Validation of PASD1 protein as an immunotherapeutic target in colorectal cancer

Abstract

Colorectal cancer (CRC) is the third commonest malignancy in Asia including Malaysia. Cancer testis antigens (CTA) which are expressed in a variety of cancers but with limited expression in normal tissues except the testis, represent an attractive approach to improve treatment options for CRC. PAS (Per ARNT Sim) domain containing 1 (PASD1) is a CTA reported to be expressed mostly in hematopoietic malignancies but has a limited expression in solid tumours.The aim of the study was to investigate the expression of PASD1 in CRC samples and assess the CD8 T cell responses, as well as the cytokine profiles upon induction with PASD1 peptides. PASD1 mRNA expression was determined via RT-PCR on paired tumour and normal tissue samples from 25 CRC patients. Tissue expression of PASD1 was assessed via immunohistochemistry in 23 CRC samples. Four immunogenic PASD1 peptides specific to HLA-A*24:02 were identified using web-based algorithms. Peripheral blood mononuclear cells from CRC patients were used to investigate the immunogenicity of these peptides in vitro using IFN-y ELISpot assay. The effects of the CD8+ T cells pulsed with PASD1 peptides against cancer cells were performed using the cytolytic and granzyme-B ELISpot cytotoxicity assays. The cytokine profiles of the co-culture between effector and target cells were also assessed. From this study, the gene and protein expression of PASD1 were detected in 20% and 17.3% of the CRC samples respectively. PASD1(4), was shown to be selectively immunogenic in CRC samples. The generated PASD1 specific cytotoxic T cells from the three CRC patients were able to lyse the SW480 cell line. Moreover, this study also demonstrated the dose-dependent increase of IL-10, IFN-y, TNF-α, sFas, sFasL, granzyme A, granzyme B and perforin in the co-culture media of CD8 T cells with SW480 cell line. Overall, the PASD1 (4) peptide with the amino acids sequence of TYCSSTVFL at the position 276-273 was identified to be relatively more immunogenic as compared to the other peptides and it represents a potential target for peptide-based immunotherapy in HLA-A*24:02 CRC.