Chaperone-mediated autophagy in disease : from disease-driven suppression to potential therapeutic reactivation

<jats:p>&lt;p dir="ltr"&gt;Alteration of the proteome through aberrant expression, stabilization, and accumulation of disease-driving proteins, is a common feature of many human diseases. Targeted elimination of these pathological proteins represents an attractive therapeutic strategy. Chaperone-mediated autophagy (CMA) is a selective lysosomal degradation pathway in which proteins containing a CMA- recognition motif are recognized and delivered to lysosomes via the rate-limiting receptor LAMP-2A, shaping proteome and regulating diverse biological processes. This thesis investigates the role of CMA and the therapeutic potential of its pharmacological restoration in two human diseases characterized by pathological proteome remodeling: metastatic cancer and pathological cardiac hypertrophy.&lt;/p&gt;&lt;p dir="ltr"&gt;A central contribution of this thesis was the generation of the first isoform- specific LAMP-2A knockout (KO) human cell lines, a model previously lacking. LAMP-2A-deficient cells enabled KO validation of anti-LAMP-2A antibodies for immunostaining, providing a rigorous tool for assessing LAMP-2A expression in clinical samples, and allowed investigation of CMA's role in both disease contexts. In cancer, we found that genetic loss of CMA promotes mesenchymal tumor growth and metastatic dissemination, increasing overall tumor aggressiveness. We showed that metastatic lesions had suppressed LAMP-2A expression relative to matched primary tumors from the same patients, correlating with acquisition of mesenchymal traits. Mechanistically, we found that CMA directly degrades pro- metastatic proteins, including components of the TGF-ß signaling pathway, and we demonstrated that CMA-loss-induced tumor growth was driven by exacerbated TGF-B signaling, identifying CMA as an endogenous barrier to metastatic progression.&lt;/p&gt;&lt;p dir="ltr"&gt;This thesis further investigates whether oncogenic signaling could be responsible for CMA suppression and if it is therapeutically reversible. While TGF-ß signaling further reduced LAMP-2A expression, revealing a reciprocal regulatory loop that reinforces CMA suppression during metastatic progression, we found that oncogenic ERK signaling was a major reversible upstream repressor of LAMP-2A expression. Transcriptomic and functional analyses further identified the FOX transcription factors FOXO1 and FOXP1 as mediators of LAMP-2A transcription downstream of ERK inhibition, providing a molecular mechanism by which oncogenic signaling suppresses CMA. In parallel, by a chemical screen, we identified GSK1059615 as a pharmacological activator of CMA that restored CMA activity and demonstrated efficacy in both in vitro and in vivo models, providing an important tool for evaluating the therapeutic potential of CMA activation.&lt;/p&gt;&lt;p dir="ltr"&gt;The last part of this thesis investigates the role of CMA in pathological cardiac hypertrophy, a context in which CMA had not been studied. We found that CMA activity was suppressed across clinical transcriptomic datasets and in rodent and human models of cardiac hypertrophy. Genetic ablation of LAMP-2A was sufficient to induce hypertrophic growth through enhanced TGF-ß signaling, demonstrating that CMA deficiency is a causal driver of pathological hypertrophy. Conversely, we showed that pharmacological restoration of CMA reversed established hypertrophy in a LAMP-2A-dependent manner in cardiomyocytes.&lt;/p&gt;&lt;p dir="ltr"&gt;Collectively, this work demonstrates a protective role for CMA in metastatic progression and cardiac hypertrophy, identifying CMA restoration as a promising therapeutic strategy.&lt;/p&gt;&lt;h3 dir="ltr"&gt;List of scientific papers&lt;/h3&gt;&lt;p dir="ltr"&gt;I. Zhou X, Shirokova V, Kaminskyy VO, &lt;b&gt;Berenger E,&lt;/b&gt; Kochetkova E, Norberg E, Genander M and Vakifahmetoglu-Norberg H (2023). Knockout validation of LAMP2A antibodies for immunostaining in human cancer cells. Autophagy. 19: 2575-2577.&lt;br&gt;&lt;a href="https://doi.org/10.1080/15548627.2023.2213515" target="_blank" rel="noreferrer"&gt;https://doi.org/10.1080/15548627.2023.2213515&lt;br&gt;&lt;br&gt;&lt;/a&gt;&lt;/p&gt;&lt;p dir="ltr"&gt;II. Zhou X*, &lt;b&gt;Berenger E*&lt;/b&gt;, Shi Y*, Shirokova V, Kochetkova E, Becirovic T, Zhang B, Kaminskyy VO, Esmaeilian Y, Hosaka K, Lindskog C, Hydbring P, Ekman S, Cao Y, Genander M, Iwanicki M, Norberg E and Vakifahmetoglu-Norberg H (2025). Chaperone-mediated autophagy regulates the metastatic state of mesenchymal tumors. EMBO Mol Med. 17: 747-774.&lt;br&gt;&lt;a href="https://doi.org/10.1038/s44321-025-00210-w" target="_blank" rel="noreferrer"&gt;https://doi.org/10.1038/s44321-025-00210-w&lt;br&gt;&lt;br&gt;&lt;/a&gt;&lt;/p&gt;&lt;p dir="ltr"&gt;III. &lt;b&gt;Berenger E*&lt;/b&gt;, Kacal M*, Maestri A, Kochetkova E, Zhang B, Sajwan S, Mannervik M, Norberg E, Kaminskyy VO and Vakifahmetoglu- Norberg H. Oncogenic ERK signaling represses chaperone- mediated autophagy through transcriptional control of LAMP-2A. [Submitted]&lt;/p&gt;&lt;p dir="ltr"&gt;IV. &lt;b&gt;Berenger E,&lt;/b&gt; Pironti G, Andersson DC, Chevillard C, Kaminskyy V, Norberg E and Vakifahmetoglu-Norberg H. Restoring chaperone- mediated autophagy reverses pathological pressure overload cardiac hypertrophy. [Manuscript]&lt;/p&gt;&lt;p dir="ltr"&gt;* These authors contributed equally.&lt;/p&gt;</jats:p>