Centrosome Amplification Drives mTORC1‑Dependent SASP and Senescence Escape via Microtubule‑Scaffolded Lysosomal Positioning in Aged Cells

Hypothesis

Centrosome amplification (CA) in aged somatic cells increases astral microtubule nucleation, which serves as a scaffold to reposition lysosomes near the perinuclear region, thereby enhancing Rag GTPase‑mediated mTORC1 activation on lysosomal surfaces. This mTORC1 surge selectively translates a subset of SASP factors (IL‑6, IL‑8, ANGPTL4) through a HIF1α‑independent, Rac1‑ROS axis, promoting a pro‑invasive, senescence‑escape phenotype that can occur even when centrosome clustering prevents multipolar mitosis.

Mechanistic Rationale

1. Microtubule nucleation as a spatial regulator – Amplified centrosomes generate excess astral microtubules that bind dynein/dynactin complexes, pulling lysosomes toward the microtubule‑organizing center. Prior work shows microtubule density correlates with lysosomal clustering and mTORC1 signaling (3).
2. Lysosomal mTORC1 drives a distinct SASP – mTORC1 phosphorylates 4EBP1 and S6K, favoring cap‑dependent translation of mRNAs with complex 5’UTRs, including those encoding IL‑6, IL-8, and ANGPTL4 (4). This bypasses the canonical NF‑κB/DNA‑damage SASP pathway described in the variant SASP via Rac1‑ROS‑HIF1α‑ANGPTL4.
3. Rac1‑ROS amplification loop – Increased microtubule nucleation also elevates Rac1 GEF activity at microtubule plus‑ends, augmenting ROS production that further stabilizes HIF1α and amplifies ANGPTL4 transcription (5).
4. Clustering uncouples mitotic death from signaling – HSET/KIFC1‑mediated centrosome clustering allows cells to bypass lethal multipolar divisions while retaining the heightened microtubule network that fuels lysosomal mTORC1 signaling, explaining why CA can coexist with proliferation or invasion rather than arrest.

Testable Predictions

• Prediction 1: In aged cells with CA, lysosomal mTORC1 activity (measured by phospho‑S6K on lysosomal fractions) will be higher than in young cells or aged cells where CA is genetically suppressed (PLK4 knockdown).
• Prediction 2: Acute reduction of microtubule nucleation using low‑dose nocodazole or HSET inhibition will disperse lysosomes, decrease mTORC1 signaling, and reduce secretion of IL‑6, IL-8, and ANGPTL4 without increasing multipolar mitosis.
• Prediction 3: Expressing a constitutively active RagC mutant will rescue SASP secretion and invasive potential in CA cells where microtubule nucleation is compromised, whereas dominant‑negative RagC will suppress SASP even with intact CA.
• Prediction 4: In vivo, aged mouse tissues treated with a lysosomal mTORC1 inhibitor (e.g., Torin1) will show diminished variant SASP markers and reduced paracrine invasion phenotypes despite persistent CA.

Experimental Approach

1. Model systems: Use primary mouse embryonic fibroblasts (MEFs) passaged to replicative senescence, aged Drosophila midgut stem cells, and human mammary epithelial cells with inducible PLK4 overexpression to generate CA.
2. Manipulate microtubule nucleation: Treat cells with 10 nM nocodazole (low dose to spare spindle integrity) or siRNA against HSET/KIFC1; assess lysosomal positioning via LAMP1 immunostaining and microtubule acetylation.
3. Readouts:
   • Lysosomal mTORC1: subcellular fractionation followed by western blot for p‑S6K (Thr389) and p‑4EBP1.
   • SASP secretion: ELISA for IL-6, IL-8, ANGPTL4 in conditioned media.
   • Senescence markers: SA‑β‑gal, p16^INK4a^.
   • Invasion: transwell Matrigel assay using conditioned media.
4. Rescue experiments: Express RagC^GTP^ (QL) or dominant‑negative RagC^GDP^ (TN) via lentivirus; assess whether SASP levels are restored or suppressed independent of microtubule nucleation.
5. In vivo validation: Treat aged mice with Torin1 (1 mg/kg i.p. three times weekly) for 4 weeks; quantify SASP cytokines in serum and histological invasion markers in liver and intestine.

If lysosomal mTORC1 activation is necessary and sufficient for the CA‑driven, pro‑invasive SASP, this hypothesis links centrosome number to metabolic signaling and provides a therapeutic window: targeting lysosomal positioning or mTORC1 could suppress the maladaptive SASP of aged cells while leaving essential mitotic clustering intact.