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N-WASP deficiency enhances oncogenic KRAS-mediated lung adenocarcinoma progression in mice

ABSTRACT: Objective: Oncogenic KRAS mutations drive 25-30% of western lung adenocarcinoma (LUAD) cases, however the mechanisms that restrain tumor progression remain poorly defined. N-Wiskott–Aldrich syndrome protein (N-WASP) regulates actin cytoskeleton dynamics and epithelial architecture, but its role in KRAS-driven lung tumorigenesis is unclear. Methods: We generated a conditional N-WASP knockout in KRASG12D-driven lung adenocarcinoma mice (N-WASPLKO/KRASG12D) using Sftpc-CreERT2, and assessed N-WASP knockdown A549 cells (A549KD) in 3D culture for tumor burden, ERK signaling, transcriptomic changes, and E-cadherin expression.Results: Conditional deletion of N-WASP in type II alveolar cells (N-WASPLKO) markedly increased tumor number and size in KRASG12D mice. The tumors isolated from these mice retained strong pro–surfactant protein C expression, confirming alveolar epithelial origin and predominantly adopted a solid growth pattern, indicative of disrupted epithelial organization. Mechanistically, N-WASP loss enhanced KRAS-driven ERK1/2 activation in lung tissue, consistent with increased mitogenic signaling. In A549 cells, N-WASP deficiency disrupted cytoskeletal architecture, promoted proliferation, and impaired cell–cell adhesion, recapitulating in vivo tumor phenotypes. Transcriptomic profiling of N-WASP–deficient lungs revealed coordinated upregulation of genes regulating proliferation, cytoskeletal remodeling, and tumor-promoting pathways, alongside downregulation of epithelial differentiation and adhesion programs.

ORGANISM(S): Mus musculus

PROVIDER: GSE343586 | GEO | 2026/09/08

REPOSITORIES: GEO

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Project description:Background: LKB1 is among the most frequently altered tumor suppressors in lung adenocarcinoma. Inactivation of Lkb1 accelerates the growth and progression of oncogenic KRAS-driven lung tumors in mouse models. However, the molecular mechanisms by which LKB1 constrains lung tumorigenesis and whether the aggressive cancer state that stems from Lkb1 deficiency can be reverted remains unknown. By bulk gene expression profiling, Lkb1 restoration promotes the expression of markers and functions of alveolar type II cells, suggesting that LKB1 may govern a cell-state transition within the neoplastic epithelial compartment. Purpose: To determine whether the restoration of Lkb1 drives changes in cell state and/or abundance within established oncogenic KRAS-driven lung tumors. Approach: To control LKB1 function in vivo, we generated an Lkb1XTR allele, which enables Cre-mediated disruption of Lkb1 expression during tumor development and subsequent FLPo-ERT2-mediated reactivation of Lkb1 within established tumors. Lung tumors were initiated in KT;Lkb1XTR/XTR (non-restorable) and KT;Lkb1XTR/XTR;FLPo-ERT2 (restorable) mice with Lenti-Cre. Following tumor development, lung tumor-bearing were treated with either corn oil vehicle or tamoxifen for two weeks prior to isolating specifically neoplastic cells by FACS for single cell RNA-seq. Results: Single cell analysis revealed that the neoplastic epithelial compartment was composed of alveolar type I- and type II-like subpopulations, a Krt8+ transitional state, an actively proliferating subpopulations, and an indeterminate subpopulation partially resembling the alveolar type II-like identity. Dynamic inference analyses indicated that the indeterminate population represents an intermediate state along the alveolar type II to alveolar type I trans-differentiation trajectory. Notably, the indeterminate cluster was more proliferative than the alveolar type II-like subpopulation and exhibited higher expression of Sox9, which is a marker of distal lung epithelial progenitors. There was a marked shift in the epithelial compartment from the indeterminate state to alveolar type II epithelial-like identity in response to Lkb1 restoration. Conclusions: LKB1 governs the transition between a proliferative progenitor-like population and mature alveolar type II-like identity within oncogenic KRAS-driven lung tumors

Project description:Lung adenocarcinoma is responsible for significant global mortality with limited effective treatments. Although some studies suggest that these tumors arise from alveolar epithelial type 2 cells (AEC2s), there is scant information regarding the early events that might occur in human AEC2s at the inception of oncogenesis. This limitation, is partially due to a lack of human model systems that recapitulate the initiation of oncogenesis in AEC2s. Unfortunately, primary AEC2s from patients are difficult to access in vivo or stably maintain in cell cultures. Hence, we sought to develop an in vitro system to model the early stages of oncogenesis utilizing human induced AEC2s (iAEC2s) generated through the directed differentiation of induced pluripotent stem cells (iPSCs). To this end, we selected a normal human iPSC line we have previously engineered to carry fluorochrome reporters targeted to lung epithelial-specific loci, NKX2-1GFP and SFTPCtdTomato that enable monitoring and purification of alveolar lung epithelial cells. To test the effects of adenocarcinoma oncogene induction in these cells, we targeted a third locus, AAVS1 using gene editing to engineer a doxycycline-inducible cassette encoding mutant KRASG12D, the most commonly found oncogene in lung adenocarcinomas. Successful induction of KRASG12D with doxycycline was demonstrated in both the targeted undifferentiated iPSCs as well as in the iAEC2s derived from these cells. We profiled the downstream effects of KRASG12D induction in iAEC2s, comparing dox vs vehicle exposed cells by cell counting, FACS for NKX2-1GFP/SFTPCtdTomato, RT-qPCR, deep proteomic and phosphoproteomic analyses, and scRNA-sequencing. Through this characterization, we found that induction of KRASG12D robustly activates MAPK signaling resulting in a shift of iAEC2s away from their mature alveolar program towards a distal lung epithelial progenitor phenotype. Successful modeling of lung adenocarcinoma with this model system has a variety of future applications, including testing unknown mechanisms for oncogenesis, discovery of novel biomarkers of disease, or development of new effective treatment methods through drug screening.

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N-WASP deficiency enhances oncogenic KRAS-mediated lung adenocarcinoma progression in mice

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