Abstract A83: Induction of PD-L1 expression by cytotoxic agents through activation of NF-kB signal
Authors: Hamanishi Junzo , Matsumura Noriomi , Jin Peng , Abiko Kaoru , Horikawa Naoki , Yamaguchi Ken , Baba Tsukasa , Murphy Susan K. , Konishi Ikuo , Mandai Masaki 
Published: August 2014

Recently, growing evidence has shown that the therapeutic efficacy of chemotherapy largely depends on the host immune system. We analyzed gene expression microarray dataset GSE13525 to examine how cytotoxic agents influence the immune system. Expression of programmed cell death 1 ligand 1 (PD-L1) was increased following carboplatin (CBDCA) treatment in the human ovarian cancer cell line 36M2 from GSE13525 (p&lt;0.05). Previously, we reported PD-L1 expression is associated with poor prognosis in ovarian cancer (Hamanishi J, PNAS, 2007), and that PD-L1 promotes progression of ovarian cancer by inducing host immune tolerance through its receptor programmed cell death 1 (PD-1) on peripheral cytotoxic T lymphocytes (Abiko K, Clin Can Res, 2013). The aim of this study was to investigate the molecular mechanism of chemotherapy-induced PD-L1 expression in ovarian cancer cells and to examine if inhibition of the PD-L1/PD-1 signal increases the efficacy of chemotherapy in ovarian cancer.

Pathway analysis of gene expression microarray data from clinical ovarian cancer samples collected before and after chemotherapy (GSE15622) showed that both paclitaxel (PTX) (n=20) and CBDCA (n=14) treatment activated the NF-kB signaling pathway (single-sample Gene Set Enrichment Analysis; p&lt;0.01, respectively). We treated human ovarian cancer cell lines (OVCAR8, ovary1847, RMG-II and SKOV3) and mouse ovarian cancer cell lines (ID8 and HM-1) with CBDCA (100uM), PTX (10uM), gemcitabine (GEM: 120uM), liposomal doxorubicine (2ug/ml) or endoxan (7uM) for 24 hours. All of these cytotoxic agents increased NF-kB-p65 and PD-L1 protein expression detected by Western blotting in all of the cell lines tested. Increased expression of PD-L1 was also confirmed using flow cytometry in HM1 and ID8 cells treated with PTX or GEM (p&lt;0.05, respectively). Following siRNA-mediated knockdown of NF-kB in HM1, ID8, ovary1847 and OVCAR8, induction of PD-L1 expression was drastically impaired by the cytotoxic agents PTX or GEM. This change was independent of IFN-gamma signaling, which is a well-known activator of PD-L1 expression, because IFN-gamma was not secreted from the treated cells, and because shRNA-mediated knock down of IFN-gamma receptor 1 also did not change PD-L1 expression.

In order to determine if PD-L1 expression on ovarian cancer cells induced by cytotoxic agents could serve as a molecular target in ovarian cancer, we performed in vivo experiments. We performed lentiviral transduction into ID8 cells using PD-L1 cDNA (ID8-PDL1), vector control (ID8-control) and microRNA targeting PD-L1 mRNA (ID8-mirPDL1). Following intraperitoneal injection of these ID8 cells into syngeneic C57/BL6 mice, we injected PTX or vehicle intraperitoneally (6 groups, n=12 in each group). The ID8-PD-L1 mice without PTX showed the worst prognosis, while ID8-mirPDL1 mice with PTX showed the best prognosis (p&lt;0.01). In an independent experiment using GEM instead of PTX, the ID8-mirPDL1 with GEM treatment again showed the best prognosis (p&lt;0.01).

In summary, we found chemotherapeutic agents upregulate PD-L1 expression through activation of NF-kB signaling. Immunotherapeutic targeting of the PD-L1/PD-1 signal combined with chemotherapy may be a promising treatment modality against ovarian cancer.

Citation Format: Junzo Hamanishi, Noriomi Matsumura, Peng Jin, Kaoru Abiko, Naoki Horikawa, Ken Yamaguchi, Tsukasa Baba, Susan K. Murphy, Ikuo Konishi, Masaki Mandai. Induction of PD-L1 expression by cytotoxic agents through activation of NF-kB signal. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr A83.