Introduction:

There is a need for identification and targeting additional mechanisms of immunosuppression in clear cell renal cell carcinoma (ccRCC). Adenosine is a known mediator of immunosuppression within the tumor microenvironment (TME). An adenosine 2A receptor antagonist has demonstrated antitumor activity in preclinical models and an early phase 1 clinical trial in humans with advanced ccRCC. We seek to identify additional regulators of purinergic signaling within the tumor microenvironment for use as a therapeutic target or clinically useful biomarker.

Methods:

We performed a differential expression of gene (DEG) analysis, with respect to disease recurrence status, on 90 genes involved in the regulation of extracellular purines and purinergic signaling using whole-gene microarray data from 236 ccRCC tumors from patients in the placebo arm of the PROTECT trial (NCT01235962; pazopanib vs placebo). The R package “limma” (version 3.32.10) was used for DEG with adjusted p values (q values) to correct for multiple comparisons testing. We validated these findings using RNA sequencing data from the TCGA-KIRC cohort. For the Kaplan-Meier analyses, gene expression data were annotated into quartiles, and disease-free survival (DFS) and overall survival (OS) association were tested using logrank testing. To determine cell populations expressing Connexin-26 (Cx26) we performed single-cell RNA sequencing of ccRCC tumors (n = 6 tumors) from MSKCC.

Results:

Among purinergic gene profiles, RNA upregulation of gap junction beta-2 (GJB2) was the most significantly associated with metastatic recurrence (q = 6.5e10^-5). GJB2 encodes for Cx26, a transmembrane protein that forms gap junctions and hemichannels in the membrane of cells to mediate the release of ATP and calcium ions from cells. This association was validated using TCGA-KIRC which demonstrated an indirect relationship between GJB2 and both DFS (p = 3.5e10^-3) and OS (p = 8e10^-4). Single-cell RNA-sequencing demonstrated an expression profile largely restricted to tumor and proximal renal epithelial cells.

Conclusion:

Cx26 upregulation is associated with worse disease outcomes in patients with ccRCC, as demonstrated in two independent patient cohorts, and is expressed on ccRCC tumor cells. Increased expression of Cx26 hemichannels in ccRCC tumor cells may lead higher intratumoral adenosine and subsequent immunosuppression. Further investigation is warranted to determine if a mechanistic connection exists between Cx26-mediated ATP release and disease progression in ccRCC and whether Cx26 could serve as a clinically useful biomarker or therapeutic target in patients with ccRCC.

Funding: NIH 5P30CA008748-56 MSK Cancer Center Support Grant (AAH)