The tumor suppressor Rb critically regulates starvation-induced stress response in C. elegans

Abstract

How animals coordinate gene expression in response to starvation is an outstanding problem closely linked to aging, obesity, and cancer. Newly hatched Caenorhabditis elegans respond to food deprivation by halting development and promoting long-term survival (L1 diapause), thereby providing an excellent model for the study of starvation response. Through a genetic search, we have discovered that the tumor suppressor Rb critically promotes survival during L1 diapause and most likely does so by regulating the expression of genes in both insulin-IGF-1 signaling (IIS)-dependent and -independent pathways mainly in neurons and the intestine. Global gene expression analyses suggested that Rb maintains the "starvation-induced" transcriptome and represses the "refeeding-induced" transcriptome, including the repression of many pathogen-, toxin-, and oxidative-stress-inducible and metabolic genes, as well as the activation of many other stress-resistant genes, mitochondrial respiratory chain genes, and potential IIS receptor antagonists. Notably, the majority of genes dysregulated in starved L1 Rb(-) animals were not found to be dysregulated in fed conditions. Altogether, these findings identify Rb as a critical regulator of the starvation response and suggest a link between functions of tumor suppressors and starvation survival. These results may provide mechanistic insights into why cancer cells are often hypersensitive to starvation treatment.

Figure 1. Tumor Suppressor Rb Functions to Promote L1 Starvation Survival by Affecting Both IIS Pathway-dependent and – independent Functions

(A) Survival rate of the lin-35/Rb(lf) and daf-18/Pten(lf). Both mutants have a significantly reduced L1 starvation survival rate. The lin-35/Rb(lf) defect can be rescued by expression of a lin-35/Rb(+) transgene. Data for additional synMuv genes are shown in Figure S1A. All data presented as mean ± SEM. (B) The mean survival calculated with the Kaplan-Meier method and log-rank statistical analyses of indicated mutants with decreased L1 starvation survival. ^ap-values were calculated by log-rank test and show the significance of the difference compared to the wild-type strain. ^bp-values were calculated to show the significance of the difference from the lin-35/Rb(lf) strain. (C) Survival curves showing that the defect of lin-35/Rb(lf) was partially suppressed by unc-31(lf); age-1(rf) and overexpression (oe) of daf-16/FOXO. All data presented as mean ± SEM. (D) daf-16(lf); lin-35/Rb(lf) double mutants displayed more severe defects than each single mutant. All data presented as mean ± SEM. Statistical analyses of data shown in (C) and (D) are reported in (B). Raw data for individual starvation survival experiments for Figure 1 are presented in Table S2.

Figure 2. Rb Regulates the Expression of Many IIS-regulated Genes, Potential IIS-regulating Genes, and Genes Regulated by Feeding and Starvation

(A) A simplified diagram of the insulin receptor pathway’s known effect on L1 starvation survival [2, 6]. (B) Number of genes that changed expression in both daf-2(rf)[19] and lin-35/Rb(lf). The overlaps are significantly greater than the numbers expected by random chance. Changes caused by daf-2(rf) for most of the overlap genes were in the opposite direction of that by lin-35/Rb(lf) (see Table S1B). rf, reduction-of-function. (C) Bar diagram showing representative genes that were dysregulated in lin-35/Rb(lf) in the opposite direction as that by daf-2(rf) [19]. (D) Expression changes of two potential IIS receptor antagonists in lin-35(Rb(lf). p-values shown in (B and C): * p < 0.05; **p < 0.01; ***p < 0.001. (E) Number of previously reported FedUP or StarvUP genes that are also regulated by Rb. The expression patterns of these overlap genes were disrupted by Rb(lf) (Tables S1C and S1D). Statistical analysis of overlap expected by random chance and p-values in (B and E) are described in Supplemental Experimental Procedures.

Figure 3. Rb Regulates the Expression of Many Genes That are Induced by Pathogen, Toxin, or Oxidative Stress, and Rb(lf) Mutants are Defective in Oxidative Stress Response

(A) Number of previously characterized pathogen (Pseudomonas aeruginosa PA14)-inducible genes [21], cry5B toxin-inducible genes [24], and oxidative stress-inducible genes [25] that are up-regulated by Rb(lf) (see Tables S1E–G). These data suggest Rb represses these genes in wild type to promote starvation survival. The calculation of overlap expected by random chance and p-values shown are described in Supplemental Experimental Procedures. (B) List of representative toxin, pathogen and/or oxidative stress inducible genes that are upregulated in Rb(lf) (See Tables S1E–G for complete lists). (C) Changes in expression of a group of glutathione transferase genes in lin-35/Rb(lf) mutants. These genes are known to be required for a normal oxidative stress response [25]. P values: * p < 0.05; **p < 0.01; ***p < 0.001. (D). Rb(lf) worms are hypersensitive to paraquat treatment, indicating that they are defective in oxidative stress response. Day 1 starved L1 animals were treated with or without 1 mM paraquat and examined for viability 60 hours later. p-values were generated by t-test. Error bars, SEM.

Figure 4. Rescue Effect of L1 Starvation Survival by Glucose Supplementation and Tissue-specific Expression of lin-35/RB

(A) Survival rate of the Rb(lf) and other indicated mutants supplemented with or without 2% glucose. All data presented as mean ± SEM. (B) Mean survival as calculated with the Kaplan-Meier method, and p-values as calculated with the log-rank statistical analyses, of indicated strains supplemented with or without 2% glucose. p-values are relative to the same strain without supplementation of glucose. Glucose supplementation has a significantly weaker rescuing effect on the L1 survival rate of lin-35/Rb(lf) than that of other mutants tested. (C) Survival rate of lin-35/Rb L1 mutant animals carrying extrachromosomal arrays expressing wild-type lin-35/Rb driven by three tissue specific promoters (intestine, hypodermis, and pan-neurons) and the lin-35 promoter. Transgenic animals were scored based upon the expression of a co-injection marker using a Leica fluorescence microscope. The average from multiple independent transgenic lines for each genotype is reported with the standard error of the mean for each time point (±SEM). (D) Mean survival rates calculated with Kaplan-Meier method. ^a,b,c p-values were calculated by log rank test showing the significance of the difference from wild type, lin-35/Rb(lf) and lin-35/Rb(lf) rgef-1P::lin-35, respectively. Raw data for individual starvation survival experiments for Figure 4 are presented in Table S2.