RGMb impacts partial epithelial‐mesenchymal transition and BMP2‐Induced ID mRNA expression independent of PD‐L2 in nonsmall cell lung cancer cells

Abstract

AbstractPD‐1/PD‐ligand‐axis immunotherapy‐mediated activation of T‐cells for cancer cell elimination is a promising treatment of nonsmall cell lung cancer (NSCLC). However, the effect of immunotherapy on intracellular signaling pathways in cancer cells still needs further delineation. Repulsive Guidance Molecule b (RGMb), a regulator of Bone Morphogenetic Proteins (BMPs) signaling, interacts with the PD‐ligand, PD‐L2, at cancer cell membranes. Accordingly, a clarification of the functions of RGMb and its relation to PD‐L2 might provide insight into NSCLC cell signaling responses to PD‐1/PD‐ligand‐axis immunotherapy. In this study, the functions of RGMb and PD‐L2 were examined using the two NSCLC cell lines HCC827 and A549. CRISPR/Cas9 was used to decrease the expression of RGMb and PD‐L2, while lentiviral vectors were used to increase their expression. Downstream effects were examined by RT‐qPCR and immunoassays. Ectopic expression of RGMb impacted BMP2‐induced expression of ID1 and ID2 messenger RNA (mRNA) independently of PD‐L2, while RGMb depletion by CRISPR/Cas9 did not affect the BMP2‐mediated induction of ID1, ID2, and ID3 mRNA. However, depletion of RGMb resulted in a partial epithelial‐mesenchymal transition (EMT) gene expression profile in HCC827 cells, which was not mimicked by PD‐L2 depletion. The results show that RGMb is a coregulator of BMP signaling and hence, ID mRNA expression and that RGMb can control the EMT balance in NSCLC cells. However, RGMb appears to exert these functions independently of PD‐L2, and accordingly, the PD‐1/PD‐ligand axis for immune surveillance in NSCLC cells.