FSTL4 interaction with Gαi proteins activates the Akt-mTOR pathway to drive malignant phenotypes in non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) presents a critical challenge clinically; thus, identifying novel drivers and reliable prognostic biomarkers is imperative. Here, we investigated the clinical relevance, cell-type-specific localization, and mechanistic role of Follistatin-like 4 (FSTL4) in NSCLC. Analysis of The Cancer Genome Atlas (TCGA) and local resected tissue samples show that FSTL4 expression is significantly elevated in NSCLC tissues compared to normal lung tissues, and serves as an independent prognostic indicator for reduced overall survival. Single-cell RNA sequencing confirmed that FSTL4 expression is predominantly and consistently localized to the malignant epithelial cell population across primary tumors and metastatic niches in lung adenocarcinoma (LUAD). Functionally, stable knockdown or CRISPR/Cas9-mediated knockout of FSTL4 across multiple NSCLC cell types suppressed key malignant phenotypes, including cell proliferation, and migration, and inducing G1-phase cell cycle arrest and triggering intrinsic apoptosis. This cytotoxic dependency was selective to malignant cells, as normal lung epithelial cells remained unaffected. Conversely, ectopic overexpression of FSTL4 enhanced these aggressive characteristics. Mechanistically, FSTL4 exerts its pro-oncogenic effects through regulating the Akt-mTOR signaling cascade. FSTL4 physically interacts with the inhibitory G-protein subunits Gɑi1 and Gɑi3, and the co-depletion of these subunits inhibited FSTL4-mediated Akt-mTOR activation. Crucially, the functional effects of FSTL4 silencing in primary human NSCLC cells were rescued by the introduction of a constitutively active Akt1 (caAkt1, S473D) construct, confirming Akt as the essential required downstream transducer. In vivo validation further demonstrated that FSTL4 silencing profoundly attenuated NSCLC xenograft tumor growth by reducing proliferation and accelerating apoptosis, mirroring the diminished Akt-mTOR activation observed in vitro. Collectively, these comprehensive findings unequivocally establish that elevated FSTL4 expression promotes malignant phenotypes of NSCLC possibly by activating Gɑi1/3-Akt-mTOR cascade.