Gene Expression, Cytokines and Responses to Chemotherapy Differed in Tumors and Cell Lines from African American and Caucasian American Colon Cancer Patients

Abstract

Background: Colorectal cancer is the third leading cause of cancer death among African Americans (AA). When compared to Caucasian Americans (CA), AA present with more advanced disease and lower survival rates. In order to explore molecular and cellular influences, an investigation was undertaken to examine if differences in tumor immunology and its relationship to chemotherapy response could be contributory to disparities observed between these populations. Methods: Gene expression of tumor, non-tumor tissues and colon cancer cell lines from AA and CA was examined by whole transcriptome sequencing and “The Cancer Genome Atlas” (TCGA) database was utilized as a validation cohort. Scores for immune cell populations in the tumors were calculated by RNA probing (NanoString). In addition, the secretion of cytokines characteristic of effector T helper cell (Th) subsets were measured by ELISA using cell supernatants and plasma from AA and CA patients. Finally, the sensitivity of colon cancer cell lines to the chemotherapy 5-Fluorouracil (5-FU) was compared by cell viability, clonogenics and protein assays (western blots). Results: Colon tumors from AA patients showed a significant increase in gene expression when compared to CA for FOXP3 (6.22 vs. 3.22), IL1B (103 vs. 11.4) and IL8 (220 vs. 28.9) (p<0.05). In contrast, among CA there was statistically higher gene expression of markers associated with antitumor activity such as GZMB (Granzyme B), IFNG and the immunotherapy targets PDL1 (CD274) and CTLA4 (p < 0.05). TCGA data validated the higher gene expression of GZMB (p=0.0002) and PDL1 (p=0.03) in CA patients when compared to AA. Notably, the immune cell populations assays indicated that AA tumors have significantly higher number of non-functional CD8+ cells (p<0.01), CD17+ mast cells (p<0.02) and increased CD4+/FOXP3+ T regulatory cells when compared to CA and AA colon cancer patients. Similarly, AA also differed from CA in cytokine production patterns in plasma (i.e. reduced IL-12, <0.005). The studied colon cancer cell lines (2 from each population) showed a significant difference in the secretion of IL8 in response to 5-FU, IL1b and TNF-a; and in the short and long term viability after 5-FU treatment (p<0.05). Conclusions: These significant differences suggest a deficiency of appropriate immune defense mechanisms in colon tumors from AA compared to CA patients. The in vitro studies showing related differences between the AA and CA colon cancer cell lines in response to 5-FU and inflammatory stimuli, supports the use of these cell lines as one type of model for studying the biological differences between AA and CA colon cancer tumors. As such, these immune differences could be selectively targeted to enhance tumor immunity through population-specific therapeutic approaches.