The CCK-B receptor: A novel target for therapy of hepatocellular carcinoma.

Abstract

466 Background: Today, the fastest growing cause of cancer-related death is hepatocellular carcinoma (HCC). Treatment with immune checkpoint antibodies has shown promise in HCC, but the response is still low at about 15%-20% as monotherapy. Our research laboratory discovered that the cholecystokinin-B receptor (CCK-BR) becomes over-expressed in the injured/ inflamed liver and is up-regulated in HCC. The primary aim of this study is to evaluate the effects of a CCK-BR antagonist, proglumide, alone and in combination with a PD-1 antibody (PD-1 Ab) on survival in HCC tumor-bearing mice and the tumor infiltrating T-cells. The secondary aim was to evaluate the frequency of CCK-BR expression in human HCC. Methods: RNA was extracted from murine RIL-175 HCC cells and subjected to qRT-PCR for gene expression of CCK-BRs, gastrin, and PD-L1. C57BL/6 mice (N = 40) were injected with RIL-175 HCC cells (100,000) subcutaneously. After one week when the mice had palpable tumors, they were divided into 4 groups (N = 10ea) of equal tumor size. Treatment included: controls-untreated drinking water; proglumide (0.1mg/ml) in drinking water; PD-1Ab (50µg, ip, days 7, 14, 21 with untreated water); and combination therapy with PD-1 Ab (as above) and proglumide water. Mice were euthanized when tumors reached a diameter of 20mm according to IACUC guidelines. Tumors were stained with a CD8 antibody to evaluate CD8+ tumor infiltrating T-cells. CCK-BR protein expression in human liver tissues and human HCC was evaluated by immunohistochemistry using a tissue microarray (US Biomax). Results: RIL-175 murine HCC cells exhibited increased expression of CCK-BR, gastrin, and PD-L1 by qRT-PCR. Survival of mice treated with the combination of proglumide and the PD-1 Ab was double that of the control mice and greater than proglumide or PD-1 Ab monotherapy. Compared to tumors of control mice, tumors of mice receiving proglumide monotherapy exhibited a 5-fold increase in CD8+ cells (P = 2.5x10-8), and there was a 12-fold increase in CD8+ T-cells in tumors of mice treated with the combination therapy (P = 4.7x10-17). In the human liver tissue microarray, immunoreactivity for the CCK-BR was negative in the normal human liver tissues and increased with the grade of HCC tumor (P = 0.0045) with 84.6% of grade-3 human HCC tissues staining positive for CCK-BR. Conclusions: The CCK-BR may provide a novel target for therapy in HCC. Treatment with the CCK-BR antagonist, proglumide, in combination with a PD-1 Ab appears to be synergistic improving the survival of mice by increasing the number of intratumoral CD8+ T-cells.