Simple SummaryLung cancer is the most common and lethal malignancy worldwide. Musashi-2 (MSI2) is an RNA-binding protein that is overexpressed in advanced NSCLC. VEGFR2 protein expression contributes to NSCLC progression and several FDA-approved drugs are used to target it in the clinic. Here, we show that MSI2 is a strong positive regulator of VEGFR2 protein levels in murine and human NSCLC cell lines. Furthermore, we found that MSI2 protein directly binds toVEGFR2andPTENmRNAs and impacts VEGFR2 downstream signaling, in part via PTEN regulation.AbstractLung cancer is the most frequently diagnosed cancer type and the leading cause of cancer-related deaths worldwide. Non-small cell lung cancer (NSCLC) represents most of the diagnoses of lung cancer. Vascular endothelial growth factor receptor-2 (VEGFR2) is a member of the VEGF family of receptor tyrosine kinase proteins, which are expressed on both endothelial and tumor cells, are one of the key proteins contributing to cancer development, and are involved in drug resistance. We previously showed that Musashi-2 (MSI2) RNA-binding protein is associated with NSCLC progression by regulating several signaling pathways relevant to NSCLC. In this study, we performed Reverse Protein Phase Array (RPPA) analysis of murine lung cancer, which suggests that VEGFR2 protein is strongly positively regulated by MSI2. Next, we validated VEGFR2 protein regulation by MSI2 in several human lung adenocarcinoma cell line models. Additionally, we found that MSI2 affected AKT signaling via negativePTENmRNA translation regulation. In silico prediction analysis suggested that bothVEGFR2andPTENmRNAs have predicted binding sites for MSI2. We next performed RNA immunoprecipitation coupled with quantitative PCR, which confirmed that MSI2 directly binds to VEGFR2 and PTEN mRNAs, suggesting a direct regulation mechanism. Finally, MSI2 expression positively correlated with VEGFR2 and VEGF-A protein levels in human lung adenocarcinoma samples. We conclude that the MSI2/VEGFR2 axis contributes to lung adenocarcinoma progression and is worth further investigations and therapeutic targeting.