Effect of Toll-Like Receptors 4, 5 and 7 during Prostate Tumor Progression in the Transgenic Adenocarcinoma of Mouse Prostate Model

Abstract

Toll-like receptors (TLRs), a family of transmembrane proteins that recognize highly conserved molecules in pathogens, have an important role on the triggering and promoting inflammation. Recent evidence showed that the stimulation of TLRs in cancer cells can inhibit or promote tumorigenesis dependently on receptors or tumor cell type. TLR signaling may play an important role in tumor development and activation of TLRs might play a double-edged sword role in the influence of tumor progression. Moreover, in vitro and in vivo studies often lead to conflicting results depending on experimental conditions. Although various TLRs have been associated with immune response and tumorigenesis in the prostate cells, little is known about the role of TLR5 and 7. The TLR4 signaling and expression in prostate cancer cells are remained controversial. In this study, association of TLR4, 5, and 7 in the progression of prostate transformation was examined using in vivo and in vitro prostate model. Chapter I study showed that expression of TLR4 and TLR5 was associated with progression of prostate transformation in the transgenic adenocarcinoma of mouse prostate (TRAMP) model. The expression of TLR4 and TLR5 was evaluated by immunohistochemisty in formalin-fixed paraffin-embedded prostate tissue from wild-type (WT) and TRAMP mice. Normal prostate tissue from WT mice showed strong expression of TLR4 and TLR5. However, TLR4 expression in the prostate tissue from TRAMP mice gradually decreased as pathologic grade became more aggressive. TLR5 expression in the prostate tissue from TRAMP mice also decreased in low-grade prostate intraepithelial neoplasia (PIN), high-grade PIN and poorly differentiated adenocarcinoma. Overall, our results suggest that decreased expression of TLR4 and TLR5may contribute to prostate tumorigenesis. TLR7 expression and function on tumorigenesis have been examined in several cancers including non-small cell lung cancer (NSCLC) and esophageal squamous cell carcinoma (SCC). However, the role of TLR7 on prostatic tumor development has yet to be clarified. Chapter II examined the expression of TLR7 during tumor progression of TRMAP mice and its role on cell growth. Strong expression of TLR7 was detected in the normal prostate epithelia of WT mice, but not in TLR7-deficient mice. In contrast, TLR7 expression was weak in TRAMP-C2 cells, as compared with murine bone marrow derived macrophages (BMDMs). Moreover, TLR7 mRNA was markedly expressed in RWPE-1 cells (non-cancerous prostate epithelial cells), but not in PC3 and DU145 (prostate cancer cells). Immunohistochemically, TLR7 expression gradually decreased in TRAMP mice depending on the pathologic grade of the prostate cells. TLR7 agonists increased both the gene and protein expression of TLR7 and promoted production of proinflammatory cytokines/chemokines and IFN-β gene expression in prostate cancer cell lines. Moreover, TLR7 agonist, loxoribine inhibited the growth and colony formation of TRAMP-C2 cells dependent of TLR7. In chapter III, anti-tumor effect of the small molecule imiquimod, also known as a TLR7 agonist, was examined in prostate cancer. Imiquimod inhibited the growth of mouse (TRAMP C2) and human (PC-3) prostate cancer cells. Treatment with imiquimod induced cell cycle arrest at the G2/M phase in TRMPA-C2 cells. Finally, imiquimod induced direct apoptosis in TRAMP-C2 cells via a mitochondrial dependent pathway. Intratumoral injection with imiquimod reduced significantly tumor growth and increased apoptotic cells in mice subcutaneously implanted with TRAMP-C2 cells. These findings suggest that TLRs (TLR4, 5, 7) may participate in tumor suppression and represent a promising class of immune response enhancers with the potential to generate an effective antitumor immune response by create a tumor microenvironment in the in vivo TRAMP model. Moreover, the stimulation of TLR7 agonist appears to be augmented by suppression of a regulatory mechanism with pro-inflammatory responses in prostate cancer. Specially, loxoribine would result in a highly efficient immune system activation and give rise to an enhanced anti-tumor activity in in vivo and in vitro prostate cancer. TLR7 signaling pathway suggests that TLR7-mediated tumor suppression by TLR7 agonists for prostate cancer supports immune-based therapies.