Senescence-Associated Secretory Phenotypes Reveal Cell-Nonautonomous Functions of Oncogenic RAS and the p53 Tumor Suppressor (original) (raw)
Figure 6
p53 Restrains the SASP
(A) CM containing factors secreted by the indicted cells were analyzed by antibody arrays and displayed, using PRE CM as the baseline. We pooled data from cells of the same genotype (p53 wild type or p53 deficient) under the same culture conditions. SEN indicates pooled data from cells originating from the same tissue (WI-38, IMR-90 from embryonic lung; and HCA-2, BJ from neonatal foreskin) and induced to senesce by REP or XRA. Pooling and averaging of highly correlated samples was performed as described for Figure 5, and details of the data processing are provided in Datasets S13–S16. The top four rows are the same top four rows in Figure 5A and are included to serve as a visual reference. The senescence inducer is given in parentheses. p53 status is indicated as either wild type (wt) or deficient (d) owing to either GSE22 expression or expression of an shRNA against p53. Manipulations are indicated in sequence, separated by a greater than symbol (>). The heat map key (right) indicates the log2-fold changes. Signals higher than the baseline are shown in yellow; signals below baseline are displayed in blue. Comparison between rows is accurately illustrated in (B) and (C) in which each genetically manipulated cell type is compared to its appropriate control baseline.
(B) Log2-fold values for SASP factors that are significantly increased, or significantly and uniquely (as indicated by double asterisks [**]) elevated, in CM from SEN cells made p53 deficient by GSE22, using untreated wild-type SEN values as the baseline. Green indicates WI-38 cells made senescent by XRA, after which p53 was subsequently inactivated by expressing GSE22 using a lentivirus; these cells do not resume proliferation (“unreverted”) upon p53 inactivation (see Figure S6). Blue indicates WI-38 in which p16 was inactivated by an shRNA, induced to senesce by XRA, then infected with the GSE22-expressing lentivirus; these cells do revert (REV) after p53 inactivation. Pink indicates HCA2 cells made SEN by XRA, then infected with GSE22 lentivirus; these cells also revert after p53 inactivation.
(C) Log2-fold values for SASP factors that are significantly increased, or significantly and uniquely (as indicated by double asterisks [**]) elevated, in CM from cells made p53-deficient (by GSE22 expression), then induced to senesce by REP, XRA, or RAS. Red indicates WI-38 and IMR90 (averaged) cells expressing GSE22, then induced to senesce by XRA or REP, using cells made SEN by XRA or REP as a baseline. Gray indicates WI-38, IMR-90, and HCA2 (averaged) expressing GSE22, then made senescent by RAS, using SEN by RAS as a baseline.
(D) WI-38 cells expressing GSE22 were induced to senesce by XRA and then immunostained for the SASP proteins IL-6 and IL-8, the senescence marker p16INK4a, and p53, which accumulates in the presence of GSE22.
(E) Comparative graphical representation of the secretory profiles of cells made senescent by XRA or REP (dotted line), RAS (black line), p53 inactivation (GSE22) followed by XRA or REP (blue line), or p53 inactivation (GSE22) followed by RAS (red line). The increased slopes (as indicated by the arrow)indicate amplified SASPs.
(F) Hierarchical cluster analysis of all the cells analyzed in (A), plus the SASP induced by RAS (see Figure 5). RAS status is indicated as either wild type (wt) or oncogenic (o) owing to expression of Ha-RASv12.
(G) WI-38 cells with wild-type (wt) or inactive (GSE) p53 were irradiated or induced to express oncogenic RAS (RAS), and CM was collected 4 or 10 d later. Soluble factors were analyzed by antibody arrays and displayed as described in Figure 1D, using PRE CM as the baseline (black column on the left; see also Figure S5C for details). Signals higher than baseline are shown in yellow; signals below baseline are in blue. n/a, not applicable.
(H) Log2-fold values for prostate epithelial cell SASP factors that are significantly or uniquely (as indicated by double asterisks [**]) elevated in CM from p53-deficient cancer cells (PC3, BPH1, and RWPE1) that were induced to senesce by XRA, compared to primary p53 wild-type cells (PrECs) that were induced to senesce by XRA.