Targeting the EP1-FasL Signaling Pathway Reduces Colon Tumor Cell Growth In Vivo

Abstract

Background: The role of cyclooxygenase-2 and its metabolite prostaglandin E2 (PGE2) in promoting colon carcinogenesis is well established. PGE2 signals through four different receptor subtypes, EP1 - EP4, and signaling through the EP1 receptor has been shown to upregulate Fas ligand (FasL/CD95L) expression in human colon carcinoma In Vitro. Accumulating evidence favors a role for FasL in tumorigenesis, possibly by inhibiting the anti-tumor immune response. Thus, targeting PGE2/EP1-induced FasL expression in tumors may have therapeutic potential. Aim: To investigate if blocking PGE2/EP1-induced FasL expression alters tumor development In Vivo. Methods: Female BALB/c mice were injected subcutaneously with CT26 murine colon tumor cells. Once tumors reached a volume of 0.5cm3, EP1 receptor specific antagonist ONO-8713 (ONO Pharmaceutical, Japan), was administered either (i) orally mixed into their daily feed (1000ppm) or (ii)by direct injection into the tumors three times a week (30mg/kg per injection). To control for the impact of FasL downregulation, another group was injected with CT26 cells stably transfected with FasL short hairpin RNA (shRNA) which suppressed FasL expression. After 51 days, tumors were excised and analysed for FasL expression by qRT-PCR and immunohistochemistry. Tumor-infiltrating lymphocytes (TIL) were phenotyped by flow cytometry. Splenocytes were tested for CTL activity after 5 days. Cell proliferation In Vitro was assessed by resazurin reduction. Results: The therapeutic efficacy of targeting tumor-expressed FasL was confirmed by a significantly reduced tumor volume (57%+/-3.4%) when the inoculated tumors had FasL suppressed in advance by shRNA. However, reduced tumor volume In Vivo was also found after administration of the EP1 receptor antagonist. Oral administration led to a mean reduction of 46%+/-18% in tumor volume compared with untreated, and direct intra-tumoral injection resulted in 74%+/-6.7% reduction. This was associated with downregulaton of FasL expression by the tumors. In contrast to the results In Vivo, EP1 receptor antagonism did not affect tumor cell proliferation In Vitro indicating an indirect mechanism for the In Vivo efficacy. This was supported by analysis of TIL which revealed reduced CD4+CD25+FOXP3+ regulatory T cells within the tumors of treated mice. In addition, splenocytes derived from ONO-8713-treated mice demonstrated increased cytotoxicity against CT26 tumor cells, relative to untreated mice. Conclusion: 1) Targeting EP1 receptor signaling reduces tumor development In Vivo. 2) The mechanism is indirect but is associated with reduced FasL expression and increased anti-tumor immune responses. The findings underscore the value of unraveling the mechanisms regulating FasL expression in the search for new therapeutic targets against cancer.