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LKB1 drives stasis and C/EBP-mediated reprogramming to an alveolar type II fate in lung cancer [scRNA-seq]

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 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 total viable cells by FACS for single cell RNA-seq. Results: Lkb1 restoration did not result in any dramatic changes in the relative abundance of stromal cell types beyond a significant increase in a rare population of putative mast cells. There was a marked shift in the epithelial compartment from an indeterminate state to an alveolar type II epithelial-like identity in response to Lkb1 restoration. Conclusions: The acute transcriptional response to Lkb1 restoration is largely limited to the neoplastic epithelial compartment.

ORGANISM(S): Mus musculus

PROVIDER: GSE179501 | GEO | 2021/12/23

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: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.

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.

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LKB1 drives stasis and C/EBP-mediated reprogramming to an alveolar type II fate in lung cancer [scRNA-seq]

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