Integrating HECW1 expression into the clinical indicators exhibits high accuracy in assessing the prognosis of patients with clear cell renal cell carcinoma

Abstracts Background Although many intratumoral biomarkers have been reported to predict clear cell renal cell carcinoma (ccRCC) patient prognosis, combining intratumoral and clinical indicators could predict ccRCC prognosis more accurately than any of these markers alone. This study mainly examined the prognostic value of HECT, C2 and WW domain-containing E3 ubiquitin protein ligase 1 (HECW1) expression in ccRCC patients in combination with established clinical indicators. Methods The expression level of HECW1 was screened out by data-independent acquisition mass spectrometry (DIA-MS) and analyzed in ccRCC patients from the The Cancer Genome Atlas (TCGA) database and our cohort. A total of 300 ccRCC patients were stochastically divided into a training cohort and a validation cohort, and real-time PCR, immunohistochemistry (IHC) and statistical analyses were employed to examine the prognostic value of HECW1 in ccRCC patients. Results The expression level of HECW1 usually decreased in human ccRCC specimens relative to control specimens in TCGA (p < 0.001). DIA-MS, Real-time PCR, and IHC analyses also showed that the majority of ccRCCs harbored decreased HECW1 expression compared with that in normal adjacent tissues (p < 0.001). Additionally, HECW1 expression was reduced in ccRCC cell lines compared with the normal renal cell line HK-2 (p < 0.001). Moreover, lower HECW1 expression was found in ccRCC patients with a higher tumor node metastasis (TNM) stage, bone metastasis, or first-line targeted drug resistance (p < 0.001). Low HECW1 expression indicated higher TNM stage, SSIGN (Stage, Size, Grade, and Necrosis) score and WHO/ISUP grade and poor prognosis in ccRCC patients (p < 0.05). Even after multivariable adjustment, HECW1, TNM stage, and SSIGN score served as independent risk factors. The c-index analysis showed that integrating intratumoral HECW1 expression into TNM stage or SSIGN score resulted in a higher c-index value than these indicators alone for predicting ccRCC patient prognosis. Conclusion HECW1 is a novel prognostic biomarker and therapeutic target in ccRCC, and integrating intratumoral HECW1 expression with established clinical indicators yields higher accuracy in assessing the postoperative prognosis of ccRCC patients.

Upregulation of ARNTL2 Predicts Poor Survival for Clear Cell Renal Cell Carcinoma and Explores its Associations with PD-L1 and Immune Infiltration

Background: Aryl hydrocarbon receptor nuclear translocator like 2 (ARNTL2) pertain to the PAS superfamily. Emerging evidences have demonstrated the carcinogenic roles of transcription factor ARNTL2 in human malignancies, while its roles in ccRCC remain elusive. We sought to explore the comprehensive roles of ARNTL2 in ccRCC and place major emphasis on its correlations with tumor immunity.Methods: The available data from GEO, TCGA and GTEx database were combined with our ccRCC patient tissues to verify the upregulation of ARNTL2, Kaplan–Meier survival curve analysis, Cox regression analyses (including univariate and multivariate) were utilized to evaluate the prognostic values of ARNTL2, the potential biological mechanisms of ARNTL2 were analyzed by using GSEA method. The ssGSEA and xCell algorithm were employed to assess the correlations of ARNTL2 expression with tumor immune microenvironment (TIME), The spearman analyses were applied to investigate the relationships between ARNTL2 expression and the tumor mutational burden (TMB), PD-L1 expression and microsatellite instability (MSI) in pan-cancer.Results: ARNTL2 was overexpressed in ccRCC and increased ARNTL2 expression strongly linked to advanced clinical stage and unfavorable overall survival. ARNTL2 was recognized as an independent prognostic marker through cox regression analyses. A prognostic nomogram was subsequently constructed to predict 1-, 3- and 5-year overall survival via integrating ARNTL2 expression with other clinicopathologic variables. GSEA analysis revealed that the focal adhesion, T cell receptor, cell cycle and JAK-STAT signaling pathway were remarkably enriched in high ARNTL2 samples. xCell analysis suggested that high expression of ARNTL2 exhibited an immune infiltration status similar to CD8+ inflamed ccRCC subtype, which characterized by a high infiltration level of CD8+ T cell and elevated expression level of the immune escape biomarkers such as PD-L1, PD-L2, PD1 and CTLA4. Further pan-cancer analysis indicated that ARNTL2 was tightly linked to TMB, MSI, PD-L1 expression, tumor immunity and poor OS in diverse cancer types.Conclusions: ARNTL2 is an independent adverse predictor of ccRCC patient survival and tightly linked to TMB, MSI, PD-L1 expression and immunity.

Activation and function of receptor tyrosine kinases in human clear cell renal cell carcinomas

Background The receptor tyrosine kinases (RTKs) play critical roles in the development of cancers. Clear cell renal cell carcinoma (ccRCC) accounts for 75% of the RCC. The previous studies on the RTKs in ccRCCs mainly focused on their gene expressions. The activation and function of the RTKs in ccRCC have not been fully investigated. Methods In the present study, we analyzed the phosphorylation patterns of RTKs in human ccRCC patient samples, human ccRCC and papillary RCC cell lines, and other kidney tumor samples using human phospho-RTK arrays. We further established ccRCC patient-derived xenograft models in nude mice and assessed the effects of RTKIs (RTK Inhibitors) on the growth of these cancer cells. Immunofluorescence staining was used to detect the localization of keratin, vimentin and PDGFRβ in ccRCCs. Results We found that the RTK phosphorylation patterns of the ccRCC samples were all very similar, but different from that of the cell lines, other kidney tumor samples, as well as the adjacent normal tissues. 9 RTKs, EGFR1–3, Insulin R, PDGFRβ, VEGFR1, VEGFR2, HGFR and M-CSFR were found to be phosphorylated in the ccRCC samples. The adjacent normal tissues, on the other hand, had predominantly only two of the 4 EGFR family members, EGFR and ErbB4, phosphorylated. What’s more, the RTK phosphorylation pattern of the xenograft, however, was different from that of the primary tissue samples. Treatment of the xenograft nude mice with corresponding RTK inhibitors effectively inhibited the Erk1/2 signaling pathway as well as the growth of the tumors. In addition, histological staining of the cancer samples revealed that most of the PDGFRβ expressing cells were localized in the vimentin-positive periepithelial stroma. Conclusions Overall, we have identified a set of RTKs that are characteristically phosphorylated in ccRCCs. The phosphorylation of RTKs in ccRCCs were determined by the growing environments. These phosphorylated/activated RTKs will guide targeting drugs development of more effective therapies in ccRCCs. The synergistical inhibition of RTKIs combination on the ccRCC suggest a novel strategy to use a combination of RTKIs to treat ccRCCs.