BackgroundInhibitory immune checkpoint proteins promote tumor immune escape and are associated with inferior patient outcome. However, the biological functions and regulatory roles of one of its members, HHLA2, in the tumor immune microenvironment have not been explored.MethodsRandomForest analyses (371 cases), qRT-PCR (15 cases), and immunohistochemical staining (189 cases) were used to validate the prognostic value of HHLA2 in hepatocellular carcinoma (HCC) patients. Bioinformatic analyses were further performed to explore the biological functions and potential signaling pathways affected by HHLA2. Moreover, ESTIMATE, single sample gene set enrichment analysis, CIBERSORT, TIMER, and other deconvolution methods were used to analyze the composition and infiltration level of immune cells. Multiplex immunofluorescence assays were employed to validate the fractions of suppressive immune cells, and HHLA2-related molecular alterations were investigated. Finally, the clinical response to chemotherapy and immune checkpoint blockade was predicted by TIDE, Submap, and several other in silico analyses.ResultsRandomForest analysis revealed that HHLA2 was the most important inhibitory immune checkpoint associated with HCC patient prognosis (relative importance = 1). Our HCC cohorts further revealed that high HHLA2 expression was an independent prognostic biomarker of shorter overall survival (P<0.01) and time to recurrence (P<0.001) for HCC patients. Bioinformatics experiments revealed that HHLA2 may accelerate the cell cycle of cancer cells. Additionally, we found that high expression of HHLA2 was associated with immune infiltrates, including some immunosuppressive cells, cytokines, chemokines, and corresponding receptors, resulting in an immunosuppressive environment. Notably, HHLA2 expression was positively correlated with the infiltration of exhausted CD8+ T cells, which was validated by immunofluorescence. Genomic alteration analyses revealed that promoter hypermethylation of HHLA2 may be associated with its low expression. More importantly, patients with high HHLA2 expression may be more sensitive to chemotherapy and have better responses to immunotherapy.ConclusionsHigh expression of HHLA2 is an independent prognostic biomarker for HCC patients. It can activate the cell cycle and foster an immunosuppressive tumor microenvironment by enriching exhausted CD8+ T cells. Promoter hypermethylation might lead to low expression of HHLA2 in HCC. Thus, targeting HHLA2 may be a practical therapeutic strategy for HCC patients in the future.

Background Hepatocellular carcinoma (HCC) is a prevalent malignancy with poor prognosis. As a cell adhesion molecule, poliovirus receptor (PVR/CD155) is abnormally overexpressed in tumour cells, and related to tumour proliferation and invasion. However, the potential role and mechanism of CD155 have not yet been elucidated in HCC. Methods Immunohistochemistry, RT‐PCR and Western blot assays were used to determine CD155 expression in HCC cell lines and tissues. Cell Counting Kit‐8 and colony formation assays were used to examine cell proliferation. Transwell and wound healing assays were used to evaluate cell migration and invasion. Cell apoptosis and cycle distribution were assessed by flow cytometry. Cox regression and Kaplan–Meier analyses were performed to explore the clinical significance of CD155. The role of CD155 in vivo was evaluated by establishing liver orthotropic xenograft mice model. RNA sequencing, bioinformatics analysis and co‐immunoprecipitation assay were used to explore the downstream signalling pathway of CD155. Results CD155 was upregulated in HCC tissues and represented a promising prognostic indicator for HCC patients ( n = 189) undergoing curative resection. High CD155 expression enhanced cell proliferation, migration and invasion, and contributed to cell survival in HCC. CD155 overexpression also induced epithelial–mesenchymal transition in HCC cells. CD155 function in HCC involved SRC/p38 MAPK signalling pathway. CD155 interacted with SRC homology‐2 domain of SRC and promoted SRC activation, further inhibiting the downstream p38 MAPK signalling pathway in HCC. Conclusions CD155 promotes HCC progression via the SRC/p38 MAPK signalling pathway. CD155 may represent a predictor for poor postsurgery prognosis in HCC patients.