PROGRAMMED DEATH LIGAND 1 (PD-L1) IMMUNE CHECKPOINT-MEDIATED STIMULATION OF MALIGNANT PHENOTYPES IN TUMOUR CELLS
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Most studies on the Programmed Death 1 (PD-1)/Programmed Death Ligand 1 (PD-L1) immune checkpoint have focused on elucidating the signaling mechanisms leading to inactivation of immune effectors. We, along with others, have shown that signaling by the PD-1/PD-L1 immune checkpoint may be bidirectional. Hence, we hypothesized that PD-1/PD-L1 signaling promotes malignant phenotypes in cancer cells, such as increased drug resistance as well as invasion and metastasis through the activation of oncogenic signaling pathways. This thesis aimed to (a) identify potential oncogenic signaling pathways downstream of PD-L1; (b) determine and characterise the mechanism of PD-1/PD-L1-induced drug resistance; and (c) examine the role of PD-1/PD-L1 signaling on cancer cell invasion and metastasis. Analysis of the TCGA-BRCA dataset indicated a correlation between PD-L1 expression and expression of various genes involved in oncogenic signaling and autophagy. Furthermore, Nanostring analysis of MDA-MB-231 cells treated with recombinant PD-1 (rPD-1) revealed a potential role for NOTCH1, WNT, MAPK and TGF-/SMAD signaling downstream of PD-L1. Since autophagy is a well-established mechanism of chemoresistance in cancer cells, we examined the contribution of this cellular process to PD-1/PD-L1-induced drug resistance. Findings revealed that exposure of human MDA-MB-231 breast cancer cells, but not DU-145 prostate cancer cells, to rPD-1 resulted in increased expression of key mediators of autophagy, including microtubule-associated protein 1A/1B-light chain 3 (LC3)-II, and Beclin-1, as well as increased autophagosome formation. Using autophagy inhibitors, we showed that drug resistance induced by reverse PD-1/PD-L1 signaling is causally linked to increased autophagy. In addition, results of our studies demonstrated a role for extracellular signal-related kinase (ERK) signaling in PD-1/PD-L1 induced malignant phenotypes. Blocking the ERK signaling pathway with U0126, a MEK inhibitor, inhibited rPD-1 induced LC3-II expression. Tumours from rPD-1 treated-4T1 mammary carcinoma-bearing mice also showed increased LC3-II expression. Our studies also show that MDA-MB-231 and DU-145 cells treated with rPD-1 demonstrate increased invasion, but not migration in vitro. Results of in vivo experiments did not support a role for PD-1/PD-L1 signaling in driving chemoresistance or metastasis. These studies reveal potential signaling pathways downstream of PD-L1 and provide a rationale for the use of PD-1/PD-L1 immune checkpoint blockers and autophagy inhibitors.