Metastatic cells are preferentially vulnerable to lysosomal inhibition.

Molecular alterations that confer phenotypic advantages to tumors can also expose specific therapeutic vulnerabilities. To search for potential treatments that would selectively affect metastatic cells, we examined the sensitivity of lineage-related human bladder cancer cell lines with different lung colonization abilities to chloroquine (CQ) or bafilomycin A1, which are inhibitors of lysosome function and autophagy. Both CQ and bafilomycin A1 were more cytotoxic in vitro to highly metastatic cells compared with their less metastatic counterparts. Genetic inactivation of macroautophagy regulators and lysosomal proteins indicated that this was due to greater reliance on the lysosome but not upon macroautophagy. To identify the mechanism underlying these effects, we generated cells resistant to CQ in vitro. Surprisingly, selection for in vitro CQ resistance was sufficient to alter gene expression patterns such that unsupervised cluster analysis of whole-transcriptome data indicated that selection for CQ resistance alone created tumor cells that were more similar to the poorly metastatic parental cells from which the metastatic cells were derived; importantly, these tumor cells also had diminished metastatic ability in vivo. These effects were mediated in part by differential expression of the transcriptional regulator ID4 (inhibitor of DNA binding 4); depletion of ID4 both promoted in vitro CQ sensitivity and restored lung colonization and metastasis of CQ-resistant cells. These data demonstrate that selection for metastasis ability confers selective vulnerability to lysosomal inhibitors and identify ID4 as a potential biomarker for the use of lysosomal inhibitors to reduce metastasis in patients.

Autophagy and disease.

As outlined in the accompanying Minireviews, autophagy is a complicated and highly regulated process that delivers cellular material to lysosomes for degrading, recycling, and generating molecules that fuel cellular metabolism. Autophagy is important for normal cellular and organismal physiology, and both increased and decreased autophagy has been associated with disease. Importantly, these connections are already being exploited to treat patients with dozens of clinical trials that aim to manipulate autophagy to treat (or prevent) disease. This Minireview discusses some of the important issues and problems to be solved if these efforts are to be successful.

Autophagy in malignant transformation and cancer progression.

Autophagy plays a key role in the maintenance of cellular homeostasis. In healthy cells, such a homeostatic activity constitutes a robust barrier against malignant transformation. Accordingly, many oncoproteins inhibit, and several oncosuppressor proteins promote, autophagy. Moreover, autophagy is required for optimal anticancer immunosurveillance. In neoplastic cells, however, autophagic responses constitute a means to cope with intracellular and environmental stress, thus favoring tumor progression. This implies that at least in some cases, oncogenesis proceeds along with a temporary inhibition of autophagy or a gain of molecular functions that antagonize its oncosuppressive activity. Here, we discuss the differential impact of autophagy on distinct phases of tumorigenesis and the implications of this concept for the use of autophagy modulators in cancer therapy.

Protein Tyrosine Phosphatase-N13 Promotes Myofibroblast Resistance to Apoptosis in Idiopathic Pulmonary Fibrosis.

RATIONALE: Idiopathic pulmonary fibrosis (IPF) is a progressive, fibrotic interstitial lung disease characterized by (myo)fibroblast accumulation and collagen deposition. Resistance to Fas-induced apoptosis is thought to facilitate (myo)fibroblast persistence in fibrotic lung tissues by poorly understood mechanisms. OBJECTIVES: To test the hypothesis that PTPN13 (protein tyrosine phosphatase-N13) is expressed by IPF lung (myo)fibroblasts, promotes their resistance to Fas-induced apoptosis, and contributes to the development of pulmonary fibrosis. METHODS: PTPN13 was localized in lung tissues from patients with IPF and control subjects by immunohistochemical staining. Inhibition of PTPN13 function in primary IPF and normal lung (myo)fibroblasts was accomplished by: 1) downregulation with TNF-α (tumor necrosis factor-α)/IFN-γ, 2) siRNA knockdown, or 3) a cell-permeable Fas/PTPN13 interaction inhibitory peptide. The role of PTPN13 in the development of pulmonary fibrosis was assessed in mice with genetic deficiency of PTP-BL, the murine ortholog of PTPN13. MEASUREMENTS AND MAIN RESULTS: PTPN13 was constitutively expressed by (myo)fibroblasts in the fibroblastic foci of patients with IPF. Human lung (myo)fibroblasts, which are resistant to Fas-induced apoptosis, basally expressed PTPN13 in vitro. TNF-α/IFN-γ or siRNA-mediated PTPN13 downregulation and peptide-mediated inhibition of the Fas/PTPN13 interaction in human lung (myo)fibroblasts promoted Fas-induced apoptosis. Bleomycin-challenged PTP-BL-/- mice, while developing inflammatory lung injury, exhibited reduced pulmonary fibrosis compared with wild-type mice. CONCLUSIONS: These findings suggest that PTPN13 mediates the resistance of human lung (myo)fibroblasts to Fas-induced apoptosis and promotes pulmonary fibrosis in mice. Our results suggest that strategies aimed at interfering with PTPN13 expression or function may represent a novel strategy to reduce fibrosis in IPF.

Regulation of Apoptosis by Autophagy to Enhance Cancer Therapy.

In cancer therapy, a principle goal is to kill cancer cells while minimizing death of normal cells. Traditional cytotoxic therapies and the newer agents that target specific signaling proteins that are critical for cancer cell growth do this by activating a specific type of programmed cell death - apoptosis. However, it has been well established that cancer cells have varying levels of responses to apoptotic stimuli, with some being close to an "apoptotic threshold" and others being further away and that this ultimately determines whether cancer therapy is successful or not. In this review, we will highlight how the underlying mechanisms that control apoptosis thresholds relate to another important homeostatic process in cell survival and cell death, autophagy, and discuss recent evidence suggesting how inhibition of autophagy can enhance the action of anti-cancer drugs by modulating the apoptotic response.

Non-cell-autonomous Effects of Autophagy Inhibition in Tumor Cells Promote Growth of Drug-resistant Cells.

Autophagy, the mechanism by which cells deliver material to the lysosome, has been associated with resistance to anticancer drugs, leading autophagy inhibition to be widely studied as a potential chemosensitization strategy for cancer cells. This strategy is based on the idea that inhibition of autophagy will increase drug sensitivity and kill more cancer cells. Here we report an unintended negative effect of this strategy. When modeling the effect of drug resistance in a heterogeneous cancer cell population, we found that autophagy inhibition in drug-sensitive tumor cells causes increased growth of drug-resistant cells in the population through a mechanism involving caspase activation and prostaglandin E2 signaling. These results emphasize the importance of understanding how autophagy manipulation in a tumor cell can have both cell-autonomous and nonautonomous effects and suggest that attempts to chemosensitize by inhibiting autophagy could be enhanced by adopting methods aimed at reducing tumor repopulation.

Autophagy and its effects: making sense of double-edged swords.

Autophagy is the mechanism by which cellular material is delivered to lysosomes and degraded. This process has become a major focus of biological and biomedical research with thousands of papers published each year and rapidly growing appreciation that autophagy affects many normal and pathological processes. However, as we learn more about this evolutionarily ancient process, we are discovering that autophagy's effects may work for both the good and the bad of an organism. Here, I discuss some of these context-dependent findings and how, as we make sense of them, we can try to apply our knowledge for practical purposes.

RIP1 negatively regulates basal autophagic flux through TFEB to control sensitivity to apoptosis.

In a synthetic lethality/viability screen, we identified the serine-threonine kinase RIP1 (RIPK1) as a gene whose knockdown is highly selected against during growth in normal media, in which autophagy is not critical, but selected for in conditions that increase reliance on basal autophagy. RIP1 represses basal autophagy in part due to its ability to regulate the TFEB transcription factor, which controls the expression of autophagy-related and lysosomal genes. RIP1 activates ERK, which negatively regulates TFEB though phosphorylation of serine 142. Thus, in addition to other pro-death functions, RIP1 regulates cellular sensitivity to pro-death stimuli by modulating basal autophagy.

Regulation of cell proliferation and migration by p62 through stabilization of Twist1.

The selective autophagy substrate p62 serves as a molecular link between autophagy and cancer. Suppression of autophagy causes p62 accumulation and thereby contributes to tumorigenesis. Here we demonstrate that autophagy deficiency promotes cell proliferation and migration through p62-dependent stabilization of the oncogenic transcription factor Twist1. p62 binds to Twist1 and inhibits degradation of Twist1. In mice, p62 up-regulation promotes tumor cell growth and metastasis in a Twist1-dependent manner. Our findings demonstrate that Twist1 is a key downstream effector of p62 in regulation of cell proliferation and migration and suggest that targeting p62-mediated Twist1 stabilization is a promising therapeutic strategy for prevention and treatment of cancer.

Autophagy Inhibition Delays Early but Not Late-Stage Metastatic Disease.

The autophagy pathway has been recognized as a mechanism of survival and therapy resistance in cancer, yet the extent of autophagy's function in metastatic progression is still unclear. Therefore, we used murine models of metastatic cancer to investigate the effect of autophagy modulation on metastasis development. Pharmacologic and genetic autophagy inhibition were able to impede cell proliferation in culture, but did not impact the development of experimentally induced 4T1 and B16-F10 metastases. Similarly, autophagy inhibition by adjuvant chloroquine (CQ) treatment did not delay metastasis in an orthotopic 4T1, tumor-resection model. However, neoadjuvant CQ treatment or genetic autophagy inhibition resulted in delayed metastasis development, whereas stimulation of autophagy by trehalose hastened development. Cisplatin was also administered either as a single agent or in combination with CQ. The combination of cisplatin and CQ was antagonistic. The effects of autophagy modulation on metastasis did not appear to be due to alterations in the intrinsic metastatic capability of the cells, as modulating autophagy had no impact on migration, invasion, or anchorage-independent growth in vitro. To explore the possibility of autophagy's influence on the metastatic microenvironment, bone marrow-derived cells (BMDCs), which mediate the establishment of the premetastatic niche, were measured in the lung and in circulation. Trehalose-treated mice had significantly more BMDCs than either vehicle- or CQ-treated mice. Autophagy inhibition may be most useful as a treatment to impede early metastatic development. However, modulating autophagy may also alter the efficacy of platinum-based therapies, requiring caution when considering combination therapies.

Measuring Autophagy in the Context of Cancer.

Autophagy plays multiple roles in the formation and progression of cancer, including both suppressive and promotive roles. It not only impacts cancer cell growth and viability directly but also has a significant role through its effects on the tumor microenvironment. Measurement of autophagy can be confusing and sometimes misleading due to the inherent difficulty of measuring both the formation and turnover of molecules involved in the autophagic process. The LC3 proteins serve as autophagosomal markers and are the basis for most of the assays used for measuring autophagy. Since each of the current assays for autophagy has significant limitations, the use of multiple assays for the analysis of autophagy in most contexts is highly advised. Here we outline three assays that are commonly used to evaluate autophagic flux in cells. These assays include the determination of LC3II formation and LC3II and p62 turnover by use of Western Blotting, quantification of LC3 puncta, and the measurement of autophagic flux using tandem labeled mCherry-GFP-LC3.

Autophagy and cancer therapy.

Autophagy is the process by which cellular material is delivered to lysosomes for degradation and recycling. There are three different types of autophagy, but macroautophagy, which involves the formation of double membrane vesicles that engulf proteins and organelles that fuse with lysosomes, is by far the most studied and is thought to have important context-dependent roles in cancer development, progression, and treatment. The roles of autophagy in cancer treatment are complicated by two important discoveries over the past few years. First, most (perhaps all) anticancer drugs, as well as ionizing radiation, affect autophagy. In most, but not all cases, these treatments increase autophagy in tumor cells. Second, autophagy affects the ability of tumor cells to die after drug treatment, but the effect of autophagy may be to promote or inhibit cell death, depending on context. Here we discuss recent research related to autophagy and cancer therapy with a focus on how these processes may be manipulated to improve cancer therapy.

Autophagy inhibition for chemosensitization and radiosensitization in cancer: do the preclinical data support this therapeutic strategy?

Recognition of the cytoprotective functions of autophagy that occur in tumor cells exposed to various forms of chemotherapy or radiation has generated intense interest in the possibility that pharmacological interference with autophagy could provide a clinical strategy for overcoming therapeutic resistance. Multiple clinical trials are currently in progress to evaluate the antimalarial agent chloroquine (generally in its clinical formulation as hydroxychloroquine) and its impact on various forms of cancer therapy. In this commentary/review, we focus on the relatively limited number of studies in the literature where chloroquine has been tested in combination with chemotherapy or radiation in experimental tumor-bearing animal models. We also present recent data from our own laboratories, in cell culture experiments as well as in vivo studies, which demonstrate that neither chloroquine nor silencing of an autophagy regulatory gene was effective in conferring radiation sensitivity in an experimental model of breast cancer. The capacity for sensitization by chloroquine appears to be quite wide-ranging, with dramatic effects for some drugs/tumor models and modest or minimal effects in others. One possible caveat is that, with only a few exceptions, experiments have generally been performed in xenograft models, thereby eliminating the involvement of the immune system, which might ultimately be proven to play a central role in determining the effectiveness of autophagy inhibition in chemosensitization or radiosensitization. Nevertheless, a careful review of the current literature suggests that caution is likely to be warranted in translating preclinical findings relating to autophagy inhibition as an adjunctive therapeutic strategy.

Autophagy regulates tumor growth and metastasis.

The role of autophagy in tumorigenesis and tumor metastasis remains poorly understood. Here we show that inhibition of autophagy stabilizes the transcription factor Twist1 through Sequestosome-1 (SQSTM1, also known as p62) and thus increases cell proliferation, migration, and epithelial-mesenchymal transition (EMT) in tumor development and metastasis. Inhibition of autophagy or p62 overexpression blocks Twist1 protein degradation in the proteasomes, while p62 inhibition enhances it. SQSTM1/p62 interacts with Twist1 via the UBA domain of p62, in a Twist1-ubiquitination-dependent manner. Lysine 175 in Twist1 is critical for Twist1 ubiquitination, degradation, and SQSTM1/p62 interaction. For squamous skin cancer and melanoma cells that express Twist1, SQSTM1/p62 increases tumor growth and metastasis in mice. Together, our results identified Twist1 as a key downstream protein for autophagy and suggest a critical role of the autophagy/p62/Twist1 axis in cancer development and metastasis.

Translational evidence that impaired autophagy contributes to arterial ageing.

Ageing causes arterial endothelial dysfunction that increases the risk of cardiovascular diseases (CVD), but the underlying mechanisms are incompletely understood. The aim of the present study was to determine the role of autophagy, the cellular process of recycling damaged biomolecules, in endothelial dysfunction with ageing. In older humans, expression of autophagy markers in arterial endothelial cells was impaired by ∼50% (P <0.05) and was associated with an ∼30% (P <0.05) reduction in arterial endothelium-dependent dilatation (EDD). Similarly, in C57BL/6 control mice ageing was associated with an ∼40% decrease (P <0.05) in arterial markers of autophagy and an ∼25% reduction (P <0.05) in EDD. In both humans and mice, impaired EDD was mediated by reduced nitric oxide (NO) bioavailability and was associated with increased oxidative stress and inflammation (P <0.05). In old mice, treatment with the autophagy-enhancing agent trehalose restored expression of autophagy markers, rescued NO-mediated EDD by reducing oxidative stress, and normalized inflammatory cytokine expression. In cultured endothelial cells, inhibition of autophagy increased oxidative stress and reduced NO production, whereas trehalose enhanced NO production via an autophagy-dependent mechanism. These results provide the first evidence that autophagy is impaired with ageing in vascular tissues. Our findings also suggest that autophagy preserves arterial endothelial function by reducing oxidative stress and inflammation and increasing NO bioavailability. Autophagy-enhancing strategies may therefore have therapeutic efficacy for ameliorating age-associated arterial dysfunction and preventing CVD.

Specificity and prognostic validation of a polyclonal antibody to detect Six1 homeoprotein in ovarian cancer.

OBJECTIVE: The presence of Six1 mRNA gene portends a poor prognosis in ovarian cancer. We describe validation of a Six1 specific antibody and evaluate its association with tumorigenicity and prognosis in ovarian cancer. METHODS: A Six1 antibody (Six1cTerm) was raised to residues downstream of the Six1 homeodomain, representing its unique C-terminus as compared to other Six family members. Cells were transfected with Six1-Six6 and Western blot was performed to demonstrate Six1 specificity. Ovarian cancer cell lines were analyzed for Six1 mRNA and Six1cTerm and tumorigenicity was evaluated. Ovarian cancer tissue microarrays (OTMA) were analyzed for Six1cTerm by immunohistochemistry and scored by two blinded observers. The metastatic tumors of 15 stage IIIC high grade serous ovarian cancers were analyzed with Six1 mRNA and Six1cTerm and expression was compared to clinical factors and survival. RESULTS: The Six1cTerm antibody is specific for Six1. Cell line tumorigenicity in SCID mice correlates with Six1 levels both by mRNA(p=0.001, Mann-Whitney U test) and by protein (presence vs. absence, p=0.05 Fischer's Exact test). Six1 protein was present in up to 54% of OTMA specimens. Six1 protein expression in omental/peritoneal metastases correlated with worsened survival in a sample (n=15) of high grade serous stage IIIC ovarian cancers (p=0.001). CONCLUSIONS: The Six1cTerm antibody is specific and able to detect Six1 in cell lines and tumor tissue. Six1 protein detection is common in ovarian cancer and is associated with tumorigenicity and poor prognosis in this group of patient samples. Six1cTerm antibody should be further validated as prognostic tool.

Modulation of pediatric brain tumor autophagy and chemosensitivity.

Brain and spinal tumors are the second most common malignancies in childhood after leukemia, and they remain the leading cause of death from childhood cancer. Autophagy is a catabolic cellular process that is thought to regulate chemosensitivity, however its role in pediatric tumors is unknown. Here we present studies in pediatric medulloblastoma cell lines (DAOY, ONS76) and atypical teratoid/rhabdoid tumor cell lines (BT-16, BT-12) to test this role. Autophagy was inhibited using siRNA against autophagy-related genes ATG12 and ATG7 or pharmacologically induced or inhibited using rapamycin and chloroquine to test the effect of autophagy on chemosensitivity. Autophagic flux was measured using Western blot analysis of LC3-II and p62 and cell viability was determined using MTS assays and clonogenic growth. We found that when pediatric brain tumor cells under starvation stress, exposed to known autophagy inducers such as rapamycin, or treated with current chemotherapeutics (lomustine, cisplatin), all stimulate autophagy. Silencing ATG12 and ATG7 or exposure to a known autophagy inhibitor, chloroquine, could inhibit this autophagy increase; however, the effect of autophagy on tumor cell killing was small. These results may have clinical relevance in the future planning of therapeutic regimens for pediatric brain tumors.

Autophagy and organelle homeostasis in cancer.

Beginning with the earliest studies of autophagy in cancer, there have been indications that autophagy can both promote and inhibit cancer growth and progression; autophagy regulation of organelle homeostasis is similarly complicated. In this review we discuss pro- and antitumor effects of organelle-targeted autophagy and how this contributes to several hallmarks of cancer, such as evading cell death, genomic instability, and altered metabolism. Typically, the removal of damaged or dysfunctional organelles prevents tumor development but can also aid in proliferation or drug resistance in established tumors. By better understanding how organelle-specific autophagy takes place and can be manipulated, it may be possible to go beyond the brute-force approach of trying to manipulate all autophagy in order to improve therapeutic targeting of this process in cancer.

Mitochondrial-derived vesicles compensate for loss of LC3-mediated mitophagy.

Mitochondria are critical metabolic and signaling hubs, and dysregulated mitochondrial homeostasis is implicated in many diseases. Degradation of damaged mitochondria by selective GABARAP/LC3-dependent macro-autophagy (mitophagy) is critical for maintaining mitochondrial homeostasis. To identify alternate forms of mitochondrial quality control that functionally compensate if mitophagy is inactive, we selected for autophagy-dependent cancer cells that survived loss of LC3-dependent autophagosome formation caused by inactivation of ATG7 or RB1CC1/FIP200. We discovered rare surviving autophagy-deficient clones that adapted to maintain mitochondrial homeostasis after gene inactivation and identified two enhanced mechanisms affecting mitochondria including mitochondrial dynamics and mitochondrial-derived vesicles (MDVs). To further understand these mechanisms, we quantified MDVs via flow cytometry and confirmed an SNX9-mediated mechanism necessary for flux of MDVs to lysosomes. We show that the autophagy-dependent cells acquire unique dependencies on these processes, indicating that these alternate forms of mitochondrial homeostasis compensate for loss of autophagy to maintain mitochondrial health.

Surgical procedures suppress autophagic flux in the kidney.

Many surgical models are used to study kidney and other diseases in mice, yet the effects of the surgical procedure itself on the kidney and other tissues have not been elucidated. In the present study, we found that both sham surgery and unilateral nephrectomy (UNX), which is used as a model of renal compensatory hypertrophy, in mice resulted in increased mammalian target of rapamycin complex 1/2 (mTORC1/2) in the remaining kidney. mTORC1 is known to regulate lysosomal biogenesis and autophagy. Genes associated with lysosomal biogenesis and function were decreased in sham surgery and UNX kidneys. In both sham surgery and UNX, there was suppressed autophagic flux in the kidney as indicated by the lack of an increase in LC3-II or autophagosomes seen on immunoblot, IF and EM after bafilomycin A1 administration and a concomitant increase in p62, a marker of autophagic cargo. There was a massive increase in pro-inflammatory cytokines, which are known to activate ERK1/2, in the serum after sham surgery and UNX. There was a large increase in ERK1/2 in sham surgery and UNX kidneys, which was blocked by the MEK1/2 inhibitor, trametinib. Trametinib also resulted in a significant decrease in p62. In summary, there was an intense systemic inflammatory response, an ERK-mediated increase in p62 and suppressed autophagic flux in the kidney after sham surgery and UNX. It is important that researchers are aware that changes in systemic pro-inflammatory cytokines, ERK1/2 and autophagy can be caused by sham surgery as well as the kidney injury/disease itself.