Drug-like sphingolipid SH-BC-893 opposes ceramide-induced mitochondrial fission and corrects diet-induced obesity.

Ceramide-induced mitochondrial fission drives high-fat diet (HFD)-induced obesity. However, molecules targeting mitochondrial dynamics have shown limited benefits in murine obesity models. Here, we reveal that these compounds are either unable to block ceramide-induced mitochondrial fission or require extended incubation periods to be effective. In contrast, targeting endolysosomal trafficking events important for mitochondrial fission rapidly and robustly prevented ceramide-induced disruptions in mitochondrial form and function. By simultaneously inhibiting ARF6- and PIKfyve-dependent trafficking events, the synthetic sphingolipid SH-BC-893 blocked palmitate- and ceramide-induced mitochondrial fission, preserved mitochondrial function, and prevented ER stress in vitro. Similar benefits were observed in the tissues of HFD-fed mice. Within 4 h of oral administration, SH-BC-893 normalized mitochondrial morphology in the livers and brains of HFD-fed mice, improved mitochondrial function in white adipose tissue, and corrected aberrant plasma leptin and adiponectin levels. As an interventional agent, SH-BC-893 restored normal body weight, glucose disposal, and hepatic lipid levels in mice consuming a HFD. In sum, the sphingolipid analog SH-BC-893 robustly and acutely blocks ceramide-induced mitochondrial dysfunction, correcting diet-induced obesity and its metabolic sequelae.

Macropinocytosis confers resistance to therapies targeting cancer anabolism.

Macropinocytic cancer cells scavenge amino acids from extracellular proteins. Here, we show that consuming necrotic cell debris via macropinocytosis (necrocytosis) offers additional anabolic benefits. A click chemistry-based flux assay reveals that necrocytosis provides not only amino acids, but sugars, fatty acids and nucleotides for biosynthesis, conferring resistance to therapies targeting anabolic pathways. Indeed, necrotic cell debris allow macropinocytic breast and prostate cancer cells to proliferate, despite fatty acid synthase inhibition. Standard therapies such as gemcitabine, 5-fluorouracil (5-FU), doxorubicin and gamma-irradiation directly or indirectly target nucleotide biosynthesis, creating stress that is relieved by scavenged nucleotides. Strikingly, necrotic debris also render macropinocytic, but not non-macropinocytic, pancreas and breast cancer cells resistant to these treatments. Selective, genetic inhibition of macropinocytosis confirms that necrocytosis both supports tumor growth and limits the effectiveness of 5-FU in vivo. Therefore, this study establishes necrocytosis as a mechanism for drug resistance.

Starving PTEN-deficient prostate cancer cells thrive under nutrient stress by scavenging corpses for their supper.

Our recent work demonstrates that inactivating mutations in phosphatase and tensin homolog (PTEN) are sufficient to drive macropinocytosis in the context of AMP-activated protein kinase (AMPK) activation. Given that blocking macropinocytosis limits PTEN-deficient prostate tumor growth, AMPK or phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) inhibitors could have therapeutic value in castration-resistant prostate cancer patients, particularly when used in combination with standard of care therapies. Abbreviations: ATG5: autophagy related 5; NHE: Na(+)/H(+) exchanger; PAK1: p21-activated kinase 1; PI3K: phosphatidylinositol-4,5-bisphosphate 3-kinase; PIP3: phosphatidylinositol (3,4,5)-trisphosphate; PIP2: phosphatidylinositol (4,5)-bisphosphate; RAC1: Rac family small GTPase 1.

Nutrient scavenging in cancer.

While cancer cell proliferation depends on access to extracellular nutrients, inadequate tumour perfusion means that glucose, amino acids and lipids are often in short supply. To overcome this obstacle to growth, cancer cells utilize multiple scavenging strategies, obtaining macromolecules from the microenvironment and breaking them down in the lysosome to produce substrates for ATP generation and anabolism. Recent studies have revealed four scavenging pathways that support cancer cell proliferation in low-nutrient environments: scavenging of extracellular matrix proteins via integrins, receptor-mediated albumin uptake and catabolism, macropinocytic consumption of multiple components of the tumour microenvironment and the engulfment and degradation of entire live cells via entosis. New evidence suggests that blocking these pathways alone or in combination could provide substantial benefits to patients with incurable solid tumours. Both US Food and Drug Administration (FDA)-approved drugs and several agents in preclinical or clinical development shut down individual or multiple scavenging pathways. These therapies may increase the extent and durability of tumour growth inhibition and/or prevent the development of resistance when used in combination with existing treatments. This Review summarizes the evidence suggesting that scavenging pathways drive tumour growth, highlights recent advances that define the oncogenic signal transduction pathways that regulate scavenging and considers the benefits and detriments of therapeutic strategies targeting scavenging that are currently under development.

PTEN Deficiency and AMPK Activation Promote Nutrient Scavenging and Anabolism in Prostate Cancer Cells.

We report that PTEN-deficient prostate cancer cells use macropinocytosis to survive and proliferate under nutrient stress. PTEN loss increased macropinocytosis only in the context of AMPK activation, revealing a general requirement for AMPK in macropinocytosis and a novel mechanism by which AMPK promotes survival under stress. In prostate cancer cells, albumin uptake did not require macropinocytosis, but necrotic cell debris proved a specific macropinocytic cargo. Isotopic labeling confirmed that macropinocytosed necrotic cell proteins fueled new protein synthesis in prostate cancer cells. Supplementation with necrotic debris, but not albumin, also maintained lipid stores, suggesting that macropinocytosis can supply nutrients other than amino acids. Nontransformed prostatic epithelial cells were not macropinocytic, but patient-derived prostate cancer organoids and xenografts and autochthonous prostate tumors all exhibited constitutive macropinocytosis, and blocking macropinocytosis limited prostate tumor growth. Macropinocytosis of extracellular material by prostate cancer cells is a previously unappreciated tumor-microenvironment interaction that could be targeted therapeutically.Significance: As PTEN-deficient prostate cancer cells proliferate in low-nutrient environments by scavenging necrotic debris and extracellular protein via macropinocytosis, blocking macropinocytosis by inhibiting AMPK, RAC1, or PI3K may have therapeutic value, particularly in necrotic tumors and in combination with therapies that cause nutrient stress. Cancer Discov; 8(7); 866-83. ©2018 AACR.See related commentary by Commisso and Debnath, p. 800This article is highlighted in the In This Issue feature, p. 781.

Shaping the multi-scale architecture of mitochondria.

Mitochondria are complex organelles with a highly regulated architecture across all levels of organization. The architecture of the inner mitochondrial membrane (IMM) provides a crucial platform for many mitochondrial functions while mitochondrial network architecture is crucial for coordinating these activities throughout the cell. This review summarizes the recent findings regarding the most important shaping factors that regulate IMM organization, how IMM architecture supports bioenergetic functions and how IMM morphology adapts to meet other physiological needs of the cell. This review also highlights recent work suggesting that the functional connectivity of mitochondrial networks can be achieved not just by matrix continuity but also by inter-mitochondrial contact sites, which generate conductive continuity within a matrix-discontinuous mitochondrial network.

Characterization of the zebrafish homolog of zipper interacting protein kinase.

Zipper-interacting protein kinase (ZIPK) is a conserved vertebrate-specific regulator of actomyosin contractility in smooth muscle and non-muscle cells. Murine ZIPK has undergone an unusual divergence in sequence and regulation compared to other ZIPK orthologs. In humans, subcellular localization is controlled by phosphorylation of threonines 299 and 300. In contrast, ZIPK subcellular localization in mouse and rat is controlled by interaction with PAR-4. We carried out a comparative biochemical characterization of the regulation of the zebrafish ortholog of ZIPK. Like the human orthologs zebrafish ZIPK undergoes nucleocytoplasmic-shuttling and is abundant in the cytoplasm, unlike the primarily nuclear rat ZIPK. Rat ZIPK, but not human or zebrafish ZIPK, interacts with zebrafish PAR-4. Mutation of the conserved residues required for activation of the mammalian orthologs abrogated activity of the zebrafish ZIPK. In contrast to the human ortholog, mutation of threonine 299 and 300 in the zebrafish ZIPK has no effect on the activity or subcellular localization. Thus, we found that zebrafish ZIPK functions in a manner most similar to the human ZIPK and quite distinct from murine orthologs, yet the regulation of subcellular localization is not conserved.

Integrating mitochondrial organization and dynamics with cellular architecture.

Mitochondrial organization, dynamics, and interactions with other intracellular structures and organelles are crucial for proper cell physiology. In this review we will discuss recent work on the significance of mitochondrial organization in regulating the size and distribution of mitochondrial DNA nucleoids and emphasize the importance of a new role for actin in regulating mitochondrial dynamics. We will also highlight new and unexpected examples of how mitochondria are integrated with many aspects of cell behavior, including cell migration, cell division, and the proper functioning of specialized cells such as neurons and immune cells. Together, these recent studies demonstrate the importance of mitochondrial organization in generating cellular architecture and vice versa.

Protein phosphatase 1 ß paralogs encode the zebrafish myosin phosphatase catalytic subunit.

BACKGROUND: The myosin phosphatase is a highly conserved regulator of actomyosin contractility. Zebrafish has emerged as an ideal model system to study the in vivo role of myosin phosphatase in controlling cell contractility, cell movement and epithelial biology. Most work in zebrafish has focused on the regulatory subunit of the myosin phosphatase called Mypt1. In this work, we examined the critical role of Protein Phosphatase 1, PP1, the catalytic subunit of the myosin phosphatase. METHODOLOGY/PRINCIPAL FINDINGS: We observed that in zebrafish two paralogous genes encoding PP1ß, called ppp1cba and ppp1cbb, are both broadly expressed during early development. Furthermore, we found that both gene products interact with Mypt1 and assemble an active myosin phosphatase complex. In addition, expression of this complex results in dephosphorylation of the myosin regulatory light chain and large scale rearrangements of the actin cytoskeleton. Morpholino knock-down of ppp1cba and ppp1cbb results in severe defects in morphogenetic cell movements during gastrulation through loss of myosin phosphatase function. CONCLUSIONS/SIGNIFICANCE: Our work demonstrates that zebrafish have two genes encoding PP1ß, both of which can interact with Mypt1 and assemble an active myosin phosphatase. In addition, both genes are required for convergence and extension during gastrulation and correct dosage of the protein products is required.