LKB1 drives stasis and C/EBP-mediated reprogramming to an alveolar type II fate in lung cancer [Secondary Tuba-seq]
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ABSTRACT: 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. Purpose: To ascertain the tumor-suppressive capacity C/EBP family members, including Cebpa, Cebpb, and Cebpd, through CRISPR/Cas9-mediated targeting in a genetically engineered mouse model of oncogenic KRAS-driven lung adenocarcinoma. Approach: Measurements of tumor size across genetic perturbations were obtained by tumor barcode sequencing (Tuba-seq; Rogers et al., 2017 Nature Methods). Briefly, lung tumors were initiated in KrasLSL-G12D/+;R26LSL-tdTomato (KT) and KT;H11LSL-Cas9 mice using a pool of Lenti-sgRNA/Cre vectors that are each modified with two-component barcodes composed of sgRNA-specific and clonal identifiers. This particular pool of Lenti-sgRNA/Cre vectors was composed of vectors targeting each of the three C/ebp paralogs individually, in pairwise combinations, and all three simultaneously. Following tumor development, the two-component lentiviral barcodes integrated within transduced populations were amplified from genomic DNA of whole-lung homogenates. The resulting amplicons were then subjected to next-generation sequencing. From the resulting reads, barcode pileups were generated and filtered prior to conversion to absolute numbers of neoplastic cells via normalization to spiked-in samples of known quantities of barcoded neoplastic cells. The resulting tumor size distributions were then analyzed by multiple statistical measures as described previously (Rogers et al., 2017 Nature Methods). Results: The targeting of Cebpa significantly increased tumor size relative to tumors initiated with vectors encoding inert sgRNAs (oncogenic KRAS only tumors). The targeting of Cebpb and Cebpd had no significant impact on tumor growth. There were no additive effects of knocking out additional paralogs on top of Cebpa. Conclusions: C/EBPa is the dominant tumor-suppressive paralog and there was no evidence of functional redundancy among C/EBPa, C/EBPb, and C/EBPd.
ORGANISM(S): Mus musculus
PROVIDER: GSE179499 | GEO | 2021/12/23
REPOSITORIES: GEO
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