Hypothesis: Centrosome Amplification Triggers HIF‑1α/Rac‑Dependent mTORC1 Reactivation to Actively Suppress Autophagy as a Survival Strategy in Aging Cells

Background

Aging cells often exhibit centrosome amplification, which disrupts microtubule-dependent autophagosome trafficking and impairs lysosome positioning [1]. Concurrently, centrosome amplification can trigger a non‑canonical senescence-associated secretory phenotype driven by Rac activation, ROS production, and HIF‑1α stabilization [3]. This HIF‑1α/Rac axis has been linked to metabolic re‑programming but its direct impact on autophagy initiation remains unexplored.

Hypothesis

We hypothesize that in aged fibroblasts, centrosome amplification actively suppresses autophagy through HIF‑1α‑dependent transcriptional up‑regulation of miR‑21 and Rac‑PAK1‑mediated inhibition of the TSC1‑TSC2 complex, leading to mTORC1 re‑activation and phosphorylation‑dependent inhibition of ULK1. This active suppression serves as a short‑term survival tactic that prevents autophagic removal of damaged centriolar satellites, thereby avoiding mitotic catastrophe, but ultimately perpetuates centrosome abnormality and genomic instability.

Mechanistic Rationale

1. HIF‑1α → miR‑21 → TSC2 down‑regulation – HIF‑1α can induce miR‑21 expression, which targets the 3′‑UTR of TSC2 mRNA, weakening the TSC1‑TSC2 GAP activity toward Rheb and allowing mTORC1 signaling to rise.
2. Rac → PAK1 → TSC2 phosphorylation – Active Rac stimulates PAK1, which phosphorylates TSC2 on serine residues that inhibit its GAP function, further enhancing mTORC1 activity.
3. mTORC1 → ULK1 inhibition – Elevated mTORC1 phosphorylates ULK1 at Ser757, blocking the initiation complex required for phagophore formation.
4. Feedback loop – Impaired autophagy prevents doryphagy of centriolar satellite proteins [2], perpetuating centrosome amplification and sustaining the HIF‑1α/Rac signal.

Testable Predictions

• Inhibition of Rac (NSC23766) or HIF‑1α (PX‑478) in centrosome‑amplified aged fibroblasts will reduce phospho‑S6K levels, increase phospho‑ULK1 (Ser555) accumulation, and restore LC3‑II turnover in the presence of bafilomycin A1.
• CRISPR‑mediated knockout of HIF‑1α will diminish miR‑21 expression, elevate TSC2 protein, and suppress mTORC1 signaling despite persistent centrosome amplification.
• Restoring autophagic flux (e.g., with rapamycin low‑dose or spermidine) in these cells will increase doryphagy of centriolar satellites, leading to a measurable reduction in γ‑tubulin‑positive centrosome number over 5‑7 days.
• Conversely, forced activation of mTORC1 (e.g., via Rheb overexpression) in young fibroblasts will phenocopy the autophagy block and induce centrosome amplification only when combined with oxidative stress.

Experimental Approach

1. Cell model – Passaged human fibroblasts (IMR‑90) exhibiting centrosome amplification (>2 γ‑tubulin foci per nucleus) and senescence markers (p16^INK4a, SA‑β‑gal).
2. Interventions – Treat with Rac inhibitor NSC23766 (50 µM), HIF‑1α inhibitor PX‑478 (10 µM), or vehicle; include HIF‑1α CRISPR‑KO line.
3. Readouts
   • Western blot for phospho‑S6K (Thr389), phospho‑ULK1 (Ser757 and Ser555), LC3‑I/II, p62, miR‑21 (qRT‑PCR), TSC2.
   • Autophagic flux assay: LC3‑II accumulation with/without bafilomycin A1 (100 nM, 4 h).
   • Immunofluorescence for γ‑tubulin (centrosome count) and centriolar satellite protein PCM1.
   • Senescence-associated secretory phenotype (IL‑6, IL‑8 ELISA).
4. Controls – Young proliferating fibroblasts, and aged fibroblasts treated with rapamycin (20 nM) as a positive autophagy inducer.

Potential Outcomes and Interpretation

• Support: Inhibitors reduce mTORC1 activity, restore ULK1 de‑phosphorylation, increase autophagic flux, and lower centrosome number; HIF‑1α KO mimics these effects.
• Refutation: Inhibition fails to alter mTORC1 signaling or autophagic flux, or centrosome number remains unchanged despite restored autophagy, indicating that the HIF‑1α/Rac axis does not directly suppress autophagy in this context.

This framework converts the correlative observation of stalled autophagy into a testable mechanistic link between centrosome excess, stress‑signaling, and nutrient‑sensing pathways, offering a clear route to validate or falsify the idea that aged cells deliberately suppress autophagy as a precarious survival strategy.