Kyoto University team clarifies mechanism behind EP2/4 inhibition of tumors ― may lead to new cancer immunotherapeutics

Cancer is the leading cause of death in Japan. On a global scale, 19.3 million new cases are diagnosed annually. Cancer immunotherapeutics have been attracting attention due to the emergence of immune checkpoint inhibitors that show dramatic efficacy. However, only about 20 to 30% of patients respond to these treatments, so research and development are underway worldwide to develop new immunotherapies.

A research group led by Professor Emeritus Shuh Narumiya, Kyoto University, has demonstrated that in an animal model of immune checkpoint inhibitor insensitive LLC1 lung cancer, the bioactive lipid Prostaglandin E2 (PGE2) promotes inflammatory response, angiogenesis, and recruitment and activation of regulatory T cells (Treg) through the receptors EP2 and EP4. The group's members included Associate Professor Dean Thumkeo and PhD student Siwakorn Punyawatthananukool, Department of Drug Discovery Medicine, Graduate School of Medicine, Kyoto University, among others. In human lung squamous cell cancer, ovarian serous cystadenocarcinoma, breast invasive cancer, and liver hepatocellular carcinoma, the expression levels of EP2 and EP4 strongly correlated with the expression levels of inflammatory response/angiogenesis-related genes and regulatory T cell mobilization/activation-related genes found in animal models, and an inverse correlation with prognosis. In other words, EP2/EP4 inhibitors may act against these human cancers by suppressing inflammation and Tregs, thereby releasing immunosuppression in the tumor microenvironment, exerting an antitumor effect.

Associate Professor Thumkeo said, "Recently, many companies such as Ono Pharmaceuticals and Eisai have been developing EP2/EP4 inhibitors, but there is a lack of scientific evidence regarding their mechanism of action, so thorough clinical trials are being conducted. Our results indicate that EP2/EP4 inhibitors are effective in human lung squamous cell cancer, ovarian serous cystadenocarcinoma, breast invasive carcinoma, and liver hepatocellular carcinoma. In the future, clinical trials focusing on specific types of cancer will likely speed up the development process." The study was published in the online edition of Cell Reports on June 7.

After confirming that EP2/EP4 inhibitor treatment significantly suppressed tumor growth in LLC1 tumors, the group examined the effects on EP2 and EP4 receptor knockout mice and observed a similar suppression of tumor growth. This result suggests that EP2 and EP4 inhibition works not through the cancer cells themselves but host-derived cells. To further investigate the mechanism, they then administered EP2/EP4 inhibitors to LLC1 cancer-bearing mice, removed the tumors, collected host-derived tumor-infiltrating immune cells by FACS, and analyzed the gene expression of a total of 31,971 of these cells using single-cell RNA sequencing. Clustering analysis was then performed according to gene expression patterns to classify the tumor-infiltrating immune cells into 15 cell populations, including myeloid-derived cells and lymphocytes. The T-cell population was further subjected to subclustering analysis and classified into four subpopulations: CD8T cells, CD4Treg cells, non-Treg CD4T cells, and amplifier T cells.

Next, gene expression changes due to EP2/EP4 inhibition were analyzed in each population. The group found that the EP2/EP4 pathway activates the NF-κB pathway in myeloid cell populations to induce the expression of inflammation and angiogenesis-related genes. However, they also found that by acting on mregDC cells (mature dendritic cells within tumors rich with immunoregulatory molecules) and CD4Treg cells, it promotes the recruitment and activation of Tregs into tumors.

The researchers also extracted survival information (5-year follow-up) and EP2 and EP4 expression levels in cancer tissues of approximately 2,500 patients with lung squamous cell cancer, breast invasive cancer, ovarian serous cystadenocarcinoma, and liver hepatocellular carcinoma who have gene expression data among the cases registered in the American Cancer Genome Atlas (TCGA). The group defined the PTGER score as the mean of the expression of EP2 and EP4. Patients were classified into two groups: those with high PTGER scores and those with low PTGER scores, with their 5-year survival curves compared.

The results suggest that PGE2-EP2/EP4 signaling acts on two opposing phenomena, activation of inflammation in myeloid cells and Treg cell-mediated immunosuppression, a novel concept.

This study's revelation that the actual role of prostaglandins in cancer and some of the molecular mechanisms that have long been suggested by epidemiological studies of aspirin, a prostaglandin synthesis inhibitor, is of great academic significance.

Journal Information
Publication: Cell Reports
Title: PGE2-EP2/EP4 signaling elicits immunosuppression by driving the mregDC-Treg axis in inflammatory tumor microenvironment
DOI: 10.1016/j.celrep.2022.110914