Abstract
Simple SummaryAutophagy is a cell survival and recycling mechanism which protects cancer cells upon therapeutic drug treatment. Here we investigated the impact of autophagy inhibition in a cancer of lymphoid origin, namely ALK-positive anaplastic large cell lymphoma (ALK + ALCL). We inhibited autophagy in two distinct cell subsets of ALK + ALCL, one of which we previously shown to possess more stem-like and tumorigenic properties. Our study found that blockage of autophagy in the stem-like subset resulted in marked drug-sensitization to crizotinib, a current therapeutic agent used to treat ALK + ALCL. We also found differential involvement of the Myconcogene in the autophagy process within the two subsets and identified its relative importance to the stem-like population. Our research suggests inhibition of autophagy alongside crizotinib preferentially targets stem-like cells, thus improving crizotinib therapy.Previously it was shown that autophagy contributes to crizotinib resistance in ALK-positive anaplastic large cell lymphoma (ALK + ALCL). We asked if autophagy is equally important in two distinct subsets of ALK + ALCL, namely Reporter Unresponsive (RU) and Reporter Responsive (RR), of which RR cells display stem-like properties. Autophagic flux was assessed with a fluorescence tagged LC3 reporter and immunoblots to detect endogenous LC3 alongside chloroquine, an autophagy inhibitor. The stem-like RR cells displayed significantly higher autophagic response upon crizotinib treatment. Their exaggerated autophagic response is cytoprotective against crizotinib, as inhibition of autophagy using chloroquine or shRNA against BECN1 or ATG7 led to a decrease in their viability. In contrast, autophagy inhibition in RU resulted in minimal changes. Since the differential protein expression of MYC is a regulator of the RU/RR dichotomy and is higher in RR cells, we asked if MYC regulates the autophagy-mediated cytoprotective effect. Inhibition of MYC in RR cells using shRNA significantly blunted crizotinib-induced autophagic response and effectively suppressed this cytoprotective effect. In conclusion, stem-like RR cells respond with rapid and intense autophagic flux which manifests with crizotinib resistance. For the first time, we have highlighted the direct role of MYC in regulating autophagy and its associated chemoresistance phenotype in ALK + ALCL stem-like cells.
Highlights
Macroautophagy, more commonly referred to as autophagy, is a process originally discovered in yeasts as a survival mechanism in the face of starvation [1,2]
The generation of Reporter Unresponsive (RU) and Reporter Responsive (RR) cell subsets derived from ALK + ALCL cell lines have been detailed previously [28]
Despite that RR cells are known to have a baseline level of GFP expression due to their intrinsic SOX2 reporter activity [32,34], the green fluorescence from pHluorin has been shown to be more sensitive to pH decreases as a result of autophagy in comparison to the green fluorescence from GFP [33]
Summary
Macroautophagy, more commonly referred to as autophagy, is a process originally discovered in yeasts as a survival mechanism in the face of starvation [1,2]. Autophagy is known to be an important homeostatic process in human cells, in which misfolded proteins, damaged organelles and intracellular pathogens are removed through the autophagosome/lysosome pathway [3,4]. In keeping with its role in chemoresistance, a number of clinical trials have shown promising results regarding the use of autophagy inhibitors (such as chloroquine and its derivatives hydroxychloroquine and 3-methyladenine) in sensitizing cancer cells to chemotherapeutic agents [11,12]. The connection between autophagy and other important aspects of cancer cell biology, such as apoptosis [2,13] and cancer stemness [14,15] have been increasingly recognized
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