High Cytoplasmic and Membranous Versus Nuclear Talin-1 Expression Associated With Malignancy Potential and Poor Survival in Clear Cell Renal Cell Carcinoma

Abstract

Using bioinformatics analysis Talin-1 protein was indicated as a potential prognostic focal adhesion factor in renal cell carcinoma (RCC). We, therefore, examined its protein expression levels and prognostic significant of Talin-1 with clinical follow-up in total of 269 tissue specimens from three important subtypes of RCC and 30 adjacent normal samples using tissue microarrays (TMAs). Then, we used combined analysis with B7-H3 to confirm that Talin-1 may be related to metastasis. The results showed that high membranous and cytoplasmic expression of Talin-1 was significantly associated with advanced nucleolar grade (P < 0.001, all), microvascular invasion (P = 0.007, P = 0.004, respectively), histological tumor necrosis (P = 0.020, P = 0.018), and invasion to Gerota’s fascia (P = 0.025, P = 0.050) in ccRCC. In addition, high membranous and cytoplasmic expression of Talin-1 was associated with significantly poorer disease-specific survival (P = 0.043, P = 0.024, respectively) and progression- free survival (P = 0.046, in cytoplasm). 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 in patients with distant metastasis. Collectively, these observations indicate that Talin-1 is an important molecule involved in the spread and progression of ccRCC when expressed particularly in cytoplasm and may serve as a novel prognostic biomarker in this subtype.

Introduction

Renal cell carcinoma (RCC) is the most common type of kidney cancer found in adults. It represents approximately 3% of adult malignancies and 90–95% of all neoplasms arising from the kidney ^1,2. The RCC incidence rates have been increasing during the decade ³. According to the American Cancer Society, an estimated 73,750 new cases (45,520 in males and 28,230 in females) and 14,830 deaths (9,860 in males and 4,970 in females) from RCC will occur in 2020 ⁴. If cancer has not metastasized, the 5-year survival rate is 65–90%, but this is lowered considerably when cancer has spread so that the 5-year survival rates for patients with stage IV disease are less than 20% ⁵. Unfortunately, ~ 20–30% of RCC patients have distant metastasis at initial diagnosis. In addition, about 25–40% of RCCs would relapse and ultimately develop into metastatic disease after resection of the primary tumor ⁶. Moreover, increasing resistance to targeted therapies have been occurred in advanced RCC patients ⁷. Therefore, prognostic and predictive biomarkers will be an urgent requirement to assist in early diagnosis and identify novel therapeutic targets for RCC.

To date, several subtypes of RCC have been defined, of which clear cell RCC (ccRCC) is the most common subtype (75–80%), followed by papillary RCC (pRCC) 15%, and chromophobe RCC (chRCC) around 5% ⁸. In this study, we have focused on all these three important histological subtypes of RCC.

High throughput proteomics was introduced as an innovative novel approach that paves the way for cancer diagnostic or prognostic biomarker discovery ⁹. The advantages of these types of studies can be the identification of effective proteins with a different expression and cellular functions in the samples in various diseases ¹⁰. Although a high level of variance exists in proteomics data, network, and enrichment analysis explore the hub genes and their related biological significance among results ¹¹. In the present study, protein-protein interaction (PPI) network analysis was performed for proteomics data of available studies that had reported proteins with significant differential expression in RCC tissue samples compared to adjacent normal tissues. Enrichment analysis of hub genes was obtained for a better understanding of influential genes in the RCC network. It was found that TLN1 (Talin-1) is one of the hub genes that could be practical as a prognostic marker in renal cancer.

Talin-1 is a ubiquitous cytoskeleton‑associated protein with a high-molecular-weight which has been shown to play an essential role in regulating the activity of the integrin family of cell adhesion proteins using coupling integrins to F-actin ^12,13. Also, Talin-1 is responsible for binding to critical adhesion molecules, including integrins, vinculin, actin, and other focal adhesion kinases (FAKs) and induce cell cytoskeletal remodeling. Therefore increased expression of Talin-1 could lead to the development of tumor and migration ^14,15. A number of studies have demonstrated that overexpression of Talin-1 was associated with advanced disease and reduced overall survival (OS) in patients, including oral squamous cell carcinoma (OSCC) ¹⁶, prostate cancer ¹⁷, colon cancer ¹⁸, and nasopharyngeal carcinoma (NPC) ¹⁹. To date, the expression levels and clinical significance of Talin-1 in the RCC tumors remain unclear. Thus, in the current study, to validate and confirm the Talin-1 protein obtained from the network analysis on results of proteomics studies in RCC samples, we have investigated the membranous, cytoplasmic, and nuclear expression levels of Talin-1 protein in three important subtypes of RCC from formalin-fixed paraffin-embedded (FFPE) tissue samples which assembled on tissue microarrays (TMAs) using the immunohistochemistry (IHC) technique. Then, we sought to determine the association between expression levels of Talin-1 protein and clinicopathological parameters, including nucleolar grade, tumor stage, microvascular invasion, and other important clinicopathological characteristics of RCC as well as survival outcomes. Finally, we used combined analysis with another marker of our previous study, namely B7-H3 ²⁰ which is useful for predicting progression and metastasis in ccRCC to confirm that Talin-1 may be related to metastasis.

Results

Discussion

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.

Conclusion

In summary, bioinformatics analysis indicated Talin-1 protein as a potential focal adhesion prognostic marker in RCC. Our finding also demonstrated that Talin-1 protein is upregulated in human RCC tissues compared to adjacent normal tissues. Further, our results for the first time revealed that the statistically significant differences between membranous and cytoplasmic Talin-1 expression in various histological subtypes of RCC. A better understanding of the histological subtypes in RCC is urgently needed for further development of treatment options. Moreover, the current study indicates that membranous and cytoplasmic Talin-1 protein expression, particularly cytoplasmic expression compared to nuclear expression plays an important role in more aggressive tumor behaviors, metastasis, and progression of ccRCC. In addition, high membranous and cytoplasmic expression of Talin-1 protein was identified as a worse prognostic variable affecting DSS or PFS in univariate analysis, indicating that Talin-1 may serve as a novel prognostic biomarker in ccRCC if follow up time more extended. However, further studies on the function and mechanism of action of Talin-1 is a requirement to provide new opportunities for therapeutic targeting of RCC.

Methods

Declarations

Authors' contributions

LS designed and supervised the work, gathered the paraffin embedded tissues, collected the patient data, prepared the information of patient survival outcomes, analyzed and interpreted the data as well as wrote the manuscript; SV performed the immunohistochemistry examinations and contributed to write the some sections of the manuscript; MA examined hematoxylin and eosin slides, marked the most representative areas in different parts of the tumor for preparing the TMAs blocks, and scored TMAs slides after immunohistochemical staining; FF performed the bioinformatic analysis and wrote the bioinformatic sections in the manuscript. ZM provided for us the laboratory equipment and some materials in this project. All authors read and approved the final manuscript.

Competing interests

The authors declare that they have no competing interests.

Ethics Approval and Consent to participate

This study was approved by the Iran University of Medical Sciences Human Research Ethics Committee in Iran (Ref no: IR.IUMS.REC.1398.938). All procedures performed in this study were in accordance with the 1964 Helsinki Declaration and its later amendments. Informed consent was obtained from all individual participants included in the study at the time of sample collection with routine consent forms.

Funding

This work was supported by the grant from Iran University of Medical Sciences (Ref no: 16289).

Additional information

Supplementary information is available for this paper.

References

1. Kantarjian, H., Koller, C. A. & Wolff, R. A. The MD Anderson manual of medical oncology (McGraw-hill, New York, NY, USA, 2011).
2. Street, W. Cancer Facts & Figs. 2019. American Cancer Society: Atlanta, GA, USA (2019).
3. Capitanio, U. et al. Epidemiology of renal cell carcinoma. European urology. 75, 74–84 (2019).
4. Siegel Rebecca, L. & Ahmedin, M. K. D. J. Cancer statistics, 2020.[J]. CA. Cancer J Clin. 70, 7–30 (2020).
5. Patil, S. et al. Improvement in overall survival of patients with advanced renal cell carcinoma: prognostic factor trend analysis from an international data set of clinical trials. The Journal of urology. 188, 2095–2100 (2012).
6. Lalani, A. K. A. et al. Systemic treatment of metastatic clear cell renal cell carcinoma in 2018: current paradigms, use of immunotherapy, and future directions. European urology. 75, 100–110 (2019).
7. Duran, I. et al. Resistance to targeted therapies in renal cancer: the importance of changing the mechanism of action. Targeted oncology. 12, 19–35 (2017).
8. Motzer, R. J. et al. Kidney cancer, version 3.2015. Journal of the National Comprehensive Cancer Network. 13, 151–159 (2015).
9. Shruthi, B. S., Palani Vinodhkumar, S. & Proteomics A new perspective for cancer.Advanced biomedical research5 (2016).
10. Pan, S. et al. High throughput proteome screening for biomarker detection. Molecular & Cellular Proteomics. 4, 182–190 (2005).
11. Wu, X., Al Hasan, M. & Chen, J. Y. Pathway and network analysis in proteomics. Journal of theoretical biology. 362, 44–52 (2014).
12. Critchley, D. Cytoskeletal proteins talin and vinculin in integrin-mediated adhesion. Biochem. Soc. Trans. 32, 831–836 (2004).
13. Kanamori, H. et al. Identification by differential tissue proteome analysis of talin-1 as a novel molecular marker of progression of hepatocellular carcinoma. Oncology. 80, 406–415 (2011).
14. Giancotti, F. G. & Ruoslahti, E. Integrin signaling. science 285, 1028–1033(1999).
15. Sakamoto, S., McCann, R. O., Dhir, R. & Kyprianou, N. Talin1 promotes tumor invasion and metastasis via focal adhesion signaling and anoikis resistance. Cancer research. 70, 1885–1895 (2010).
16. Lai, M. T. et al. Talin-1 overexpression defines high risk for aggressive oral squamous cell carcinoma and promotes cancer metastasis. The Journal of pathology. 224, 367–376 (2011).
17. Xu, N. et al. Upregulation of Talin-1 expression associates with advanced pathological features and predicts lymph node metastases and biochemical recurrence of prostate cancer.Medicine95 (2016).
18. Bostanci, O. et al. A novel screening test for colon cancer: Talin-1. Eur Rev Med Pharmacol Sci. 18, 2533–2537 (2014).
19. Xu, Y. F. et al. High expression of Talin-1 is associated with poor prognosis in patients with nasopharyngeal carcinoma. BMC cancer. 15, 332 (2015).
20. Zanjani, L. S. et al. Cytoplasmic expression of B7-H3 and membranous EpCAM expression are associated with higher grade and survival outcomes in patients with clear cell renal cell carcinoma.Annals of Diagnostic Pathology, 151483 (2020).
21. Maziveyi, M. & Alahari, S. K. Cell matrix adhesions in cancer: the proteins that form the glue. Oncotarget. 8, 48471 (2017).
22. Alizadeh, A. M., Shiri, S. & Farsinejad, S. Metastasis review: from bench to bedside. Tumor biology. 35, 8483–8523 (2014).
23. Yoon, H., Dehart, J. P., Murphy, J. M. & Lim, S. T. S. Understanding the roles of FAK in cancer: inhibitors, genetic models, and new insights. Journal of Histochemistry & Cytochemistry. 63, 114–128 (2015).
24. Aye, J. M. et al. The effects of focal adhesion kinase and platelet-derived growth factor receptor beta inhibition in a patient-derived xenograft model of primary and metastatic Wilms tumor. Oncotarget. 10, 5534 (2019).
25. Tai, G., Hohenstein, P. & Davies, J. A. FAK–Src signalling is important to renal collecting duct morphogenesis: discovery using a hierarchical screening technique. Biology open. 2, 416–423 (2013).
26. Béraud, C. et al. Targeting FAK scaffold functions inhibits human renal cell carcinoma growth. International journal of cancer. 137, 1549–1559 (2015).
27. Murphy, J. M., Rodriguez, Y. A., Jeong, K., Ahn, E. Y. E. & Lim, S. T. S. Targeting focal adhesion kinase in cancer cells and the tumor microenvironment.Experimental & Molecular Medicine,1–10(2020).
28. Zhou, J., Yi, Q. & Tang, L. The roles of nuclear focal adhesion kinase (FAK) on Cancer: a focused review. Journal of Experimental & Clinical Cancer Research. 38, 250 (2019).
29. Golubovskaya, M. V. Focal adhesion kinase as a cancer therapy target. Anti-Cancer Agents in Medicinal Chemistry (Formerly Current Medicinal Chemistry-Anti-Cancer Agents). 10, 735–741 (2010).
30. Ljungberg, B. et al. EAU guidelines on renal cell carcinoma: 2014 update. European urology. 67, 913–924 (2015).
31. Lang, H. et al. Prognostic value of microscopic venous invasion in renal cell carcinoma: long-term follow-up. European urology. 46, 331–335 (2004).
32. Bedke, J. et al. Microvascular and lymphovascular tumour invasion are associated with poor prognosis and metastatic spread in renal cell carcinoma: a validation study in clinical practice. BJU Int. 121, 84–92 (2018).
33. Ito, K. et al. Tumor necrosis is a strong predictor for recurrence in patients with pathological T1a renal cell carcinoma. Oncology letters. 9, 125–130 (2015).
34. Zhang, L. et al. Tumor necrosis as a prognostic variable for the clinical outcome in patients with renal cell carcinoma: a systematic review and meta-analysis. BMC cancer. 18, 1–13 (2018).
35. Laird, A. et al. Differential expression of prognostic proteomic markers in primary tumour, venous tumour thrombus and metastatic renal cell cancer tissue and correlation with patient outcome. PloS one. 8, e60483 (2013).
36. Thompson, R. H. et al. Metastatic renal cell carcinoma risk according to tumor size. The Journal of urology. 182, 41–45 (2009).
37. Ahmad, A. A. et al. Papillary Renal Cell Carcinomas Rewire Glutathione Metabolism and Are Deficient in Both Anabolic Glucose Synthesis and Oxidative Phosphorylation. Cancers. 11, 1298 (2019).
38. Flem-Karlsen, K., Fodstad, Ã., Tan, M. & Nunes-Xavier, C. E. B7-H3 in cancer–beyond immune regulation. Trends in Cancer. 4, 401–404 (2018).
39. Li, M. et al. Overexpression of B7-H3 in CD14 + monocytes is associated with renal cell carcinoma progression. Med. Oncol. 31, 349 (2014).
40. Ji, L., Jiang, F., Cui, X. & Qin, C. Talin1 knockdown prohibits the proliferation and migration of colorectal cancer cells via the EMT signaling pathway. Oncology letters. 18, 5408–5416 (2019).
41. Zhang, J. L., Qian, Y. B., Zhu, L. X. & Xiong, Q. R. Talin1, a valuable marker for diagnosis and prognostic assessment of human hepatocelluar carcinomas. Asian Pac J Cancer Prev. 12, 3265–3269 (2011).
42. Youns, M. M., Wahab, A., Hassan, A. H. A., Attia, M. S. & Z. A. & Serum talin-1 is a potential novel biomarker for diagnosis of hepatocellular carcinoma in Egyptian patients. Asian Pacific journal of cancer prevention. 14, 3819–3823 (2013).
43. Slater, M., Cooper, M. & Murphy, C. The cytoskeletal proteins α-actinin, Ezrin, and talin are de-expressed in endometriosis and endometrioid carcinoma compared with normal uterine epithelium. Applied Immunohistochemistry & Molecular Morphology. 15, 170–174 (2007).
44. Vizcaíno, J. A. et al. 2016 update of the PRIDE database and its related tools. Nucleic acids research. 44, D447–D456 (2016).
45. Koch, E. et al. Transcriptome-proteome integration of archival human renal cell carcinoma biopsies enables identification of molecular mechanisms. American Journal of Physiology-Renal Physiology. 316, F1053–F1067 (2019).
46. Xiao, Y. et al. Decreased mitochondrial DNA content drives OXPHOS dysregulation in chromophobe renal cell carcinoma.Cancer Research(2020).
47. Doncheva, N. T., Morris, J. H., Gorodkin, J. & Jensen, L. J. Cytoscape StringApp: network analysis and visualization of proteomics data. Journal of proteome research. 18, 623–632 (2018).
48. Shannon, P. et al. Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome research. 13, 2498–2504 (2003).
49. Pletscher-Frankild, S., Pallejà, A., Tsafou, K., Binder, J. X. & Jensen, L. J. DISEASES: Text mining and data integration of disease–gene associations. Methods. 74, 83–89 (2015).
50. Queralt-Rosinach, N., Piñero, J., Bravo, Ã., Sanz, F. & Furlong, L. I. DisGeNET-RDF: harnessing the innovative power of the Semantic Web to explore the genetic basis of diseases. Bioinformatics. 32, 2236–2238 (2016).
51. Kuleshov, M. V. et al. Enrichr: a comprehensive gene set enrichment analysis web server 2016 update. Nucleic acids research. 44, W90–W97 (2016).
52. Bindea, G. et al. ClueGO: a Cytoscape plug-in to decipher functionally grouped gene ontology and pathway annotation networks. Bioinformatics. 25, 1091–1093 (2009).
53. Ayob, A. Z. & Ramasamy, T. S. Cancer stem cells as key drivers of tumour progression. Journal of biomedical science. 25, 1–18 (2018).
54. Sidiropoulos, K. et al. Reactome enhanced pathway visualization. Bioinformatics. 33, 3461–3467 (2017).
55. Slenter, D. N. et al. WikiPathways: a multifaceted pathway database bridging metabolomics to other omics research. Nucleic acids research. 46, D661–D667 (2018).
56. Chandrashekar, D. S. et al. UALCAN: a portal for facilitating tumor subgroup gene expression and survival analyses. Neoplasia. 19, 649–658 (2017).
57. Trpkov, K. et al. Handling and staging of renal cell carcinoma: the International Society of Urological Pathology Consensus (ISUP) conference recommendations. The American journal of surgical pathology 37, 1505–1517(2013).
58. Delahunt, B. et al. The International Society of Urological Pathology (ISUP) grading system for renal cell carcinoma and other prognostic parameters. The American journal of surgical pathology. 37, 1490–1504 (2013).
59. Moch, H., Cubilla, A. L., Humphrey, P. A., Reuter, V. E. & Ulbright, T. M. The 2016 WHO classification of tumours of the urinary system and male genital organs—part A: renal, penile, and testicular tumours. European urology. 70, 93–105 (2016).
60. Jourdan, F. et al. Tissue microarray technology: validation in colorectal carcinoma and analysis of p53, hMLH1, and hMSH2 immunohistochemical expression. Virchows Arch. 443, 115–121 (2003).
61. Langer, R. et al. Prognostic significance of expression patterns of c-erbB-2, p53, p16INK4A, p27KIP1, cyclin D1 and epidermal growth factor receptor in oesophageal adenocarcinoma: a tissue microarray study. Journal of clinical pathology 59, 631–634(2006).
62. Picarda, E., Ohaegbulam, K. C. & Zang, X. Molecular pathways: targeting B7-H3 (CD276) for human cancer immunotherapy. Clin. Cancer Res. 22, 3425–3431 (2016).