Citrullination of proteins is associated with a number of autoimmune diseases. Protein Arginine Deiminases (PADs) are a family of Ca2+ dependent enzymes that under cellular stress post-translationally convert arginine to citrulline within protein substrates to generate self-modified neo-antigens. It has been shown that presentation of citrullinated peptides on MHC class II stimulates CD4 T cells to mediate potent anti-tumor immunity (1). In this study we focus on the role of the PADI2 and PADI4 family members in colorectal cancer. Using a tissue microarray of colorectal cancers and compiling a comprehensive database of clinicopathological variables, the expression of PADI2 and PADI4 was assessed by immunohistochemistry in a study cohort. This data was used to correlate PADI2 and PADI4 expression with patient survival.
In the study cohort 462 colorectal tumors were stained for PADI2 and PADI4. Of these 171 (37%) and 231 (50%) could not be evaluated for PADI2 and PADI4 staining respectively due to the absence of enough tissue core or no evaluable tumor cells (i.e. all stroma) in the core. Of the 291 evaluable colorectal tumors stained with a PADI2 specific antibody, only 18/291 (6.2%) tumors failed to stain. A further 153/291 (52.5%) stained weakly, 102/291 (35.1%) moderate and 18/291 (6.2%) stained strongly. Of the 231 evaluable colorectal tumors stained with a PADI4 specific antibody, no tumors failed to stain. All cases stained strongly for PADI4 expression within the nucleus. In the cytoplasm 63/231 (27.3%) stained weakly, 143/231 (61.9%) moderate and 25/231 (10.8%) stained strongly.
PADI2 expression did not correlate with any clinicopathological variables whereas nuclear but not cytoplasmic PADI4 showed a strong association with histological type (p = 0.008). Kaplan-Meier analysis showed there was a correlation of PADI2 and cytoplasmic PADI4 expression with improved survival. Expression of PADI2 gave an increase in survival time from 44.8 months (95% CI 24.3 to 65.4) to 76.2 months (95% CI 69.9 to 82.4, log rank test, p = 0.012). Expression of cytoplasmic PADI4 increased survival time from 57.9 months (95% CI 43.6 to 72.3) to 77.3 months (95% CI 69.6 to 85.1, log rank test, p = 0.012). No significant correlation was observed between PADI2 and the cytoskeletal protein Vimentin or the glycolytic enzyme α-enolase both reported to be citrullinated by PAD enzymes. PADI2 expression was significantly associated with expression of the Nuclear antigen Ki67 (p = 0.046) a cellular marker for proliferation. Nuclear PADI4 significantly correlated with the cytoplasmic glycolytic enzyme α-enolase only (p = 0.001) and cytoplasmic PADI4 was highly significantly associated with α-enolase located in both the cytoplasm (p<0.001) and shorter nuclear form (p<0.001) known as MBP-1, a transcription factor known to downregulate the activity of the c-myc proto-oncogene. Cytoplasmic PADI4 expression also significantly correlated with expression of the anti-apoptotic protein Bcl2 (p = 0.028). Multivariate analysis using Cox regression showed that PADI2 (p = 0.040)/Cytoplasmic PADI4 (p = 0.036), the stress related protein MICA (p = 0.006/0.022), vascular invasion (p<0.001, 0.040) and tumor stage (p<0.001/<0.001) are all independent markers of good prognosis in colorectal cancer. These results are consistent with the hypothesis that stressed tumor cells present citrullinated epitopes allowing their growth to be controlled by T cells.
1. Brentville VA, Metheringham RL, Gunn B, Symonds P, Daniels I, Gijon M, Cook K, Xue W, Durrant LG (2016). Citrullinated vimentin presented on MHC-II in tumor cells is a target for CD4+ T cell-mediated antitumor immunity. Cancer Research 2016 Feb 1;76(3):548-60
Citation Format: R. Metheringham, M. Gijon, I. Daniels, K. Cook, P. Symonds, T. Pitt, W. Xue, V. Brentville, L. Durrant. Protein arginine deiminase enzymes which citrullinate epitopes for MHC II presentation are independent predictors of survival in colorectal cancer [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A015.