ABSTRACT
While the majority of phosphatidylinositol-4, 5-bisphosphate (PI-4, 5-P2) in mammalian cells is generated by the conversion of phosphatidylinositol-4-phosphate (PI-4-P) to PI-4, 5-P2, a small fraction can be made by phosphorylating phosphatidylinositol-5-phosphate (PI-5-P). The physiological relevance of this second pathway is not clear. Here, we show that deletion of the genes encoding the two most active enzymes in this pathway, Pip4k2a and Pip4k2b, in the liver of mice causes a large enrichment in lipid droplets and in autophagic vesicles during fasting. These changes are due to a defect in the clearance of autophagosomes that halts autophagy and reduces the supply of nutrients salvaged through this pathway. Similar defects in autophagy are seen in nutrient-starved Pip4k2a-/-Pip4k2b-/- mouse embryonic fibroblasts and in C. elegans lacking the PI5P4K ortholog. These results suggest that this alternative pathway for PI-4, 5-P2 synthesis evolved, in part, to enhance the ability of multicellular organisms to survive starvation.
Subject(s)
Autophagy/physiology , Fasting/metabolism , Lipid Metabolism/physiology , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Animals , Autophagosomes/metabolism , Caenorhabditis elegans/metabolism , Cell Line , Fibroblasts/metabolism , HEK293 Cells , Humans , Liver/metabolism , Mice , Phosphatidylinositol Phosphates/metabolism , Signal Transduction/physiologyABSTRACT
In this issue of Molecular Cell, Malek et al. (2017) describe a novel HPLC-MS method permitting separation of PI(3,4)P2 and PI(4,5)P2, a technical issue hindering the phosphoinositide signaling field. They use this method to uncover a new target and critical role for PTEN in cancer.
Subject(s)
Neoplasms , Phosphatidylinositol 3-Kinases , Humans , PTEN Phosphohydrolase , Phosphatidylinositols , Signal TransductionABSTRACT
Deleted in Liver Cancer 1 (DLC1) is a RHO GTPase-activating protein (GAP) that negatively regulates RHO. Through its GAP activity, it modulates the actin cytoskeleton network and focal adhesion dynamics, ultimately leading to suppression of cell invasion and metastasis. Despite its presence in various structural and signaling components, little is known about how the activity of DLC1 is regulated at focal adhesions. Here we show that EGF stimulation activates the GAP activity of DLC1 through a concerted mechanism involving DLC1 phosphorylation by MEK/ERK and its subsequent dephosphorylation by protein phosphatase 2A (PP2A) and inhibition of focal adhesion kinase by MEK/ERK to allow the binding between DLC1 and PP2A. Phosphoproteomics and mutation studies revealed that threonine 301 and serine 308 on DLC1, known previously to be mutated in certain cancers, are required for DLC1-PP2A interaction and the subsequent activation of DLC1 upon their dephosphorylation. The intricate interplay of this "MEK/ERK-focal adhesion kinase-DLC1-PP2A" quartet provides a novel checkpoint in the spatiotemporal control of cell spreading and cell motility.
Subject(s)
Epidermal Growth Factor/pharmacology , Focal Adhesion Kinase 1/metabolism , Focal Adhesions/drug effects , GTPase-Activating Proteins/metabolism , Protein Phosphatase 2/metabolism , Tumor Suppressor Proteins/metabolism , Blotting, Western , Cell Movement/drug effects , Cell Proliferation/drug effects , HEK293 Cells , HeLa Cells , Humans , Immunoprecipitation , MAP Kinase Kinase 1/metabolism , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Phosphorylation , Proteome/analysis , Signal Transduction , Wound HealingABSTRACT
Phosphoinositides are essential signaling molecules. The PI5P4K family of phosphoinositide kinases and their substrates and products, PI5P and PI4,5P2, respectively, are emerging as intracellular metabolic and stress sensors. We performed an unbiased screen to investigate the signals that these kinases relay and the specific upstream regulators controlling this signaling node. We found that the core Hippo pathway kinases MST1/2 phosphorylated PI5P4Ks and inhibited their signaling in vitro and in cells. We further showed that PI5P4K activity regulated several Hippo- and YAP-related phenotypes, specifically decreasing the interaction between the key Hippo proteins MOB1 and LATS and stimulating the YAP-mediated genetic program governing epithelial-to-mesenchymal transition. Mechanistically, we showed that PI5P interacted with MOB1 and enhanced its interaction with LATS, thereby providing a signaling connection between the Hippo pathway and PI5P4Ks. These findings reveal how these two important evolutionarily conserved signaling pathways are integrated to regulate metazoan development and human disease.
Subject(s)
Adaptor Proteins, Signal Transducing , Hippo Signaling Pathway , Protein Serine-Threonine Kinases , Signal Transduction , Transcription Factors , YAP-Signaling Proteins , Humans , Protein Serine-Threonine Kinases/metabolism , Protein Serine-Threonine Kinases/genetics , Hippo Signaling Pathway/genetics , Adaptor Proteins, Signal Transducing/metabolism , Adaptor Proteins, Signal Transducing/genetics , Transcription Factors/metabolism , Transcription Factors/genetics , YAP-Signaling Proteins/metabolism , YAP-Signaling Proteins/genetics , Transcriptional Activation , Phosphorylation , HEK293 Cells , Epithelial-Mesenchymal Transition , Phosphoproteins/metabolism , Phosphoproteins/genetics , Animals , Serine-Threonine Kinase 3 , Intracellular Signaling Peptides and Proteins/metabolism , Intracellular Signaling Peptides and Proteins/geneticsABSTRACT
Phosphatidylinositol (PI)regulating enzymes are frequently altered in cancer and have become a focus for drug development. Here, we explore the phosphatidylinositol-5-phosphate 4-kinases (PI5P4K), a family of lipid kinases that regulate pools of intracellular PI, and demonstrate that the PI5P4Kα isoform influences androgen receptor (AR) signaling, which supports prostate cancer (PCa) cell survival. The regulation of PI becomes increasingly important in the setting of metabolic stress adaptation of PCa during androgen deprivation (AD), as we show that AD influences PI abundance and enhances intracellular pools of PI-4,5-P2. We suggest that this PI5P4Kα-AR relationship is mitigated through mTORC1 dysregulation and show that PI5P4Kα colocalizes to the lysosome, the intracellular site of mTORC1 complex activation. Notably, this relationship becomes prominent in mouse prostate tissue following surgical castration. Finally, multiple PCa cell models demonstrate marked survival vulnerability following stable PI5P4Kα inhibition. These results nominate PI5P4Kα as a target to disrupt PCa metabolic adaptation to castrate resistance.
Subject(s)
Prostatic Neoplasms, Castration-Resistant , Receptors, Androgen , Animals , Humans , Male , Mice , Androgen Antagonists , Androgens/metabolism , Cell Line, Tumor , Mechanistic Target of Rapamycin Complex 1/metabolism , Prostatic Neoplasms, Castration-Resistant/metabolism , Receptors, Androgen/metabolism , Signal TransductionABSTRACT
We present Multi-miR, a microRNA-embedded shRNA system modeled after endogenous microRNA clusters that enables simultaneous expression of up to three or four short hairpin RNAs (shRNAs) from a single promoter without loss of activity, enabling robust combinatorial RNA interference (RNAi). We further developed complementary all-in-one vectors that are over one log-scale more sensitive to doxycycline-mediated activation in vitro than previous methods and resistant to shRNA inactivation in vivo. We demonstrate the utility of this system for intracranial expression of shRNAs in a glioblastoma model. Additionally, we leverage this platform to target the redundant RAF signaling node in a mouse model of KRAS-mutant cancer and show that robust combinatorial synthetic lethality efficiently abolishes tumor growth.
Subject(s)
MicroRNAs , Mice , Animals , MicroRNAs/genetics , RNA Interference , Genetic Vectors , RNA, Small Interfering/genetics , Promoter Regions, GeneticABSTRACT
While organelles are individual compartments with specialized functions, it is becoming clear that organellar communication is essential for maintaining cellular homeostasis. This cooperation is carried out by various interactions taking place on the membranes of organelles. The membranes themselves contain a multitude of proteins and lipids that mediate these connections and one such class of molecules facilitating these relations are the phospholipids. There are several phospholipids, but the focus of this perspective is on a minor group called the phosphoinositides and specifically, phosphatidylinositol 4,5-bisphosphate (PI-4,5-P2). This phosphoinositide, on intracellular membranes, is largely generated by the non-canonical Type II PIPKs, namely, Phosphotidylinositol-5-phosphate-4-kinases (PI5P4Ks). These evolutionarily conserved enzymes are emerging as key stress response players in cells. Further, PI5P4Ks have been shown to modulate pathways by regulating organelle crosstalk, revealing roles in preserving metabolic homeostasis. Here we will attempt to summarize the functions of the PI5P4Ks and their product PI-4,5-P2 in facilitating inter-organelle communication and how they impact cellular health as well as their relevance to human diseases.
ABSTRACT
PI5P4Ks are a class of phosphoinositide kinases that phosphorylate PI-5-P to PI-4,5-P2. Distinct localization of phosphoinositides is fundamental for a multitude of cellular functions. Here, we identify a role for peroxisomal PI-4,5-P2 generated by the PI5P4Ks in maintaining energy balance. We demonstrate that PI-4,5-P2 regulates peroxisomal fatty acid oxidation by mediating trafficking of lipid droplets to peroxisomes, which is essential for sustaining mitochondrial metabolism. Using fluorescent-tagged lipids and metabolite tracing, we show that loss of the PI5P4Ks significantly impairs lipid uptake and ß-oxidation in the mitochondria. Further, loss of PI5P4Ks results in dramatic alterations in mitochondrial structural and functional integrity, which under nutrient deprivation is further exacerbated, causing cell death. Notably, inhibition of the PI5P4Ks in cancer cells and mouse tumor models leads to decreased cell viability and tumor growth, respectively. Together, these studies reveal an unexplored role for PI5P4Ks in preserving metabolic homeostasis, which is necessary for tumorigenesis.
Subject(s)
Carcinogenesis/genetics , Mitochondria/genetics , Neoplasms/metabolism , Peroxisomes/metabolism , Phosphotransferases (Alcohol Group Acceptor)/genetics , Animals , Cell Line, Tumor , Energy Metabolism/genetics , Female , Homeostasis/genetics , Humans , Lipid Droplets/metabolism , Lipid Metabolism/genetics , Male , Mice , Mitochondria/metabolism , Mitochondria/ultrastructure , Neoplasms/genetics , Neoplasms/pathology , Peroxisomes/geneticsABSTRACT
Today, the importance of autophagy in physiological processes and pathological conditions is undeniable. Initially, autophagy merely was described as an evolutionarily conserved mechanism to maintain metabolic homeostasis in times of starvation; however, in recent years it is now apparent that autophagy is a powerful regulator of many facets of cellular metabolism, that its deregulation contributes to various human pathologies, including cancer and neurodegeneration, and that its modulation has considerable potential as a therapeutic approach. Different lipid species, including sphingolipids, sterols, and phospholipids, play important roles in the various steps of autophagy. In particular, there is accumulating evidence indicating the minor group of phospholipids called the phosphoinositides as key modulators of autophagy, including the signaling processes underlying autophagy initiation, autophagosome biogenesis and maturation. In this review, we discuss the known functions to date of the phosphoinositides in autophagy and attempt to summarize the kinases and phosphatases that regulate them as well as the proteins that bind to them throughout the autophagy program. We will also provide examples of how the control of phosphoinositides and their metabolizing enzymes is relevant to understanding many human diseases.
Subject(s)
Autophagy , Phosphatidylinositols/metabolism , Animals , Humans , Huntington Disease/genetics , Huntington Disease/metabolism , Myopathies, Structural, Congenital/genetics , Myopathies, Structural, Congenital/metabolism , Neoplasms/genetics , Neoplasms/metabolism , Oculocerebrorenal Syndrome/genetics , Oculocerebrorenal Syndrome/metabolism , Phosphatidylinositols/geneticsABSTRACT
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.
Subject(s)
AMP-Activated Protein Kinases/metabolism , Nutrients/metabolism , PTEN Phosphohydrolase/genetics , Pinocytosis , Prostatic Neoplasms/metabolism , Stress, Physiological , Animals , Gene Deletion , Humans , Male , Mice , Mice, Inbred C57BLABSTRACT
Oncogenic mutations drive anabolic metabolism, creating a dependency on nutrient influx through transporters, receptors, and macropinocytosis. While sphingolipids suppress tumor growth by downregulating nutrient transporters, macropinocytosis and autophagy still provide cancer cells with fuel. Therapeutics that simultaneously disrupt these parallel nutrient access pathways have potential as powerful starvation agents. Here, we describe a water-soluble, orally bioavailable synthetic sphingolipid, SH-BC-893, that triggers nutrient transporter internalization and also blocks lysosome-dependent nutrient generation pathways. SH-BC-893 activated protein phosphatase 2A (PP2A), leading to mislocalization of the lipid kinase PIKfyve. The concomitant mislocalization of the PIKfyve product PI(3,5)P2 triggered cytosolic vacuolation and blocked lysosomal fusion reactions essential for LDL, autophagosome, and macropinosome degradation. By simultaneously limiting access to both extracellular and intracellular nutrients, SH-BC-893 selectively killed cells expressing an activated form of the anabolic oncogene Ras in vitro and in vivo. However, slower-growing, autochthonous PTEN-deficient prostate tumors that did not exhibit a classic Warburg phenotype were equally sensitive. Remarkably, normal proliferative tissues were unaffected by doses of SH-BC-893 that profoundly inhibited tumor growth. These studies demonstrate that simultaneously blocking parallel nutrient access pathways with sphingolipid-based drugs is broadly effective and cancer selective, suggesting a potential strategy for overcoming the resistance conferred by tumor heterogeneity.
Subject(s)
Enzyme Activators/pharmacology , Neoplasm Proteins/antagonists & inhibitors , Neoplasms/drug therapy , Protein Phosphatase 2/antagonists & inhibitors , Sphingolipids/pharmacology , Animals , Biological Transport, Active/drug effects , Cell Line, Tumor , Drug Screening Assays, Antitumor , Humans , Mice , Mice, Knockout , Neoplasm Proteins/metabolism , Neoplasms/metabolism , Neoplasms/pathology , Phosphatidylinositol 3-Kinases/metabolism , Phosphatidylinositol Phosphates/metabolism , Protein Phosphatase 2/metabolismABSTRACT
Deleted in Liver Cancer-1 (DLC1) is a RhoGTPase-activating protein (GAP) and a tumor suppressor often downregulated in cancers. It is localized to the focal adhesions (FAs) and its absence leads to enhanced cell migration, invasion, and metastasis. Although DLC1 interacts with focal adhesion kinase (FAK), talin, and tensin, its role in focal adhesions dynamics remains unclear. We examined the effect of DLC1 in Human Foreskin Fibroblasts and determined its localization, dynamics and impact on paxillin by Fluorescence Recovery After Photobleaching at both nascent and mature focal adhesions. During early cell spreading, DLC1 is preferentially localized at the inner/mature adhesions whereas phosphorylated paxillin occupies the outer/nascent FAs. In addition, DLC1 downregulates paxillin turnover in a process, that does not require its GAP activity. Instead, it requires the presence of FAK. Acting in concert, both DLC1 and FAK could provide a unique spatio-temporal mechanism to regulate paxillin function in tissue homeostasis.