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Unbiased genomic analysis of multiple stages of lung cancer development

ABSTRACT: To uncover the gene expression alterations that occur during lung cancer progression, we interrogated the gene expression state of neoplastic cells at different stages of malignant progression. We initiated tumors in KrasLSL-G12D/+;p53flox/flox;R26LSL-tdTomato (KPT) mice with a pool of barcoded lentiviral-Cre vectors and purified Tomatopositive cancer cells away from the diverse and variable stromal cell populations. Five to nine months after tumor initiation, cancer cells were isolated from individual primary tumors and metastases using fluorescence-activated cell sorting. Sequencing of the barcode region of the integrated lentiviral vectors established primary tumor-metastasis and metastasis-metastasis relationships. Tumor barcoding allowed us to unequivocally distinguish non-metastatic primary tumors (TnonMet) from those primary tumors that had seeded metastases (TMet). We profiled 10 TnonMet samples as well as TMet and metastasis (Met) samples representing 12 metastatic events. To examine additional earlier stages of lung cancer development, we also analyzed premalignant cells from hyperplasias that develop in KPT mice shortly after tumor initiation (KPT-Early; KPT-E), as well as tumors from KrasG12D;R26LSL-tdTomato (KT) mice which rarely gain metastatic ability

ORGANISM(S): Mus musculus

PROVIDER: GSE84447 | GEO | 2017/02/06

SECONDARY ACCESSION(S): PRJNA329195

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. 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.

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. Purpose: To assess the impact of CRISPR/Cas9-mediated targeting of a subset of LKB1-dependent genes and C/EBP factors on tumor size in the context of 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 a series of LKB1-dependent genes as well a subset of C/EBP transciription factors. 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: Targeting of Lkb1 dramatically increased lung tumor size relative to inert sgRNAs. Little to no impact on tumor size was observed upon targeting LKB1-dependent genes. However, simultaneous targeting of Cebpa/b/d resulted in a significant increase in tumor size. Conclusions: A subset of C/EBP transcription factors constrain oncogenic KRAS-driven lung tumor growth.

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Unbiased genomic analysis of multiple stages of lung cancer development

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