasm, and nucleus with a range of intensities from weak to strong as well as the percentage of staining area. Further, there was a statistically significant association between the membranous and cytoplasmic expression of Talin-1 protein but not in nuclear expression and the RCC subtypes. Subsequently, a statistically significant difference was observed in the median levels of membranous and cytoplasmic Talin-1 protein expression between ccRCC and pRCC (type I & II) as well as pRCC and chRCC subtype, indicating that expression of Talin-1 protein in membrane and cytoplasm versus nucleus are more important in subtypes of RCC and also these expression patterns vary among the histological RCC subtypes, thus this may affect on the prognostic value of them and treatment options.
IHC analysis of human RCCs compared to adjacent normal tissue samples demonstrated that Talin-1 protein expression is upregulated in RCCs. The present study also considered the expression of Talin-1 on UALCAN database, containing a large amount of proteomics data which showed that increased expression of Talin-1 in ccRCCs rather than normal samples. Thus, these results are in line with our findings of Talin-1 protein expression using IHC on ccRCCs.
The advantage of analyzing the expression and subcellular distribution of Talin-1 in RCCs is, the protein in different subcellular localization may have a specific function. Our finding showed that membranous and cytoplasmic expression of Talin-1 protein is positively associated with the important clinicopathological parameters, including advanced nucleolar grade, MVI, histological TN, invasion to Gerota’s fascia, and renal pelvis, while in nuclear expression, there was no any association between expression of Talin-1 protein and clinicopathological features in ccRCC cases. Importantly, our results exhibited the increased expression of the median membranous and cytoplasmic of Talin-1 protein in the high grade of RCC compared with the low grade of RCC and also increased expression in patients with MVI present rather than MVI absent which exhibited the association of membranous and cytoplasmic Talin-1 protein expression with the aggressiveness of ccRCC. In addition, in this study nucleolar grade was found as an independent prognostic factor for DSS and PFS in membranous and cytoplasmic Talin-1 protein expression in multivariate analysis. After the tumor stage, one the strongest prognosticators of survival in patients with RCC is the nucleolar grade, and tumors with a high nucleolar grade display a more aggressive phenotype; thus they are associated with local invasion and distant metastasis 30. Previous studies demonstrated that MVI is related to cancer progression and survival in RCC and is the most significant prognostic variable for prognostication of metastatic spread and survival outcome independent from macrovascular invasion 31,32. Histological TN also has proposed to be a sign of tumor aggressiveness that generally leads to poor survival outcomes 33. A recently systematic review and meta-analysis study suggested that histological TN is associated with DSS, OS, RFS (recurrence-free survival), and PFS of RCC patients and may serve as a predictor of poor prognosis in RCC patients 34. Moreover, in this study tumor size and tumor stage were found as prognostic variables in univariate analysis that depicted the associations between these parameters and more aggressive tumor behaviors. The tumor stage is one of the most factors for predicting tumor progression and recurrence in RCC 35 and tumor size has shown significantly associated with the risk of metastasis 36. Therefore, these results indicated that membranous and cytoplasmic Talin-1 protein expression compared with nuclear expression are related to the degree of malignancy and progression of disease in ccRCC cases.
Our results from Kaplan-Meier survival curves in ccRCC cases showed that higher membranous and cytoplasmic Talin-1 protein expression is associated with significantly worsened DSS as well as worsened DSS or PFS compared to low Talin-1 protein expression, respectively. In addition, ccRCC patients, who expressed a high level of membranous and cytoplasmic Talin-1, had shorter 5-year DSS or PFS compared with those with low expression. However, the pattern of Talin-1 protein expression was not a predictor of survival in multivariate analysis, indicating the long term follow-up is needed to increase the events and prognosis.
Investigations have shown that increased expression of Talin-1 could lead to the progression of tumor cell adhesion and migration because of Talin-1 as a FA protein is able to mediate integrin interaction with ECM and can bind to a variety of adhesion molecules and induce cell cytoskeleton remodeling 37. B7-H3, also a member of the B7 family of immunoregulatory proteins which is overexpressed in many types of malignancies and is linked to poor prognosis, increased tumor grade, and metastasis 38. Previous studies have been reported a correlation between expression levels of B7-H3 and progression of disease in ccRCC 20,39. To confirm that whether Talin-1 is related to metastasis, we used the combined analysis of both Talin-1 protein and our previous data of B7-H3 protein in ccRCC. The co-expression of cytoplasmic Talin-1 High/B7-H3 High was observed to be associated with more aggressive tumor behavior and metastasis. Further, Tumor size and nucleolar grade were the significant risk factors affecting the DSS or PFS which depict tumor aggressiveness. Moreover, increased cytoplasmic expression of Talin-1 High/B7-H3 High compared to the other phenotypes was associated with poor prognosis and progression of the disease, especially in patients with distant metastasis, therefore, our findings revealed that increased cytoplasmic expression of Talin-1 is associated with invasiveness and metastasis in ccRCC.
Several previous studies revealed that overexpression of Talin-1 is associated with advanced disease and poor prognosis. Lai et al confirmed that increased expression of Talin-1 in OSCC is associated with poor prognosis and knockdown of Talin-1 expression inhibited ability of proliferation and invasion 16. In a recent study by Ji Ling et al. using IHC analysis on colorectal cancer (CRC) and adjacent normal tissue demonstrated that Talin-1 protein expression is upregulated in CRC and knockdown of Talin-1 reduces the proliferation and migration via the epithelial–mesenchymal transition (EMT) signaling pathway 40. Xu N et al. showed that the upregulation of Talin-1 protein in prostate cancer is significantly associated with advanced pathological parameters and predicts lymph node metastases and biochemical recurrence 17. In NPC, authors exhibited that high Talin-1 expression is associated with significantly poorer OS 19. Our present data further is in line with these previous studies and suggest that Talin-1 protein is an important molecule involved in the spread and progression of ccRCC. However, Talin-1 has a different expression in other cancers such as hepatocellular carcinoma (HCC). A previous study by Kanamori et al. 13 showed that high-level expression of Talin-1 in HCC tissues while another study demonstrated that Talin-1 is downregulated in HCC 41, while others showed that expression levels of Talin-1 in serum is significantly higher 42. In addition, Talin was found to be completely absent in endometriosis and endometrioid carcinomas 43. Therefore, it seems that expression levels of Talin-1 are different in various cancers and more investigations are needed to explore the exact mechanism and function of Talin-1 for finding the new ways for therapeutic targeting.
In the present study, for the first time, we showed that membranous, cytoplasmic, and nuclear Talin-1 protein expression in other important subtypes of RCC. We found no significant association between Talin-1 protein and clinicopathological features and patients' outcomes in pRCC cases. In chRCCs, a significant association was observed between membranous Talin-1 protein expression and increased tumor stage. More investigations need to be performed to support these findings using a larger number of cases of each pRCC and chRCC subtypes.