[Impact of chaperone-mediated autophagy on bilirubin-induced damage of mouse microglial cells]. / åˆ†å­ä¼´ä¾£ä»‹å¯¼çš„è‡ªå™¬å¯¹èƒ†çº¢ç´ è¯±å¯¼çš„å°é¼ å°èƒ¶è´¨ç»†èƒžæŸä¼¤çš„å½±å“.

OBJECTIVES: To investigate the effect of chaperone-mediated autophagy (CMA) on the damage of mouse microglial BV2 cells induce by unconjugated bilirubin (UCB). METHODS: The BV2 cell experiments were divided into two parts. (1) For the CMA activation experiment: control group (treated with an equal volume of dimethyl sulfoxide), QX77 group (treated with 20 µmol/L QX77 for 24 hours), UCB group (treated with 40 µmol/L UCB for 24 hours), and UCB+QX77 group (treated with both 20 µmol/L QX77 and 40 µmol/L UCB for 24 hours). (2) For the cell transfection experiment: LAMP2A silencing control group (treated with an equal volume of dimethyl sulfoxide), LAMP2A silencing control+UCB group (treated with 40 µmol/L UCB for 24 hours), LAMP2A silencing group (treated with an equal volume of dimethyl sulfoxide), and LAMP2A silencing+UCB group (treated with 40 µmol/L UCB for 24 hours). The cell viability was assessed using the modified MTT method. The expression levels of p65, nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3), and cysteinyl aspartate specific proteinase-1 (caspase-1) were detected by Western blot. The relative mRNA expression levels of the inflammatory cytokines interleukin (IL)-1ß, IL-6, and tumor necrosis factor-α (TNF-α) were determined by real-time quantitative polymerase chain reaction. Levels of IL-6 and TNF-α in the cell culture supernatant were measured using ELISA. The co-localization of heat shock cognate protein 70 with p65 and NLRP3 was detected by immunofluorescence. RESULTS: Compared to the UCB group, the cell viability in the UCB+QX77 group increased, and the expression levels of inflammation-related proteins p65, NLRP3, and caspase-1, as well as the mRNA relative expression levels of IL-1ß, IL-6, and TNF-α and levels of IL-6 and TNF-α decreased (P<0.05). Compared to the control group, there was co-localization of heat shock cognate protein 70 with p65 and NLRP3 in both the UCB and UCB+QX77 groups. After silencing the LAMP2A gene, compared to the LAMP2A silencing control+UCB group, the LAMP2A silencing+UCB group showed increased expression levels of inflammation-related proteins p65, NLRP3, and caspase-1, as well as increased mRNA relative expression levels of IL-1ß, IL-6, and TNF-α and levels of IL-6 and TNF-α (P<0.05). CONCLUSIONS: CMA is inhibited in UCB-induced BV2 cell damage, and activating CMA may reduce p65 and NLRP3 protein levels, suppress inflammatory responses, and counteract bilirubin neurotoxicity.

Chaperone-Mediated Autophagy Controls Proteomic and Transcriptomic Pathways to Maintain Glioma Stem Cell Activity.

Chaperone-mediated autophagy (CMA) is a homeostatic process essential for the lysosomal degradation of a selected subset of the proteome. CMA activity directly depends on the levels of LAMP2A, a critical receptor for CMA substrate proteins at the lysosomal membrane. In glioblastoma (GBM), the most common and aggressive brain cancer in adulthood, high levels of LAMP2A in the tumor and tumor-associated pericytes have been linked to temozolomide resistance and tumor progression. However, the role of LAMP2A, and hence CMA, in any cancer stem cell type or in glioblastoma stem cells (GSC) remains unknown. In this work, we show that LAMP2A expression is enriched in patient-derived GSCs, and its depletion diminishes GSC-mediated tumorigenic activities. Conversely, overexpression of LAMP2A facilitates the acquisition of GSC properties. Proteomic and transcriptomic analysis of LAMP2A-depleted GSCs revealed reduced extracellular matrix interaction effectors in both analyses. Moreover, pathways related to mitochondrial metabolism and the immune system were differentially deregulated at the proteome level. Furthermore, clinical samples of GBM tissue presented overexpression of LAMP2, which correlated with advanced glioma grade and poor overall survival. In conclusion, we identified a novel role of CMA in directly regulating GSCs activity via multiple pathways at the proteome and transcriptome levels. SIGNIFICANCE: A receptor of chaperone-mediated autophagy regulates glioblastoma stem cells and may serve as a potential biomarker for advanced tumor grade and poor survival in this disease.

The Role of Chaperone-Mediated Autophagy in Bortezomib Resistant Multiple Myeloma.

Background: Multiple myeloma (MM) remains incurable despite high-dose chemotherapy, autologous stem cell transplants and novel agents. Even with the improved survival of MM patients treated with novel agents, including bortezomib (Bz), the therapeutic options in relapsed/refractory MM remain limited. The majority of MM patients eventually develop resistance to Bz, although the mechanisms of the resistance are poorly understood. Methods: Lysosomal associated membrane protein 2A (LAMP2A) mRNA and protein expression levels were assessed in ex vivo patient samples and a Bz-resistant MM cell line model by in real-rime PCR, western blotting and immunohistochemistry. In vitro modelling of chaperone-mediated autophagy (CMA) activity in response to ER stress were assessed by western blotting and confocal microscopy. The effects of CMA inhibition on MM cell viability and Bz sensitivity in MM cells were assessed by Annexin V/7AAD apoptosis assays using flow cytometry. Results: In this study, there is evidence that CMA, a chaperone-mediated protein degradation pathway, is upregulated in Bz-resistant MM and the inhibition of CMA sensitises resistant cells to Bz. The protein levels of LAMP2A, the rate-limiting factor of the CMA pathway, are significantly increased in MM patients resistant to Bz and within our Bz-resistant cell line model. Bz-resistant cell lines also possessed higher basal CMA activity than the Bz-sensitive parent cell line. In MM cell lines, CMA activity was upregulated in response to ER stress induced by Bz. The inhibition of CMA sensitises Bz-resistant cells to Bz and the combination of CMA inhibition and Bz in vitro had a more cytotoxic effect on myeloma cells than Bz alone. Conclusion: In summary, the upregulation of CMA is a potential mechanism of resistance to Bz and a novel target to overcome Bz-resistant MM.

A perspective on the role of autophagy in cancer.

Autophagy refers to a ubiquitous set of catabolic pathways required to achieve proper cellular homeostasis. Aberrant autophagy has been implicated in a multitude of diseases including cancer. In this review, we highlight pioneering and groundbreaking research that centers on delineating the role of autophagy in cancer initiation, proliferation and metastasis. First, we discuss the autophagy-related (ATG) proteins and their respective roles in the de novo formation of autophagosomes and the subsequent delivery of cargo to the lysosome for recycling. Next, we touch upon the history of cancer research that centers upon ATG proteins and regulatory mechanisms that control an appropriate autophagic response and how these are altered in the diseased state. Then, we discuss the various discoveries that led to the idea of autophagy as a double-edged sword when it comes to cancer therapy. This review also briefly narrates how different types of autophagy-selective macroautophagy and chaperone-mediated autophagy, have been linked to different cancers. Overall, these studies build upon a steadfast trajectory that aims to solve the monumentally daunting challenge of finding a cure for many types of cancer by modulating autophagy either through inhibition or induction.

Increased LAMP2A levels correlate with a shorter disease-free survival of HER2 negative breast cancer patients and increased breast cancer cell viability.

Chaperone Mediated Autophagy (CMA) is a selective autophagy pathway deregulated in many cancers. In this study, we were aiming at understanding the importance of CMA in breast cancer. To this end, we examined the expression of the CMA markers HSP8 and LAMP2A in different breast cancer cell lines and found a wide range of LAMP2A expression levels across the cell lines analyzed. Next, we applied a specific immunohistochemical staining protocol to a tissue microarray derived from a cohort of 365 breast cancer patients. Therefore, we were able to find a correlation of high LAMP2A but not HSPA8 (HSC70) with worse disease free survival in patients with HER2 negative tumors (p = 0.026) which was independent prognostic parameter from pT category, pN category and grading in a multivariate model (HR = 1.889; 95% CI = 1.039-3.421; p = 0.037). In line, low LAMP2A levels decrease proliferation of the breast cancer cell lines T47D and MCF-7 in vitro. Our data suggest that LAMP2A supports a more severe breast cancer cell phenotype.

Metformin activates chaperone-mediated autophagy and improves disease pathologies in an Alzheimer disease mouse model.

Chaperone-mediated autophagy (CMA) is a lysosome-dependent selective degradation pathway implicated in the pathogenesis of cancer and neurodegenerative diseases. However, the mechanisms that regulate CMA are not fully understood. Here, using unbiased drug screening approaches, we discover Metformin, a drug that is commonly the first medication prescribed for type 2 diabetes, can induce CMA. We delineate the mechanism of CMA induction by Metformin to be via activation of TAK1-IKKα/ß signaling that leads to phosphorylation of Ser85 of the key mediator of CMA, Hsc70, and its activation. Notably, we find that amyloid-beta precursor protein (APP) is a CMA substrate and that it binds to Hsc70 in an IKKα/ß-dependent manner. The inhibition of CMA-mediated degradation of APP enhances its cytotoxicity. Importantly, we find that in the APP/PS1 mouse model of Alzheimer's disease (AD), activation of CMA by Hsc70 overexpression or Metformin potently reduces the accumulated brain Aß plaque levels and reverses the molecular and behavioral AD phenotypes. Our study elucidates a novel mechanism of CMA regulation via Metformin-TAK1-IKKα/ß-Hsc70 signaling and suggests Metformin as a new activator of CMA for diseases, such as AD, where such therapeutic intervention could be beneficial.

Discovery of LAMP-2A as potential biomarkers for glioblastoma development by modulating apoptosis through N-CoR degradation.

BACKGROUND: Lysosome-associated membrane protein type 2A (LAMP-2A) is the key component of chaperone-mediated autophagy (CMA), a cargo-selective lysosomal degradation pathway. Aberrant LAMP-2A expression and CMA activation have been demonstrated in various human malignancies. The study focusing on the intrinsic role of LAMP-2A and CMA in glioblastoma (GBM), and downstream mechanism could provide valuable insight into the pathogenesis and novel therapeutic modality of GBM. METHODS: The levels of LAMP-2A, nuclear receptor co-repressor (N-CoR), unfolded protein response (UPR) and apoptosis were examined in clinical samples. LAMP-2A siRNA and shRNA were constructed to manipulate CMA activation. The role of CMA and downstream mechanism through degradation of N-CoR and arresting UPR mediated apoptosis were explored in GBM cells and nude mouse xenograft model. RESULTS: Elevated LAMP-2A and associated decreased N-CoR expression were observed in GBM as compared with peritumoral region and low-grade glioma. Inhibited UPR and apoptosis were observed in GBM with high LAMP-2A expression. In vitro study demonstrated co-localization and interaction between LAMP-2A and N-CoR. LAMP-2A silencing up-regulated N-CoR and aroused UPR pathway, leading to apoptosis, while N-CoR silencing led to an opposite result. In vivo study further confirmed that LAMP-2A inhibition arrested tumor growth by promoting apoptosis. CONCLUSIONS: Our results demonstrated the central role of CMA in mediating N-CoR degradation and protecting GBM cells against UPR and apoptosis, and provided evidence of LAMP-2A as potential biomarker. Further research focusing on CMA with other tumorigenic process is needed and selective modulators of LAMP-2A remain to be investigated to provide a novel therapeutic strategy for GBM. Video Abstract.

Inhibition of chaperone­mediated autophagy reduces tumor growth and metastasis and promotes drug sensitivity in colorectal cancer.

Chaperone­mediated autophagy (CMA) is a selective type of autophagy whereby a specific subset of intracellular proteins is targeted to the lysosome for degradation. The present study investigated the mechanisms underlying the response and resistance to 5­fluorouracil (5­FU) in colorectal cancer (CRC) cell lines. In engineered 5­FU­resistant CRC cell lines, a significant elevation of lysosome­associated membrane protein 2A (LAMP2A), which is the key molecule in the CMA pathway, was identified. High expression of LAMP2A was found to be responsible for 5­FU resistance and to enhance PLD2 expression through the activation of NF­κB pathway. Accordingly, loss or gain of function of LAMP2A in 5­FU­resistant CRC cells rendered them sensitive or resistant to 5­FU, respectively. Taken together, the results of the present study suggested that chemoresistance in patients with CRC may be mediated by enhancing CMA. Thus, CMA is a promising predictor of chemosensitivity to 5­FU treatment and anti­CMA therapy may be a novel therapeutic option for patients with CRC.

Chaperone-mediated autophagy affects tumor cell proliferation and cisplatin resistance in esophageal squamous cell carcinoma.

BACKGROUND: Chaperone-mediated autophagy (CMA) is a lysosomal degradation pathway of selective soluble proteins. Lysosomal membrane associated protein 2a (LAMP2a) is the lysosomal membrane receptor of CMA and influences CMA activity. Although it has been suggested that higher expression of LAMP2a is associated with more advanced tumor node metastasis (TNM) stages and shorter survival time in patients with esophageal squamous cell carcinoma (ESCC), the underlying mechanism has not been known yet. METHODS: In this study, we modulated the activity of CMA through LAMP2a or small molecular compounds in human ESCC cells to investigate its role in ESCC. RESULTS: We found that down-regulating the activity of CMA could inhibit the proliferation and colony formation of ESCC cells as well as increase their sensitivity to cisplatin. CONCLUSIONS: Our results promote better understanding of how CMA affects human ESCC and provide a new therapeutic target against ESCC through down-regulating LAMP2a.

Chaperone-mediated autophagy sustains haematopoietic stem-cell function.

The activation of mostly quiescent haematopoietic stem cells (HSCs) is a prerequisite for life-long production of blood cells1. This process requires major molecular adaptations to allow HSCs to meet the regulatory and metabolic requirements for cell division2-4. The mechanisms that govern cellular reprograming upon stem-cell activation, and the subsequent return of stem cells to quiescence, have not been fully characterized. Here we show that chaperone-mediated autophagy (CMA)5, a selective form of lysosomal protein degradation, is involved in sustaining HSC function in adult mice. CMA is required for protein quality control in stem cells and for the upregulation of fatty acid metabolism upon HSC activation. We find that CMA activity in HSCs decreases with age and show that genetic or pharmacological activation of CMA can restore the functionality of old mouse and human HSCs. Together, our findings provide mechanistic insights into a role for CMA in sustaining quality control, appropriate energetics and overall long-term HSC function. Our work suggests that CMA may be a promising therapeutic target for enhancing HSC function in conditions such as ageing or stem-cell transplantation.

Chaperone-Mediated Autophagy Markers LAMP2A and HSC70 Are Independent Adverse Prognostic Markers in Primary Resected Squamous Cell Carcinomas of the Lung.

LAMP2A and HSC70 are crucial players in chaperone-mediated autophagy (CMA), a targeted, lysosome-dependent protein degradation pathway. Elevated LAMP2A levels, indicative of increased CMA activity, are observed in several malignancies, and CMA downregulation may be exploited therapeutically. We evaluated the impact of LAMP2A and HSC70 in pulmonary squamous cell carcinomas (pSQCC). Antibodies were validated by knockdown and overexpression experiments using three different cell lines. Expression levels in tissue were analyzed by immunohistochemistry in a cohort of 336 consecutive pSQCC using tissue microarrays. There was no significant correlation between the two markers among each other and no association with pathological parameters (TNM categories, grading). However, both high LAMP2A and HSC70 expression were associated with worse outcome, including overall survival (OS; p = 0.012 and p = 0.001) and disease free survival (DFS; p = 0.049 and p = 0.036). In multivariate analysis, both markers and a combination of them were independent adverse prognostic factors for OS (LAMP2Ahigh: HR = 2.059; p < 0.001; HSC70high: HR = 1.987; p < 0.001; LAMP2Ahigh/HSC70high: HR = 1.529; p < 0.001) and DFS (LAMP2Ahigh: HR = 1.709; p = 0.004; HSC70high: HR = 1.484; p = 0.027; LAMP2Ahigh/HSC70high: HR = 1.342, p < 0.001). The negative prognostic impact of high LAMP2A and HSC70 and their variable expression in pSQCC may justify the use of these proteins as potential biomarkers for future CMA-inhibiting therapies.

The Role of Chaperone-Mediated Autophagy in Cell Cycle Control and Its Implications in Cancer.

The cell cycle involves a network of proteins that modulate the sequence and timing of proliferation events. Unregulated proliferation is the most fundamental hallmark of cancer; thus, changes in cell cycle control are at the heart of malignant transformation processes. Several cellular processes can interfere with the cell cycle, including autophagy, the catabolic pathway involved in degradation of intracellular constituents in lysosomes. According to the mechanism used to deliver cargo to the lysosome, autophagy can be classified as macroautophagy (MA), microautophagy (MI), or chaperone-mediated autophagy (CMA). Distinct from other autophagy types, CMA substrates are selectively recognized by a cytosolic chaperone, one-by-one, and then addressed for degradation in lysosomes. The function of MA in cell cycle control, and its influence in cancer progression, are already well-established. However, regulation of the cell cycle by CMA, in the context of tumorigenesis, has not been fully addressed. This review aims to present and debate the molecular mechanisms by which CMA can interfere in the cell cycle, in the context of cancer. Thus, cell cycle modulators, such as MYC, hypoxia-inducible factor-1 subunit alpha (HIF-1α), and checkpoint kinase 1 (CHK1), regulated by CMA activity will be discussed. Finally, the review will focus on how CMA dysfunction may impact the cell cycle, and as consequence promote tumorigenesis.

Chaperone-mediated Autophagy Governs Progression of Papillary Thyroid Carcinoma via PPARγ-SDF1/CXCR4 Signaling.

CONTEXT: Papillary thyroid carcinoma (PTC) is the most common endocrine malignancy. Chaperone-mediated autophagy (CMA), 1 type of autophagy, is thought to promote or suppress cancer development in different cancer types. However, the effect of CMA on PTC development and the underlying mechanisms remain unknown. OBJECTIVE: To determine whether CMA plays implied critical roles in the development of PTC. DESIGN: We investigated the association between CMA and PTC development in PTC tissues and normal thyroid tissues by detecting the key protein of CMA, lysosome-associated membrane protein type 2A (LAMP2A), using quantitative polymerase chain reaction (PCR) and immunohistochemistry, which were further validated in the TGCA dataset. The effect of CMA on PTC development was studied by cell proliferation, migration, and apoptosis assays. The underlying mechanisms of peroxisome proliferator-activated receptor γ (PPARγ)-stromal cell-derived factor 1 (SDF1)/ C-X-C motif chemokine receptor 4 (CXCR4) signaling were clarified by western blotting, quantitative PCR, and rescue experiments. Knockdown and tamoxifen were used to analyze the effect of estrogen receptor (ER) α on CMA. RESULTS: Our study confirmed that CMA, indicated by LAMP2A expression, was significantly increased in PTC tumor tissues and cell lines, and was associated with tumor size and lymph node metastasis of patients. Higher CMA in PTC promoted tumor cell proliferation and migration, thereby promoting tumor growth and metastasis. These effects of CMA on PTC were exerted by decreasing PPARγ protein expression to enhance SDF1 and CXCR4 expression. Furthermore, CMA was found positively regulated by ERα signaling in PTC. CONCLUSION: Our investigation identified CMA regulated by ERα promoting PTC tumor progression that enhanced tumor cell proliferation and migration by targeting PPARγ-SDF1/CXCR4 signaling, representing a potential target for treatment of PTC.

Pros and Cons of Chaperone-Mediated Autophagy in Cancer Biology.

Autophagy contributes to cellular quality control and energetics through lysosomal breakdown and recycling of essential cellular components. Chaperone-mediated autophagy (CMA) adds to these autophagic functions the ability to timely and selectively degrade single tagged proteins to terminate their cellular function and, in this way, participate in the regulation of multiple cellular processes. Many cancer cells upregulate CMA for protumorigenic and prosurvival purposes. However, growing evidence supports a physiological role for CMA in limiting malignant transformation. Understanding the mechanisms behind this functional switch of CMA from antioncogenic to pro-oncogenic is fundamental for targeting CMA in cancer treatment. We summarize current understanding of CMA functions in cancer biology and discuss the basis for its context-dependent dual role in oncogenesis.

Targetome analysis of chaperone-mediated autophagy in cancer cells.

Chaperone-mediated autophagy (CMA) is a lysosomal degradation pathway of select soluble proteins. Nearly one-third of the soluble proteins are predicted to be recognized by this pathway, yet only a minor fraction of this proteome has been identified as CMA substrates in cancer cells. Here, we undertook a quantitative multiplex mass spectrometry approach to study the proteome of isolated lysosomes in cancer cells during CMA-activated conditions. By integrating bioinformatics analyses, we identified and categorized proteins of multiple cellular pathways that were specifically targeted by CMA. Beyond verifying metabolic pathways, we show that multiple components involved in select biological processes, including cellular translation, was specifically targeted for degradation by CMA. In particular, several proteins of the translation initiation complex were identified as bona fide CMA substrates in multiple cancer cell lines of distinct origin and we show that CMA suppresses cellular translation. We further show that the identified CMA substrates display high expression in multiple primary cancers compared to their normal counterparts. Combined, these findings uncover cellular processes affected by CMA and reveal a new role for CMA in the control of translation in cancer cells. Abbreviations: 6-AN: 6-aminonicotinamide; ACTB: actin beta; AR7: atypical retinoid 7; CHX: cycloheximide; CMA: chaperone-mediated autophagy; CQ: chloroquine; CTS: cathepsins; DDX3X: DEAD-box helicase 3 X-linked; EEF2: eukaryotic translation elongation factor 2; EIF4A1: eukaryotic translation initiation factor 4A1; EIF4H: eukaryotic translation initiation factor 4H; GEO: Gene Expression Omnibus; GO: Gene Ontology; GSEA: gene set enrichment analysis; HK2: hexokinase 2; HSPA8/HSC70: heat shock protein family A (Hsp70) member 8; LAMP: lysosomal-associated membrane protein; LDHA: lactate dehydrogenase A; NES: normalized enrichment score; NFKBIA: NFKB inhibitor alpha; PCA: principle component analysis; PQ: paraquat; S.D.: standard deviation; SUnSET: surface sensing of translation; TMT: tandem mass tags; TOMM40/TOM40: translocase of outer mitochondrial membrane 40.