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

Abstract

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.

Keywords: Clear cell renal cell carcinoma; Disease progression; HECW1; Prognostic marker.

Fig. 1

HECW1 expression is decreased in clear cell renal cell carcinoma. (a), The protein expression of HECW1 in ccRCC samples was determined by Data-independent acquisition mass spectrometry (DIA-MS) assay. (b) The expression of HECW1 in ccRCC tissues (Tumor, n = 508) and normal adjacent tissues (Normal, n = 72) from The Cancer Genome Atlas (TCGA) datasets was analyzed. The normalized read counts are shown in boxplots to compare the expression differences. (c) The expression of HECW1 between ccRCC tumor tissues (n = 69) and normal adjacent tissues (n = 69) from TCGA datasets was analyzed the same way as described above. (d) Real-time PCR was used to detect the relative mRNA expression of HECW1 in tumor samples and their paired normal adjacent tissues from ccRCC patients (n = 60). (e) Representative images of hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC) staining for HECW1 in ccRCC tissues and normal adjacent tissues are presented (scale bar = 20 μm). The expression of HECW1 evaluated by the H-score method (for details, see Materials and Methods) in corresponding tissues is shown, and the values are represented as the mean ± SD (***p < 0.001; Wilcoxon test)

Fig. 2

HECW1 expression is negatively associated with tumor stage, bone metastasis, and targeted drug resistance in ccRCC. (a) Real-time PCR was used to detect the mRNA expression of HECW1 in HK-2, 786-O or 769-P, and Caki-1 cells. (b) Representative images of H&E and IHC staining for HECW1 in ccRCC tissues with different tumor node metastasis (TNM) stages are shown (scale bar = 20 μm). The expression of HECW1 in ccRCC specimens was evaluated by the H-score method, and the values are represented as the mean ± SD. (c) Representative images of H&E and IHC staining for HECW1 in tissues of localized ccRCC and ccRCC with bone metastasis are shown (scale bar = 20 μm). The expression of HECW1 in ccRCC specimens was evaluated by the H-score method, and values are represented as the mean ± SD. (d-e) Real-time PCR was employed to detect the mRNA expression of HECW1 in 786-O-SR (d) and 786-O-PR (e) cells. (f-g) Representative images of H&E and IHC staining for HECW1 in sunitinib-resistant (f) or pazopanib-resistant (g) orthotopic ccRCC specimens are presented (scale bar = 20 μm). Values are represented as the mean ± SD. All p values are defined as ***p < 0.001

Fig. 3

Low HECW1 expression is predictive of unfavorable clinicopathological characteristics and poor postoperative prognosis in ccRCC patients. (a) A flow chart of the present study is shown. (b) Representative images of H&E and IHC staining of HECW1 in ccRCC specimens are presented (scale bar = 20 μm). The expression level of HECW1 in ccRCC was evaluated by the H-score method. (c) A time-dependent receiver operating characteristic (ROC) analysis was used to determine the optimum cut-off value of HECW1 in the randomized training cohort (at a 3:2 ratio). (d-g) Kaplan-Meier curves for the overall survival (OS) and progression-free survival (PFS) of ccRCC patients were analyzed according to HECW1 expression in the randomized training cohort (d, e) and validation cohort (f, g) (at a 3:2 ratio)