Overexpression of HHLA2 in human clear cell renal cell carcinoma is significantly associated with poor survival of the patients

It is well known that human clear cell renal cell carcinoma (ccRCC) is a highly immunogenic and chemo-resistant tumor. Recently, emerging data suggest that the immune checkpoint blockade therapy is an important breakthrough in the treatment against ccRCC. HHLA2, a recently reported member of B7 family, is uniquely expressed in humans but not in mice, and it plays an important role in the functional inhibition of CD4 and CD8 T cells. Herein, we aimed to study the clinical implications of HHLA2 expression in human ccRCC and its potential regulatory role in the biological functions of the cancer cells.

In the present study, we examined HHLA2 expression in human ccRCC tissues and analyzed the clinical implications as well as prognostic value. The intervention of HHLA2 in human ccRCC cell lines ACHN and 786-O was performed and its effect on the cellular function of the cells was also analyzed. We also identified the differentially expressed genes upon HHLA2 knockdown in ccRCC cell lines by using gene microarray analysis.

We found that higher HHLA2 mRNA expression level in human ccRCC tissues compared with that in adjacent normal tissues based on TCGA data, and the HHLA2 expression at mRNA level was positively and significantly correlated with PD-L1, PD-L2, B7-H6, but negatively and significantly correlated with B7-H3. Moreover, our immunohistochemistry study showed that the staining intensity of HHLA2 in human ccRCC tissues was significantly higher than that in the adjacent normal tissues, and the overall survival rate of ccRCC patients with higher HHLA2 expression was significantly poorer than that of the patients with lower HHLA2 expression. Higher expression of HHLA2 in ccRCC tissues was positively and significantly associated with larger tumor size and advanced TNM stage. The COX model revealed that the parameters including patient’s age, TNM stage and HHLA2 expression level could be used as the independent risk factors respectively for the prognostic prediction of the patients. Our cellular study showed that upon knockdown of HHLA2 expression in human ccRCC cell lines, the cell viability, the migration and the invasion ability were significantly inhibited, while the cell cycle arrest at G1 phase was induced and the expressions of Cyclin D1, c-Myc and Cyclin E1 were decreased. In addition, according to the microarray data, the expressions of epithelia-to-mesenchymal transition markers, such as E-cadherin, N-cadherin and Vimentin, were significantly changed after knockdown of HHLA2 expression.

Our findings indicated that HHLA2 was involved in the progression of human ccRCC and could be used as an important prognostic predictor for this malignancy.