CASK promotes non-small cell lung cancer growth through coordinated regulation of EGFR expression, trafficking, and p21 expression

Background: Calcium/calmodulin-dependent serine protein kinase (CASK), a member of the membrane-associated guanylate kinase (MAGUK) family, functions as a multifunctional scaffold protein by engaging diverse binding partners. Although CASK has been implicated in tumorigenesis, its molecular role in the pathogenesis of non-small cell lung Cancer (NSCLC) remains poorly characterized.

Methods: CASK expression was assessed by immunohistochemistry in lung adenocarcinoma tissues and matched non-tumor samples. Bioinformatics analyses were performed using Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) datasets. Functional studies were conducted in epidermal growth factor receptor (EGFR) wild-type H1299 cells and EGFR exon 19-deleted PC9 cells following CASK silencing. RNA-Seq was used to identify differentially expressed genes, and assays of cell growth, cell cycle progression, EGFR trafficking, and downstream signaling were performed.

Results: CASK expression was significantly elevated in early-stage NSCLC, and higher levels correlated with reduced overall and first progression survival. Silencing CASK inhibited cell growth by blocking the G1-S transition and inducing p21waf1/Cip1, a key mediator of cell cycle arrest, at both transcript and protein levels. Growth suppression was rescued by treatment with a p21 inhibitor UC2288, confirming its central role. Elevated p21 in CASK-deficient cells was reversed by EGFR, Akt, and ERK inhibitors, suggesting that CASK is involved in downregulating p21 via suppressing EGFR/ERK-dependent transcription and EGFR/AKT-dependent protein stabilization. Although p53 is constitutively involved in upregulation of p21 gene expression, the findings from the p53 inhibitor and p21 promoter activity assay excluded the role of p53 in CASK silencing-induced p21 upregulation. Furthermore, CASK modulated the autocrine EGFR loop. CASK silencing enhanced EGFR expression through gene transcription and post-transcriptional protein stabilization and promoted EGFR activation by upregulating TGF-α in p53-, ERK-, and AKT-dependent manners. Notably, CASK loss did not affect EGFR trafficking to early endosomes but delayed its transition to late endosomes in an ERK-dependent fashion, thereby reducing receptor degradation.

Conclusions: We identified CASK as a previously unrecognized driver of NSCLC growth and a potential prognostic biomarker. By regulating EGFR trafficking to late endosomes and attenuating Akt and ERK signaling, CASK suppresses p21 expression and promotes NSCLC cell proliferation, revealing a novel proliferation regulator in NSCLC.