Outcome of early clinical trials of the combination of hydroxychloroquine with chemotherapy in cancer

The premise of inhibiting autophagy to overcome resistance to chemotherapy has been investigated in 5 clinical phase I trials combining hydroxychloroquine with vorinostat, temsirolimus, temozolomide, or bortezomib. These studies have provided a number of insights relating to the tolerability of the combination treatments. In addition, these studies should provide guidance in the planning and design of future trials to directly determine whether the strategy of autophagy inhibition could prove useful in the treatment of various malignancies.     

Dual inhibition of glycolysis and autophagy as a therapeutic strategy in the treatment of Ehrlich ascites carcinoma

Cancer cells have extra biosynthetic demands to sustain cell growth and redox homeostasis. Glycolysis and autophagy are crucial to fuel and recycle these biosynthetic demands. This plasticity of cancer cell metabolism participates in therapy resistances. The current study was designed to assess the therapeutic efficacy of dual targeting of glycolysis and autophagy in cancer. Using 3‐bromopyruvate (3‐BP; antiglycolytic inhibitor) and hydroxychloroquine (HCQ; autophagy inhibitor), we demonstrate their antitumor activity in Ehrlich ascites carcinoma (EAC)‐bearing mice. A combination of 3‐BP and HCQ significantly decreases tumor ascitic volume and cell count as compared with the EAC group and individual treatment groups. The enhanced antitumor activity is accompanied by hexokinase inactivation, inhibition of cellular protective autophagy, elevated antioxidant activity, and reduced oxidative stress levels. Together, these results suggest targeting both pathways in cancer as an effective therapeutic strategy. Further studies are required to validate this strategy in different cancer models and preclinical trials.     

Targeting autophagy

In recent years, tremendous progress has been made toward unveiling the mechanism of autophagy and its exploitation by many different cancer types. This year the American Association for the Advancement of Science held a one day Symposium on Autophagy: An Emerging Therapeutic Target in Human Disease in Vancouver, British Columbia and brought together experts in cell biology, drug discovery, and clinical translation to share their research findings and prospects. Currently, autophagy is being investigated on several fronts, from modulation of gene expression to in vivo studies, and more recently clinical trials in cancer. Key topics of discussion were determining which stage of autophagy would be the ideal target for inhibition to produce the highest impact, and which cancers or cancer subtypes would be the most sensitive to autophagy inhibitors; the answers to these questions may be a turning point in cancer therapy research.     

Unfolding the role of autophagy in the cancer metabolism

Autophagy is considered an indispensable process that scavenges toxins, recycles complex macromolecules, and sustains the essential cellular functions. In addition to its housekeeping role, autophagy plays a substantial role in many pathophysiological processes such as cancer. Certainly, it adapts cancer cells to thrive in the stress conditions such as hypoxia and starvation. Cancer cells indeed have also evolved by exploiting the autophagy process to fulfill energy requirements through the production of metabolic fuel sources and fundamentally altered metabolic pathways. Occasionally autophagy as a foe impedes tumorigenesis and promotes cell death. The complex role of autophagy in cancer makes it a potent therapeutic target and has been actively tested in clinical trials. Moreover, the versatility of autophagy has opened new avenues of effective combinatorial therapeutic strategies. Thereby, it is imperative to comprehend the specificity of autophagy in cancer-metabolism. This review summarizes the recent research and conceptual framework on the regulation of autophagy by various metabolic pathways, enzymes, and their cross-talk in the cancer milieu, including the implementation of altered metabolism and autophagy in clinically approved and experimental therapeutics.     

Nelfinavir, a new anti-cancer drug with pleiotropic effects and many paths to autophagy

The development of cancer drugs is slow and costly. One approach to accelerate the availability of new drugs is to reposition drugs approved for other indications as anti-cancer agents. HIV protease inhibitors (HIV PIs) are useful in treating HIV infection and cause toxicities in humans that are similar to those observed when the kinase Akt, a target for cancer therapy, is inhibited. To test whether HIV PIs inhibited Akt and cancer cell proliferation, we screened 6 HIV PIs and found that three, ritonavir, saquinavir and nelfinavir, inhibit the growth of over 60 cancer cell lines derived from 9 different tumor types; Nelfinavir is the most potent. Nelfinavir causes caspase-dependent apoptosis and non-apoptotic death, as well as endoplasmic reticulum (ER) stress and autophagy. Nelfinavir blocks growth factor receptor activation and decreases growth factor-induced and endogenous Akt signaling. In vivo, nelfinavir inhibits tumor growth and upregulates markers of ER stress, autophagy and apoptosis. Nelfinavir is currently being tested in cancer patients in Phase I clinical trials where biomarkers will be assessed. Current studies are focused on measuring autophagy in clinical specimens and identifying combination strategies that will exploit the induction of autophagy and increase the effectiveness of nelfinavir.     

Nelfinavir,a new anti-cancer drug with pleiotropic effects and many paths to autophagy

The development of cancer drugs is slow and costly. One approach to accelerate the availability of new drugs is to reposition drugs approved for other indications as anti-cancer agents. HIV protease inhibitors (HIV PIs) are useful in treating HIV infection and cause toxicities in humans that are similar to those observed when the kinase Akt, a target for cancer therapy, is inhibited. To test whether HIV PIs inhibited Akt and cancer cell proliferation, we screened 6 HIV PIs and found that three, ritonavir, saquinavir and nelfinavir, inhibit the growth of over 60 cancer cell lines derived from 9 different tumor types; Nelfinavir is the most potent. Nelfinavir causes caspase-dependent apoptosis and non-apoptotic death, as well as endoplasmic reticulum (ER) stress and autophagy. Nelfinavir blocks growth factor receptor activation and decreases growth factor-induced and endogenous Akt signaling. In vivo, nelfinavir inhibits tumor growth and upregulates markers of ER stress, autophagy and apoptosis. Nelfinavir is currently being tested in cancer patients in Phase I clinical trials where biomarkers will be assessed. Current studies are focused on measuring autophagy in clinical specimens and identifying combination strategies that will exploit the induction of autophagy and increase the effectiveness of nelfinavir.     

PLK1 (polo like kinase 1) inhibits MTOR complex 1 and promotes autophagy

Mechanistic target of rapamycin complex 1 (MTORC1) and polo like kinase 1 (PLK1) are major drivers of cancer cell growth and proliferation, and inhibitors of both protein kinases are currently being investigated in clinical studies. To date, MTORC1′s and PLK1′s functions are mostly studied separately, and reports on their mutual crosstalk are scarce. Here, we identify PLK1 as a physical MTORC1 interactor in human cancer cells. PLK1 inhibition enhances MTORC1 activity under nutrient sufficiency and in starved cells, and PLK1 directly phosphorylates the MTORC1 component RPTOR/RAPTOR in vitro. PLK1 and MTORC1 reside together at lysosomes, the subcellular site where MTORC1 is active. Consistent with an inhibitory role of PLK1 toward MTORC1, PLK1 overexpression inhibits lysosomal association of the PLK1-MTORC1 complex, whereas PLK1 inhibition promotes lysosomal localization of MTOR. PLK1-MTORC1 binding is enhanced by amino acid starvation, a condition known to increase autophagy. MTORC1 inhibition is an important step in autophagy activation. Consistently, PLK1 inhibition mitigates autophagy in cancer cells both under nutrient starvation and sufficiency, and a role of PLK1 in autophagy is also observed in the invertebrate model organism Caenorhabditis elegans. In summary, PLK1 inhibits MTORC1 and thereby positively contributes to autophagy. Since autophagy is increasingly recognized to contribute to tumor cell survival and growth, we propose that cautious monitoring of MTORC1 and autophagy readouts in clinical trials with PLK1 inhibitors is needed to develop strategies for optimized (combinatorial) cancer therapies targeting MTORC1, PLK1, and autophagy.     

Autophagy blockade enhances HDAC inhibitors' pro-apoptotic effects: potential implications for the treatment of a therapeutic-resistant malignancy

Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive, highly metastatic, poor prognosis tumors for which effective therapeutic strategies are currently lacking. We summarize recent work focusing on preclinical evaluation of histone deacetylase inhibitors (HDACis) for the treatment of MPNST. HDACis are a novel drug class with anti-cancer therapeutic promise. Using human MPNST cell lines and xenograft models we found that a MPNST subset is highly sensitive to HDACis, whereas a fraction is relatively resistant. HDACis were found to induce autophagy in all MPNST cells in vitro and in vivo; in "sensitive" MPNST cells autophagy occurs in concert with apoptosis, whereas unopposed autophagy develops in "resistant" cells. Genetic and chemical autophagy blockade significantly enhances HDACi-induced apoptotic cell death in both resistant and sensitive cells. Combined chloroquine and HDACi treatment abrogates growth of human MPNST xenografts and lung metastases. The potential role of autophagy in cancer therapeutic response remains controversial; however, our study supports HDACi-induced autophagy as a MPNST survival mechanism. These data also imply that the consequences of drug-induced autophagy may be compound-type, tumor-type, or even molecular context-dependent, suggesting a complex crosstalk between autophagy and apoptosis. Clinical trials evaluating HDACis with autophagy blockade for therapy of MPNST therefore merit consideration.     

Inhibition of autophagy attenuates pancreatic cancer growth independent of TP53/TRP53 status

Basal levels of autophagy are elevated in most pancreatic ductal adenocarcinomas (PDAC). Suppressing autophagy pharmacologically using chloroquine (CQ) or genetically with RNAi to essential autophagy genes inhibits human pancreatic cancer growth in vitro and in vivo, which presents possible treatment opportunities for PDAC patients using the CQ-derivative hydroxychloroquine (HCQ). Indeed, such clinical trials are ongoing. However, autophagy is a complex cellular mechanism to maintain cell homeostasis under stress. Based on its biological role, a dual role of autophagy in tumorigenesis has been proposed: at tumor initiation, autophagy helps maintain genomic stability and prevent tumor initiation; while in advanced disease, autophagy degrades and recycles cellular components to meet the metabolic needs for rapid growth. This model was proven to be the case in mouse lung tumor models. However, in contrast to prior work in various PDAC model systems, loss of autophagy in PDAC mouse models with embryonic homozygous Trp53 deletion does not inhibit tumor growth and paradoxically increases progression. This raised concerns whether there may be a genotype-dependent reliance of PDAC on autophagy. In a recent study, our group used a Trp53 heterozygous mouse PDAC model and human PDX xenografts to address the question. Our results demonstrate that autophagy inhibition was effective against PDAC tumors irrespective of TP53/TRP53 status.     

细胞自噬在植物碳氮营养中作用的研究进展

自噬（autophagy）是真核生物细胞通过形成自噬体,回收利用胞内物质,维持细胞健康的高通量亚细胞降解途径。随着酵母和动物自噬研究的深入,植物自噬也受到越来越多的关注。近期的研究揭示了植物自噬的基本机制及其生理意义,也发现了植物特有的自噬形式与自噬相关基因。该文主要综述了自噬在植物碳、氮营养中的作用。     

The Bcl-2 Homology Domain 3 Mimetic Gossypol Induces Both Beclin 1-dependent and Beclin 1-independent Cytoprotective Autophagy in Cancer Cells

Gossypol, a natural Bcl-2 homology domain 3 mimetic compound isolated from cottonseeds, is currently being evaluated in clinical trials. Here, we provide evidence that gossypol induces autophagy followed by apoptotic cell death in both the MCF-7 human breast adenocarcinoma and HeLa cell lines. We first show that knockdown of the Bcl-2 homology domain 3-only protein Beclin 1 reduces gossypol-induced autophagy in MCF-7 cells, but not in HeLa cells. Gossypol inhibits the interaction between Beclin 1 and Bcl-2 (B-cell leukemia/lymphoma 2), antagonizes the inhibition of autophagy by Bcl-2, and hence stimulates autophagy. We then show that knockdown of Vps34 reduces gossypol-induced autophagy in both cell lines, and consistent with this, the phosphatidylinositol 3-phosphate-binding protein WIPI-1 is recruited to autophagosomal membranes. Further, Atg5 knockdown also reduces gossypol-mediated autophagy. We conclude that gossypol induces autophagy in both a canonical and a noncanonical manner. Notably, we found that gossypol-mediated apoptotic cell death was potentiated by treatment with the autophagy inhibitor wortmannin or with small interfering RNA against essential autophagy genes (Vps34, Beclin 1, and Atg5). Our findings support the notion that gossypol-induced autophagy is cytoprotective and not part of the cell death process induced by this compound.     

Autophagy and human diseases

Autophagy is a major intracellular degradative process that delivers cytoplasmic materials to the lysosome for degradation. Since the discovery of autophagy-related (Atg) genes in the 1990s, there has been a proliferation of studies on the physiological and pathological roles of autophagy in a variety of autophagy knockout models. However, direct evidence of the connections between ATG gene dysfunction and human diseases has emerged only recently. There are an increasing number of reports showing that mutations in the ATG genes were identified in various human diseases such as neurodegenerative diseases, infectious diseases, and cancers. Here, we review the major advances in identification of mutations or polymorphisms of the ATG genes in human diseases. Current autophagy-modulating compounds in clinical trials are also summarized.     

Proteomic analysis revealed association of aberrant ROS signaling with suberoylanilide hydroxamic acid-induced autophagy in Jurkat T-leukemia cells

Suberoylanilide hydroxamic acid (SAHA) is a newly emerging histone deacetylase inhibitor (HDACi) and has been approved in phase II clinical trials for treating patients with cutaneous T-cell lymphoma. Autophagy is a conserved self-digestion process that degrades cytoplasmic materials and recycles long-lived proteins and organelles within cells. In this study, we demonstrate that SAHA stimulates autophagy in Jurkat T-leukemia cells, which was evidenced by the appearance of autophagic vacuoles, formation of acidic vesicular organelles, recruitment of LC3-II to the autophagosomes and conversion of LC3-I to LC3-II. Moreover, SAHA treatment upregulated expression of Beclin 1 and Atg7 and promoted formation of the Atg12-Atg5 conjugate. Furthermore, inhibition of autophagy by chloroquine (CQ) enhanced SAHA-induced apoptosis. To determine the underlying mechanism of SAHA-induced autophagy, two complementary proteomic approaches (2-DE and SILAC), coupled with ESI-Q-TOF MS/MS analysis are utilized to profile differentially expressed proteins between control and SAHA-treated Jurkat T-leukemia cells. In total, 72 proteins were identified with significant alterations. Cluster analysis of the changed proteins reveal several groups of enzymes associated with energy metabolism, anti-oxidative stress and cellular redox control, which suggested an abnormal reactive oxygen species (ROS) production in SAHA-treated Jurkat T-leukemia cells. These observations were further confirmed by ROS chemiluminescence assay. Mechanistic studies revealed that SAHA-triggered autophagy was mediated by ROS production, which could be attenuated by N-acetyl cysteine (NAC), a ROS inhibitor. Finally, we illustrated that Akt-mTOR signaling, a major suppressive cascade of autophagy, was inactivated by SAHA treatment. Taken together, our study identifies autophagy as a reaction to counter increased ROS and is thus involved as a cellular prosurvival mechanism in response to SAHA treatment.     

Eating on the fly: function and regulation of autophagy during cell growth, survival and death in Drosophila

Significant progress has been made over recent years in defining the normal progression and regulation of autophagy, particularly in cultured mammalian cells and yeast model systems. However, apart from a few notable exceptions, our understanding of the physiological roles of autophagy has lagged behind these advances, and identification of components and features of autophagy unique to higher eukaryotes also remains a challenge. In this review we describe recent insights into the roles and control mechanisms of autophagy gained from in vivo studies in Drosophila. We focus on potential roles of autophagy in controlling cell growth and death, and describe how the regulation of autophagy has evolved to include metazoan-specific signaling pathways. We discuss genetic screening approaches that are being used to identify novel regulators and effectors of autophagy, and speculate about areas of research in this system likely to bear fruit in future studies.     

Autophagy blockade enhances HDAC inhibitors’ pro-apoptotic effects

Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive, highly metastatic, poor prognosis tumors for which effective therapeutic strategies are currently lacking. We summarize recent work focusing on preclinical evaluation of histone deacetylase inhibitors (HDACis) for the treatment of MPNST. HDACis are a novel drug class with anti-cancer therapeutic promise. Using human MPNST cell lines and xenograft models we found that a MPNST subset is highly sensitive to HDACis, whereas a fraction is relatively resistant. HDACis were found to induce autophagy in all MPNST cells in vitro and in vivo; in “sensitive” MPNST cells autophagy occurs in concert with apoptosis, whereas unopposed autophagy develops in “resistant” cells. Genetic and chemical autophagy blockade significantly enhances HDACi-induced apoptotic cell death in both resistant and sensitive cells. Combined chloroquine and HDACi treatment abrogates growth of human MPNST xenografts and lung metastases. The potential role of autophagy in cancer therapeutic response remains controversial; however, our study supports HDACi-induced autophagy as a MPNST survival mechanism. These data also imply that the consequences of drug-induced autophagy may be compound-type, tumor-type, or even molecular context-dependent, suggesting a complex crosstalk between autophagy and apoptosis. Clinical trials evaluating HDACis with autophagy blockade for therapy of MPNST therefore merit consideration.     

Development of selective nutrient deprivation as a system to study autophagy induction and regulation in neurons

Autophagy is emerging as a fundamentally important pathway for countering misfolded protein stress in the central nervous system. Indeed, many studies suggest that upregulation of a properly functioning macroautophagy pathway can be neuroprotective in neurodegenerative disorders characterized by the production of toxic protein conformers. Despite these advances, little is known about how autophagy is regulated in neurons. To directly study neuronal autophagy, we developed a primary neuron culture system where we can induce autophagy by withdrawal of a key supplement from the culture medium. We recently reported that the absence of insulin from the culture medium induces autophagy in this primary neuron system, and that the neuronal autophagy activation is mTOR-dependent. Further studies indicate that our nutrient-deprivation method of autophagy induction yields normally functioning and fully progressing autophagy based upon treatment with lysosomal inhibitors. As this method of autophagy induction can protect neurons from proteotoxic cell death, our findings suggest that an understanding of how to turn on autophagy in neurons could translate into a viable approach for treating neurodegenerative proteinopathies. However, before therapeutic applications can be realized, the pathways regulating neuronal autophagy need to be defined. As highlighted herein, our system for autophagy induction should contribute to efforts aimed at understanding the regulatory basis of autophagy activation in neurons.     

Autophagy modulator scoring system: a user-friendly tool for quantitative analysis of methodological integrity of chemical autophagy modulator studies

ABSTRACT

Over the past 20 years (1999–2019), we have witnessed a rapid increase in publications involving chemical macroautophagy/autophagy modulators. However, an overview of the methodologies used in these studies is still lacking, and methodology flaws are frequently observed in some reports. To provide an objective and quantitative analysis of studies involving autophagy modulators, we present an Autophagy Modulator Scoring System (AMSS), which is designed to evaluate methodological integrity. AMSS-A includes the autophagy characterization by 4 aspects, namely, autophagosome quantification, autophagy-related biochemical changes, autophagy substrate degradation, and autophagic flux. AMSS-B contains the pharmacological and functional characteristics of chemical autophagy modulators, including lysosomal function, drug targets, autophagy-dependent pharmacological effects, and validation in multiple cell lines and in vivo models. Our analysis shows that of the 385 studies reporting chemical autophagy modulators, only 142 single studies had examined all 4 aspects of autophagy characterization in AMSS-A, and only 10 out of 142 studies had fulfilled all the AMSS criteria in a single study. A comprehensive analysis of the methodologies used in all the studies was made, along with a summary of studies that demonstrated the highest methodological integrity based on AMSS ranking. To test the reliability of the AMSS, a co-efficiency analysis of scores and co-citation values in the co-citation network was performed, and a significant co-efficiency was obtained. Collectively, AMSS provides insight into the methodological integrity of autophagy modulators studies and also offers a user-friendly toolkit to help choose appropriate assays to characterize autophagy modulators.

Abbreviations: 3-MA: 3-methyladenine; AMSS: Autophagy Modulator Scoring System; ATG: autophagy-related; BAF: bafilomycin A₁; BECN1: beclin 1; CQ: chloroquine; GFP: green fluorescent protein; LC3: microtubule associated protein 1 light chain 3; mRFP: monomeric red fluorescent protein; MTOR: mechanistic target of rapamycin kinase; PtdIns3K: phosphatidylinositol 3-kinase; PtdIns3P: phosphatidylinositol-3-phosphate     

The role of autophagy in doxorubicin-induced cardiotoxicity

Doxorubicin (Dox) is an effective chemotherapeutic agent, however, its use is limited by cardiotoxicity. The mechanisms causing cardiotoxicity have not been clearly elucidated, but known to involve, at least in part, oxidative stress, mitochondrial dysfunction and apoptosis. More recently, it has been suggested that dysregulation of autophagy may also play an important role in Dox-induced cardiotoxicity. Autophagy has dual functions. Under physiological conditions, autophagy is essential for optimal cellular function and survival by ridding the cell of damaged or unwanted proteins and organelles. Under pathological conditions, autophagy may be stimulated in order to protect the cell from stress stimuli or, alternatively, to contribute to cell death. Thus, appropriate regulation of autophagy can be a matter of life or death. The role of autophagy in Dox-induced cardiotoxicity has recently been explored, however, conflicting reports on the effects of Dox on autophagy and its role in cardiotoxicity exist. Most, but not all, of the studies conclude that Dox upregulates cardiac autophagy and contributes to the pathogenesis of Dox-induced toxicity. Dox may induce autophagy by suppressing the expression of GATA4 and/or S6K1, which may directly or indirectly regulate expression of essential autophagy genes such as Atg12, Atg5, Beclin1 and Bcl-2. Interestingly, the Dox-induced autophagic response may be species specific as Dox treatment has been shown to stimulate autophagy in rat models, but suppress autophagy in mouse models. Additional studies will elucidate this possibility.     

Programmed autophagy in the Drosophila fat body is induced by ecdysone through regulation of the PI3K pathway

Eukaryotic cells catabolize their own cytoplasm by autophagy in response to amino acid starvation and inductive signals during programmed tissue remodeling and cell death. The Tor and PI3K signaling pathways have been shown to negatively control autophagy in eukaryotes, but the mechanisms that link these effectors to overall animal development and nutritional status in multicellular organisms remain poorly understood. Here, we reveal a complex regulation of programmed and starvation-induced autophagy in the Drosophila fat body. Gain-of-function genetic analysis indicated that ecdysone receptor signaling induces programmed autophagy whereas PI3K signaling represses programmed autophagy. Genetic interaction studies showed that ecdysone signaling downregulates PI3K signaling and that this represents the effector mechanism for induction of programmed autophagy. Hence, these studies link hormonal induction of autophagy to the regulatory function of the PI3K signaling pathway in vivo.     

Eating on the fly: Function and regulation of autophagy during cell growth,survival and death in Drosophila

Significant progress has been made over recent years in defining the normal progression and regulation of autophagy, particularly in cultured mammalian cells and yeast model systems. However, apart from a few notable exceptions, our understanding of the physiological roles of autophagy has lagged behind these advances, and identification of components and features of autophagy unique to higher eukaryotes also remains a challenge. In this review we describe recent insights into the roles and control mechanisms of autophagy gained from in vivo studies in Drosophila. We focus on potential roles of autophagy in controlling cell growth and death, and describe how the regulation of autophagy has evolved to include metazoan-specific signaling pathways. We discuss genetic screening approaches that are being used to identify novel regulators and effectors of autophagy, and speculate about areas of research in this system likely to bear fruit in future studies.