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Abstract
Disrupting functional protein homeostasis is an established therapeutic strategy for certain tumors. Ongoing studies are evaluating autophagy inhibition for overcoming chemotherapeutic resistance to such therapies by neutralizing lysosomal pH. New and sensitive methods to monitor autophagy in patients are needed to improve trial design and interpretation. We report that mitochondrial-damaged breast cancer cells and rat breast tumors accumulate p53-positive protein aggregates that resist lysosomal degradation. These aggregates were localized to enzymatically-active autolysosomes that were degrading autophagosomes and the autophagic receptor proteins TAX1BP1 and NDP52. NDP52 was identified to associate with aggregated proteins and knocking down NDP52 led to the accumulation of protein aggregates. TAX1BP1 was identified to partly localize with aggregates, and knocking down TAX1BP1 enhanced aggregate formation, suppressed autophagy, impaired NDP52 autophagic degradation and induced cell death. We propose that quantifying aggregates and autophagic receptors are two potential methods to evaluate autophagy and lysosomal degradation, as confirmed using primary human tumor samples. Collectively, this report establishes protein aggregates and autophagy receptors, TAX1BP1 and NDP52, as potential endpoints for monitoring autophagy during drug development and clinical studies.
Highlights
Inhibition of proteasome function is a standard treatment component for certain cancers like multiple myeloma[1], but has limited activity in patients with metastatic breast cancer[2]
Protein aggregates in the MTA-treated breast cancer cell lines did increase as compared to the nontreated cancer cells or the non-cancerous cell line (Fig. 1a) and prolonging the treatment resulted in further aggregate accumulation (Supplementary Fig. 1b)
CNG gels revealed that large (>450 kDa) protein bands were only detectable in the insoluble fractions from MTA-treated cells (Fig. 1d) and micro-flow imaging (MFI) demonstrated a quantifiable increase in sub-visible particles 1–5 μm in size within cells subjected to Bortezomib, carbonyl cyanide m-chlorophenyl hydrazine (CCCP), and MTAs (Fig. 1e)
Summary
Inhibition of proteasome function is a standard treatment component for certain cancers like multiple myeloma[1], but has limited activity in patients with metastatic breast cancer[2]. New methods to accurately characterize autophagy in patients is a critical gap in this field and limits the interpretation of such trials[6]. We investigate protein aggregates and the autophagic mechanism to identify mechanisticallyrelevant targets and methods that may improve the clinical assessment of autophagy. Selective autophagy is the recognition of specific cargo, such as protein aggregates (aggrephagy) and dysfunctional mitochondria (mitophagy)[8]. Autophagic receptor proteins facilitate selective cargo recognition by utilizing ubiquitin binding domains to recognize poly-ubiquitinated misfolded proteins and a LC3 binding domain to associate with the autophagosome[9,10]. Several mammalian autophagic receptors are known to recognize poly-ubiquitinated proteins on the mitochondria for mitophagy[11], but the ability to recognize aggregated proteins independent of the mitochondria remains unclear. Clinical trials have heavily focused on p62, a selective autophagic receptor
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