Genome-Wide and Phenotypic Evaluation of Stem Cell Progenitors Derived From Gprc5a-Deficient Murine Lung Adenocarcinoma With Somatic Kras Mutations

Abstract

Lung adenocarcinomas (LUADs) with somatic mutations in the KRAS oncogene comprise the most common molecular subtype of lung cancer in smokers and present with overall dismal prognosis and resistance to most therapies. Our group recently demonstrated that tobacco carcinogen-exposed mice with knockout of the airway lineage G-protein coupled receptor, Gprc5a , develop LUADs with somatic mutations in Kras . Earlier work has suggested that cancer stem cells (CSCs) play crucial roles in clonal evolution of tumors and in therapy resistance. To date, our understanding of CSCs in LUADs with somatic Kras mutations remains lagging. Here we derived CSCs (as spheres in 3D cultures) with self-renewal properties from a murine Kras -mutant LUAD cell line we previously established from a tobacco carcinogen-exposed Gprc5a −/− mouse. Using syngeneic Gprc5a −/− models, we found that these CSCs, compared to their parental isoforms, exhibited increased tumorigenic potential in vivo , particularly in female animals. Using whole-transcriptome sequencing coupled with pathways analysis and confirmatory PCR, we identified gene features ( n = 2,600) differentially expressed in the CSCs compared to parental cells and that were enriched with functional modules associated with an augmented malignant phenotype including stemness, tumor-promoting inflammation and anti-oxidant responses. Further, based on in silico predicted activation of GSK3β in CSCs, we found that tideglusib, an irreversible inhibitor of the kinase, exhibited marked anti-growth effects in the cultured CSCs. Our study underscores molecular cues in the pathogenesis of Kras -mutant LUAD and presents new models to study the evolution, and thus high-potential targets, of this aggressive malignancy.

Acetylsalicylic acid and salicylic acid present anticancer properties against melanoma by promoting nitric oxide-dependent endoplasmic reticulum stress and apoptosis

“…The ability of cultured cancer cells to grow as nonadherent spheroids is a potent predictor of tumor growth and is currently used to evaluate potential anticancer agents [28][29][30][31][32] . Three-dimensional in vitro cell culture techniques enable modeling of tumors and their microenvironments to aid in cancer drug discovery [33][34][35][36][37][38] .…”

Anti-Tumor Effects of Biomimetic Sulfated Glycosaminoglycans on Lung Adenocarcinoma Cells in 2D and 3D In Vitro Models

“…In addition, the qRT-PCR preliminary results of the cultivated spheres (n = 1) showed that the increase in the sulfation of biomimetic GAGs led to the decrease in the stemness properties of CSCs by down-regulation of some CSCs' markers ( Figure S5) [49][50][51][52][53][54][55][56]. Results showed downregulation of ALDH1A1 and ALDH3A1 in H1792 cells and Aldh1a1 in MDA-F471 cells, which are known to be overexpressed in KRAS-mutant LUAD CSCs, the downregulation of CCL20 in H1792 and MDA-F471, which plays a major role in the growth and metastasis of lung cancer cells, and the downregulation of Alcam and Tnf in MDA-F471, epithelial cell marker and proinflammatory cytokine, respectively [48]. These findings are consistent with other studies that stated the effects of anti-tumor agents on the CSC population using sphere-forming assay [46][47][48][49][50]57,58].…”

“…Results showed downregulation of ALDH1A1 and ALDH3A1 in H1792 cells and Aldh1a1 in MDA-F471 cells, which are known to be overexpressed in KRAS-mutant LUAD CSCs, the downregulation of CCL20 in H1792 and MDA-F471, which plays a major role in the growth and metastasis of lung cancer cells, and the downregulation of Alcam and Tnf in MDA-F471, epithelial cell marker and proinflammatory cytokine, respectively [48]. These findings are consistent with other studies that stated the effects of anti-tumor agents on the CSC population using sphere-forming assay [46][47][48][49][50]57,58]. Furthermore, they motivate further investigations to determine the ability of AlgSulf n to target and affect CSCs growth and the pathway that is affected by the AlgSulf n .…”

“…Targeting CSCs is one of the aims of targeted cancer therapy given the evidence that supports the role of CSCs in tumorigenesis and tumor re-occurrence [46,47]. Based on a previous study from our team that showed the significant expression of CSC-properties in H1792 and MDA-F471 spheres at G2 and G5 [48], we analyzed the sphere-forming potentials of AlgSulf n -treated LUAD cells at G2. Our results verified that the increase in the DS of AlgSulf n significantly decreased the sphere formation capacity of human and murine LUAD cells where the SFU and the size of spheres at G2 decreased.…”

“…This sensitivity may be because the murine cell line was developed from a lung adenocarcinoma in a mouse exposed to tobacco carcinogen, which made this cell line more aggressive. Another possible cause is the mutational landscape in the murine tumor cells, MDA-F471 cells carry Kras G12D variants, Kras gene amplification and very high somatic mutation burden while the H1792 cell line carries KRAS G12C variant and other co-occurring driver mutations as well as lower somatic mutation burden compared to the murine cell line [48]. These genomic differences may also extend to GF/receptor signaling pathways between the human and murine cells and thus, their anti-proliferative and migratory responsiveness to the sulfated biomimetic GAGs in monolayer and sphere assays [57].…”