Proteomic analysis of pRb loss highlights a signature of decreased mitochondrial oxidative phosphorylation

Abstract

The retinoblastoma tumor suppressor (pRb) 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 acutely ablated Rb in adult mice and conducted a quantitative analysis of RNA and proteomic changes in the colon and lungs, where Rb KO was sufficient or insufficient to induce ectopic proliferation, respectively. As expected, Rb KO 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 Rb KO tissues showed a striking decrease in proteins with mitochondrial functions. Accordingly, RB1 inactivation in human cells decreased both mitochondrial mass and oxidative phosphorylation (OXPHOS) function. RB KO cells showed decreased mitochondrial respiratory capacity and the accumulation of hypopolarized mitochondria. Additionally, RB/Rb loss altered mitochondrial pyruvate oxidation from 13 C-glucose through the TCA cycle in mouse tissues and cultured cells. Consequently, RB KO 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.

Increased mitochondrial function downstream from KDM5A histone demethylase rescues differentiation in pRB-deficient cells

“…However, analysis of Rb1 −/− embryos that were rescued from embryonic lethality with either wild-type placentas or low levels of the Rb1 gene showed marked skeletal muscle defects at birth (Zacksenhaus et al 1996;de Bruin et al 2003;MacPherson et al 2003;Wu et al 2003), thus underscoring the functional importance of pRB as a regulator of muscle differentiation and development. Consistent with in vivo findings, Rb1 −/− mouse embryonic fibroblasts (MEFs) did not respond to the forced expression of the myogenic differentiation antigen (MyoD) (Novitch et al 1996). Lysine (K)-specific demethylase 5A (KDM5A; also known as RBP2 and JARID1A) was isolated in a genetic screen with pRB derivatives deficient in interaction with E2F but retaining the ability to induce differentiation, suggesting that induction of differentiation is an E2F-independent pRB function (Benevolenskaya et al 2005).…”

RB1: a prototype tumor suppressor and an enigma

“…Proteomic profiles of RB1 mutant mouse tissues show that changes in the levels of proliferation proteins are not a uniform feature of RB1 mutant tissues or the major proteomic effect of pRB loss (Nicolay et al 2015). Substantial differences between the effects of pRB loss on mRNA and protein levels suggest that post-transcriptional controls are likely to play a significant role in determining the ultimate effects of RB1 inactivation.…”

“…RB1 mutation (either alone or in conjunction with other pRB family members) causes change in metabolic pathways. These alterations include reduced mitochondrial respiration, reduced activity in the electron transport chain, changes in mitochondrial polarity, and altered flux from glucose or glutamine in RB1 mutant cells (Sankaran et al 2008;Clem and Chesney 2012;Nicolay et al , 2015Reynolds et al 2014; for review, see Lopez-Mejia and Fajas 2015). Indeed, proteomic studies show that changes in mitochondrial function are a major feature of RB1 mutant mouse tissues (Nicolay et al 2015).…”

“…These alterations include reduced mitochondrial respiration, reduced activity in the electron transport chain, changes in mitochondrial polarity, and altered flux from glucose or glutamine in RB1 mutant cells (Sankaran et al 2008;Clem and Chesney 2012;Nicolay et al , 2015Reynolds et al 2014; for review, see Lopez-Mejia and Fajas 2015). Indeed, proteomic studies show that changes in mitochondrial function are a major feature of RB1 mutant mouse tissues (Nicolay et al 2015). While the mechanistic basis for these metabolic changes has not been fully elucidated, these results are consistent with reports showing that E2F and RB proteins bind directly to promoters of genes encoding important regulators of metabolic flux, oxidative phosphorylation, and mitochondrial function (Cam et al 2004;Hsieh et al 2008;Chicas et al 2010;Blanchet et al 2011;Ambrus et al 2013).…”

Exploring cancer metabolism using stable isotope-resolved metabolomics (SIRM)

“…Unlike oncogenic KRAS, increased [ 13 C]Glc uptake and lactate pro-duction was not observed in tissues derived from RB knock-out mice or RB-silenced epithelial cells (77). Instead, loss of pRb enhances Glc entry into the Krebs cycle, as evidenced by an increase in PDH-mediated production of [ 13 C 2 ]citrate (77).…”