Expression and signaling of Toll-like receptor 4 (TLR4) and MyD88 in ovarian carcinoma cells

e16508 Background: TLR4, expressed by the cells of the immune system play a role in the protection of the host against pathogens. TLRs are also expressed on human cancer cells, but their role in tumor growth is unknown. The aim of this study was to correlate the presence of TLR4 and MyD88 expression with clinicopathologic outcome in patients with ovarian cancer and to analyze the consequences of signaling via the TLR4/MyD88 pathway in ovarian cancer cell lines. Methods: Tumor specimens from 41 patients with ovarian carcinoma were evaluated for TLR4 and MyD88 by immunohistochemistry and correlated with clinical and pathologic disease features. TLR4/MyD88 expression in OVCAR3, SKOV3, and A2780 was determined using RT-PCR, WB, and immunohistochemistry. NF-kB translocation to nucleus was measured by confocal microscopy. Culture supernatants were tested for levels of cytokines in Luminex-based assays. Proliferation of cancer cells was measured in the CFSE assays. Their sensitivity to paclitaxel (PLX) was measured by Annexin V binding. Western Blot analysis was used to measure activation of the PI3K/Akt, IRAK 1, IRAK 4, and TRIF. Results: In ovarian cancer patients TLR4 and MyD88 expression by the tumor was observed in 100% and 83% of tissues, respectively. The expression of MyD88 was associated with shorter progression-free survival (42 vs 31 months, p < 0.05). Ex vivo studies showed that TLR4 was expressed on OVCAR3, SKOV3, and A2780 cell lines, while A2780 did not expressed MyD88. In MyD88+ tumor cells, LPS increased proliferation (PI 17 vs 8, p < 0.05), activated NF-kB pathway and promoted cytokine production (IL-8, IL-6, RANTES, VEGF and MCP-1). LPS and PLX binding to TLR4 on MyD88+ cells induced activation of PI3K/Akt, IRAK4, and IRAK1, but decreased expression of pro-apoptotic TRIF. In contrast, in MyD88(-) cells LPS did not induce proliferation and neither LPS nor PLX induced secretion of pro-inflammatory cytokines. Further, no changes were noted in IRAK1 expression, but strong signal was observed for TRIF. TLR4+/MyD88+ tumor cells showed grater resistance to PLX. Conclusions: Our ex vivo studies elucidate the molecular mechanisms involved in TLR4/MyD88 signaling. Ligation via TLR4 leads to tumor growth, release of proinflammatory cytokines and induction of resistance to PLX-induced apoptosis. No significant financial relationships to disclose.