HSPA8 chaperone complex drives chaperone-mediated autophagy regulation in acute promyelocytic leukemia cell differentiation.

INTRODUCTION: Acute myeloid leukemia (AML) is a cancer of the hematopoietic system characterized by hyperproliferation of undifferentiated cells of the myeloid lineage. While most of AML therapies are focused towards tumor debulking, all-trans retinoic acid (ATRA) induces neutrophil differentiation in the AML subtype acute promyelocytic leukemia (APL). Macroautophagy has been extensively investigated in the context of various cancers and is often dysregulated in AML where it can have context-dependent pro- or anti-leukemogenic effects. On the contrary, the implications of chaperone-mediated autophagy (CMA) on the pathophysiology of diseases are still being explored and its role in AML has remains elusive. METHODS: We took advantages of human AML primary samples and databases to analyze CMA gene expression and activity. Furthermore, we used ATRA-sensitive (NB4) and -resistant (NB4-R1) cells to further dissect a potential function for CMA in ATRA-mediated neutrophil differentiation. NB4-R1 cells are unique in that they do respond to retinoic acid transcriptionally, but do not mature in response to retinoid signaling alone unless maturation is triggered by adding cAMP. RESULTS: Here, we report that CMA related mRNA transcripts are significantly higher expressed in immature hematopoietic cells as compared to neutrophils, contrasting the macroautophagy gene expression patterns. Accordingly, lysosomal degradation of an mCherry-KFERQ CMA reporter decreases during ATRA-induced differentiation of APL cells. On the other hand, using NB4-R1 cells we found that macroautophagy flux primed ATRA resistant NB4-R1 cells to differentiate upon ATRA treatment, but reduced association of lysosome-associated membrane protein type 2A (LAMP-2A) and heat shock protein family A (Hsp70) member 8 (HSPA8), which are necessary for complete neutrophil maturation. Accordingly, depletion of HSPA8 attenuated CMA activity and facilitated APL cell differentiation. In contrast, maintaining high CMA activity by ectopic expression of LAMP-2A impeded APL differentiation. CONCLUSION: Overall, our findings suggest that APL neutrophil differentiation requires CMA inactivation and that this pathway predominantly depends on HSPA8 and is possibly assisted by other co-chaperones.

Isoform-specific, Semi-quantitative Determination of Highly Homologous Protein Levels via CRISPR-Cas9-mediated HiBiT Tagging.

Many protein families consist of multiple highly homologous proteins, whether they are encoded by different genes or originating from the same genomic location. Predominance of certain isoforms has been linked to various pathological conditions, such as cancer. Detection and relative quantification of protein isoforms in research are commonly done via immunoblotting, immunohistochemistry, or immunofluorescence, where antibodies against an isoform-specific epitope of particular family members are used. However, isoform-specific antibodies are not always available, making it impossible to decipher isoform-specific protein expression patterns. Here, we describe the insertion of the versatile 11 amino acid HiBiT tag into the genomic location of the protein of interest. This tag was developed and is distributed by Promega (Fitchburg, WI, USA). This protocol describes precise and specific protein expression analysis of highly homologous proteins through expression of the HiBiT tag, enabling protein expression quantification when specific antibodies are missing. Protein expression can be analyzed through traditional methods such as western blotting or immunofluorescence, and also in a luciferase binary reporter system, allowing for reliable and fast relative expression quantification using a plate reader. Graphical overview.

Immunohistochemical Detection of the Chaperone-Mediated Autophagy Markers LAMP2A and HSPA8 in Formalin-Fixed and Paraffin-Embedded Tissues.

Autophagy is crucial for maintaining cellular homeostasis and its deregulation is involved in disease development, including cancer. The key players of chaperone-mediated autophagy (CMA), a particular selective subtype of autophagy, are HSPA8 and LAMP2A. Both proteins can be immunohistochemically detected in formalin-fixed paraffin-embedded (FFPE) tissue. LAMP2A is frequently overexpressed in a variety of cancers where it likely supports cancer cell survival and resistance to anti-cancer therapies in a context-dependent manner. Here we present the immunohistochemical staining protocol of antibodies against LAMP2A and HSPA8, using an automated staining system, suitable for routine diagnostics. Additionally, we also suggest a staining evaluation method.

TBK1 is part of a galectin 8 dependent membrane damage recognition complex and drives autophagy upon Adenovirus endosomal escape.

Intracellular pathogens cause membrane distortion and damage as they enter host cells. Cells perceive these membrane alterations as danger signals and respond by activating autophagy. This response has primarily been studied during bacterial invasion, and only rarely in viral infections. Here, we investigate the cellular response to membrane damage during adenoviral entry. Adenoviruses and their vector derivatives, that are an important vaccine platform against SARS-CoV-2, enter the host cell by endocytosis followed by lysis of the endosomal membrane. We previously showed that cells mount a locally confined autophagy response at the site of endosomal membrane lysis. Here we describe the mechanism of autophagy induction: endosomal membrane damage activates the kinase TBK1 that accumulates in its phosphorylated form at the penetration site. Activation and recruitment of TBK1 require detection of membrane damage by galectin 8 but occur independently of classical autophagy receptors or functional autophagy. Instead, TBK1 itself promotes subsequent autophagy that adenoviruses need to take control of. Deletion of TBK1 reduces LC3 lipidation during adenovirus infection and restores the infectivity of an adenovirus mutant that is restricted by autophagy. By comparing adenovirus-induced membrane damage to sterile lysosomal damage, we implicate TBK1 in the response to a broader range of types of membrane damage. Our study thus highlights an important role for TBK1 in the cellular response to adenoviral endosome penetration and places TBK1 early in the pathway leading to autophagy in response to membrane damage.

Hexokinase 3 enhances myeloid cell survival via non-glycolytic functions.

The family of hexokinases (HKs) catalyzes the first step of glycolysis, the ATP-dependent phosphorylation of glucose to glucose-6-phosphate. While HK1 and HK2 are ubiquitously expressed, the less well-studied HK3 is primarily expressed in hematopoietic cells and tissues and is highly upregulated during terminal differentiation of some acute myeloid leukemia (AML) cell line models. Here we show that expression of HK3 is predominantly originating from myeloid cells and that the upregulation of this glycolytic enzyme is not restricted to differentiation of leukemic cells but also occurs during ex vivo myeloid differentiation of healthy CD34+ hematopoietic stem and progenitor cells. Within the hematopoietic system, we show that HK3 is predominantly expressed in cells of myeloid origin. CRISPR/Cas9 mediated gene disruption revealed that loss of HK3 has no effect on glycolytic activity in AML cell lines while knocking out HK2 significantly reduced basal glycolysis and glycolytic capacity. Instead, loss of HK3 but not HK2 led to increased sensitivity to ATRA-induced cell death in AML cell lines. We found that HK3 knockout (HK3-null) AML cells showed an accumulation of reactive oxygen species (ROS) as well as DNA damage during ATRA-induced differentiation. RNA sequencing analysis confirmed pathway enrichment for programmed cell death, oxidative stress, and DNA damage response in HK3-null AML cells. These signatures were confirmed in ATAC sequencing, showing that loss of HK3 leads to changes in chromatin configuration and increases the accessibility of genes involved in apoptosis and stress response. Through isoform-specific pulldowns, we furthermore identified a direct interaction between HK3 and the proapoptotic BCL-2 family member BIM, which has previously been shown to shorten myeloid life span. Our findings provide evidence that HK3 is dispensable for glycolytic activity in AML cells while promoting cell survival, possibly through direct interaction with the BH3-only protein BIM during ATRA-induced neutrophil differentiation.

Role of cardiolipins, mitochondria, and autophagy in the differentiation process activated by all-trans retinoic acid in acute promyelocytic leukemia.

The role played by lipids in the process of granulocytic differentiation activated by all-trans retinoic acid (ATRA) in Acute-Promyelocytic-Leukemia (APL) blasts is unknown. The process of granulocytic differentiation activated by ATRA in APL blasts is recapitulated in the NB4 cell-line, which is characterized by expression of the pathogenic PML-RARα fusion protein. In the present study, we used the NB4 model to define the effects exerted by ATRA on lipid homeostasis. Using a high-throughput lipidomic approach, we demonstrate that exposure of the APL-derived NB4 cell-line to ATRA causes an early reduction in the amounts of cardiolipins, a major lipid component of the mitochondrial membranes. The decrease in the levels of cardiolipins results in a concomitant inhibition of mitochondrial activity. These ATRA-dependent effects are causally involved in the granulocytic maturation process. In fact, the ATRA-induced decrease of cardiolipins and the concomitant dysfunction of mitochondria precede the differentiation of retinoid-sensitive NB4 cells and the two phenomena are not observed in the retinoid-resistant NB4.306 counterparts. In addition, ethanolamine induced rescue of the mitochondrial dysfunction activated by cardiolipin deficiency inhibits ATRA-dependent granulocytic differentiation and induction of the associated autophagic process. The RNA-seq studies performed in parental NB4 cells and a NB4-derived cell population, characterized by silencing of the autophagy mediator, ATG5, provide insights into the mechanisms underlying the differentiating action of ATRA. The results indicate that ATRA causes a significant down-regulation of CRLS1 (Cardiolipin-synthase-1) and LPCAT1 (Lysophosphatidylcholine-Acyltransferase-1) mRNAs which code for two enzymes catalyzing the last steps of cardiolipin synthesis. ATRA-dependent down-regulation of CRLS1 and LPCAT1 mRNAs is functionally relevant, as it is accompanied by a significant decrease in the amounts of the corresponding proteins. Furthermore, the decrease in CRLS1 and LPCAT1 levels requires activation of the autophagic process, as down-regulation of the two proteins is blocked in ATG5-silenced NB4-shATG5 cells.

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

[This corrects the article DOI: 10.1155/2020/8506572.].

Chaperone-Mediated Autophagy Markers LAMP2A and HSPA8 in Advanced Non-Small Cell Lung Cancer after Neoadjuvant Therapy.

In recent years autophagy has attracted the attention of researchers from many medical fields, including cancer research, and certain anti-macroautophagy drugs in combination with cytotoxic or targeted therapies have entered clinical trials. In the present study, we focused on a less explored subtype of autophagy, i.e., chaperone-mediated autophagy (CMA), with the key proteins LAMP2A and HSPA8 (HSC70), and their immunohistochemical evaluation with previously extensively validated antibodies. We were interested in whether the marker expression is influenced by the antecedent therapy, and its correlation with survival on a cohort of patients with non-small cell lung cancer (NSCLC) after neoadjuvant therapy and matched primary resected tumors. In concordance with our previous study, we did not find any intratumoral heterogeneity, nor correlation between the two parameters, nor correlation between the markers and any included pathological parameters. Surprisingly, the expression of both markers was also independent to tumor response or administered neoadjuvant treatment. In the survival analysis, the results were only significant for LAMP2A, where higher levels were associated with longer 5-year overall survival and disease-free survival for the mixed group of adenocarcinomas and squamous cell carcinomas (p < 0.0001 and p = 0.0019 respectively) as well as the squamous cell carcinoma subgroup (p = 0.0001 and p = 0.0001 respectively). LAMP2A was also an independent prognostic marker in univariate and multivariate analysis.

Lysosomes in acute myeloid leukemia: potential therapeutic targets?

Lysosomes, since their discovery, have been primarily known for degrading cellular macromolecules. However, in recent studies, they have begun to emerge as crucial regulators of cell homeostasis. They are at the crossroads of catabolic and anabolic pathways and are intricately involved in cellular trafficking, nutrient signaling, energy metabolism, and immune regulation. Their involvement in such essential cellular functions has renewed clinical interest in targeting the lysosome as a novel way to treat disease, particularly cancer. Acute myeloid leukemia (AML) is an aggressive blood cancer with a low survival probability, particularly in older patients. The genomic landscape of AML has been extensively characterized but few targeted therapies (with the exception of differentiation therapy) can achieve a long-term cure. Therefore, there is an unmet need for less intensive and more tolerable therapeutic interventions. In this review, we will give an overview on the myriad of functions performed by lysosomes and their importance in malignant disease. Furthermore, we will discuss their relevance in hematopoietic cells and different ways to potentially target them in AML.

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.

The Multifaceted Functions of Autophagy in Breast Cancer Development and Treatment.

Macroautophagy (herein referred to as autophagy) is a complex catabolic process characterized by the formation of double-membrane vesicles called autophagosomes. During this process, autophagosomes engulf and deliver their intracellular content to lysosomes, where they are degraded by hydrolytic enzymes. Thereby, autophagy provides energy and building blocks to maintain cellular homeostasis and represents a dynamic recycling mechanism. Importantly, the clearance of damaged organelles and aggregated molecules by autophagy in normal cells contributes to cancer prevention. Therefore, the dysfunction of autophagy has a major impact on the cell fate and can contribute to tumorigenesis. Breast cancer is the most common cancer in women and has the highest mortality rate among all cancers in women worldwide. Breast cancer patients often have a good short-term prognosis, but long-term survivors often experience aggressive recurrence. This phenomenon might be explained by the high heterogeneity of breast cancer tumors rendering mammary tumors difficult to target. This review focuses on the mechanisms of autophagy during breast carcinogenesis and sheds light on the role of autophagy in the traits of aggressive breast cancer cells such as migration, invasion, and therapeutic resistance.

ALK inhibition activates LC3B-independent, protective autophagy in EML4-ALK positive lung cancer cells.

ALK inhibitors effectively target EML4-ALK positive non-small cell lung cancer, but their effects are hampered by treatment resistance. In the present study, we asked whether ALK inhibition affects autophagy, and whether this may influence treatment response. Whereas the impact of targeted therapies on autophagic activity previously have been assessed by surrogate marker proteins such as LC3B, we here thoroughly examined effects on functional autophagic activity, i.e. on the sequestration and degradation of autophagic cargo, in addition to autophagic markers. Interestingly, the ALK inhibitor Ceritinib decreased mTOR activity and increased GFP-WIPI1 dot formation in H3122 and H2228 EML4-ALK+ lung cancer cells, suggesting autophagy activation. Moreover, an mCherry-EGFP-LC3B based assay indicated elevated LC3B carrier flux upon ALK inhibition. In accordance, autophagic cargo sequestration and long-lived protein degradation significantly increased upon ALK inhibition. Intriguingly, autophagic cargo flux was dependent on VPS34 and ULK1, but not LC3B. Co-treating H3122 cells with Ceritinib and a VPS34 inhibitor or Bafilomycin A1 resulted in reduced cell numbers. Moreover, VPS34 inhibition reduced clonogenic recovery of Ceritinib-treated cells. In summary, our results indicate that ALK inhibition triggers LC3B-independent macroautophagic flux in EML4-ALK+ cells to support cancer cell survival and clonogenic growth.

Reducing FASN expression sensitizes acute myeloid leukemia cells to differentiation therapy.

Fatty acid synthase (FASN) is the only human lipogenic enzyme available for de novo fatty acid synthesis and is often highly expressed in cancer cells. We found that FASN mRNA levels were significantly higher in acute myeloid leukemia (AML) patients than in healthy granulocytes or CD34+ hematopoietic progenitors. Accordingly, FASN levels decreased during all-trans retinoic acid (ATRA)-mediated granulocytic differentiation of acute promyelocytic leukemia (APL) cells, partially via autophagic degradation. Furthermore, our data suggest that inhibition of FASN expression levels using RNAi or (-)-epigallocatechin-3-gallate (EGCG) accelerated the differentiation of APL cell lines and significantly re-sensitized ATRA refractory non-APL AML cells. FASN reduction promoted translocation of transcription factor EB (TFEB) to the nucleus, paralleled by activation of CLEAR network genes and lysosomal biogenesis. Together, our data demonstrate that inhibition of FASN expression in combination with ATRA treatment facilitates granulocytic differentiation of APL cells and may extend differentiation therapy to non-APL AML cells.

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 LIM Protein Ajuba Augments Tumor Metastasis in Colon Cancer.

Colorectal cancer, along with its high potential for recurrence and metastasis, is a major health burden. Uncovering proteins and pathways required for tumor cell growth is necessary for the development of novel targeted therapies. Ajuba is a member of the LIM domain family of proteins whose expression is positively associated with numerous cancers. Our data shows that Ajuba is highly expressed in human colon cancer tissue and cell lines. Publicly available data from The Cancer Genome Atlas shows a negative correlation between survival and Ajuba expression in patients with colon cancer. To investigate its function, we transduced SW480 human colon cancer cells, with lentiviral constructs to knockdown or overexpress Ajuba protein. The transcriptome of the modified cell lines was analyzed by RNA sequencing. Among the pathways enriched in the differentially expressed genes, were cell proliferation, migration and differentiation. We confirmed our sequencing data with biological assays; cells depleted of Ajuba were less proliferative, more sensitive to irradiation, migrated less and were less efficient in colony formation. In addition, loss of Ajuba expression decreased the tumor burden in a murine model of colorectal metastasis to the liver. Taken together, our data supports that Ajuba promotes colon cancer growth, migration and metastasis and therefore is a potential candidate for targeted therapy.

Progress and Challenges in The Use of MAP1LC3 as a Legitimate Marker for Measuring Dynamic Autophagy In Vivo.

Tremendous efforts have been made these last decades to increase our knowledge of intracellular degradative systems, especially in the field of autophagy. The role of autophagy in the maintenance of cell homeostasis is well documented and the existence of defects in the autophagic machinery has been largely described in diseases and aging. Determining the alterations occurring in the many forms of autophagy that coexist in cells and tissues remains complicated, as this cellular process is highly dynamic in nature and can vary from organ to organ in the same individual. Although autophagy is extensively studied, its functioning in different tissues and its links with other biological processes is still poorly understood. Several assays have been developed to monitor autophagy activity in vitro, ex vivo, and in vivo, based on different markers, the use of various inhibitors and activators, and distinct techniques. This review emphasizes the methods applied to measure (macro-)autophagy in tissue samples and in vivo via a protein, which centrally intervenes in the autophagy pathway, the microtubule-associated protein 1A/1B-light chain 3 (MAP1LC3), which is the most widely used marker and the first identified to associate with autophagosomal structures. These approaches are presented and discussed in terms of pros and cons. Some recommendations are provided to improve the reliability of the interpretation of results.

Assessing Autophagy in Archived Tissue or How to Capture Autophagic Flux from a Tissue Snapshot.

Autophagy is a highly conserved degradation mechanism that is essential for maintaining cellular homeostasis. In human disease, autophagy pathways are frequently deregulated and there is immense interest in targeting autophagy for therapeutic approaches. Accordingly, there is a need to determine autophagic activity in human tissues, an endeavor that is hampered by the fact that autophagy is characterized by the flux of substrates whereas histology informs only about amounts and localization of substrates and regulators at a single timepoint. Despite this challenging task, considerable progress in establishing markers of autophagy has been made in recent years. The importance of establishing clear-cut autophagy markers that can be used for tissue analysis cannot be underestimated. In this review, we attempt to summarize known techniques to quantify autophagy in human tissue and their drawbacks. Furthermore, we provide some recommendations that should be taken into consideration to improve the reliability and the interpretation of autophagy biomarkers in human tissue samples.

Inhibition of UBE2L6 attenuates ISGylation and impedes ATRA-induced differentiation of leukemic cells.

Ubiquitin/ISG15-conjugating enzyme E2L6 (UBE2L6) is a critical enzyme in ISGylation, a post-translational protein modification that conjugates the ubiquitin-like modifier, interferon-stimulated gene 15 (ISG15), to target substrates. Previous gene expression studies in acute promyelocytic leukemia (APL) cells showed that all-trans-retinoic acid (ATRA) altered the expression of many genes, including UBE2L6 (200-fold) and other members of the ISGylation pathway. Through gene expression analyses in a cohort of 98 acute myeloid leukemia (AML) patient samples and in primary neutrophils from healthy donors, we found that UBE2L6 gene expression is reduced in primary AML cells compared with normal mature granulocytes. To assess whether UBE2L6 expression is important for leukemic cell differentiation-two cell line models were employed: the human APL cell line NB4 and its ATRA-resistant NB4R counterpart, as well as the ATRA-sensitive human AML HL60 cells along with their ATRA-resistant subclone-HL60R. ATRA strongly induced UBE2L6 in NB4 APL cells and in ATRA-sensitive HL60 AML cells, but not in the ATRA-resistant NB4R and HL60R cells. Furthermore, short hairpin (sh)RNA-mediated UBE2L6 depletion in NB4 cells impeded ATRA-mediated differentiation, suggesting a functional role for UBE2L6 in leukemic cell differentiation. In addition, ATRA induced ISG15 gene expression in NB4 APL cells, leading to increased levels of both free ISG15 protein and ISG15 conjugates. UBE2L6 depletion attenuated ATRA-induced ISG15 conjugation. Knockdown of ISG15 in NB4 APL cells inhibited ISGylation and also attenuated ATRA-induced differentiation. In summary, we demonstrate the functional importance of UBE2L6 in ATRA-induced neutrophil differentiation of APL cells and propose that this may be mediated by its catalytic role in ISGylation.