Marina N. Sharifi

Autophagy in cancer metastasis

Autophagy is a highly conserved self-degradative process that has a key role in cellular stress responses and survival. Recent work has begun to explore the function of autophagy in cancer metastasis, which is of particular interest given the dearth of effective therapeutic options for metastatic disease. Autophagy is induced upon progression of various human cancers to metastasis and together with data from genetically engineered mice and experimental metastasis models, a role for autophagy at nearly every phase of the metastatic cascade has been identified. Specifically, autophagy has been shown to be involved in modulating tumor cell motility and invasion, cancer stem cell viability and differentiation, resistance to anoikis, epithelial-to-mesenchymal transition, tumor cell dormancy and escape from immune surveillance, with emerging functions in establishing the pre-metastatic niche and other aspects of metastasis. In this review, we provide a general overview of how autophagy modulates cancer metastasis and discuss the significance of new findings for disease management.

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 angiogenesis—two 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 dysfunction—caused by defects in mitophagy—can promote the Warburg effect and tumor progression, and suggest better approaches to stratifying TNBC for treatment.

p62/SQSTM1 accumulation in squamous cell carcinoma of head and neck predicts sensitivity to phosphatidylinositol 3-kinase pathway inhibitors.

The phosphoinositol-3 kinase (PI3K) pathway is highly dysregulated in squamous cell carcinoma of the head and neck (SCCHN). While inhibitors of the PI3K/AKT pathway are being developed in cancer, their efficacy does not appear to be related to the presence of mutations or amplification in pathway genes. The PI3K pathway is a major regulator of macro-autophagy, an evolutionarily conserved catabolic process that degrades cellular materials to promote cellular homeostasis and survival under stress. Employing a panel of SCCHN cell lines, we observed a significant correlation between the activity of PI3K/AKT inhibitors and their ability to induce autophagy. More specifically, resistance to these inhibitors was associated with accumulation of p62/SQSTM1, a pleotropic protein that is consumed during autophagy, while loss of autophagy was, for the first time, found to be due to silencing of an essential autophagy gene, ATG7. Moreover, modulating ATG7 and p62/SQSTM1 could regulate sensitivity to PI3K/AKT inhibitors, underscoring a mechanistic link between autophagy and drug sensitivity. Analysis of human tissues revealed progressive accumulation of p62/SQSTM1 in a significant proportion of cancer samples compared to normal tissue, suggesting that defective autophagy has relevance to SCCHN. These findings are further validated by analysis of TCGA data confirming homozygous deletion and mRNA down-regulation of ATG7 in 10.0% of SCCHN samples. Taken together, these data indicate that p62/SQSTM1 levels modulate sensitivity to PI3K/AKT inhibitors; cancers vary in their capacity to undergo autophagy through epigenetic modification and, when deficient, accumulate p62/SQSTM1; and expression of autophagy-related proteins may serve as markers for resistance to PI3K/AKT inhibitors in SCCHN.

Measuring Autophagy in Stressed Cells

Macro-autophagy is a major catabolic process in the cell used to degrade protein aggregates, dysfunctional organelles and intracellular pathogens that would otherwise become toxic. Autophagy also generates energy and metabolites for the cell through recycling of degraded autophagosomal cargo, which can be particularly important for cell viability under stress. The significance of changes in the rates of autophagic flux for cellular function and disease is being increasingly appreciated, and interest in measuring autophagy in different experimental systems is growing accordingly. Here, we describe key methodologies used in the field to measure autophagic flux, including monitoring LC3 processing by western blot, fluorescent cell staining, and flow cytometry, in addition to changes in the levels or posttranslational modifications of other autophagy markers, such as p62/Sqstm1 and the Atg5-Atg12 conjugate. We also describe what cellular stresses may be used to induce autophagy and how to control for changes in the rates of autophagic flux as opposed to inhibition of flux. Finally, we detail available techniques to monitor autophagy in vivo.

Novel insights into how autophagy regulates tumor cell motility

ABSTRACT Metastasis requires tumor cells to overcome a series of challenges to successfully travel to and colonize new microenvironments. As an adaptive (or maladaptive) response to stress, macroautophagy/autophagy has garnered increasing interest with respect to cancer metastasis, supported by clinical observations of increased autophagic flux in distant metastases relative to primary tumors. Recently, we identified a new role for autophagy in tumor cell motility through the turnover of focal adhesions, large multi-protein structures that link extracellular matrix-bound integrins to the cytoskeleton. The disassembly of focal adhesions at the cell rear is critical to forward movement and successful migration/invasion. We demonstrated that the focal adhesion protein PXN (paxillin), which serves as a crucial scaffolding and signal integrator, binds directly to LC3B through a conserved LC3-interacting region (LIR) motif to stimulate focal adhesion disassembly and metastasis and that this interaction is further promoted by oncogenic SRC.

Functions of autophagy in the tumor microenvironment and cancer metastasis

Macro‐autophagy is an ancient and highly conserved self‐degradative process that plays a homeostatic role in normal cells by eliminating organelles, pathogens, and protein aggregates. Autophagy, as it is routinely referred to, also allows cells to maintain metabolic sufficiency and survive under conditions of nutrient stress by recycling the by‐products of autophagic degradation, such as fatty acids, amino acids, and nucleotides. Tumor cells are more reliant than normal cells on autophagy for survival in part due to their rapid growth rate, altered metabolism, and nutrient‐deprived growth environment. How this dependence of tumor cells on autophagy affects their progression to malignancy and metastatic disease is an area of increasing research focus. Here, we review recent work identifying critical functions for autophagy in tumor cell migration and invasion, tumor stem cell maintenance and therapy resistance, and cross‐talk between tumor cells and their microenvironment.

Autophagic degradation of focal adhesions underlies metastatic cancer dissemination

ABSTRACT Autophagy inhibition is being evaluated as a novel therapeutic strategy in multiple tumor types, but little is known about its implications for metastatic dissemination. We recently reported that autophagic degradation of paxillin through direct interaction with the autophagy protein LC3B is required for focal adhesion disassembly, Src-stimulated tumor cell motility, and metastasis.

Abstract 316: Modifying autophagy through combination treatments as a potential therapeutic strategy in head and neck squamous cell carcinoma (HNSCC)

Introduction: Autophagy, a catabolic process, can play a crucial role in cancer cell survival. Modifying this process has shown to be an effective mechanism of inducing cell death. In order to explore this concept, we induced autophagy with one agent (Akt-inhibitor MK-2206), and decreased autophagosomal trafficking with other agents (Paclitaxel and Nocadazole) by disrupting the microtubule networks. We hypothesized that by modifying autophagy through this combination, it would lead to an accumulation of autophagosomes and eventual cell death in a synergistic manner. Methods: SCC 61 and Cal 27 HNSCC cell lines were used to explore this hypothesis. SCC 61 cells were transduced with an m-Cherry GFP LC3B vector in order to detect autophagosomes under both fixed and live cell confocal microscopy. Cell viability and apoptosis assays were performed. A siRNA ATG 7 KO SCC 61 cell line was used to detect if in fact autophagy was important for this therapeutic approach. Results: There was an accumulation of autophagosomes with the combination of an Akt inhibitor and microtubule disrupters. A decrease in fusion of autophagosome to lysosome was seen when cells were treated with an agent, which disrupted the microtubule networks (Paclitaxel) but even more dramatically when treated with the combination (MK-2206 and Paclitaxel). Moreover, accumulation of autophagosomes occurred prior to induction of apoptosis with combination treatment. These effects were decreased with siRNA ATG 7 KO in SCC 61 cells, which were more resistant to combination treatment compared to control. Discussion: Manipulating autophagy with Akt inhibition (induction) and Paclitaxel (decreasing autophagic trafficking) leads to an accumulation of autophagosomes and eventual cell death in a synergistic manner. This therapeutic approach of altering the normal autophagic process warrants further investigation. Citation Format: Omar G. Ahmed, Wen-Liang Kuo, Tai-Fen Wei, Jeannette S. Messer, Marina Sharifi, Madhavi Nagilla, Kay Macleod, Ezra E.W. Cohen. Modifying autophagy through combination treatments as a potential therapeutic strategy in head and neck squamous cell carcinoma (HNSCC). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 316. doi:10.1158/1538-7445.AM2014-316

Abstract 321: Autophagy is required for focal adhesion turnover, tumor cell motility and metastasis

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Macro-autophagy is a catabolic process important for degradation of damaged organelles, protein aggregates and intracellular recycling of proteins and lipids. Autophagy is thought to suppress tumor initiation by promoting genome stability and limiting necrosis and inflammation, but conversely is utilized by tumor cells to survive nutrient stress, hypoxia, and cytotoxic therapies, promoting growth of established tumors. However, whether autophagy impacts the metastatic process is not well understood. To examine the role of autophagy in breast cancer metastasis, essential autophagy genes atg5 and atg7 were stably knocked down the aggressive and metastatic 4T1 mouse mammary tumor cell line using shRNA. Inhibition of autophagy did not reduce cell proliferation or survival under stress in vitro or primary tumor growth in vivo, but markedly reduced metastasis to the lungs and liver. Surprisingly, this was not due reduced survival in the circulation or at the secondary site, as atg5 and atg7-deficient cells were able to colonize the lungs to the same extent as control cells when injected into the circulation in a tail vein experimental metastasis assay, suggesting that inhibition of autophagy impairs escape from the primary tumor in this model. Indeed, inhibition of autophagy prevented cell spreading, migration and invasion through collagen in transwell assays of 4T1 cells, MDA-MB-231 human breast cancer cells, and B16.F10 melanoma cells. This was associated with failure of RhoA-mediated protrusion, impaired focal adhesion disassembly, and accumulation of the key focal adhesion component paxillin. We show that paxillin is degraded by autophagy, and siRNA-mediated knockdown of paxillin in autophagy-deficient cells rescued their spreading and motility, suggesting that impaired focal adhesion disassembly and migration in autophagy-deficient cells was the result of failure of autophagic degradation of paxillin. Intriguingly, active RhoA is localized at autophagosomes, and inhibition of RhoA activity inhibits not only cellular protrusion but also autophagic flux in metastatic cells, suggesting that RhoA may be involved in upstream regulation of autophagic degradation of paxillin to promote focal adhesion disassembly. In sum, we have identified a novel and unexpected function for autophagy in focal adhesion disassembly and tumor cell motility that is required for metastasis in vivo. This suggests that while autophagy may also promote the survival of disseminating tumor cells during metastasis, autophagy is in fact required for escape from the primary tumor. Citation Format: Marina N. Sharifi, Christopher Collier, Lauren Drake, Hong Chen, Stephanie Mui, Kay F. Macleod. Autophagy is required for focal adhesion turnover, tumor cell motility and metastasis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 321. doi:10.1158/1538-7445.AM2014-321

Abstract 4316: BNIP3 suppresses mammary tumorigenesis and metastasis through negative regulation of Warburg effect and HIF-1α

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA BNIP3 is a hypoxia-inducible protein involved in mitochondrial dynamics and mitophagy that is up-regulated at pre-malignant stages of various human cancers, including breast cancer but down-regulated at later stages of tumorigenesis. To examine the role of BNip3 in tumor progression, we crossed BNip3 null mice to the MMTV-PyMT mouse model of breast cancer. Loss of BNip3 promoted primary tumor growth in vivo and tumor cell proliferation in vitro and was associated with increased aerobic glycolysis and reduced mitochondrial respiration. BNip3 null tumor cells also showed increased invasive properties that were cell intrinsic and transplantable to wild-type host mice. Consistently, there was reduced latency to lung metastasis in the MMTV-PyMT;BNip3-/- mice and increased numbers of metastases compared to control mice. BNip3 null tumors exhibited increased mitochondrial mass, but a greater fraction of mitochondria were dysfunctional with reduced membrane potential and decreased metabolite uptake compared to BNip3 wild-type tumor cells. Consistently, increased proliferation of BNip3 null tumor cells was associated with increased carbon flux from glucose to lipid and nucleic acids. Interestingly, although mitochondria were less efficient at uptake of pyruvate and glutamine, the rate of production of Krebs cycle intermediates was increased. This indicates that defective mitochondria due to inefficient mitophagy can induce the Warburg effect, as originally proposed by Warburg himself. Loss of BNip3 also resulted in more hypoxic tumors that expressed increased levels of Hif-1α and its target genes and showed increased angiogenesis, although blood vessels were abnormal possibly explaining increased tumor hypoxia, thus suggesting a negative feedback loop between Hif-1α and its target gene, BNip3. Inhibition of glycolysis or inhibition of HIF activity reduced proliferation in BNip3 null tumor cells to the levels observed in control cells. Significantly, BNip3 null tumor cells underwent autophagic flux as efficiently as wild-type cells demonstrating for the first time that inhibition of mitophagy has a distinct tumorigenic consequence to general inactivation of autophagy, as reported elsewhere. These results demonstrate that BNip3 has tumor and metastasis suppressor properties required to maintain mitochondrial integrity, promote oxidative metabolism and mitigate against the metastasis promoting activities of hypoxia and glycolytic metabolism. Citation Format: Aparajita Hoskote Chourasia, Kristin Tracy, Michelle Boland, Marina Sharifi, Kay F. Macleod. BNIP3 suppresses mammary tumorigenesis and metastasis through negative regulation of Warburg effect and HIF-1α. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4316. doi:10.1158/1538-7445.AM2014-4316