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.

Characterization of HNRNPA1 mutations defines diversity in pathogenic mechanisms and clinical presentation.

Mutations in HNRNPA1 encoding heterogeneous nuclear ribonucleoprotein (hnRNP) A1 are a rare cause of amyotrophic lateral sclerosis (ALS) and multisystem proteinopathy (MSP). hnRNPA1 is part of the group of RNA-binding proteins (RBPs) that assemble with RNA to form RNPs. hnRNPs are concentrated in the nucleus and function in pre-mRNA splicing, mRNA stability, and the regulation of transcription and translation. During stress, hnRNPs, mRNA, and other RBPs condense in the cytoplasm to form stress granules (SGs). SGs are implicated in the pathogenesis of (neuro-)degenerative diseases, including ALS and inclusion body myopathy (IBM). Mutations in RBPs that affect SG biology, including FUS, TDP-43, hnRNPA1, hnRNPA2B1, and TIA1, underlie ALS, IBM, and other neurodegenerative diseases. Here, we characterize 4 potentially novel HNRNPA1 mutations (yielding 3 protein variants: *321Eext*6, *321Qext*6, and G304Nfs*3) and 2 known HNRNPA1 mutations (P288A and D262V), previously connected to ALS and MSP, in a broad spectrum of patients with hereditary motor neuropathy, ALS, and myopathy. We establish that the mutations can have different effects on hnRNPA1 fibrillization, liquid-liquid phase separation, and SG dynamics. P288A accelerated fibrillization and decelerated SG disassembly, whereas *321Eext*6 had no effect on fibrillization but decelerated SG disassembly. By contrast, G304Nfs*3 decelerated fibrillization and impaired liquid phase separation. Our findings suggest different underlying pathomechanisms for HNRNPA1 mutations with a possible link to clinical phenotypes.

Higher-order organization of biomolecular condensates.

A guiding principle of biology is that biochemical reactions must be organized in space and time. One way this spatio-temporal organization is achieved is through liquid-liquid phase separation (LLPS), which generates biomolecular condensates. These condensates are dynamic and reactive, and often contain a complex mixture of proteins and nucleic acids. In this review, we discuss how underlying physical and chemical processes generate internal condensate architectures. We then outline the diverse condensate architectures that are observed in biological systems. Finally, we discuss how specific condensate organization is critical for specific biological functions.

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.

Active and dynamic mitochondrial S-depalmitoylation revealed by targeted fluorescent probes.

The reversible modification of cysteine residues by thioester formation with palmitate (S-palmitoylation) is an abundant lipid post-translational modification (PTM) in mammalian systems. S-palmitoylation has been observed on mitochondrial proteins, providing an intriguing potential connection between metabolic lipids and mitochondrial regulation. However, it is unknown whether and/or how mitochondrial S-palmitoylation is regulated. Here we report the development of mitoDPPs, targeted fluorescent probes that measure the activity levels of "erasers" of S-palmitoylation, acyl-protein thioesterases (APTs), within mitochondria of live cells. Using mitoDPPs, we discover active S-depalmitoylation in mitochondria, in part mediated by APT1, an S-depalmitoylase previously thought to reside in the cytosol and on the Golgi apparatus. We also find that perturbation of long-chain acyl-CoA cytoplasm and mitochondrial regulatory proteins, respectively, results in selective responses from cytosolic and mitochondrial S-depalmitoylases. Altogether, this work reveals that mitochondrial S-palmitoylation is actively regulated by "eraser" enzymes that respond to alterations in mitochondrial lipid homeostasis.

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.

Autophagy Promotes Focal Adhesion Disassembly and Cell Motility of Metastatic Tumor Cells through the Direct Interaction of Paxillin with LC3.

Autophagy is a conserved catabolic process that plays a housekeeping role in eliminating protein aggregates and organelles and is activated during nutrient deprivation to generate metabolites and energy. Autophagy plays a significant role in tumorigenesis, although opposing context-dependent functions of autophagy in cancer have complicated efforts to target autophagy for therapeutic purposes. We demonstrate that autophagy inhibition reduces tumor cell migration and invasion in vitro and attenuates metastasis in vivo. Numerous abnormally large focal adhesions (FAs) accumulate in autophagy-deficient tumor cells, reflecting a role for autophagy in FA disassembly through targeted degradation of paxillin. We demonstrate that paxillin interacts with processed LC3 through a conserved LIR motif in the amino-terminal end of paxillin and that this interaction is regulated by oncogenic SRC activity. Together, these data establish a function for autophagy in FA turnover, tumor cell motility, and metastasis.

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.

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.

Molecular rationale for the use of PI3K/AKT/mTOR pathway inhibitors in combination with crizotinib in ALK-mutated neuroblastoma.

Mutations in the ALK tyrosine kinase receptor gene represent important therapeutic targets in neuroblastoma, yet their clinical translation has been challenging. The ALK(F1174L) mutation is sensitive to the ALK inhibitor crizotinib only at high doses and mediates acquired resistance to crizotinib in ALK-translocated cancers. We have shown that the combination of crizotinib and an inhibitor of downstream signaling induces a favorable response in transgenic mice bearing ALK(F1174L)/MYCN-positive neuroblastoma. Here, we investigated the molecular basis of this effect and assessed whether a similar strategy would be effective in ALK-mutated tumors lacking MYCN overexpression. We show that in ALK-mutated, MYCN-amplified neuroblastoma cells, crizotinib alone does not affect mTORC1 activity as indicated by persistent RPS6 phosphorylation. Combined treatment with crizotinib and an ATP-competitive mTOR inhibitor abrogated RPS6 phosphorylation, leading to reduced tumor growth and prolonged survival in ALK(F1174L)/MYCN-positive models compared to single agent treatment. By contrast, this combination, while inducing mTORC1 downregulation, caused reciprocal upregulation of PI3K activity in ALK-mutated cells expressing wild-type MYCN. Here, an inhibitor with potency against both mTOR and PI3K was more effective in promoting cytotoxicity when combined with crizotinib. Our findings should enable a more precise selection of molecularly targeted agents for patients with ALK-mutated tumors.

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.