Feedback loops involving ERK, AMPK and TFEB generate non-genetic heterogeneity that allows cells to evade anoikis

Abstract

AbstractSome solid tumor cells escape death triggered by matrix-deprivation and cause cancer spread through metastatic growth. The role of phenotypic plasticity in this adaptation remains unknown. We recently identified a double-negative feedback loop between pAMPK (phospho-AMPK) and pAkt (phospho-Akt) that regulates the switch between attached and detached states of cancer cells. In this study, we show that matrix-detachment itself can give rise to two subpopulations with varying ERK signaling levels and autophagy flux. Cells with elevated ERK activity show autophagy maturation arrest leading to anoikis, whereas those with low ERK activity overcome this block and generate anchorage-independent colonies. Investigating upstream, we show a novel role of AMPK-mediated phosphorylation of PEA15 in inhibiting ERK activity by reducing the formation of MEK-ERK complex. Consequently, cells with higher AMPK activity have lower phospho-ERK, and this heterogeneity is reflected in vivo. Exploring downstream, we demonstrate that ERK inhibition leads to upregulation of TFEB, a major regulator of lysosome biogenesis and autophagy. Overexpression of TFEB not only rescues the defect in autophagy flux, but also re-inforces AMPK signaling, thus revealing a positive feedback loop between AMPK and TFEB. Mathematical modelling of this loop shows that it can give rise to two distinct cellular phenotypes – pAMPKhigh/TFEBhigh/pERKlow and pAMPKlow/TFEBlow/pERKhigh – and phenotype switching, thus offering a mechanistic basis for our observations for non-genetic heterogeneity in anoikis adaptation. Significantly, we observed these heterogeneous cell states in patient-derived circulating tumor cells also. Thus, our work unravels a novel feedback loop that can generate non-genetic heterogeneity within matrix-detached cancer cells; targeting such loops may offer novel therapeutic approaches for restricting metastasis and improving therapeutic efficacy.