Autophagy in major human diseases.

Autophagy is a core molecular pathway for the preservation of cellular and organismal homeostasis. Pharmacological and genetic interventions impairing autophagy responses promote or aggravate disease in a plethora of experimental models. Consistently, mutations in autophagy-related processes cause severe human pathologies. Here, we review and discuss preclinical data linking autophagy dysfunction to the pathogenesis of major human disorders including cancer as well as cardiovascular, neurodegenerative, metabolic, pulmonary, renal, infectious, musculoskeletal, and ocular disorders.

The Integrated Stress Response in Pancreatic Development, Tissue Homeostasis, and Cancer.

Present in all eukaryotic cells, the integrated stress response (ISR) is a highly coordinated signaling network that controls cellular behavior, metabolism, and survival in response to diverse stresses. The ISR is initiated when any 1 of 3 stress-sensing kinases (protein kinase R-like endoplasmic reticulum kinase [PERK], general control non-derepressible 2 [GCN2], double-stranded RNA-dependent protein kinase [PKR], heme-regulated eukaryotic translation initiation factor 2α kinase [HRI]) becomes activated to phosphorylate the protein translation initiation factor eukaryotic translation initiation factor 2α (eIF2α), shifting gene expression toward a comprehensive rewiring of cellular machinery to promote adaptation. Although the ISR has been shown to play an important role in the homeostasis of multiple tissues, evidence suggests that it is particularly crucial for the development and ongoing health of the pancreas. Among the most synthetically dynamic tissues in the body, the exocrine and endocrine pancreas relies heavily on the ISR to rapidly adjust cell function to meet the metabolic demands of the organism. The hardwiring of the ISR into normal pancreatic functions and adaptation to stress may explain why it is a commonly used pro-oncogenic and therapy-resistance mechanism in pancreatic ductal adenocarcinoma and pancreatic neuroendocrine tumors. Here we review what is known about the key roles that the ISR plays in the development, homeostasis, and neoplasia of the pancreas.

Mitophagy in tumorigenesis and metastasis.

Cells use mitophagy to remove dysfunctional or excess mitochondria, frequently in response to imposed stresses, such as hypoxia and nutrient deprivation. Mitochondrial cargo receptors (MCR) induced by these stresses target mitochondria to autophagosomes through interaction with members of the LC3/GABARAP family. There are a growing number of these MCRs, including BNIP3, BNIP3L, FUNDC1, Bcl2-L-13, FKBP8, Prohibitin-2, and others, in addition to mitochondrial protein targets of PINK1/Parkin phospho-ubiquitination. There is also an emerging link between mitochondrial lipid signaling and mitophagy where ceramide, sphingosine-1-phosphate, and cardiolipin have all been shown to promote mitophagy. Here, we review the upstream signaling mechanisms that regulate mitophagy, including components of the mitochondrial fission machinery, AMPK, ATF4, FoxOs, Sirtuins, and mtDNA release, and address the significance of these pathways for stress responses in tumorigenesis and metastasis. In particular, we focus on how mitophagy modulators intersect with cell cycle control and survival pathways in cancer, including following ECM detachment and during cell migration and metastasis. Finally, we interrogate how mitophagy affects tissue atrophy during cancer cachexia and therapy responses in the clinic.

Autophagy, cancer stem cells and drug resistance.

Autophagy is a cellular survival mechanism that is induced by cancer therapy, among other stresses, and frequently contributes to cancer cell survival during long periods of dormancy and the eventual outgrowth of metastatic disease. Autophagy degrades large cellular structures that, once broken down, contribute to cellular survival through the recycling of their constituent metabolites. However, the extent to which this fuel function of autophagy is key to its role in promoting stemness, dormancy and drug resistance remains to be determined. Other roles for autophagy in determining cell fate more directly through targeted degradation of key transcription factors, such as p53 and FoxO3A, or by enforcing a reversible quiescent growth arrest, are discussed in this review. This review also highlights the need to parse out the roles of different forms of selective autophagy in stemness, CD44 expression and dormancy that, for example, are increasingly being attributed explicitly to mitophagy. The clinical relevance of this work and how an increased understanding of functions of autophagy in stemness, dormancy and drug resistance could be manipulated for increased therapeutic benefit, including eliminating minimal residual disease and preventing metastasis, are discussed. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Expanding perspectives on the significance of mitophagy in cancer.

Mitophagy is a selective mode of autophagy in which mitochondria are specifically targeted for degradation at the autophagolysosome. Mitophagy is activated by stresses such as hypoxia, nutrient deprivation, DNA damage, inflammation and mitochondrial membrane depolarization and plays a role in maintaining mitochondrial integrity and function. Defects in mitophagy lead to mitochondrial dysfunction that can affect metabolic reprogramming in response to stress, alter cell fate determination and differentiation, which in turn affects disease incidence and etiology, including cancer. Here, we discuss how different mitophagy adaptors and modulators, including Parkin, BNIP3, BNIP3L, p62/SQSTM1 and OPTN, are regulated in response to physiological stresses and deregulated in cancers. Additionally, we explore how these different mitophagy control pathways coordinate with each other. Finally, we review new developments in understanding how mitophagy affects stemness, cell fate determination, inflammation and DNA damage responses that are relevant to understanding the role of mitophagy in cancer.

Mitophagy defects arising from BNip3 loss promote mammary tumor progression to metastasis.

BNip3 is a hypoxia-inducible protein that targets mitochondria for autophagosomal degradation. We report a novel tumor suppressor role for BNip3 in a clinically relevant mouse model of mammary tumorigenesis. BNip3 delays primary mammary tumor growth and progression by preventing the accumulation of dysfunctional mitochondria and resultant excess ROS production. In the absence of BNip3, mammary tumor cells are unable to reduce mitochondrial mass effectively and elevated mitochondrial ROS increases the expression of Hif-1α and Hif target genes, including those involved in glycolysis and angiogenesis­two processes that are also markedly increased in BNip3-null tumors. Glycolysis inhibition attenuates the growth of BNip3-null tumor cells, revealing an increased dependence on autophagy for survival. We also demonstrate that BNIP3 deletion can be used as a prognostic marker of tumor progression to metastasis in human triple-negative breast cancer (TNBC). These studies show that mitochondrial dysfunction­caused by defects in mitophagy­can promote the Warburg effect and tumor progression, and suggest better approaches to stratifying TNBC for treatment.

In Brief: Mitophagy: mechanisms and role in human disease.

Mitophagy is a selective form of macro-autophagy in which mitochondria are specifically targeted for autophagic degradation. Mitophagy plays an important role in cellular homeostasis by eliminating dysfunctional mitochondria and reducing mitochondrial mass as an adaptive response to stress. Cells execute mitophagy through several non-redundant mechanisms, including the PINK1/Parkin partnership, which modulates turnover of depolarized mitochondria, and stress-induced BNIP3, NIX, and FUNDC1 molecular adaptors, which interact directly with LC3 to promote mitophagy. These pathways are deregulated in human diseases, including cancer, neurodegeneration, metabolic disorders, muscle atrophy, ageing, and inflammation, reflecting the importance of mitophagy as a cellular housekeeping function. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Small molecules inhibit STAT3 activation, autophagy, and cancer cell anchorage-independent growth.

Triple-negative breast cancers (TNBCs) lack the signature targets of other breast tumors, such as HER2, estrogen receptor, and progesterone receptor. These aggressive basal-like tumors are driven by a complex array of signaling pathways that are activated by multiple driver mutations. Here we report the discovery of 6 (KIN-281), a small molecule that inhibits multiple kinases including maternal leucine zipper kinase (MELK) and the non-receptor tyrosine kinase bone marrow X-linked (BMX) with single-digit micromolar IC50s. Several derivatives of 6 were synthesized to gain insight into the binding mode of the compound to the ATP binding pocket. Compound 6 was tested for its effect on anchorage-dependent and independent growth of MDA-MB-231 and MDA-MB-468 breast cancer cells. The effect of 6 on BMX prompted us to evaluate its effect on STAT3 phosphorylation and DNA binding. The compound's inhibition of cell growth led to measurements of survivin, Bcl-XL, p21WAF1/CIP1, and cyclin A2 levels. Finally, LC3B-II levels were quantified following treatment of cells with 6 to determine whether the compound affected autophagy, a process that is known to be activated by STAT3. Compound 6 provides a starting point for the development of small molecules with polypharmacology that can suppress TNBC growth and metastasis.

Tumour suppressor gene function in carcinoma-associated fibroblasts: from tumour cells via EMT and back again?

Recent reports indicate that inactivation of the RB, TP53 or PTEN tumour suppressor genes is detected in tumour stroma of oropharyngeal, breast and other human cancers. Mouse models have validated the tumour-promoting effects of deleting Rb, Pten or p53 in fibroblasts that converts them from normal fibroblasts to carcinoma associated fibroblasts (CAFs). The tumour-promoting activity of CAFs in these contexts was associated with increased paracrine signaling to tumour cells through production of specific growth factors, chemokines and MMPs by CAFs. The conversion of NOFs into CAFs through acquisition of specific mutations, such as loss of tumour suppressors, or deregulated expression of microRNAs or key epigenetic events, can clearly occur independently of genetic and epigenetic changes in tumour cells but an alternative source of CAFs that is being reconsidered is that CAFs derive from the tumour cells by EMT. Recent mouse models employing lineage-tracing techniques have suggested that this can take place in vivo and the extent to which this is relevant more broadly is discussed.

Context-dependent roles for autophagy in myeloid cells in tumor progression.

Autophagy is known to suppress tumor initiation by removing genotoxic stresses in normal cells. Conversely, autophagy is also known to support tumor progression by alleviating metabolic stresses in neoplastic cells. Centered on this pro-tumor role of autophagy, there have been many clinical trials to treat cancers through systemic blocking of autophagy. Such systemic inhibition affects both tumor cells and non-tumor cells, and the consequence of blocked autophagy in non-tumor cells in the context of tumor microenvironment is relatively understudied. Here, we examined the effect of autophagy-deficient myeloid cells on the progression of autophagy-competent tumors. We found that blocking autophagy only in myeloid cells modulated tumor progression markedly but such effects were context dependent. In a tumor implantation model, the growth of implanted tumor cells was substantially reduced in mice with autophagy-deficient myeloid cells; T cells infiltrated deeper into the tumors and were responsible for the reduced growth of the implanted tumor cells. In an oncogene-driven tumor induction model, however, tumors grew faster and metastasized more in mice with autophagy-deficient myeloid cells. These data demonstrate that the autophagy status of myeloid cells plays a critical role in tumor progression, promoting or suppressing tumor growth depending on the context of tumor-myeloid cell interactions. This study indicates that systemic use of autophagy inhibitors in cancer therapy may have differential effects on rates of tumor progression in patients due to effects on myeloid cells and that this warrants more targeted use of selective autophagy inhibitors in a cancer therapy in a clinical setting.

DISSECTING TUMOR TRANSCRIPTIONAL HETEROGENEITY FROM SINGLE-CELL RNA-SEQ DATA BY GENERALIZED BINARY COVARIANCE DECOMPOSITION.

Profiling tumors with single-cell RNA sequencing (scRNA-seq) has the potential to identify recurrent patterns of transcription variation related to cancer progression, and so produce new therapeutically-relevant insights. However, the presence of strong inter-tumor heterogeneity often obscures more subtle patterns that are shared across tumors, some of which may characterize clinically-relevant disease subtypes. Here we introduce a new statistical method to address this problem. We show that this method can help decompose transcriptional heterogeneity into interpretable components - including patient-specific, dataset-specific and shared components relevant to disease subtypes - and that, in the presence of strong inter-tumor heterogeneity, our method can produce more interpretable results than existing widely-used methods. Applied to data from three studies on pancreatic cancer adenocarcinoma (PDAC), our method produces a refined characterization of existing tumor subtypes (e.g. classical vs basal), and identifies a new gene expression program (GEP) that is prognostic of poor survival independent of established prognostic factors such as tumor stage and subtype. The new GEP is enriched for genes involved in a variety of stress responses, and suggests a potentially important role for the integrated stress response in PDAC development and prognosis.

Pancreatic tumors exhibit myeloid-driven amino acid stress and upregulate arginine biosynthesis.

Nutrient stress in the tumor microenvironment requires cancer cells to adopt adaptive metabolic programs for survival and proliferation. Therefore, knowledge of microenvironmental nutrient levels and how cancer cells cope with such nutrition is critical to understand the metabolism underpinning cancer cell biology. Previously, we performed quantitative metabolomics of the interstitial fluid (the local perfusate) of murine pancreatic ductal adenocarcinoma (PDAC) tumors to comprehensively characterize nutrient availability in the microenvironment of these tumors. Here, we develop Tumor Interstitial Fluid Medium (TIFM), a cell culture medium that contains nutrient levels representative of the PDAC microenvironment, enabling us to study PDAC metabolism ex vivo under physiological nutrient conditions. We show that PDAC cells cultured in TIFM adopt a cellular state closer to that of PDAC cells present in tumors compared to standard culture models. Further, using the TIFM model, we found arginine biosynthesis is active in PDAC and allows PDAC cells to maintain levels of this amino acid despite microenvironmental arginine depletion. We also show that myeloid derived arginase activity is largely responsible for the low levels of arginine in PDAC tumors. Altogether, these data indicate that nutrient availability in tumors is an important determinant of cancer cell metabolism and behavior, and cell culture models that incorporate physiological nutrient availability have improved fidelity to in vivo systems and enable the discovery of novel cancer metabolic phenotypes.

Lipid droplet turnover at the lysosome inhibits growth of hepatocellular carcinoma in a BNIP3-dependent manner.

Hepatic steatosis is a major etiological factor in hepatocellular carcinoma (HCC), but factors causing lipid accumulation leading to HCC are not understood. We identify BNIP3 (a mitochondrial cargo receptor) as an HCC suppressor that mitigates against lipid accumulation to attenuate tumor cell growth. Targeted deletion of Bnip3 decreased tumor latency and increased tumor burden in a mouse model of HCC. This was associated with increased lipid in bnip3-/- HCC at early stages of disease, while lipid did not accumulate until later in tumorigenesis in wild-type mice, as Bnip3 expression was attenuated. Low BNIP3 expression in human HCC similarly correlated with increased lipid content and worse prognosis than HCC expressing high BNIP3. BNIP3 suppressed HCC cell growth by promoting lipid droplet turnover at the lysosome in a manner dependent on BNIP3 binding LC3. We have termed this process "mitolipophagy" because it involves the coordinated autophagic degradation of lipid droplets with mitochondria.

Autophagy: cellular and molecular mechanisms.

Autophagy is a self-degradative process that is important for balancing sources of energy at critical times in development and in response to nutrient stress. Autophagy also plays a housekeeping role in removing misfolded or aggregated proteins, clearing damaged organelles, such as mitochondria, endoplasmic reticulum and peroxisomes, as well as eliminating intracellular pathogens. Thus, autophagy is generally thought of as a survival mechanism, although its deregulation has been linked to non-apoptotic cell death. Autophagy can be either non-selective or selective in the removal of specific organelles, ribosomes and protein aggregates, although the mechanisms regulating aspects of selective autophagy are not fully worked out. In addition to elimination of intracellular aggregates and damaged organelles, autophagy promotes cellular senescence and cell surface antigen presentation, protects against genome instability and prevents necrosis, giving it a key role in preventing diseases such as cancer, neurodegeneration, cardiomyopathy, diabetes, liver disease, autoimmune diseases and infections. This review summarizes the most up-to-date findings on how autophagy is executed and regulated at the molecular level and how its disruption can lead to disease.

Autophagy: assays and artifacts.

Autophagy is a fundamental and phylogenetically conserved self-degradation process that is characterized by the formation of double-layered vesicles (autophagosomes) around intracellular cargo for delivery to lysosomes and proteolytic degradation. The increasing significance attached to autophagy in development and disease in higher eukaryotes has placed greater importance on the validation of reliable, meaningful and quantitative assays to monitor autophagy in live cells and in vivo in the animal. To date, the detection of processed LC3B-II by western blot or fluorescence studies, together with electron microscopy for autophagosome formation, have been the mainstays for autophagy detection. However, LC3 expression levels can vary markedly between different cell types and in response to different stresses, and there is also concern that over-expression of tagged versions of LC3 to facilitate imaging and detection of autophagy interferes with the process itself. In addition, the realization that it is not sufficient to monitor static levels of autophagy but to measure 'autophagic flux' has driven the development of new or modified approaches to detecting autophagy. Here, we present a critical overview of current methodologies to measure autophagy in cells and in animals.

ULK1 promotes mitophagy via phosphorylation and stabilization of BNIP3.

UNC51-like kinase-1 (ULK1) is the catalytic component of the autophagy pre-initiation complex that stimulates autophagy via phosphorylation of ATG14, BECLN1 and other autophagy proteins. ULK1 has also been shown to specifically promote mitophagy but the mechanistic basis of how has remained unclear. Here we show that ULK1 phosphorylates the BNIP3 mitochondrial cargo receptor on a critical serine residue (S17) adjacent to its amino terminal LIR motif. ULK1 similarly phosphorylates BNIP3L on S35. Phosphorylation of BNIP3 on S17 by ULK1 promotes interaction with LC3 and mitophagy. ULK1 interaction also promotes BNIP3 protein stability by limiting its turnover at the proteasome. The ability of ULK1 to regulate BNIP3 protein stability depends on an intact "BH3" domain and deletion of its "BH3" domain reduces BNIP3 turnover and increases BNIP3 protein levels independent of ULK1. In summary ULK1 promotes mitophagy by both stabilization of BNIP3 protein and via phosphorylation of S17 to stimulate interaction with LC3.

BNIP3-dependent mitophagy promotes cytosolic localization of LC3B and metabolic homeostasis in the liver.

Mitophagy formed the basis of the original description of autophagy by Christian de Duve when he demonstrated that GCG (glucagon) induced macroautophagic/autophagic turnover of mitochondria in the liver. However, the molecular basis of liver-specific activation of mitophagy by GCG, or its significance for metabolic stress responses in the liver is not understood. Here we show that BNIP3 is required for GCG-induced mitophagy in the liver through interaction with processed LC3B; an interaction that is also necessary to localize LC3B out of the nucleus to cytosolic mitophagosomes in response to nutrient deprivation. Loss of BNIP3-dependent mitophagy caused excess mitochondria to accumulate in the liver, disrupting metabolic zonation within the liver parenchyma, with expansion of zone 1 metabolism at the expense of zone 3 metabolism. These results identify BNIP3 as a regulator of metabolic homeostasis in the liver through its effect on mitophagy and mitochondrial mass distribution.Abbreviations: ASS1, arginosuccinate synthetase; BNIP3, BCL2/adenovirus E1B interacting protein 3; CV, central vein; GCG - glucagon; GLUL, glutamate- ammonia ligase (glutamine synthetase); HCQ, hydroxychloroquine; LIR, LC3-interacting region; MAP1LC3B/LC3B, microtubule-associated protein 1 light chain 3 beta; mtDNA:nucDNA, ratio of mitochondrial DNA to nuclear DNA; PV, periportal vein; TOMM20, translocase of outer mitochondrial membrane protein 20.

BNIP3 is an RB/E2F target gene required for hypoxia-induced autophagy.

Hypoxia and nutrient deprivation are environmental stresses governing the survival and adaptation of tumor cells in vivo. We have identified a novel role for the Rb tumor suppressor in protecting against nonapoptotic cell death in the developing mouse fetal liver, in primary mouse embryonic fibroblasts, and in tumor cell lines. Loss of pRb resulted in derepression of BNip3, a hypoxia-inducible member of the Bcl-2 superfamily of cell death regulators. We identified BNIP3 as a direct target of pRB/E2F-mediated transcriptional repression and showed that pRB attenuates the induction of BNIP3 by hypoxia-inducible factor to prevent autophagic cell death. BNIP3 was essential for hypoxia-induced autophagy, and its ability to promote autophagosome formation was enhanced under conditions of nutrient deprivation. Knockdown of BNIP3 reduced cell death, and remaining deaths were necrotic in nature. These studies identify BNIP3 as a key regulator of hypoxia-induced autophagy and suggest a novel role for the RB tumor suppressor in preventing nonapoptotic cell death by limiting the extent of BNIP3 induction in cells.

Autophagy and cancer cell metabolism.

Autophagy is an ancient catabolic process used by cells to clear excess or dysfunctional organelles and large subcellular structures and thus performs an important housekeeping role for the cell. Autophagy is acutely sensitive to nutrient availability and is upregulated at a transcriptional and posttranslational level in response to nutrient deprivation. This serves to promote turnover of cellular content and recycling of nutrients for continued growth and survival. While important for most normal tissues, tumor cells appear to be particularly dependent on autophagy for survival under ischemic or therapeutic stress, and in response to loss of matrix attachment; autophagy is upregulated markedly in cancers as they progress to malignancy. Ras-driven tumors appear to be particularly dependent on autophagy and thus inhibition of autophagy is being pursued as a productive clinical approach for such cancers. However, this enthusiasm needs to be offset against possible negative effects of autophagy inhibition on normal tissue function and on limiting antitumor immune responses. In addressing all of these topics, we focus in on understanding how autophagy is induced by nutrient stress, its role in recycling metabolites for growing tumors, how selective forms of autophagy, such as mitophagy and ribophagy contribute specifically to tumorigenesis, how autophagy in the tumor microenvironment and throughout the animal affects access of the tumor to nutrients, and finally how different oncogenic pathways may determine which tumors respond to autophagy inhibition and which ones will not.

Oncogenic KRAS Induces NIX-Mediated Mitophagy to Promote Pancreatic Cancer.

Activating KRAS mutations are found in nearly all cases of pancreatic ductal adenocarcinoma (PDAC), yet effective clinical targeting of oncogenic KRAS remains elusive. Understanding of KRAS-dependent PDAC-promoting pathways could lead to the identification of vulnerabilities and the development of new treatments. We show that oncogenic KRAS induces BNIP3L/NIX expression and a selective mitophagy program that restricts glucose flux to the mitochondria and enhances redox capacity. Loss of Nix restores functional mitochondria to cells, increasing demands for NADPH reducing power and decreasing proliferation in glucose-limited conditions. Nix deletion markedly delays progression of pancreatic cancer and improves survival in a murine (KPC) model of PDAC. Although conditional Nix ablation in vivo initially results in the accumulation of mitochondria, mitochondrial content eventually normalizes via increased mitochondrial clearance programs, and pancreatic intraepithelial neoplasia (PanIN) lesions progress to PDAC. We identify the KRAS-NIX mitophagy program as a novel driver of glycolysis, redox robustness, and disease progression in PDAC. SIGNIFICANCE: NIX-mediated mitophagy is a new oncogenic KRAS effector pathway that suppresses functional mitochondrial content to stimulate cell proliferation and augment redox homeostasis. This pathway promotes the progression of PanIN to PDAC and represents a new dependency in pancreatic cancer.This article is highlighted in the In This Issue feature, p. 1143.