Tumorigenesis in tuberous sclerosis complex is autophagy and p62/sequestosome 1 (SQSTM1)-dependent

Abstract

Tuberous sclerosis complex (TSC) is a tumor suppressor syndrome characterized by benign tumors in multiple organs, including the brain and kidney. TSC-associated tumors exhibit hyperactivation of mammalian target of rapamycin complex 1 (mTORC1), a direct inhibitor of autophagy. Autophagy can either promote or inhibit tumorigenesis, depending on the cellular context. The role of autophagy in the pathogenesis and treatment of the multisystem manifestations of TSC is unknown. We found that the combination of mTORC1 and autophagy inhibition was more effective than either treatment alone in inhibiting the survival of tuberin (TSC2)-null cells, growth of TSC2-null xenograft tumors, and development of spontaneous renal tumors in Tsc2(+/-) mice. Down-regulation of Atg5 induced extensive central necrosis in TSC2-null xenograft tumors, and loss of one allele of Beclin1 almost completely blocked macroscopic renal tumor formation in Tsc2(+/-) mice. Surprisingly, given the finding that lowering autophagy blocks TSC tumorigenesis, genetic down-regulation of p62/sequestosome 1 (SQSTM1), the autophagy substrate that accumulates in TSC tumors as a consequence of low autophagy levels, strongly inhibited the growth of TSC2-null xenograft tumors. These data demonstrate that autophagy is a critical component of TSC tumorigenesis, suggest that mTORC1 inhibitors may have autophagy-dependent prosurvival effects in TSC, and reveal two distinct therapeutic targets for TSC: autophagy and the autophagy target p62/SQSTM1.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Survival of TSC2-null cells is dependent on autophagy. (A) Immunoblot analysis of LC3-II and p62/SQSTM1 in Tsc2+/+ and Tsc2−/− MEFs. (B) Transmission electron microscopy of Tsc2+/+ and Tsc2−/− MEFs. (Insets) Representative autophagosomes and autolysosomes under higher magnification. (Scale bar: 2 μM.) The graph shows number of autophagosomes per cell. *P < 0.05. (C) Immunoblot analysis of p62/SQSTM1 and tuberin in Tsc2+/+ and Tsc2−/− MEFs grown in normoxic (21% O2) or hypoxic (1% O2) conditions for 48 h. (D) Immunoblot analysis of p62/SQSTM1 in Tsc2+/+ and Tsc2−/− MEFs treated with 20 nM rapamycin or vehicle for 24 h. (E) OCR measured using a Seahorse Bioscience XF24 analyzer after treatment of angiomyolipoma-derived TSC2-null 621-101 cells with CQ (10 μM), rapamycin (20 nM), or both for 24 h. Cellular respiration rate per million cells (OCR, pmol/min/million cells) is shown. Basal OCR (white bars) was measured for 20 min. Resting OCR (black bars) was measured in the presence of the ATP synthase inhibitor oligomycin (5 mM). Each data point represents mean ± SD; n = 6. *P < 0.05. (F) ATP levels measured using the PerkinElmer ATPlite assay system after treatment of ELT3 cells cultured in DMEM + 10% FBS with CQ (10 μM), rapamycin (20 nM), or both for 24 h. *P < 0.05. (G) Morphology of TSC2-null ELT3 cells cultured in serum-free, glutamine-free, and low-glucose (1 g/L) media after treatment with CQ (10 μM), rapamycin (20 nM), or both for 24 h. (H) Percentage of propidium iodide-positive ELT3 cells cultured and treated as described for 24 h. *P < 0.05.

Fig. 2.

Dual inhibition of autophagy and mTORC1 is more effective than inhibition of either agent alone in suppressing TSC2-null tumor development. (A) SCID mice bearing established TSC2-null ELT3 xenograft tumors were treated with vehicle control or CQ (50 mg/kg/d for 2 wk; n = 12 per group). *P < 0.05. (B and C) Nude mice bearing established Tsc2−/− MEF xenograft tumors were treated with vehicle control (n = 4), CQ (50 mg/kg/d) (n = 4) (B), rapamycin (6 mg/kg/d), or rapamycin (6 mg/kg/d) plus CQ (50 mg/kg/d) for 2 wk (n = 8 per group) (C). *P < 0.05. (D) Tsc2+/− mice in the A/J genetic background were treated with vehicle or with rapamycin (3 or 6 mg/kg, 3 times/wk), CQ (50 mg/kg/d), or both for 4–8 wk. Macroscopic kidney tumors were scored at age 5 mo. *P < 0.05.

Fig. 3.

ATG5 is required for TSC2-null xenograft tumor cell survival. (A) Immunoblot analysis of GFP and p62/SQSTM1 in Tsc2−/− MEFs infected with GFP-control or GFP-Atg5 shRNA. (B) Whole-mount H&E staining of size-matched xenograft tumors of Tsc2−/− MEFs with GFP-control or GFP-Atg5 shRNA. The percentage of necrotic area was determined by a blinded investigator using H&E-stained sections. *P < 0.05. (C) Immunoblot analysis of GFP in tumors from B.

Fig. 4.

Allelic disruption of Beclin1 suppresses spontaneous renal tumorigenesis in Tsc2 heterozygous mice. (A) The kidney with the most severe lesions from each genotype is shown; arrows indicate cystadenomas. (B) Macroscopic tumor scores from WT (wt), Tsc2+/− (T2+/−), Beclin1+/− (B1+/−), and Tsc2+/−Beclin1+/− (T2+/−B1+/−) littermates (n = 8 kidneys per genotype). No tumors were observed in WT or Beclin1+/− mice. *P < 0.05. (C) Representative H&E-stained lesions. N, normal kidney. T, cystadenomas. (Original magnification, 20×.) (D) Microscopic tumor scores. *P < 0.05.

Fig. 5.

p62/SQSTM1 is required for TSC2-null tumor development. (A) Immunoblot analysis of p62/SQSTM1 and tuberin (TSC2) in matched pairs of angiomyolipoma (T1–T3) and adjacent normal kidney (N1–N3). (B) Immunohistochemical analysis of p62/SQSTM1 accumulation in LAM nodules, which are positive for muscle actin. (Original magnification, 20× and 60×.) N, normal lung. L, LAM cells. (C) Immunoblot analysis of p62/SQSTM1 and LC3-II in Tsc2−/− MEFs stably infected with lentiviral control or p62/SQSTM1 shRNA. (D) Cell growth as assessed by the ATPlite cell viability assay. (E) Immunoblot analysis of phospho-p44/42 MAPK (Thr202/Try204) and phospho-IKKα/β (Ser176/180) in Tsc2−/− MEFs stably infected with lentiviral control or p62/SQSTM1 shRNA. (F) Kaplan–Meier plot of the percentage of tumor-free mice after inoculation with Tsc2−/− MEFs stably infected with lentiviral control or p62/SQSTM1 shRNA. *P < 0.05, Mantel–Cox log-rank test. (G) Immunoblot analysis of p62/SQSTM1 in xenograft tumors from F.