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RNA-seq analysis of acute Rb1 deletion in adult mouse tissues

ABSTRACT: Abstract:The pRb tumor suppressor protein associates with chromatin and regulates gene expression. Numerous studies have identified Rb-dependent RNA signatures, but the proteomic effects of Rb-loss are largely unexplored. We have acutely ablated Rb in adult mice and conducted quantitative analysis of RNA and proteomic changes in colon and lung, where Rb-loss was sufficient or insufficient to induce ectopic proliferation respectively. As expected, RbKO caused similar increases in classic pRb/E2F-regulated transcripts in both tissues, but unexpectedly their protein products increased only in the colon, consistent with its increased proliferative index. Thus, these protein changes induced by Rb-loss are coupled with proliferation, but uncoupled from transcription. The proteomic changes in common between RbKO tissues showed a striking decrease in proteins with mitochondrial functions. Accordingly, RB1 inactivation in human cells decreased both mitochondrial mass and OXPHOS function. RBKO cells showed decreased mitochondrial respiratory capacity and the accumulation of hypopolarized mitochondria. Additionally, RB/Rb-loss altered mitochondrial pyruvate oxidation from 13C-Glucose through the TCA cycle in both cells and mouse tissues. Consequently, RBKO cells have an enhanced sensitivity to mitochondrial stress conditions. In summary, proteomic analyses provide a new perspective on Rb/RB1 mutation, highlighting the importance of pRb for mitochondrial function and suggesting vulnerabilities for treatment.

ORGANISM(S): Mus musculus

PROVIDER: GSE71924 | GEO | 2015/09/11

SECONDARY ACCESSION(S): PRJNA292461

REPOSITORIES: GEO

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Project description:Abstract:The pRb tumor suppressor protein associates with chromatin and regulates gene expression. Numerous studies have identified Rb-dependent RNA signatures, but the proteomic effects of Rb-loss are largely unexplored. We have acutely ablated Rb in adult mice and conducted quantitative analysis of RNA and proteomic changes in colon and lung, where Rb-loss was sufficient or insufficient to induce ectopic proliferation respectively. As expected, RbKO caused similar increases in classic pRb/E2F-regulated transcripts in both tissues, but unexpectedly their protein products increased only in the colon, consistent with its increased proliferative index. Thus, these protein changes induced by Rb-loss are coupled with proliferation, but uncoupled from transcription. The proteomic changes in common between RbKO tissues showed a striking decrease in proteins with mitochondrial functions. Accordingly, RB1 inactivation in human cells decreased both mitochondrial mass and OXPHOS function. RBKO cells showed decreased mitochondrial respiratory capacity and the accumulation of hypopolarized mitochondria. Additionally, RB/Rb-loss altered mitochondrial pyruvate oxidation from 13C-Glucose through the TCA cycle in both cells and mouse tissues. Consequently, RBKO cells have an enhanced sensitivity to mitochondrial stress conditions. In summary, proteomic analyses provide a new perspective on Rb/RB1 mutation, highlighting the importance of pRb for mitochondrial function and suggesting vulnerabilities for treatment. Design: Two tissues (lung and colon) from adult control (Rb1 wild type) or variable (Rb1 acute knockout). We examined the consequences of pRb loss in an in vivo model. Mice were bred to introduce a tamoxifen inducible form of Cre recombinase (ROSA26Sortm1(cre/Esr1)Tyj ) into the genetic backround of either Rb wild type (Rb+/+) or Rb floxed (Rbf/f) alleles. These animals were then injected with tamoxifen, ablating Rb in Rbf/f mice and generating RbKO tissues. Lung and colon tissues were harvested 96 hrs after the tamoxifen injections and analyzed. Each cohort used 3 biological replicates. Methods: RNA extraction: Ninety six hours following the last injection of tamoxifen mice were processed were sacrificed and dissected tissues flash frozen in liquid nitrogen for RNA extraction. RNA was extracted using the TRIzol-choloroform method and then DNase treated. RNA quality was validated using a bioanalyzer. Library construction and RNA-squencing was performed at the MGH NextGeneration Sequencing facility. Three mice were used per genotype, taking matched tissues from each mouse. Methods: Alignment: Sequenced libraries were mapped to the mouse genome (mm10) using TOPHAT (Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks, Trapnell C et al, Nature protocols 2012, 7, 562-578, doi:10.1038/nprot.2012.016). A RefSeq GTF file was used to annotate the mapped transcripts. FPKM matrixes and differentially regulated gene lists were generated using the Cufflinks-Cuffmerge-Cuffdiff pipeline (Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks, Trapnell C et al, Nature protocols 2012, 7, 562-578, doi:10.1038/nprot.2012.016).

Project description:Chemotherapy resistance is one of the reasons for the loss of the eye in retinoblastoma (RB) pa-tients. RB chemotherapy resistance has been investigated in different cell culture models like WERI RB1. Furthermore, chemotherapy resistant RB subclones like the etoposide resistant WERI ETOR cell line have been established to improve understanding of chemotherapy resistance in RB. Ob-jective of this study was to characterize the cell line models of an etoposide sensitive WERI RB1 and its etoposide resistant subclone WERI ETOR by proteomics analysis. Subsequently, quantitative proteomic data served for correlation analysis with known drug perturbation profiles. . WERI RB1 and WERI ETOR were cultured and prepared for quantitative mass spectrometry. Comparative proteomic profiling was performed with electrospray ionization tandem mass spectrometry in data-independent acquisition mode (SWATH). The raw SWATH files were processed using neural networks in library free mode along with machine learning algorithms. Pathway enrichment was performed using the REACTOME pathway resource and correlated to the Molecular Signature Database (MSigDB) hallmark gene set collections for functional annotation. Also, a drug connec-tivity analysis using the L1000 database was used to correlate the mechanism-of-action (MOA) for different anticancer reagents to WERI RB1 / WERI ETOR signatures. A total 4,756 proteins were identified across all samples which revealed a distinct clustering between groups. Of these pro-teins, 64 proteins were significantly altered (q < 0.05 & log2FC |>2|, 22% higher in WERI ETOR). Pathway analysis showed an enriched metabolic pathway for retinoid metabolism and transport pathway in WERI ETOR and for sphingolipid de novo biosynthesis in WERI RB1. In addition, this study showed similar protein signatures of topoisomerase inhibitors and WERI ETOR as well as of ATPase inhibitors, acetylcholine receptor antagonists and VEGFR inhibitors and WERI RB1. In this study, WERI RB1 and WERI ETOR were characterized as a cell line model for chemotherapy re-sistance in RB by using data-independent mass spectrometry. The global proteome revealed the activation of sphingolipid de novo biosynthesis in WERI RB1 and potential treatment options for etoposide resistant RB.

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RNA-seq analysis of acute Rb1 deletion in adult mouse tissues

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