Age‑Dependent Rewiring of Autophagic Cargo Priority Drives Field Cancerization via Selective MMR Depletion
Hypothesis
Aging reprograms the selective autophagy machinery in colonic epithelial cells to favor degradation of soluble mismatch‑repair (MMR) proteins over damaged mitochondria, creating a permissive environment for mitochondrial field defects and spontaneous microsatellite instability that fuels field cancerization.
Mechanistic Rationale
- With age, lysosomal acidification declines and cytosolic NAD+ drops, altering the activity of autophagy receptors such as p62/SQSTM1 and NBR1 that recognize ubiquitin‑linked cargo.
- These receptors exhibit higher affinity for K63‑linked ubiquitin chains prevalent on MMR proteins under oxidative stress, whereas mitophagy receptors (e.g., BNIP3, NIX) rely more on phospho‑ubiquitin signals that become less accessible as mitochondrial membrane potential depolarizes.
- Consequently, under nutrient stress, autophagosomes sequester MMR proteins first, depleting their functional pool while damaged mitochondria persist, sustaining the COX‑deficient gradient observed in normal‑appearing mucosa.
Predictions
- In colonocytes from donors >65 y, the ratio of MMR protein to TOMM20 within LC3‑II‑positive autophagosomes will be significantly higher than in donors <45 y under identical starvation conditions.
- Acute inhibition of p62 will restore mitophagy precedence and increase mitochondrial clearance without affecting bulk autophagy flux.
- Forced overexpression of a degradation‑resistant MMR variant (e.g., MLH1‑K->R ubiquitin‑site mutant) will mitigate age‑associated COX deficiency and reduce spontaneous microsatellite alterations in cultured colon organoids.
Experimental Design
- Obtain colonic biopsies from age‑stratified donors (20‑35, 50‑65, >75 y) with no histological dysplasia.
- Isolate primary colonic epithelial cells and subject them to EBSS starvation for 2 h.
- Perform LC3 immuno‑pull‑down, followed by quantitative Western blot or targeted mass spectrometry for MMR proteins (MLH1, MSH2, MSH6, PMS2) and mitochondrial markers (TOMM20, COXIV).
- Parallel cultures treated with p62 siRNA or the NBR1‑blocking peptide to test receptor contribution.
- Measure mitochondrial respiration (Seahorse XF) and COX activity, alongside microsatellite instability PCR panels.
- Validate findings in human colon‑derived organoids transduced with lentiviral constructs expressing ubiquitin‑site‑mutant MLH1 or CRISPR‑knock‑in of a phospho‑deficient BNIP3.
Potential Outcomes and Interpretation
- Confirmation of an elevated MMR:mitochondrial cargo ratio in aged autophagosomes would substantiate the hypothesis that autophagy selectivity shifts with age.
- Rescue of mitophagy and reduction of field‑defect markers upon p62 knockdown would indicate receptor‑driven cargo competition as a mechanistic driver.
- Lack of age‑dependent cargo shift, or failure of MMR stabilization to affect mitochondrial parameters, would falsify the proposed hierarchy rewiring and redirect focus to alternative sources of field cancerization.
Implications
If validated, this mechanism links two hallmarks of aging—mitochondrial decay and mismatch‑repair erosion—through a common regulatory node, offering a tractable target (autophagy receptor modulation) for preventive interventions aimed at slowing colonic field progression before neoplastic transformation.