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The role of chaperone-mediated autophagy in drug resistance

Abstract

In the search for alternatives to overcome the challenge imposed by drug resistance development in cancer treatment, the modulation of autophagy has emerged as a promising alternative that has achieved good results in clinical trials. Nevertheless, most of these studies have overlooked a novel and selective type of autophagy: chaperone-mediated autophagy (CMA). Following its discovery, research into CMA’s contribution to tumor progression has accelerated rapidly. Therefore, we now understand that stress conditions are the primary signal responsible for modulating CMA in cancer cells. In turn, the degradation of proteins by CMA can offer important advantages for tumorigenesis, since tumor suppressor proteins are CMA targets. Such mutual interaction between the tumor microenvironment and CMA also plays a crucial part in establishing therapy resistance, making this discussion the focus of the present review. Thus, we highlight how suppression of LAMP2A can enhance the sensitivity of cancer cells to several drugs, just as downregulation of CMA activity can lead to resistance in certain cases. Given this panorama, it is important to identify selective modulators of CMA to enhance the therapeutic response.

Keywords:
Chaperone-mediated autophagy; cancer; resistance

Introduction

Cancer encompasses a diverse range of approximately 277 distinct disease types (Demir Cetinkaya and Biray Avci, 2022Demir Cetinkaya B and Biray Avci C (2022) Molecular perspective on targeted therapy in breast cancer: A review of current status. Med Oncol 39:149.), and it is one of the most extensively studied conditions in any area of science. The clonal evolutionary concept of cancer progression was introduced in 1976. This model essentially posits that genetically unstable cells display several gene mutations, and selective pressures - arising from both endogenous or exogenous factors, such as chemotherapeutic treatment - foster the growth and survival of subpopulations possessing a biological fitness advantage (Nowell, 1976Nowell PC (1976) The clonal evolution of tumor cell populations. Science 194:23-28. ). This phenomenon contributes to the molecular complexity of cancer and the pronounced heterogeneity found within each type of cancer. Certainly, this divergency lies at the core of the challenge in achieving high effectiveness in healing and therapeutic processes for patients, primarily due to acquired resistance achieved by natural selection during cancer development. Therefore, cancer ranks as the second leading global cause of death among children and adults worldwide (Naghavi et al., 2017Naghavi M, Abajobir AA, Abbafati C, Abbas KM, Abd-Allah F, Abera SF, Aboyans V, Adetokunboh O, Ärnlöv J, Afshin A et al. (2017) Global, regional, and national age-sex specific mortality for 264 causes of death, 1980-2016: A systematic analysis for the Global Burden of Disease Study 2016. Lancet 390:1151-1210. ), and its prevalence is on the rise.

In fact, cancer poses a significant health challenge for humanity as a whole, prompting extensive studies majorly focusing on therapeutic drug development. However, one of the critical hurdles encountered during cancer therapy is the emergence of resistance mechanisms. Some examples of resistance mechanisms that cancer cells can employ are alterations of efflux or influx pumping of the drug to the cell, DNA repair pathways activation, immune evasion, and metabolic adaptation (Holohan et al., 2013Holohan C, Van Schaeybroeck S, Longle DB and Johnston PG (2013) Cancer drug resistance: An evolving paradigm. Nat Rev Cancer 13:714-726.). Thus, given that cancer cells exhibit abnormal nutrient requirements in contrast to normal cells, pathways that control cell metabolism and growth are an interesting target to be studied as potential therapeutic alternatives and ways to overcome resistance. In this context, the autophagy process, which involves the cellular recycling of components, has gained significant attention over the past years. Modulating autophagic pathways present a promising avenue for targeted cancer therapies, as it can influence the survival and growth of cancer cells by controlling their nutrient use and stress responses.

Given the substantial data regarding the role of autophagy in supporting tumor growth and promote resistance (Levy et al., 2017Levy JMM, Zahedi S, Griesinger AM, Morin A, Davies KD, Aisner DL, Kleinschmidt-DeMasters BK, Fitzwalter BE, Goodall ML, Thorburn J et al. (2017) Autophagy inhibition overcomes multiple mechanisms of resistance to BRAF inhibition in brain tumors. Elife 6:e19671.; Mele et al., 2020Mele L, Del Vecchio V, Liccardo D, Prisco C, Schwerdtfeger M, Robinson N, Desiderio V, Tirino V, Papaccio G and La Noce M (2020) The role of autophagy in resistance to targeted therapies. Cancer Treat Rev 88:102043.; Noman et al., 2020Noman MZ, Parpal S, Van Moer K, Xiao M, Yu Y, Viklund J, De Milito A, Hasmim M, Andersson M, Amaravadi RK et al. (2020) Inhibition of Vps34 reprograms cold into hot inflamed tumors and improves anti-PD-1/PD-L1 immunotherapy. Sci Adv 6:eaax7881.), researchers are actively investigating how to modulate it to enhance and overcome antitumor treatments, with promising results emerging from clinical trials, but also United States Food and Drug Administration (FDA) approved agents (reviewed in Mohsen et al., 2022Mohsen S, Sobash PT, Algwaiz GF, Nasef N, Al-Zeidaneen SA and Karim NA (2022) Autophagy agents in clinical trials for cancer therapy: A brief review. Curr Oncol 29:1695-1708. ). Until now, the only FDA-approved autophagy inhibitors are Chloroquine and Hydroxychloroquine, both being used to treat Malaria (Ganguli et al., 2014Ganguli A, Choudhury D, Datta S, Bhattacharya S and Chakrabarti G (2014) Inhibition of autophagy by chloroquine potentiates synergistically anti-cancer property of artemisinin by promoting ROS dependent apoptosis. Biochimie 107:338-349.; Manic et al., 2014Manic G, Obrist F, Kroemer G, Vitale I and Galluzzi L (2014) Chloroquine and hydroxychloroquine for cancer therapy. Mol Cell Oncol 1:e29911. ; De Sanctis et al., 2023De Sanctis JB, Charris J, Blanco Z, Ramírez H, Martínez GP and Mijares MR (2023) Molecular mechanisms of chloroquine and hydroxychloroquine used in cancer therapy. Anticancer Agents Med Chem 23:1122-1144.). A more novel pathway of the NEDD-8 inhibitory agent, Pevonedistat, is undergoing clinical trials. Pevonedistat (MLN4924) has been shown to increase cell apoptosis and autophagy via neddylation (Soucy et al., 2010Soucy TA, Dick LR, Smith PG, Milhollen MA and Brownell JE (2010) The NEDD8 conjugation pathway and its relevance in cancer biology and therapy. Genes Cancer 1:708-716. ). Therefore, unlike Chloroquine/Hydroxychloroquine, Pevonedistat acts as a pro-apoptotic and autophagy inducer rather than an inhibitor.

Considering these aforementioned developments, there is a substantial expanse of this yet unexplored field towards the domain of autophagy modulation, targeting an enhanced response to cancer treatment. Nevertheless, the ongoing trials are primarily oriented towards the modulation of only one form of autophagy, known as macroautophagy (MA) (Deretic, 2008Deretic V (2008) Autophagosome and phagosome. Methods Mol Biol 445:10.; Hu et al., 2008Hu C, Solomon VR, Ulibarri G and Lee H (2008) The efficacy and selectivity of tumor cell killing by Akt inhibitors are substantially increased by chloroquine. Bioorg Med Chem 16:7888-7893.) overlooking a very important form of autophagy named Chaperone-Mediated Autophagy (CMA). Such limited approach accentuates the vast prospects for advancing cancer treatment through a comprehensive understanding of these complex pathways, that we aim to better explore in this review.

Discovery of selective lysosomal degradation: CMA

In 1963, Christian de Duve used the term “autophagy”, derived from the Greek for “self-eating”, to describe the presence of single or double-membrane vesicles containing pieces of cytoplasm and organelles in various states of degradation (De Duve and Wattiaux, 1966De Duve C and Wattiaux R (1966) Functions of lysosomes. Annu Rev Physiol 28:435-492. ). Currently, it is known that autophagy is a mechanism by which cytoplasmic material is delivered to the lysosome for degradation and recycling, being a crucial physiological process for sustaining eukaryotic cells homeostasis (Ohsumi, 2014Ohsumi Y (2014) Historical landmarks of autophagy research. Cell Res 24:9-23. ; Parzych and Klionsky, 2014Parzych KR and Klionsky DJ (2014) An overview of autophagy: Morphology, mechanism, and regulation. Antioxid Redox Signal 20:460-473. ). Different from the ubiquitin-proteasome system (UPS) (Nandi et al., 2006Nandi D, Tahiliani P, Kumar A and Chandu D (2006) The ubiquitin-proteasome system. J Biosci 31:137-155. ), autophagy is not limited to protein degradation, but also plays a central role in cell metabolism, in addition to the degradation of other types of biomolecules such as carbohydrates and lipids, organelles and even some pathogens (Parzych and Klionsky, 2014Parzych KR and Klionsky DJ (2014) An overview of autophagy: Morphology, mechanism, and regulation. Antioxid Redox Signal 20:460-473. ). Thus, autophagy is related to several biological events, which occur in response to stressful situations but also under physiological conditions to support normal cellular functions. Some of these include catabolizing the degradation and recycling of intracellular components by the elimination of defective proteins and organelles assuring nutrient and energy balance, cellular stress response, prevention of abnormal protein aggregate accumulation, removal of intracellular pathogens, and DNA repair and cell death (Galluzzi et al., 2017Galluzzi L, Baehrecke EH, Ballabio A, Boya P, Pedro JMB, Cecconi F, Choi AM, Chu CT, Codogno P, Colombo MI et al. (2017) Molecular definitions of autophagy and related processes. EMBO J 36:1811-1836. ).

The fundamental significance of autophagy for health and longevity has been confirmed by numerous studies suggesting that various autophagic processes are compromised with aging (Kaushik et al., 2021Kaushik S, Tasset I, Arias E, Pampliega O, Wong E, Martinez-Vicente M and Cuervo AM (2021) Autophagy and the hallmarks of aging. Ageing Res Rev 72:101468.). Moreover, autophagy has been linked to several human health conditions, including cancer (Ogier-Denis and Codogno, 2003Ogier-Denis E and Codogno P (2003) Autophagy: A barrier or an adaptive response to cancer. Biochim Biophys Acta 1603:113-128. ; White, 2015White E (2015) The role for autophagy in cancer. J Clin Invest 125:42-46. ). As autophagy can promote cell survival during stress conditions, in the context of cancer treatment, this process can have a dual role, either pro-survival or pro-death, depending on the different stages of tumorigenesis. On the one hand, if by breaking down cellular components and recycling them, cancer cells can sustain their energy needs and reduce the toxic effects of treatment, enabling them to survive and recover (Amaravadi, 2008Amaravadi RK (2008) Autophagy-induced tumor dormancy in ovarian cancer. J Clin Invest 118:3837-3840.; Laddha et al., 2014Laddha SV, Ganesan S, Chan CS and White E (2014) Mutational landscape of the essential autophagy gene BECN1 in human cancers. Mol Cancer Res 12:485-490.; Strohecker and White, 2014Strohecker AM and White E (2014) Autophagy promotes BrafV600E-driven lung tumorigenesis by preserving mitochondrial metabolism. Autophagy 10:384-385. ; Guo et al., 2016Guo JY, Teng X, Laddha SV, Ma S, Van Nostrand SC, Yang Y, Khor S, Chan CS, Rabinowitz JD and White E (2016) Autophagy provides metabolic substrates to maintain energy charge and nucleotide pools in Ras-driven lung cancer cells. Genes Dev 30:1704-1717.; Yamamoto et al., 2020Yamamoto K, Venida A, Yano J, Biancur DE, Kakiuchi M, Gupta S, Sohn ASW, Mukhopadhyay S, Lin EY, Parker SJ et al. (2020) Autophagy promotes immune evasion of pancreatic cancer by degrading MHC-I. Nature 581:100-105. ), on the other hand, excessive or prolonged autophagy can lead to autophagic programmed cell death, contributing to treatment-induced cell death (Liang et al., 1999Liang XH, Jackson S, Seaman M, Brown K, Kempkes B, Hibshoosh H and Levine B (1999) Induction of autophagy and inhibition of tumorigenesis by beclin 1. Nature 402:672-676.; Qu et al., 2003Qu X, Yu J, Bhagat G, Furuya N, Hibshoosh H, Troxel A, Rosen J, Eskelinen EL, Mizushima N, Ohsumi Y et al. (2003) Promotion of tumorigenesis by heterozygous disruption of the beclin 1 autophagy gene. J Clin Invest 112:1809-1820. ; Mathew et al., 2009Mathew R, Karp CM, Beaudoin B, Vuong N, Chen G, Chen HY, Bray K, Reddy A, Bhanot G, Gelinas C et al. (2009) Autophagy suppresses tumorigenesis through elimination of p62. Cell 137:1062-1075.).

As mentioned previously, the understanding of the mechanism and pathophysiology of autophagy has expanded over the last few decades. Initially, the term ‘autophagy’ was used to refer only to MA, but we now know that it refers to three distinct types, named: MA, CMA and Microautophagy (MI) (Duan and Tong, 2021Duan X and Tong C (2021) Autophagy in Drosophila and Zebrafish. Adv Exp Med Biol 1208:33-356.). Although these types share a commonality in degrading damaged cellular components through lysosomes, their approaches to transporting substrates to the lysosome differ greatly (Yang and Klionsky, 2010Yang Z and Klionsky DJ (2010) Mammalian autophagy: Core molecular machinery and signaling regulation. Curr Opin Cell Biol 22:124-131. ).

In mammals, MA is the most comprehensively researched process and it starts with the formation of a double-membrane vesicle known as an autophagosome that absorbs molecules or organelles found in the cytoplasm. It then merges with the lysosome giving rise to the autolysosome, resulting in the cargo degradation (Feng et al., 2014Feng Y, He D, Yao Z and Klionsky DJ (2014) The machinery of macroautophagy. Cell Res 24:24-41.). Microautophagy is the pathway that has received the least attention. It works by invaginating a portion of the cytoplasm through deformation of the lysosomal membrane (Mijaljica et al., 2011Mijaljica D, Prescott M and Devenish RJ (2011) Microautophagy in mammalian cells: Revisiting a 40-year-old conundrum. Autophagy 7:673-682.; Parzych and Klionsky, 2014Parzych KR and Klionsky DJ (2014) An overview of autophagy: Morphology, mechanism, and regulation. Antioxid Redox Signal 20:460-473. ). These two pathways function by simultaneously sequestering different cytosolic components, thus lacking the capacity to degrade biomolecules selectively and individually.

Unlike the other types of autophagy pathways, CMA is a selective form specialized in protein degradation, which is based on its individual translocation through the lysosomal membrane after recognizing a specific sequence motif (KFERQ-like) (Orenstein and Cuervo, 2010Orenstein SJ and Cuervo AM (2010) Chaperone-mediated autophagy: Molecular mechanisms and physiological relevance. Semin Cell Dev Biol 21:719-726. ; Kaushik and Cuervo, 2018Kaushik S and Cuervo AM (2018) The coming of age of chaperone-mediated autophagy. Nat Rev Mol Cell Biol 19:365-381. ; Auzmendi-Iriarte and Matheu, 2021Auzmendi-Iriarte J and Matheu A (2021) Impact of chaperone-mediated autophagy in brain aging: Neurodegenerative diseases and glioblastoma. Front Aging Neurosci 12:630743.). Importantly, approximately 40% of cytosolic proteins contain the amino acid sequence corresponding to this motif (Tekirdag and Cuervo, 2018Tekirdag K and Cuervo AM (2018) Chaperone-mediated autophagy and endosomal microautophagy: Joint by a chaperone. J Biol Chem 293:5414-5424. ). Structurally, KFERQ refers to a pentapeptide sequence containing a lysine residue; one of the four hydrophobic amino acids (phenylalanine, valine, leucine or isoleucine); glutamic acid or aspartic acid; arginine at the beginning or end of the sequence; glutamine (Fred Dice, 1990Fred Dice J (1990) Peptide sequences that target cytosolic proteins for lysosomal proteolysis. Trends Biochem Sci 15:305-309.). Post-translational modifications, like phosphorylation or acetylation, can provide the necessary charge and complete the motif, even if only four of the five amino acids are present in the protein by constitution (Bandyopadhyay et al., 2008Bandyopadhyay U, Kaushik S, Varticovski L and Cuervo AM (2008) The chaperone-mediated autophagy receptor organizes in dynamic protein complexes at the lysosomal membrane. Mol Cell Biol 28:5747-5763.).

Of notice, the involvement of co-chaperones is a crucial aspect for the CMA mechanism (Figure 1) to function efficiently, as they play a key role in regulating other chaperones that are necessary for the lysosomal degradation process (Agarraberes and Dice, 2001Agarraberes FA and Dice JF (2001) A molecular chaperone complex at the lysosomal membrane is required for protein translocation. J Cell Sci 114:2491-2499.). In the cytosol, substrate recognition is carried out by the 70 kDa heat shock cognate protein (Hsc70 chaperone), also known as HSPA8 (heat shock protein family A [Hsp70] member 8). Hsc70 binds to the KFERQ-like motif present in the target protein (Chiang et al., 1989Chiang HL, Terlecky SR, Plant CP and Dice JF (1989) A role for a 70-kilodalton heat shock protein in lysosomal degradation of intracellular proteins. Science 246:382-385.), and the complex is subsequently targeted to the surface of the lysosomal membrane. At this stage, the lysosome-associated membrane protein type 2A (LAMP2A), a receptor for CMA substrates (Cuervo and Dice, 1996Cuervo AM and Dice JF (1996) A receptor for the selective uptake and degradation of proteins by lysosomes. Science 273:501-503.), binds to the substrate and multimerizes to enable its translocation into the lysosomal lumen (Bandyopadhyay et al., 2008Bandyopadhyay U, Kaushik S, Varticovski L and Cuervo AM (2008) The chaperone-mediated autophagy receptor organizes in dynamic protein complexes at the lysosomal membrane. Mol Cell Biol 28:5747-5763.). Meanwhile, the chaperone Hsc90 maintains the stability of LAMP2A and prevents the target proteins from refolding and hindering transport. After substrate-receptor binding, the chaperone present in the lysosomal lumen (lys-hsc70) helps the cargo translocation for degradation. After complete substrate degradation in the lysosomal matrix, LAMP2A can return to its initial state and bind to new proteins, allowing the translocation and degradation cycle to continue (Agarraberes and Dice, 2001Agarraberes FA and Dice JF (2001) A molecular chaperone complex at the lysosomal membrane is required for protein translocation. J Cell Sci 114:2491-2499.; Orenstein and Cuervo, 2010Orenstein SJ and Cuervo AM (2010) Chaperone-mediated autophagy: Molecular mechanisms and physiological relevance. Semin Cell Dev Biol 21:719-726. ).

Figure 1 -
CMA mechanism. The CMA pathway is initiated by recognition of the target protein in the cytosol by binding of the Hsc70 chaperone to the KFERQ motif present in the substrate (1). Subsequently, the substrate is directed to the surface of the lysosome membrane and encounters LAMP2A (2), which forms a multimeric complex (3), allowing protein translocation into the lysosomal lumen (4). Upon complete protein degradation (5), LAMP2A returns to its monomeric state (6) to initiate new processes and translocations. Created with BioRender.

Thus, de novo synthesis of LAMP2A is not mandatory for the initiation of a new degradation cycle. The lysosomal abundance of this protein can be modulated by changes in its stability, organization and dynamics in the lysosomal membrane, properties orchestrated by proteins associated with the lysosome, such as the GFAP/EF1α pair and the mTORC2/AKT1/PHLPP1 axis (Arias and Cuervo, 2020Arias E and Cuervo AM (2020) Pros and cons of chaperone-mediated autophagy in cancer biology. Trends Endocrinol Metab 31:53-66. ). Importantly, the LAMP2A protein is essential for the proper functioning of the CMA, as several studies have identified it as the key protein in the pathway. As a consequence, the levels of LAMP2A present in the lysosomal membrane regulate the rate of CMA performance (Park et al., 2015Park C, Suh Y and Cuervo AM (2015) Regulated degradation of Chk1 by chaperone-mediated autophagy in response to DNA damage. Nat Commun 6:6823. ). Hence, modulation of LAMP2A is one of the methods used to study CMA.

All eyes on the CMA: Experimental approaches to this selective autophagy

Despite the well-defined specificities of CMA in relation to the other types of autophagy, one must consider that in most cells, including tumor cells, there is a crosstalk between the three autophagic pathways and there may be compensation between them in the event of deficiency or malfunction (Massey et al., 2006Massey AC, Kaushik S, Sovak G, Kiffin R and Cuervo AM (2006) Consequences of the selective blockage of chaperone-mediated autophagy. Proc Natl Acad Sci U S A 103:5805-5810.; Schneider et al., 2015Schneider JL, Villarroya J, Diaz-Carretero A, Patel B, Urbanska AM, Thi MM, Villarroya F, Santambrogio L and Cuervo AM (2015) Loss of hepatic chaperone-mediated autophagy accelerates proteostasis failure in aging. Aging Cell 14:249-264. ). Because of this extensive connection between the different autophagic pathways, specific methods for assessing CMA activity are needed in order to understand its role, whether in a pathological or physiological condition or in response to possible therapeutic interventions (Arias and Cuervo, 2020Arias E and Cuervo AM (2020) Pros and cons of chaperone-mediated autophagy in cancer biology. Trends Endocrinol Metab 31:53-66. ). Initially, the search for methods to assess and measure CMA was more complex than for MA because the structural characteristics at the molecular level were not fully understood. But as demonstrated, there is now a foundation for a wide range of methods to study CMA, providing new tools to help researchers in the search for CMA modulation as a therapeutic target (Hubert et al., 2022Hubert V, Weiss S, Rees AJ and Kain R (2022) Modulating chaperone-mediated autophagy and its clinical applications in cancer. Cells 11:2562.). The evaluation methods for CMA are categorized as shown in Table 1. It is noteworthy that some techniques have intrinsic limitations and may require additional complementary methods.

Table 1 -
Experiments for CMA evaluation.

The most frequent methods used to study changes in CMA activity are immunoblotting and imaging, in which antibodies that can specifically recognize LAMP2A distinguishing from its variants LAMP2B and LAMP2C are used. These experiments, known as steady-state assays, can evaluate the overall activity of CMA. This category includes fluorescence assays, the use of immunogold experiments in tissues to quantify the presence of CMA activity within lysosomes, and the evaluation of lysosomal levels of CMA components (such as hsc70 and LAMP2A). There are many assays available to measure CMA activity. One of the strategies is the evaluation of lysosomal levels of LAMP2A (colocalized with hsc70 and associated with Lys-HSC70), since increased rates of CMA often correlates with high levels of LAMP2A (Cuervo and Dice, 1996Cuervo AM and Dice JF (1996) A receptor for the selective uptake and degradation of proteins by lysosomes. Science 273:501-503.; Agarraberes and Dice, 2001Agarraberes FA and Dice JF (2001) A molecular chaperone complex at the lysosomal membrane is required for protein translocation. J Cell Sci 114:2491-2499.). However, functional assays, such as intracellular protein degradation, photoconvertible CMA reporters, and isolated lysosome in vitro/in vivo methods provide a more accurate examination of CMA activity over time (Patel and Cuervo, 2015Patel B and Cuervo AM (2015) Methods to study chaperone-mediated autophagy. Methods 75:133-140. ).

Besides that, pulse and chase experiments are very powerful tools to measure CMA, as it uses a radiolabeled amino acid together with inhibitors that target lysosomal proteases or alternative autophagic pathways (like MA) (Kaushik and Cuervo, 2018Kaushik S and Cuervo AM (2018) The coming of age of chaperone-mediated autophagy. Nat Rev Mol Cell Biol 19:365-381. ). Thus, if protein degradation is more sensitive to lysosome inhibitors such as leupeptin and ammonium chloride (NH4Cl) and insensitive to MA inhibitors such 3-MA, it is considered to be a CMA-dependent process (Fuertes et al., 2003Fuertes G, Martín De Llano JJ, Villarroya A, Rivett AJ and Knecht E (2003) Changes in the proteolytic activities of proteasomes and lysosomes in human fibroblasts produced by serum withdrawal, amino-acid deprivation and confluent conditions. Biochem J 375:75-86. ).

The dual role of CMA in cancer

Physiologically, CMA is responsible for the degradation of misfolded and damaged proteins preventing cellular proteotoxicity (Jackson and Hewitt, 2016Jackson MP and Hewitt EW (2016) Cellular proteostasis: Degradation of misfolded proteins by lysosomes. Essays Biochem 60:173-180.; Kaushik and Cuervo, 2018Kaushik S and Cuervo AM (2018) The coming of age of chaperone-mediated autophagy. Nat Rev Mol Cell Biol 19:365-381. ). This autophagic pathway, like the others, is activated by stressors such as hypoxia, oxidative stress, tissue remodeling, nutrient deprivation and in response to genotoxic insults (Kiffin et al., 2004Kiffin R, Christian C, Knecht E and Cuervo AM (2004) Activation of chaperone-mediated autophagy during oxidative stress. Mol Biol Cell 15:4829-4840.; Park et al., 2015Park C, Suh Y and Cuervo AM (2015) Regulated degradation of Chk1 by chaperone-mediated autophagy in response to DNA damage. Nat Commun 6:6823. ). Therefore, impairment of the degradation of specific substrates by CMA may alter several cellular processes resulting, for instance, in cell sensitivity, genomic instability and defects in DNA maintenance and repair (Gomes et al., 2017 b Gomes LR, Menck CFM and Leandro GS (2017b) Autophagy roles in the modulation of DNA repair pathways. Int J Mol Sci 18:2351.).

Interestingly, several studies have shown that CMA can act as a tumor suppressor, preserving genomic stability and regulating the levels of proto-oncogenic (Gomes et al., 2017 a Gomes LR, Menck CFM and Cuervo AM (2017a) Chaperone-mediated autophagy prevents cellular transformation by regulating MYC proteasomal degradation. Autophagy 13:928-940. ; Arias and Cuervo, 2020Arias E and Cuervo AM (2020) Pros and cons of chaperone-mediated autophagy in cancer biology. Trends Endocrinol Metab 31:53-66. ), thus preventing malignant transformation. On the other hand, in tumor cells, CMA may act as an important precursor of tumorigenesis: it is known that certain features of the tumor microenvironment can stimulate the positive regulation of CMA, resulting in the modulation of proteins important for cancer development (Han et al., 2017Han Q, Deng Y, Chen S, Chen R, Yang M, Zhang Z, Sun X, Wang W, He Y, Wang F et al. (2017) Downregulation of ATG5-dependent macroautophagy by chaperone-mediated autophagy promotes breast cancer cell metastasis. Sci Rep 7:4759.; Arias and Cuervo, 2020Arias E and Cuervo AM (2020) Pros and cons of chaperone-mediated autophagy in cancer biology. Trends Endocrinol Metab 31:53-66. ). Therefore, although CMA plays a crucial role in cells in healthy conditions, in the oncogenic context, which is the focus of this review, modulation of this pathway is observed in tumor development, survival and progression (Kon et al., 2011Kon M, Kiffin R, Koga H, Chapochnick J, Macian F, Varticovski L and Cuervo AM (2011) Chaperone-mediated autophagy is required for tumor growth. Sci Transl Med 3:109ra117.).

What regulates CMA

Tumor cells often adapt to cope with environments featuring stress signaling. In fact, tumor microenvironment imposes a variety of challenges for cells, including hypoxia, a scarcity of growth factors and particular nutrients, and weakened substrate adhesion. Consequently, over time, only the best adjusted cells will survive, a process usually referred as a “potentiated state” (Arias and Cuervo, 2020Arias E and Cuervo AM (2020) Pros and cons of chaperone-mediated autophagy in cancer biology. Trends Endocrinol Metab 31:53-66. ). Importantly, stress conditions are the primary signal responsible for the activation of CMA (Kaushik and Cuervo, 2018Kaushik S and Cuervo AM (2018) The coming of age of chaperone-mediated autophagy. Nat Rev Mol Cell Biol 19:365-381. ).

In this sense, one of the most well-documented stress situations related to CMA activation is nutrient restriction. Typically, MA is triggered in the initial hours following serum removal, while CMA activity progressively rises, peaking around 10 hours post serum withdrawal in cultured cells or 3 days in animals (Li et al., 2011Li W, Yang Q and Mao Z (2011) Chaperone-mediated autophagy: Machinery, regulation and biological consequences. Cell Mol Life Sci 68:749-763. ). One of the pioneering studies addressing this topic was carried out by Cuervo’s group, reporting the activation of a selective metabolic pathway for lysosomal proteolysis in rat livers when subjected to extended food restriction (Cuervo et al., 1995Cuervo AM, Knecht E, Terlecky SR and Dice JF (1995) Activation of a selective pathway of lysosomal proteolysis in rat liver by prolonged starvation. Am J Physiol 269:C1200-C1208.). Such findings are important once cells might benefit by transitioning to a more selective degradation, allowing essential proteins to remain in the cytosol while targeting less critical ones for breakdown (Orenstein and Cuervo, 2010Orenstein SJ and Cuervo AM (2010) Chaperone-mediated autophagy: Molecular mechanisms and physiological relevance. Semin Cell Dev Biol 21:719-726. ).

Another documented stressor that can activate CMA is hypoxia, a condition characterized by inadequate oxygen availability in tissues, leading to a range of adaptive responses in cells, among them autophagy. The triggering of CMA during hypoxia may assist cells in selectively regulating the protein pool, removing potentially harmful or unnecessary proteins while preserving those essential for adaptation and survival under low oxygen conditions (Daskalaki et al., 2018Daskalaki I, Gkikas I and Tavernarakis N (2018) Hypoxia and selective autophagy in cancer development and therapy. Front Cell Dev Biol 6:104. ). Moreover, other stress signals, such as DNA damage, are crucial for CMA activation and it has been shown that a failure in its activation can lead to the accumulation of damage (Park et al., 2015Park C, Suh Y and Cuervo AM (2015) Regulated degradation of Chk1 by chaperone-mediated autophagy in response to DNA damage. Nat Commun 6:6823. ).

Finally, the redox status of cells governs CMA activity, which is believed to be an important mechanism to remove oxidized proteins. Additionally, the existing evidence suggests that the application of antioxidants can, to some extent or entirely, reverse or modulate autophagy, emphasizing the role of CMA in the elimination of oxidized proteins (Levonen et al., 2014Levonen AL, Hill BG, Kansanen E, Zhang J and Darley-Usmar VM (2014) Redox regulation of antioxidants, autophagy, and the response to stress: Implications for electrophile therapeutics. Free Radic Biol Med 71:196-207.). NRF2 (nuclear factor erythroid 2-related factor 2) is a protein that plays a pivotal role in regulating the cellular response to stress, especially oxidative stress, a primary form of stress to which tumor cells are subjected. Under physiological conditions, NRF2 is bound to a protein named KEAP-1, which tags it for degradation. In the presence of oxidative stress, NRF2 dissociates from Kelch‐like ECH‐associated protein 1 (KEAP1), translocates into the cell nucleus, and binds to antioxidant response elements (AREs) (De Souza et al., 2022De Souza I, Ramalho MCC, Guedes CB, Osawa IYA, Monteiro LKS, Gomes LR and Rocha CRR (2022) Ferroptosis modulation: Potential therapeutic target for glioblastoma treatment. Int J Mol Sci 23:6879.; Almeida Lima et al., 2023Almeida Lima K, Osawa IYA, Ramalho MCC, de Souza I, Guedes CB, Souza Filho CHD, Monteiro LKS, Latancia MT and Rocha CRR (2023) Temozolomide resistance in glioblastoma by NRF2: Protecting the evil. Biomedicines 11:1081.). It has been shown that NRF2 binds to the AREs in the LAMP2 gene, regulating its cellular levels. As a result, both overexpression of NRF2 or its pharmacological activation led to increased levels of LAMP2A and, subsequently, higher CMA activity. In the same study, the authors demonstrated that in mice knocked out for NRF2, CMA was impaired in lysosomes (Pajares et al., 2018Pajares M, Rojo AI, Arias E, Díaz-Carretero A, Cuervo AM and Cuadrado A (2018) Transcription factor NFE2L2/NRF2 modulates chaperone-mediated autophagy through the regulation of LAMP2A. Autophagy 14:1310-1322. ).

Recently, another study highlighted the role of NRF2 in the activation of CMA. In essence, the authors demonstrate the formation of an NRF2-CMA axis in the form of a positive feedback loop to enhance the antioxidant response and protect cells, achieved through a CMA-depended KEAP1 degradation. The main novelty presented in the study is that CMA regulates NRF2, which combined with previous findings, supports the conclusion that this regulation is reciprocal (Zhu et al., 2022Zhu L, He S, Huang L, Ren D, Nie T, Tao K, Xia L, Lu F, Mao Z and Yang Q (2022) Chaperone-mediated autophagy degrades Keap1 and promotes Nrf2-mediated antioxidative response. Aging Cell 21:e13616.).

It is important to note that CMA activity can also be regulated by several signaling pathways that may be altered in the context of cancer (Hubert et al., 2022Hubert V, Weiss S, Rees AJ and Kain R (2022) Modulating chaperone-mediated autophagy and its clinical applications in cancer. Cells 11:2562.). The first signaling mechanism identified in CMA activation was the NFAT (calcium-regulated phosphatase) pathway, which provided unique insights into CMA activation in response to oxidative stress. During T cell activation, the production of reactive oxygen species (ROS) stimulates the transcription factor NFAT1 to bind to LAMP2 proximal promoter region causing the upregulation of LAMP2A (Valdor et al., 2014Valdor R, Mocholi E, Botbol Y, Guerrero-Ros I, Chandra D, Koga H, Gravekamp C, Cuervo AM and Macian F (2014) Chaperone-mediated autophagy regulates T cell responses through targeted degradation of negative regulators of T cell activation. Nat Immunol 15:1046-1054. ). Another example is the Endoplasmic reticulum (ER) stress-induced activation of the p38 MAPK signaling pathway leading to a dual phosphorylation of LAMP2A which activates CMA, termed the ERICA pathway for “ER-stress-induced CMA” (Li et al., 2017Li W, Zhu J, Dou J, She H, Tao K, Xu H, Yang Q and Mao Z (2017) Phosphorylation of LAMP2A by p38 MAPK couples ER stress to chaperone-mediated autophagy. Nat Commun 8:1763.). In addition, Anguiano and coworkers showed the transcriptional inhibition of LAMP2A through the signaling of retinoic acid receptor alpha (RARα) (Anguiano et al., 2013). Also, the mTORC2/AKT1/PHLPP1 axis coordinates the dynamic assembly and disassembly of LAMP2A into multimers, through the phosphatase PHLPP1 inhibition of mTORC2 function, thereby blocking the activation of AKT and promoting the formation of LAMP2A multimers (Arias et al., 2015Arias E, Koga H, Diaz A, Mocholi E, Patel B and Cuervo AM (2015) Lysosomal mTORC2/PHLPP1/Akt regulate chaperone-mediated autophagy. Mol Cell 59:270-284. ). Finally, a lysosome-associated form of the GFAP (glial fibrillary acidic protein) and EF1α (elongation factor 1α) also modulated CMA activity in response to oxidative stress (Assaye and Gizaw, 2022Assaye MA and Gizaw ST (2022) Chaperone-mediated autophagy and its implications for neurodegeneration and cancer. Int J Gen Med 15:5635-5649. ).

In light of this, it becomes clear that the regulation of CMA serves as a pivotal response mechanism to various stress conditions, particularly those prevalent in tumoral environments, rather than genetic alterations in the CMA machinery itself. In fact, mutations in the LAMP2 gene, which encodes the LAMP2A protein, have only been associated with Danon disease, a severe condition that is characterized by skeletal and cardiac myopathy, as well as cognitive impairment, and no increase in cancer susceptibility (Morell et al., 2016Morell C, Bort A, Vara-Ciruelos D, Ramos-Torres Á, Altamirano-Dimas M, Díaz-Laviada I and Rodríguez-Henche N (2016) Up-regulated expression of LAMP2 and autophagy activity during neuroendocrine differentiation of prostate cancer LNCaP Cells. PLoS One 11:e0162977. ). In contrast, genetic alterations in pathways that control CMA, mentioned above, are frequently found in cancer. These include somatic NRF2 and KEAP1 mutations, hypermethylation of KEAP1 and amplification of NRF2, which culminates in a constitutive NRF2 activation; gain-of-function mutations of the canonical transient receptor potential channel (TRPC6) that leads to enhanced NFAT signaling; mutations or overexpression of genes that regulate p38 MAPK activity; chromosomal translocations involving the RARα locus; and mutations on a subunit required for mTORC2 activity (mLST8) that leads to oncogenic mTORC2-AKT activation (Parrado et al., 2000Parrado A, Chomienne C and Padua RA (2000) Retinoic acid receptor alpha (RAralpha) mutations in human leukemia. Leuk Lymphoma 39:271-282. ; Pan et al., 2013Pan MG, Xiong Y and Chen F (2013) NFAT gene family in inflammation and cancer. Curr Mol Med 13:543-554. ; Pouremamali et al., 2022Pouremamali F, Pouremamali A, Dadashpour M, Soozangar N and Jeddi F (2022) An update of Nrf2 activators and inhibitors in cancer prevention/promotion. Cell Commun Signal 20:100. ; Chen et al., 2023Chen Y, Jiao D, He H, Sun H, Liu Y, Shi Q, Zhang P, Li Y, Mo R, Gao K et al. (2023) Disruption of the Keap1-mTORC2 axis by cancer-derived Keap1/mLST8 mutations leads to oncogenic mTORC2-AKT activation. Redox Biol 67:102872. ). However, even though mutations in these pathways have the potential to consequently lead to alterations in CMA activity, there is still no clear evidence linking these mutations to their impact on CMA in cancer cells.

In general, alterations in CMA principal components observed in cancer are associated with changes in Hsc70 and LAMP2A protein and mRNA levels, not mutations in their genes (Rios et al., 2021Rios J, Sequeida A, Albornoz A and Budini M (2021) Chaperone mediated autophagy substrates and components in cancer. Front Oncol 10:614677. ). And when those levels are found elevated in malignant cells indicating that CMA is activated, this type of autophagy can degrade key proteins required for tumor growth and development. Thus, in addition to being influenced by the tumor microenvironment, CMA is also capable of regulating it (Figure 2).

Figure 2 -
CMA is influenced by and regulates the tumor microenvironment. The stress condition of the tumor microenvironment is the primary signal for the activation of CMA. Specifically, nutrient restriction, hypoxia and NRF2 pathway are well-documented CMA activators. On the other hand, CMA degrades important proteins such as KEAP-1, N-Cor, p53, RND3 and HSD17B4, which supports the tumor microenvironment, resulting in the progression of cancer by increasing NRF2, promoting cell survival and growth, sustaining the Warburg effect, regulating the cell cycle, and promoting cell invasion and migration. Created with BioRender.

What CMA regulates

The selectivity of CMA in degrading specific proteins involved in several cellular processes confers regulatory function to this autophagic pathway (Tekirdag and Cuervo, 2018Tekirdag K and Cuervo AM (2018) Chaperone-mediated autophagy and endosomal microautophagy: Joint by a chaperone. J Biol Chem 293:5414-5424. ). Notably, in the context of cancer, multiple CMA substrate proteins were found to be deregulated in a wide range of cancer cell lines (Figure 2). It has been reported that upregulation of CMA favors the survival and proliferation of cancer cells, promoting tumor growth (Kon et al., 2011Kon M, Kiffin R, Koga H, Chapochnick J, Macian F, Varticovski L and Cuervo AM (2011) Chaperone-mediated autophagy is required for tumor growth. Sci Transl Med 3:109ra117.; Saha, 2012Saha T (2012) LAMP2A overexpression in breast tumors promotes cancer cell survival via chaperone-mediated autophagy. Autophagy 8:1643-1656. ) and that enhanced CMA activity is a common feature among different cancer cell lines and human tumors independently of the MA status. In addition, several studies have shown that CMA has a pro-oncogenic function by helping the cancer cells to cope with stress conditions often found in the tumor microenvironment, and higher energetic demand supported by aerobic glycolysis to sustain increased proliferative capacity (Kon et al., 2011Kon M, Kiffin R, Koga H, Chapochnick J, Macian F, Varticovski L and Cuervo AM (2011) Chaperone-mediated autophagy is required for tumor growth. Sci Transl Med 3:109ra117.). In this sense, it is well documented that in most cancer cells there is a NRF2 upregulation which acts as protective mechanism in order to promote tumor progression and chemotherapy resistance (Almeida Lima et al., 2023Almeida Lima K, Osawa IYA, Ramalho MCC, de Souza I, Guedes CB, Souza Filho CHD, Monteiro LKS, Latancia MT and Rocha CRR (2023) Temozolomide resistance in glioblastoma by NRF2: Protecting the evil. Biomedicines 11:1081.). Interestingly, it was demonstrated that under oxidative stress CMA is activated and promotes the degradation of KEAP1, the negative regulator of the nuclear NRF2, elevating the levels of NRF2 and inducing the transcription of several antioxidant genes, as well as LAMP2A gene expression, which further enhances CMA activity (Zhu et al., 2022Zhu L, He S, Huang L, Ren D, Nie T, Tao K, Xia L, Lu F, Mao Z and Yang Q (2022) Chaperone-mediated autophagy degrades Keap1 and promotes Nrf2-mediated antioxidative response. Aging Cell 21:e13616.).

Moreover, CMA can promote tumor progression by degrading tumor suppressors such as the nuclear receptor co-repressor (N-CoR), an essential transcriptional factor known to negatively modulate proteins involved in several oncogenic pathways (Ali et al., 2011Ali AB, Nin DS, Tam J and Khan M (2011) Role of chaperone mediated autophagy (CMA) in the degradation of misfolded N-CoR protein in non-small cell lung cancer (NSCLC) cells. PLoS One 6:e25268.). The degradation of misfolded N-CoR by CMA led to survival and growth of NSCLC cells, through attenuation of misfolded N-CoR-induced ER stress and possible oncogenic signaling pathways activation, what could be prevented by LAMP2A silencing in these cells (Ali et al., 2011Ali AB, Nin DS, Tam J and Khan M (2011) Role of chaperone mediated autophagy (CMA) in the degradation of misfolded N-CoR protein in non-small cell lung cancer (NSCLC) cells. PLoS One 6:e25268.). In addition, another example is the SMAD3 protein, a member of the SMAD (mothers against decapentaplegic) family that acts as an intracellular signal transducer and transcriptional factor induced by TGF-β (transforming growth factor-beta). Likewise, SMAD3 downregulation by CMA augmented proliferation and invasion of glioma cells, supporting the negative correlation between SMAD3 expression and tumor development reported in previous studies (Liu et al., 2022Liu H, Yong Y, Li X, Ye P, Tao K, Peng G, Mo M, Guo W, Chen X, Luo Y et al. (2022) Chaperone-mediated autophagy regulates cell growth by targeting SMAD3 in glioma. Neurosci Bull 38:637-651.).

On the other hand, CMA plays a significant role as a tumor suppressor in non-tumorigenic cells. For instance, MYC degradation is dependent on the dephosphorylation at the Ser62 residue performed by the protein phosphatase 2A (PP2A) and counteracted by CIP2A. A study pointed out that CMA targets CIP2A, leading to its degradation via the ubiquitin-proteasome system, which in turn prevents MYC-driven malignant transformation of normal fibroblasts (Gomes et al., 2017 a Gomes LR, Menck CFM and Cuervo AM (2017a) Chaperone-mediated autophagy prevents cellular transformation by regulating MYC proteasomal degradation. Autophagy 13:928-940. ). Similarly, it was previously reported that hexokinase 2 (HK2), a key glycolytic enzyme upregulated in various cancer cells (Shinohara et al., 1994Shinohara Y, Yamamoto K, Kogure K, Ichihara J and Terada H (1994) Steady state transcript levels of the type II hexokinase and type 1 glucose transporter in human tumor cell lines. Cancer Lett 82:27-32. ; Mathupala et al., 2001Mathupala SP, Rempel A and Pedersen PL (2001) Glucose catabolism in cancer cells: Identification and characterization of a marked activation response of the type II hexokinase gene to hypoxic conditions. J Biol Chem 276:43407-43412.; Patra et al., 2013Patra KC, Wang Q, Bhaskar PT, Miller L, Wang Z, Wheaton W, Chandel N, Laakso M, Muller WJ, Allen EL et al. (2013) Hexokinase 2 is required for tumor initiation and maintenance and its systemic deletion is therapeutic in mouse models of cancer. Cancer Cell 24:213-228. ) and required for oncogenic transformation and tumor development (Patra et al., 2013Patra KC, Wang Q, Bhaskar PT, Miller L, Wang Z, Wheaton W, Chandel N, Laakso M, Muller WJ, Allen EL et al. (2013) Hexokinase 2 is required for tumor initiation and maintenance and its systemic deletion is therapeutic in mouse models of cancer. Cancer Cell 24:213-228. ), undergoes degradation through CMA (Xia et al., 2015Xia HG, Najafov A, Geng J, Galan-Acosta L, Han X, Guo Y, Shan B, Zhang Y, Norberg E, Zhang T et al. (2015) Degradation of HK2 by chaperone-mediated autophagy promotes metabolic catastrophe and cell death. J Cell Biol 210:705-716. ).

Furthermore, once established a tumor, CMA can slow down cancer progression by reducing the levels of tumor promoters commonly overexpressed in many tumors. For instance, CMA can degrade mutant p53, known to be involved with proliferation, resistance to apoptosis, invasiveness and migration in cancer cells (Vakifahmetoglu-Norberg et al., 2013Vakifahmetoglu-Norberg H, Kim M, Xia HG, Iwanicki MP, Ofengeim D, Coloff JL, Pan L, Ince TA, Kroemer G, Brugge JS et al. (2013) Chaperone-mediated autophagy degrades mutant p53. Genes Dev 27:1718-1730. ). Another report suggested that Galectin-3 (Gal3), an anti-apoptotic and oncogenic protein, can be degraded by CMA upon c-Abl/Arg tyrosine kinases inhibition. Silencing of Gal3 and c-Abl/Arg rendered MCF7 cells more susceptible to apoptosis, resulting in reduced tumor growth (Li et al., 2010Li X, Ma Q, Wang J, Liu X, Yang Y, Zhao H, Wang Y, Jin Y, Zeng J, Li J et al. (2010) c-Abl and Arg tyrosine kinases regulate lysosomal degradation of the oncoprotein Galectin-3. Cell Death Differ 17:1277-1287). Likewise, the epidermal growth factor receptor pathway substrate 8 (Eps8), implicated in tumor promotion and metastasis, was proposed as a CMA substrate in human cancer cells (Welsch et al., 2010Welsch T, Younsi A, Disanza A, Rodriguez JA, Cuervo AM, Scita G and Schmidt J (2010) Eps8 is recruited to lysosomes and subjected to chaperone-mediated autophagy in cancer cells. Exp Cell Res 316:1914-1924. ).

Thus, since the first identified CMA substrate, the protein RNase A (McElligott et al., 1985McElligott MA, Miao P and Dice JF (1985) Lysosomal degradation of ribonuclease A and ribonuclease S-protein microinjected into the cytosol of human fibroblasts. J Biol Chem 260:11986-11993.), the list of formally validated or proposed CMA substrates continues to grow and includes metabolic enzymes, transcription factors, cell cycle regulators, proteins involved in the early steps of cellular translation, in cell survival or death and immune system, pro-oncogenic proteins and tumor suppressor proteins, among others.

How the CMA gives an edge to cancer

Since several CMA targets, from tumor suppressor proteins to oncogenes, are involved in different cellular functions, the degradation of proteins by CMA in the context of cancer can offer important advantages for tumor development (Robert et al., 2019Robert G, Jacquel A and Auberger P (2019) Chaperone-mediated autophagy and its emerging role in hematological malignancies. Cells 8:1260. ). In the initial phases of the conversion of a healthy cell to a cancerous one, specific metabolic procedures undergo modifications. These changes result in anaerobic glycolysis becoming the preferred mode for energy production rather than oxidative phosphorylation, regardless of the availability of oxygen. This preference results in increased glucose intake owing to the reduced energy efficiency of anaerobic glycolysis. Simultaneously, it triggers extensive formation of lactate and other metabolites, which have been demonstrated to boost proliferation and consequently promote tumor growth (Arias and Cuervo, 2020Arias E and Cuervo AM (2020) Pros and cons of chaperone-mediated autophagy in cancer biology. Trends Endocrinol Metab 31:53-66. ). This significant pro-tumorigenic occurrence was first identified by Otto Warburg in the 1920s (which is why it became known as the Warburg effect) and it has been found that CMA plays a role in promoting it (Tang et al., 2017Tang Y, Wang XW, Liu ZH, Sun YM, Tang YX and Zhou DH (2017) Chaperone-mediated autophagy substrate proteins in cancer. Oncotarget 8:51970-51985. ).

In lung cancer and melanoma cells, it was found that CMA is essential for sustaining the Warburg effect by degrading p53 and preventing its inhibitory role on the transcription of glycolytic enzymes, such as glyceraldehyde-3-phosphate dehydrogenase and aldolase. Experimental validation occurred through the confirmation of an energy deficit following CMA blockade, resulting in decreased proliferation and increased cell death (Kon et al., 2011Kon M, Kiffin R, Koga H, Chapochnick J, Macian F, Varticovski L and Cuervo AM (2011) Chaperone-mediated autophagy is required for tumor growth. Sci Transl Med 3:109ra117.). Additionally, the acetylated form of the embryonic M2 isoform of pyruvate kinase, which is more prevalent in cancer cells, is selectively degraded by CMA promoting the accumulation of glycolytic intermediates that enhance cell proliferation and growth (Lv et al., 2011Lv L, Li D, Zhao D, Lin R, Chu Y, Zhang H, Zha Z, Liu Y, Li Z, Xu Y et al. (2011) Acetylation targets the M2 isoform of pyruvate kinase for degradation through chaperone-mediated autophagy and promotes tumor growth. Mol Cell 42:719-730.).

Unlike these two scenarios where tumor growth is reduced by blocking CMA - due to the decrease of glycolytic flux and/or the accumulation of glycolytic intermediates - CMA activation can, in certain cases, also cause a metabolic crisis (Tasset and Cuervo, 2016Tasset I and Cuervo AM (2016) Role of chaperone-mediated autophagy in metabolism. FEBS J 283:2403-2413. ). Hexokinase-II (HK2), a crucial catalyst in glucose metabolism and necessary for tumorigenesis, is also a substrate of CMA, which is why upregulation of CMA also has been proposed as an effective strategy for inducing a metabolic crisis and subsequent cell death in cancer cells. However, the KFERQ motif of HK2 is hidden in the protein when there is a glucose molecule, hindering its degradation by CMA. Another impediment for the degradation of HK2 by CMA is the phosphorylation of this enzyme on Thr473, something that is frequent in breast cancer. This elucidates the potential of HK2 inhibition in reducing cancer cell proliferation (Xia et al., 2015Xia HG, Najafov A, Geng J, Galan-Acosta L, Han X, Guo Y, Shan B, Zhang Y, Norberg E, Zhang T et al. (2015) Degradation of HK2 by chaperone-mediated autophagy promotes metabolic catastrophe and cell death. J Cell Biol 210:705-716. ; Yang et al., 2018Yang T, Ren C, Qiao P, Han X, Wang L, Lv S, Sun Y, Liu Z, Du Y and Yu Z (2018) PIM2-mediated phosphorylation of hexokinase 2 is critical for tumor growth and paclitaxel resistance in breast cancer. Oncogene 37:5997-6009. ).

Besides being involved in the execution of the Warburg effect in tumor cells, CMA also controls the levels of executors of the cell cycle, providing significant benefits to cancer cells in terms of proliferation rates (Andrade-Tomaz et al., 2020Andrade-Tomaz M, de Souza I, Rocha CRR and Gomes LR (2020) The role of chaperone-mediated autophagy in cell cycle control and its implications in cancer. Cells 9:2140.). One of the essential executors of cell cycle process that is degraded through CMA is p73, a transcription factor that has the ability to promote cell cycle arrest and induce apoptosis. A recent study by Nguyen et al. (2020Nguyen D, Yang K, Chiao L, Deng Y, Zhou X, Zhang Z, Zeng SX and Lu H (2020) Inhibition of tumor suppressor p73 by nerve growth factor receptor via chaperone-mediated autophagy. J Mol Cell Biol 12:700-712.) demonstrated that this degradation is mediated by the nerve growth factor receptor (NGFR). Also, the hypoxia inducible factor 1 subunit alpha (HIF-1α) is a transcription factor that is regulated by CMA and is involved in negatively regulating DNA replication under hypoxic conditions (Goda et al., 2003Goda N, Ryan HE, Khadivi B, McNulty W, Rickert RC and Johnson RS (2003) Hypoxia-inducible factor 1α is essential for cell cycle arrest during hypoxia. Mol Cell Biol 23:359-369.).

Another advantage that CMA can offer by degrading a substrate involved in the cell cycle, such as CHK1, is that it prevents cell cycle arrest in G2 in response to DNA damage, which leads to heightened susceptibility to genotoxic stress, as CHK1 accumulates and DNA damage increases (Park et al., 2015Park C, Suh Y and Cuervo AM (2015) Regulated degradation of Chk1 by chaperone-mediated autophagy in response to DNA damage. Nat Commun 6:6823. ). CMA also regulates the Rho family GTPase (RND3), which can hinder proliferation through control of the cell cycle. RND3 decreases MYC’s transcriptional activity and expression. This reveals the vital role of CMA in the cell cycle machine, as MYC impacts various gene regulators of the cell cycle, such as cyclins, cyclin-dependent kinases (CDKs) and EF2 transcription factor (Andrade-Tomaz et al., 2020Andrade-Tomaz M, de Souza I, Rocha CRR and Gomes LR (2020) The role of chaperone-mediated autophagy in cell cycle control and its implications in cancer. Cells 9:2140.).

As well as having an impact on cell cycle and consequently on cell proliferation, CMA is also capable of affecting the migratory capacity of tumor cells and promoting their dissemination (Arias and Cuervo, 2020Arias E and Cuervo AM (2020) Pros and cons of chaperone-mediated autophagy in cancer biology. Trends Endocrinol Metab 31:53-66. ). A recently published study highlights YAP1 (yes-associated protein 1) and IL6ST (interleukin 6 cytokine family signal transducer) as novel targets of CMA. Both substrates are related to an increase in cell migration besides proliferation in human hepatocellular carcinoma (HCC) and hepatocyte cell lines. The knockdown of LAMP2A led to increased cell proliferation and migration in these cell lines, likely as a result of upregulated YAP1 and IL6ST, providing evidence for the tumor-suppressive effect of CMA (Desideri et al., 2023Desideri E, Castelli S, Dorard C, Toifl S, Grazi GL, Ciriolo MR and Baccarini M (2023) Impaired degradation of YAP1 and IL6ST by chaperone-mediated autophagy promotes proliferation and migration of normal and hepatocellular carcinoma cells. Autophagy 19:152-162.). Conversely, CMA has also been implicated in promoting tumor metastasis in lung and breast cancer cells (Kon et al., 2011Kon M, Kiffin R, Koga H, Chapochnick J, Macian F, Varticovski L and Cuervo AM (2011) Chaperone-mediated autophagy is required for tumor growth. Sci Transl Med 3:109ra117.; Han et al., 2017Han Q, Deng Y, Chen S, Chen R, Yang M, Zhang Z, Sun X, Wang W, He Y, Wang F et al. (2017) Downregulation of ATG5-dependent macroautophagy by chaperone-mediated autophagy promotes breast cancer cell metastasis. Sci Rep 7:4759.). The molecular mechanisms behind CMA-dependent metastasis remain largely unknown, but studies indicate that the degradation of the HSD17B4 protein by CMA is responsible for this invasive and migratory property (Arias and Cuervo, 2020Arias E and Cuervo AM (2020) Pros and cons of chaperone-mediated autophagy in cancer biology. Trends Endocrinol Metab 31:53-66. ).

Therefore, it is clear that CMA emerges as an indispensable mechanism in the development and progression of cancer cells as it offers essential edges for these cells. Studies using animal models support this finding, as mice with selective hepatic CMA inhibition displayed a higher incidence of spontaneous hepatic tumors with age (Hubert et al., 2022Hubert V, Weiss S, Rees AJ and Kain R (2022) Modulating chaperone-mediated autophagy and its clinical applications in cancer. Cells 11:2562.). But perhaps the greatest impact of CMA activity is revealed after the onset of cancer, since CMA is closely related to the development of resistance to cancer therapy.

The role of the CMA in cancer treatment

CMA and chemotherapy

The most prevalent approaches to treat cancer encompass surgical procedures, radiation therapy, and chemotherapy, with the specific treatment modality being determined by cancer type and its severity (Mansoori et al., 2017Mansoori B, Mohammadi A, Davudian S, Shirjang S and Baradaran B (2017) The different mechanisms of cancer drug resistance: A brief review. Adv Pharm Bull 7:339-348. ). Despite considerable advancements in cancer research, chemotherapy remains a promising option for cancer treatment. Nevertheless, a significant number of cancerous cells develop resistance to chemotherapeutic drugs, which significantly contributes to tumor progression and recurrence. This resistance currently stands as the main limiting factor in 90% of metastatic cancer treatment, impairing the chances of cure from this disease. Consequently, it is of extreme importance to further investigate drug resistant cancers to facilitate the development of novel therapeutic interventions (Emran et al., 2022Emran T, Bin Shahriar A, Mahmud AR, Rahman T, Abir MH, Siddiquee Mohd F-R, Ahmed H, Rahman N, Nainu F, Wahyudin E et al. (2022) Multidrug resistance in cancer: Understanding molecular mechanisms, immunoprevention and therapeutic approaches. Front Oncol 12:891652.).

Drug resistance represents a multifaceted challenge, as cancers can develop resistance via different mechanisms (Holohan et al., 2013Holohan C, Van Schaeybroeck S, Longle DB and Johnston PG (2013) Cancer drug resistance: An evolving paradigm. Nat Rev Cancer 13:714-726.; Vasan et al., 2019Vasan N, Baselga J and Hyman DM (2019) A view on drug resistance in cancer. Nature 575:299-309. ). Chemotherapeutic agents increase damage on various cellular components, promoting the accumulation of misfolded proteins and damaged organelles. Paradoxically, instead of inducing the death of cancer cells, these molecules can be degraded into substances that may sustain metabolism and support further growth and survival of tumor cells via the autophagy pathway. This process ultimately promotes resistance to therapeutic drugs. The mechanisms underlying resistance can be classified into two categories: one linked to high basal autophagy flux in certain tumor cell types, resulting in intrinsic resistance to chemotherapy, and the other associated with a gradual increase in autophagic flux in response to prolonged chemotherapy, thereby promoting acquired drug resistance (Lippert et al., 2008Lippert TH, Ruoff HJ and Volm M (2008) Intrinsic and acquired drug resistance in malignant tumors. The main reason for therapeutic failure. Arzneimittelforschung 58:261-264. ; Yang et al., 2022Yang L, Xie HJ, Li YY, Wang X, Liu XX and Mai J (2022) Molecular mechanisms of platinumbased chemotherapy resistance in ovarian cancer (Review). Oncol Rep 47:82.).

Extensive research has established a connection between drug resistance and upregulated autophagy (Sui et al., 2013Sui X, Chen R, Wang Z, Huang Z, Kong N, Zhang M, Han W, Lou F, Yang J, Zhang Q et al. (2013) Autophagy and chemotherapy resistance: A promising therapeutic target for cancer treatment. Cell Death Dis 4:e838. ). Beyond its role in eliminating damaged organelles, autophagy flux can serve as a cellular mechanism for overcoming environmental stress, acting as a protective mechanism that promotes tumor growth (Sui et al., 2013Sui X, Chen R, Wang Z, Huang Z, Kong N, Zhang M, Han W, Lou F, Yang J, Zhang Q et al. (2013) Autophagy and chemotherapy resistance: A promising therapeutic target for cancer treatment. Cell Death Dis 4:e838. ). In certain cases, the selectivity of CMA can further contribute to drug resistance since some proteins can only be transported and degraded via this pathway (Rios et al., 2021Rios J, Sequeida A, Albornoz A and Budini M (2021) Chaperone mediated autophagy substrates and components in cancer. Front Oncol 10:614677. ). Notably, elevated levels of LAMP2A have been associated with poor survival rates in non-small cell lung cancer (NSCLC) (Ichikawa et al., 2020Ichikawa A, Fujita Y, Hosaka Y, Kadota T, Ito A, Yagishita S, Watanabe N, Fujimoto S, Kawamoto H, Saito N et al. (2020) Chaperone-mediated autophagy receptor modulates tumor growth and chemoresistance in non-small cell lung cancer. Cancer Sci 111:4154-4165.). The blockade of CMA has been identified as a key driver of resistance, primarily due to its involvement in the modulation of several factors involved in the regulation of transcription, translation, and cell cycle control (Andrade-Tomaz et al., 2020Andrade-Tomaz M, de Souza I, Rocha CRR and Gomes LR (2020) The role of chaperone-mediated autophagy in cell cycle control and its implications in cancer. Cells 9:2140.). Consequently, managing the CMA pathway may hold promise as a therapeutic approach, particularly for patients who are ineligible for surgery and depend only on chemotherapy and radiotherapy.

Proof of concept for the therapeutic potential of targeting CMA with chemotherapeutic drugs has been demonstrated across various cancer types through the genetic modulation of LAMP2A. Recent studies have revealed that LAMP2A regulates malignancy by regulating apoptosis, and the suppression of LAMP2A enhances the sensitivity of cancer cells to several drugs, such as cisplatin, doxorubicin, 5-fluorouracil, bortezomib, among others (Huang et al., 2016Huang Z, Zhou L, Chen Z, Nice EC and Huang C (2016) Stress management by autophagy: Implications for chemoresistance. Int J Cancer 139:23-32.; Karagounis et al., 2016Karagounis IV, Kalamida D, Mitrakas A, Pouliliou S, Liousia MV, Giatromanolaki A and Koukourakis MI (2016) Repression of the autophagic response sensitises lung cancer cells to radiation and chemotherapy. Br J Cancer 115:312-321. ; Ichikawa et al., 2020Ichikawa A, Fujita Y, Hosaka Y, Kadota T, Ito A, Yagishita S, Watanabe N, Fujimoto S, Kawamoto H, Saito N et al. (2020) Chaperone-mediated autophagy receptor modulates tumor growth and chemoresistance in non-small cell lung cancer. Cancer Sci 111:4154-4165.). On the other hand, downregulation of CMA activity can be sufficient to increase HIF-1α levels and lead to temozolomide resistance in glioblastoma (Lo Dico et al., 2018Lo Dico A, Martelli C, Diceglie C, Lucignani G and Ottobrini L (2018) Hypoxia-inducible factor-1α activity as a switch for glioblastoma responsiveness to temozolomide. Front Oncol 8:249., 2021Lo Dico A, Martelli C, Diceglie C and Ottobrini L (2021) The multifaceted role of CMA in glioma: Enemy or ally? Int J Mol Sci 22:2217. ), indicating a dual role of CMA in cancer, which needs to be further investigated in order to overcome drug resistance.

It is important to note that the term “resistance” is typically used to describe stable alterations in the cell (such as mutations) that alter the cellular response. However, emerging evidence suggests that non-genetic modifications, such as transient phenotypic adaptations, also play a role in the acquisition of resistance to chemotherapy, even though these adaptations are more generally referred to as “tolerance” mechanisms (Salgia and Kulkarni, 2018Salgia R and Kulkarni P (2018) The genetic/non-genetic duality of drug ‘resistance’ in cancer. Trends Cancer 4:110-118. ). Given this inconsistency in nomenclature, adaptations in CMA activity in response to the tumor microenvironment, which are related to the level of expression of LAMP2A rather than mutations in its gene, that could be considered involved in the development of “tolerance” to treatment are here described as triggers of “resistance” to chemotherapy, noting the predominance of the use of this term in the studies that investigated the effect of CMA in response to treatment and are summarized in Table 2.

Table 2 -
CMA impact on cancer treatment.

Cisplatin and CMA

Cisplatin is a well-established chemotherapeutic agent that forms covalent bonds with DNA bases, resulting in DNA adducts. It induces diverse DNA lesions that block transcription and replication, triggering intricate intracellular signaling cascades in an effort to eliminate these lesions. In cases of compromised repair mechanisms or excessive damage, the cells undergo apoptosis (Dasari and Tchounwou, 2014Dasari S and Tchounwou P (2014) Cisplatin in cancer therapy: Molecular mechanisms of action. Eur J Pharmacol 740:364-378.). Cisplatin has exhibited notable efficacy against a broad spectrum of solid tumors, including testicular, ovarian, lung, bladder, cervical, and head and neck neoplasms (Dasari and Tchounwou, 2014Dasari S and Tchounwou P (2014) Cisplatin in cancer therapy: Molecular mechanisms of action. Eur J Pharmacol 740:364-378.). Nevertheless, occurrences of treatment-related side effects as inherent and acquired resistance persist as a significant hurdle in cisplatin-based anticancer therapy, posing a challenge throughout the treatment cycles (Rocha et al., 2018Rocha CRR, Silva MM, Quinet A, Cabral- Neto JB and Menck CFM (2018) DNA repair pathways and cisplatin resistance: An intimate relationship. Clinics (São Paulo) 73:e478s. ).

Damaged or misfolded proteins resulting from cisplatin activity can be recognized by LAMP2A and subsequently translocated for degradation in lysosomes (Cuervo and Wong, 2014Cuervo AM and Wong E (2014) Chaperone-mediated autophagy: Roles in disease and aging. Cell Res 24:92-104. ). In fact, LAMP2A levels were found to be elevated in most cisplatin resistant cells, indicating that high CMA activity can be considered as a predictive factor for the resistance to platinum-based chemotherapy. Thus, it was observed that CMA blockade conferred cisplatin therapeutic advantages to lung cancer cells in vitro and in vivo, leading to higher cleaved caspase-3 and lower cyclin D (Karagounis et al., 2016Karagounis IV, Kalamida D, Mitrakas A, Pouliliou S, Liousia MV, Giatromanolaki A and Koukourakis MI (2016) Repression of the autophagic response sensitises lung cancer cells to radiation and chemotherapy. Br J Cancer 115:312-321. ; Ichikawa et al., 2020Ichikawa A, Fujita Y, Hosaka Y, Kadota T, Ito A, Yagishita S, Watanabe N, Fujimoto S, Kawamoto H, Saito N et al. (2020) Chaperone-mediated autophagy receptor modulates tumor growth and chemoresistance in non-small cell lung cancer. Cancer Sci 111:4154-4165.). CMA inhibition also overcame cisplatin resistance in esophageal squamous cell carcinoma cells (Cao et al., 2021Cao D, Shan D, Yan W, Zhang Z, Song Q, Jiang Y, Zhang X, Zhang Z, Wang Z, Wang Y et al. (2021) Chaperone-mediated autophagy affects tumor cell proliferation and cisplatin resistance in esophageal squamous cell carcinoma. Thorac Cancer 12:1048-1057. ).

Doxorubicin and CMA

Doxorubicin, an anthracycline widely used in treating several solid tumors, exerts its anticancer effects by intercalating with DNA or covalently binding to proteins involved in DNA replication and transcription. It leads to protein synthesis inhibition and apoptosis induction. It also interacts with mitochondrial DNA, disrupting essential mitochondrial functions (Yang et al., 2014Yang F, Teves SS, Kemp CJ and Henikoff S (2014) Doxorubicin, DNA torsion, and chromatin dynamics. Biochim Biophys Acta 1845:84-89. ). Recent studies have indicated that doxorubicin initiates cellular changes consistent with autophagy induction (Koleini and Kardami, 2017Koleini N and Kardami E (2017) Autophagy and mitophagy in the context of doxorubicin-induced cardiotoxicity. Oncotarget 8:46663-46680.), and CMA has been identified as playing a pivotal role in doxorubicin resistance (Huang et al., 2020Huang PS, Lin YH, Chi HC, Tseng YH, Chen CY, Lin TK, Yeh CT and Lin KH (2020) Dysregulated FAM215A stimulates LAMP2 expression to confer drug-resistant and malignant in human liver cancer. Cells 9:961. ; Saha, 2012Saha T (2012) LAMP2A overexpression in breast tumors promotes cancer cell survival via chaperone-mediated autophagy. Autophagy 8:1643-1656. ).

In HCC, doxorubicin is broadly used, however, patients often develop resistance. Notably, long non-coding RNA FAM215A has been found to interact with LAMP2A, preventing its ubiquitination in HCC cells, thereby promoting the accumulation of LAMP2A and high CMA activity, which leads to doxorubicin resistance. LAMP2A has been associated with tumor growth and recurrence in HCC, and its downregulation has been shown to reduce proliferation and viability upon doxorubicin treatment (Huang et al., 2020Huang PS, Lin YH, Chi HC, Tseng YH, Chen CY, Lin TK, Yeh CT and Lin KH (2020) Dysregulated FAM215A stimulates LAMP2 expression to confer drug-resistant and malignant in human liver cancer. Cells 9:961. ). Doxorubicin is also commonly employed in the treatment of early-stage, node-positive, HER2-positive, and metastatic breast cancer. Overcoming drug resistance and minimizing toxicity in this type of cancer has proven to be particularly challenging. It has been shown that breast cancer cells deficient in LAMP2A have increased sensitivity to doxorubicin, marked by higher levels of reactive oxygen species (ROS) and apoptosis compared to wild-type cells (Saha, 2012Saha T (2012) LAMP2A overexpression in breast tumors promotes cancer cell survival via chaperone-mediated autophagy. Autophagy 8:1643-1656. ).

Temozolomide and CMA

Temozolomide (TMZ) is a FDA-approved oral alkylating agent for use as a first-line treatment for glioblastoma multiforme. TMZ effectively crosses the blood-brain barrier and methylates purine bases of DNA (specifically O6-guanine, N7-guanine, and N3-adenine), ultimately leading to cell death. While TMZ has extended median patient survival rates, treatment failure has been largely associated with tumor drug resistance. Notably, in contrast to previously mentioned chemotherapeutic drugs, the downregulation of CMA has been identified as a significant contributor to resistance against TMZ in gliomas (Zhang et al., 2012Zhang J, Stevens MF and Bradshaw TD (2012) Temozolomide: Mechanisms of action, repair and resistance. Curr Mol Pharmacol 5:102-114. ; Ortiz et al., 2021Ortiz R, Perazzoli G, Cabeza L, Jiménez-Luna C, Luque R, Prados J and Melguizo C (2021) Temozolomide: An updated overview of resistance mechanisms, nanotechnology advances and clinical applications. Curr Neuropharmacol 19:513-537. ). In fact, it was demonstrated that CMA plays a pivotal role in the degradation of HIF-1α, a factor directly associated with glioma malignancy and resistance (Lo Dico et al., 2018Lo Dico A, Martelli C, Diceglie C, Lucignani G and Ottobrini L (2018) Hypoxia-inducible factor-1α activity as a switch for glioblastoma responsiveness to temozolomide. Front Oncol 8:249., 2021Lo Dico A, Martelli C, Diceglie C and Ottobrini L (2021) The multifaceted role of CMA in glioma: Enemy or ally? Int J Mol Sci 22:2217. ). Furthermore, the silencing of HSC70 or PHLPP1 has also led to resistance characteristics in TMZ-treated cells, similar to the outcomes observed in LAMP2A-silenced cells. Interestingly, mitochondrial ROS release induces CMA activation, which is essential for the toxicity caused by TMZ (Lo Dico et al., 2019Lo Dico A, Salvatore D, Martelli C, Ronchi D, Diceglie C, Lucignani G and Ottobrini L (2019) Intracellular redox-balance involvement in temozolomide resistance-related molecular mechanisms in glioblastoma. Cells 8:1315. ).

5-fluorouracil and CMA

Among the different types of chemotherapeutic agents, those with antimetabolic activity are popular, specially 5-fluorouracil (5-FU), which acts as a thymidylate synthetase inhibitor. 5-FU is considered an important treatment option for colorectal cancer (CRC), however, the development of a resistance is quite frequent (Vodenkova et al., 2020Vodenkova S, Buchler T, Cervena K, Veskrnova V, Vodicka P and Vymetalkova V (2020) 5-fluorouracil and other fluoropyrimidines in colorectal cancer: Past, present and future. Pharmacol Ther 206:107447.). In this matter, it was demonstrated that elevated CMA activity can be related to loss of sensitivity to 5-FU in in vitro studies with CRC cells, being that process strongly related to the activation of the NF-kB pathway and the resulting enhanced production of PLD2, an enzyme associated with tumor progression and worse prognostics (Xuan et al., 2021Xuan Y, Zhao S, Xiao X, Xiang L and Zheng HC (2021) Inhibition of chaperonemediated autophagy reduces tumor growth and metastasis and promotes drug sensitivity in colorectal cancer. Mol Med Rep 23:360. ). In that sense, it was observed that cell lines with higher LAMP2A concentrations presented faster and increased growth, as well as elevated PLD2 expression and were more resistant to treatment with 5-FU (Xuan et al., 2021Xuan Y, Zhao S, Xiao X, Xiang L and Zheng HC (2021) Inhibition of chaperonemediated autophagy reduces tumor growth and metastasis and promotes drug sensitivity in colorectal cancer. Mol Med Rep 23:360. ).

CMA and radiotherapy

Although chemotherapy constitutes an important facet of cancer treatment, it is not the sole one, the establishment of combinatorial treatment schemes involving chemotherapy and radiotherapy is frequent. Irradiation seeks to induce cell death, as well as suppresses tumor growth by inducing DNA damage in cancer cells through the application of high doses of radiation. Radiotherapy is one of the first-line treatments for solid cancers, such as lung, breast and esophageal cancer. However, evasion mechanisms can lead to the emergence of resistance and consequent unsuccessful treatment, as well as potential disease recurrence (Wu et al., 2023Wu Y, Song Y, Wang R and Wang T (2023) Molecular mechanisms of tumor resistance to radiotherapy. Mol Cancer 22:96. ).

In this scenario, autophagic processes constitute an important tool for the onset of resistance, as they allow the disposal of the various damaged structures generated in the irradiation process. Based on an analysis of LAMP2A and LC3A levels, markers for lysosomes and autophagosomes respectively, a study showed that the presence of a more intense autophagic activity favored a radiotherapy-resistant phenotype in prostate carcinoma cells (Koukourakis et al., 2015Koukourakis MI, Kalamida D, Mitrakas A, Pouliliou S, Kalamida S, Sivridis E and Giatromanolaki A (2015) Intensified autophagy compromises the efficacy of radiotherapy against prostate cancer. Biochem Biophys Res Commun 461:268-274.). Similar results were observed in a study that analyzed the same markers, as well as LC3B, p62, TFEB in lung cancer cell lines (Karagounis et al., 2016Karagounis IV, Kalamida D, Mitrakas A, Pouliliou S, Liousia MV, Giatromanolaki A and Koukourakis MI (2016) Repression of the autophagic response sensitises lung cancer cells to radiation and chemotherapy. Br J Cancer 115:312-321. ).

In addition, an in vitro study using HCC cell lines showed that this process could favor resistance to irradiation due to the negative modulation of HMGB1. This protein would bind to LAMP2A and then be degraded by the CMA pathway, which led to a reduction in p53, an extremely important factor in inhibiting tumor growth (Wu et al., 2017Wu JH, Guo JP, Shi J, Wang H, Li LL, Guo B, Liu DX, Cao Q and Yuan ZY (2017) CMA down-regulates p53 expression through degradation of HMGB1 protein to inhibit irradiation-triggered apoptosis in hepatocellular carcinoma. World J Gastroenterol 23:2308-2317. ).

Given presented results, the inhibition of LAMP2A has been pointed out as an alternative to sensitizing neoplastic cells to radiotherapy and increasing treatment efficiency.

CMA and photodynamic therapy

Another promising anticancer treatment is photodynamic therapy (PDT), which has been approved by the FDA and is currently undergoing numerous clinical trials. PDT is a treatment that combines light and a photosensitizer to generate highly cytotoxic ROS, primarily in the form of singlet oxygen. These ROS react with cellular molecules, ultimately leading to organelle damage and cell death. To generate ROS, PDT employs photosensitizers (PS) that are excited by visible light at power levels that do not harm healthy tissue (Bartusik-Aebisher et al., 2021Bartusik-Aebisher D, Ożóg Ł and Aebisher D (2021) Alternative methods of photodynamic therapy and oxygen consumption measurements: A review. Biomed Pharmacother 134:111095.). Given that CMA functions as an effective defense mechanism against ROS-induced injury, it has been observed that PDT treatment triggers the recruitment of the CMA machinery to lysosomes in photosensitized cells. Inhibition of LAMP2A significantly increased the sensitivity of mouse embryonic fibroblasts to a wide range of PDT doses. Additionally, LAMP2 deficiency contributed to the activation of caspase-3 and cleavage of PARP, indicating a crucial mechanism of resistance to PDT via CMA activity (Dewaele et al., 2011Dewaele M, Martinet W, Rubio N, Verfaillie T, de Witte PA, Piette J and Agostinis P (2011) Autophagy pathways activated in response to PDT contribute to cell resistance against ROS damage. J Cell Mol Med 15:1402-1414.).

The challenge of specific CMA modulation

Despite the numerous connections between CMA and cancer biology, and the aforementioned evidence suggesting that modulation of CMA holds great potential for improving the therapeutic response of various types of cancer, the absence of chemical selective modulators for CMA presents a challenge to the therapeutic translatability of these findings, hindering the application of these positive results in clinical practice.

Until now, studies investigating the therapeutic value of CMA modulation in the context of cancer have relied solely on genetic modulation of LAMP2A to establish this proof of concept, however such approach faces significant experimental and clinical limitations (Arias and Cuervo, 2020Arias E and Cuervo AM (2020) Pros and cons of chaperone-mediated autophagy in cancer biology. Trends Endocrinol Metab 31:53-66. ). Even in in vitro assays, an important limitation of the genetic modulation of LAMP2A is that it does not allow the study of acute inhibition of CMA. This is due to the lengthy half-life of the protein, which mandates a wait time of at least five days for a significant reduction in LAMP2A levels to occur (Patel and Cuervo, 2015Patel B and Cuervo AM (2015) Methods to study chaperone-mediated autophagy. Methods 75:133-140. ). Thus, despite the significant progress made within gene therapy, it faces hindrances in terms of technical, ethical, political, and financial factors (Das et al., 2015Das SK, Menezes ME, Bhatia S, Wang XY, Emdad L, Sarkar D and Fisher PB (2015) Gene therapies for cancer: strategies, challenges and successes. J Cell Physiol 230:259-271.; Shahryari et al., 2019Shahryari A, Saghaeian Jazi M, Mohammadi S, Razavi Nikoo H, Nazari Z, Hosseini ES, Burtscher I, Mowla SJ and Lickert H (2019) Development and clinical translation of approved gene therapy products for genetic disorders. Front Genet 10:868. ).

Although it is evident that finding chemically specific modulators of CMA is a top priority, there are still numerous challenges to overcome to achieve this goal. One of the primary unknowns that makes the development of effective and targeted chemical compounds very challenging is the lack of information about the timeline of CMA, which transitions from a physiological and protective mechanism to a detrimental and potent pro-tumorigenic (Hubert et al., 2022Hubert V, Weiss S, Rees AJ and Kain R (2022) Modulating chaperone-mediated autophagy and its clinical applications in cancer. Cells 11:2562.). Nevertheless, the main obstacle for this development is the lack of exclusive and “druggable” compounds for CMA. This is because most of the key compounds in this pathway are multifunctional proteins that are involved in other essential cellular processes, often leading to significant levels of toxicity (Anguiano et al., 2013Anguiano J, Garner TP, Mahalingam M, Das BC, Gavathiotis E and Cuervo AM (2013) Chemical modulation of chaperone-mediated autophagy by retinoic acid derivatives. Nat Chem Biol 9:374-382.). For instance, the blockage of hsc70 also impacts other crucial mechanisms of protein folding and aggregation, such as e-MI, MA, and endocytosis (Patel and Cuervo, 2015Patel B and Cuervo AM (2015) Methods to study chaperone-mediated autophagy. Methods 75:133-140. ).

The most exclusive component of CMA is LAMP2A and, therefore, this protein is usually targeted in studies involving CMA (Kaushik and Cuervo, 2018Kaushik S and Cuervo AM (2018) The coming of age of chaperone-mediated autophagy. Nat Rev Mol Cell Biol 19:365-381. ). However, due to the high homology (nearly 85%) of LAMP2A with the other splicing variants of the LAMP2 gene (LAMP2B and LAMP2C) it makes a difficult target for chemical modulators that do not have the same precision as gene modulation techniques. So, such modulators may act non-specifically on the other isoforms and therefore affect other cellular functions, including MA, biogenesis, and cholesterol trafficking (Massey et al., 2006Massey AC, Kaushik S, Sovak G, Kiffin R and Cuervo AM (2006) Consequences of the selective blockage of chaperone-mediated autophagy. Proc Natl Acad Sci U S A 103:5805-5810.; Valdor et al., 2014Valdor R, Mocholi E, Botbol Y, Guerrero-Ros I, Chandra D, Koga H, Gravekamp C, Cuervo AM and Macian F (2014) Chaperone-mediated autophagy regulates T cell responses through targeted degradation of negative regulators of T cell activation. Nat Immunol 15:1046-1054. ).

CMA inhibitors

Inhibiting CMA activity is a potential therapeutic approach in cancer as CMA is abnormally upregulated in many cancers and required for optimal tumor growth and metastasis (Li et al., 2018Li W, Dou J, Yang J, Xu H and She H (2018) Targeting chaperone-mediated autophagy for disease therapy. Curr Pharmacol Rep 4:261-275.). As it is evident from Table 2, drug resistance in cancer cells can often be overcome through CMA inhibition. Unfortunately, the absence of a selective chemical inhibitor of CMA is the major barrier for translating these experimental findings into treatments for oncological patients (Du et al., 2017Du C, Huang D, Peng Y, Yao Y, Zhao Y, Yang Y, Wang H, Cao L, Zhu WG and Gu J (2017) 5-Fluorouracil targets histone acetyltransferases p300/CBP in the treatment of colorectal cancer. Cancer Lett 400:183-193.). Thus, no clinical trial has yet selectively targeted CMA for the treatment of any cancer, that is we still face many challenges in finding drugs that can selectively modulate CMA to maximize therapeutic effects and minimize toxicity in clinical use.

The main challenges relie in designing selective molecules that comprise only the CMA activity, without affecting other autophagic pathways. Once, inhibiting MA under certain conditions may cause tumorigenesis and metastasis, it is crucial to ensure that no such adverse effects occur (Han et al., 2017Han Q, Deng Y, Chen S, Chen R, Yang M, Zhang Z, Sun X, Wang W, He Y, Wang F et al. (2017) Downregulation of ATG5-dependent macroautophagy by chaperone-mediated autophagy promotes breast cancer cell metastasis. Sci Rep 7:4759.). Thus, although the initial screening studies of Finn et al. (2005Finn PF, Mesires NT, Vine M and Dice JF (2005) Effects of small molecules on chaperone-mediated autophagy. Autophagy 1:141-145.) identified molecules capable of inhibiting the CMA process, including cycloheximide and anisomycin, it has already been demonstrated that the activity of these protein synthesis inhibitors is unsuitable for specifically studying the effect of CMA inhibition (Finn et al., 2005Finn PF, Mesires NT, Vine M and Dice JF (2005) Effects of small molecules on chaperone-mediated autophagy. Autophagy 1:141-145.; Patel and Cuervo, 2015Patel B and Cuervo AM (2015) Methods to study chaperone-mediated autophagy. Methods 75:133-140. ).

The latest study to search for a CMA inhibitor has identified Polyphyllin D (PPD) as a compound that inhibits the interaction between hsc70 and LAMP2A, as well as the homomultimerisation of LAMP2A, which limits tumor growth in NSCLC cells. Nevertheless, PPD exhibited an impact on MA as it obstructs the STX17-SNAP29-VAMP8 signaling pathway, which prevents a compensatory regulation of MA after the inhibition of CMA. Hence, it cannot be deemed selective for CMA (Dong et al., 2023Dong R, Qin C, Yin Y, Han L, Xiao C, Wang K, Wei R, Xia Y and Kong L (2023) Discovery of a potent inhibitor of chaperone‐mediated autophagy that targets the HSC70-LAMP2A interaction in non‐small cell lung cancer cells. Br J Pharmacol. DOI:10.1111/bph.16165.
https://doi.org/10.1111/bph.16165...
).

Overall, most articles concerning the chemical inhibition of CMA use compounds that aim to target the proteolytic activity of lysosomes. Due to this, other forms of autophagy, in addition to CMA, are also disrupted. It is worth noting that recent clinical interventions have concentrated on using hydroxychloroquine, an intralysosomal proteolysis-inhibiting compound that alters the pH of the lysosome, as a means of intervening in the MA pathway with the aim of anti-cancer strategies. Although the degradation of substances by CMA once they are internalized in the lysosomes is not limited by pH, the continued rise in lysosomal pH results in destabilizing the luminal form of hsc70, that is vital for substrate translocation to the intralysosomal compartment. Consequently, hydroxychloroquine also leads to the inhibition of CMA. Therefore, future research should focus not only on finding the first chemical selective inhibitors of CMA but also on gaining a better understanding of how CMA blockage contributes to the beneficial effects seen in clinical trials with classical MA inhibitors, like hydroxychloroquine (Wang and Mao, 2014Wang G and Mao Z (2014) Chaperone-mediated autophagy: Roles in neurodegeneration. Transl Neurodegener 3:20. ; Arias and Cuervo, 2020Arias E and Cuervo AM (2020) Pros and cons of chaperone-mediated autophagy in cancer biology. Trends Endocrinol Metab 31:53-66. ). Since the individual contribution of macroautophagy and CMA to the overall involvement of autophagy in the response to chemotherapy in clinical level remains unclear due to the absence of specific modulators for CMA.

Conclusions

This review highlighted the great progress made in understanding the mechanisms underlying CMA over the past decades. We have discussed the primary methods available for studying it, the characteristics of the tumor microenvironment that promote CMA modulation, the proteins regulated by CMA that are pivotal for cancer development, the advantages that CMA can confer on neoplastic cells, and specially, the interplay between CMA and the development of therapy resistance in cancer. As our knowledge regarding this topic increases, it has become clear that CMA’s modulation may improve the therapeutic response to various types of cancer grows in direct proportion. However, without a doubt, the biggest obstacle in CMA modulation to be translated into clinics the scarcity of selective chemical modulators of this pathway.

In this scenario, the presented subject still has a long way to improve in order to reflect the knowledge developed in the sphere of basic research into proper clinical treatments regarding the modulation of autophagy, going beyond chloroquine. This process constitutes a possible alternative treatment scheme for patients who face resistance to traditional chemotherapy agents. Therefore, CMA is an emerging and exciting research area, that holds potential to be an alternative route to improve cancer treatment.

Acknowledgements

This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP #2022/03130-1; #2022/02200-6; #2021/11597-4; #2022/14035-0; #2023/02349-2) (São Paulo, Brazil), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (Brasília, Brazil).

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  • *
    These authors contributed equally to this work

Edited by

Associate Editor:

Carlos F. M. Menck

Publication Dates

  • Publication in this collection
    31 May 2024
  • Date of issue
    2024

History

  • Received
    06 Nov 2023
  • Accepted
    19 Apr 2024
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