Osteosarcoma cell intrinsic PD-L2 signals promote invasion and metastasis via the RhoA-ROCK-LIMK2 and autophagy pathways

Abstract

Known as co-stimulatory molecule, programmed death ligand-2 (PD-L2) contributes to T-cell exhaustion by interaction with programmed death-1 (PD-1) receptor, but its tumor cell-intrinsic signal effects have been little investigated. PD-L2 expression was detected by immunohistochemistry in 18 pairs of primary osteosarcoma tissues and matching lung metastasis tissues. We also investigated the effects of PD-L2 knockdown on osteosarcoma both in vitro and in vivo. In our study, PD-L2 expression was elevated in lung metastases compared with primary osteosarcoma according to an immunohistochemistry assay. Wound-healing and transwell assays revealed that PD-L2 knockdown leaded to inhibition of migration and invasion of human osteosarcoma cells in vitro. Mechanistically, we demonstrated that PD-L2 knockdown attenuated migration and invasion by inactivating RhoA-ROCK-LIMK2 signaling, suppressing epithelial–mesenchymal transition (EMT), and inhibiting autophagy by decreasing beclin-1 expression. In support of these observations, beclin-1 knockdown also inhibited activation of the RhoA-ROCK-LIMK2 pathway, leading to autophagy inhibition-induced blockade of migration and invasion. Depletion of PD-L2 in KHOS cells markedly weakens pulmonary metastatic potential in vivo by orthotopic transplantation of nude mice. Our study reveals a pro-metastatic functional mechanism for PD-L2 in osteosarcoma. Furthermore, we demonstrate a regulatory role for PD-L2 on autophagy, as well as a relationship between autophagy and metastasis in osteosarcoma, which may represent a potential therapeutic target for osteosarcoma.

Biological Function of PD-L2 and Correlation With Overall Survival in Type II Endometrial Cancer

“…PD-L1 regulates cancer cell resistance to apoptosis (22), cell proliferation, AKT/mTOR signaling pathway, and autophagy in ovarian cancer cells (13,15), while in vitro data, describing an intrinsic role of PD-L2 in cancer cells, are scarce. A recent study demonstrates that cell-intrinsic PD-L2 signals promote invasion and metastasis through the Rhoa-ROCK-LIMK2 and positively regulate autophagy pathways in osteosarcoma cells (14). In this study, we demonstrate that PD-L2 increases migration of the mixed type I/II PC-EM004b cell line, while the opposite effects were observed by PD-L2 silencing in the Ishikawa cell line, a type I model that expresses high levels of PD-L2.…”

“…PD-1 ligands are transmembrane proteins that binds PD-1 expressed on the cellular surface of activated T and B cells, monocytes, natural killer, and dendritic cells (23,35). Recent studies showed also an intracellular distribution, suggesting that these proteins might have an unexpected function, different from what has previously been described for its membranous counterpart (13,14,36,37). It has been demonstrated that cytoplasmic PD-L1 levels in SKOV3 and HO8910 ovarian cancer cell lines are high, and in SKOV3 cells, cytoplasmic PD-L1 increased cancer cell growth and migration (36).…”

“…Emerging evidence shows that PD-L1 and PD-L2 also activate tumor-intrinsic functions (13,14,22). PD-L1 regulates cancer cell resistance to apoptosis (22), cell proliferation, AKT/mTOR signaling pathway, and autophagy in ovarian cancer cells (13,15), while in vitro data, describing an intrinsic role of PD-L2 in cancer cells, are scarce.…”

“…However, the prognostic value of PD-1 ligands is still debated, and their role, when expressed in the tumor microenvironment, has not been fully elucidated yet (9). It is known that PD-L1 and PD-L2 interaction with PD-1 receptor contributes to the strong inhibition of T-cell antitumor effects, resulting in immune escape (10,13,14). Indeed, previous studies showed that PD-L1 exerts tumor crucial cell-intrinsic signals for pathogenesis, including epithelial-mesenchymal transition, autophagy, resistance against proapoptotic stimuli, regulation of glucose metabolism, and activation of tumor mTOR/AKT signaling, supporting cancer cell proliferation and survival (13,15).…”

“…PD-L1 blocking antibodies have been approved for clinical use (15), while current and ongoing studies are trying to improve clinical responses for EC (16). So far, compared with PD-L1, the functional role of PD-L2 in cancer cells has been scarcely investigated (14). However, in patients with solid cancer, a meta-analysis suggested that PD-L2 might be involved in promoting tumor metastasis and predicts unfavorable prognosis, mainly in hepatocellular carcinomas (17).…”