PTEN Signaling at the Tumor: Immune Interface

Abstract

The phosphatase and tensin homolog, PTEN, modulates a wide array of signaling processes that control cell metabolism and proliferation. Preclinical studies in gliomas and prostate carcinomas found that loss of the Pten gene imparts a senescent phenotype with diminished response to radiation and suppressed anti-tumor immunity. We utilized two tumor models of Pten mutation and analyzed immunologically relevant phenotypic changes, including after administration of radiation. Cell cycle distribution was analyzed on HeLa cells with PTEN knockdown following a single fraction of 10 Gy radiation. To further assess radiation responsiveness, Pten wild type and knockout mouse TSA breast cancer cells were treated with 5 Gy of radiation and evaluated for colony formation. HMGB1 release was measured as a surrogate of immunogenic cell death: specifically, TSA cells expressing a recombinant HMGB1-RFA fusion protein were transfected with Pten siRNA and treated with a single fraction of radiation, after which secreted levels of the fusion protein were quantified. Additionally, Pten+/+ and Pten-/- TSA cells were treated with a single fraction of radiation, and PD-L1 expression was analyzed by qPCR 24 hours later. Cell cycle analysis revealed a defect of the DNA damage checkpoint in PTEN knockdown HeLa cells. Following radiation, 64% of control HeLa cells were arrested at G2/M compared to only 34% of PTEN knockdown cells. Furthermore, irradiated Pten-/- TSA cells showed more resilient colony formation than Pten+/+ cells. The analysis of HMGB1 as a surrogate of immunogenic cell death demonstrated a 27% relative decrease of secreted HMGB1 following a single fraction of high dose radiation in Pten-/- cells, as compared to the Pten+/+ population (P < 0.0001). In addition, gene expression analysis of Pten+/+ and Pten-/- TSA cells revealed a 2-fold baseline increase in PD-L1 expression in Pten knockout cells. PTEN loss is a frequent alteration in cancer. We found that PTEN deficiency in tumor cells results in a reduced checkpoint response and more resilient colony formation following radiation, suggesting increased resistance. Furthermore, decreased secretion of HMGB1 and increased gene expression of PD-L1 in the Pten-/- cells are predictive of an immune resistant phenotype. Given these features, PTEN deficient tumor cells may be more prone to survive radiation therapy, even with an accumulating DNA damage. This trait, together with their expression of PD-L1, may suggest utility in employing anti PD-L1 immunotherapy. Further characterization of the impact of PTEN mutation on tumor responses to radiation and immunotherapy will help guide future treatment strategies in patients with associated mutations.