Cellular senescence is an important mechanism of tumor regression upon c-Myc inactivation

Abstract

Oncogene-induced senescence is an important mechanism by which normal cells are restrained from malignant transformation. Here we report that the suppression of the c-Myc (MYC) oncogene induces cellular senescence in diverse tumor types including lymphoma, osteosarcoma, and hepatocellular carcinoma. MYC inactivation was associated with prototypical markers of senescence, including acidic beta-gal staining, induction of p16INK4a, and p15INK4b expression. Moreover, MYC inactivation induced global changes in chromatin structure associated with the marked reduction of histone H4 acetylation and increased histone H3 K9 methylation. Osteosarcomas engineered to be deficient in p16INK4a or Rb exhibited impaired senescence and failed to exhibit sustained tumor regression upon MYC inactivation. Similarly, only after lymphomas were repaired for p53 expression did MYC inactivation induce robust senescence and sustained tumor regression. The pharmacologic inhibition of signaling pathways implicated in oncogene-induced senescence including ATM/ATR and MAPK did not prevent senescence associated with MYC inactivation. Our results suggest that cellular senescence programs remain latently functional, even in established tumors, and can become reactivated, serving as a critical mechanism of oncogene addiction associated with MYC inactivation.

Fig. 1.

MYC inactivation induces cellular senescence in primary tumors in vivo. (A) Acidic β-gal (β-gal) staining was conducted for primary hepatocelullar carcinoma and lymphoma 2 days and 4 days after MYC inactivation. Primary MYC-induced hepatocellular carcinoma was transplanted to SCID mice s.c (14). (B) Tumor tissues were stained with acidic β-gal (22) or hematoxylin and eosin at different time points upon MYC inactivation.

Fig. 2.

MYC inactivation induces expression of cellular senescence markers in vitro and in vivo. (A) (Top and Middle) Acidic β-gal staining was conducted for osteosarcoma and lymphoma cell lines before and after MYC inactivation for 48 and 24 h, respectively. Means and standard deviations of the percentages of SA-β-gal-positive cells are indicated. (Bottom) s.c.-injected MYC-induced lymphoma cell line 6780 with MYC on or 3 days after MYC inactivation stained with eosin and β-gal activities. (B) Real-time PCR for p15INK4b and p21CIP were done in an osteosaroma cell line in vitro (Upper) and hepatocellular carcinoma in vivo (Lower) normalized by GAPDH. (C) Western blots with osteosarcoma cells with MYC on or MYC off conditions at various time points for p16INK4a expression.

Fig. 3.

MYC inactivation induces global changes in chromatin structure. Bone tumor cells generated from mice were cultured in vitro and treated with 20 ng/ml doxycycline to inactivate MYC expression. (A) Heterochromatin formation was shown by DAPI staining at 48 h after MYC inactivation. (B–D) Immunofluorescence staining with anti-acetyl histone H4 (B), anti-acetyl histone H3 antibody (C), and anti-trimethyl K9 histone H3 antibody (D) upon MYC inactivation for 0, 6, 12, 24, and 48 h. There was a great reduction of MYC expression after 4 h of treatment of doxycycline (SI Fig. 7). (E) Western blots for MYC on and MYC off bone tumor cells in vitro and transplanted primary hepatocullar carcinoma in vivo probed with antibodies against MYC, acetyl histone H4, acetyl histone H3, trimethyl K4 histone H3, and α-tubulin.

Fig. 4.

Loss of p53, Rb, or p16INK4a affects cellular senescence and tumor regression upon MYC inactivation. (A) Acidic β-gal staining for MYC, p53^−/−/MYC, and p53 restored lymphomas in MYC on and MYC off after 24 h. Means and standard deviations of the percentages of SA-β-gal-positive cells are indicated. Bioluminescence imaging of tumor cell elimination of p53-negative versus p53-restored lymphomas before and after MYC inactivation. (B) Luciferase-labeled lymphoma cells (10⁷) were inoculated into syngeneic mice, then imaged by bioluminescence imaging on the day of MYC inactivation (MYC On) and 9 days after MYC inactivation (MYC Off). (C) Acidic β-gal staining for osteosarcoma cells infected with scramble shRNA, Rb shRNA, p16INK4a shRNA, or p53 shRNA in MYC on and MYC off for 48 h. The cells were counterstained with eosin. (D) Tumor-free survival of mice that had been injected with 10⁵ bone tumor cells infected with vector control or RB or p16INK4a shRNA vectors. (E) MYC-induced lymphoma cells were treated with or without 5 mM caffeine 1 h before turning MYC off. SA-β-Gal staining was performed 24 h later after MYC inactivation. Means and standard deviations of the percentages of SA-β-gal-positive cells are indicated. (F) MYC-induced lymphomas were treated with the ERK1/2 pathway inhibitor (U0126, 10 μM) or p38-MAPK pathway inhibitor (SB203580, 10 μM) for 48 h with MYC off conditions. (G) Cells were collected for SA-β-gal staining. A model for OIS and oncogene inactivation-induced senescence is illustrated here.