Bidirectional transport of amino acids regulates mTOR and autophagy.

Amino acids are required for activation of the mammalian target of rapamycin (mTOR) kinase which regulates protein translation, cell growth, and autophagy. Cell surface transporters that allow amino acids to enter the cell and signal to mTOR are unknown. We show that cellular uptake of L-glutamine and its subsequent rapid efflux in the presence of essential amino acids (EAA) is the rate-limiting step that activates mTOR. L-glutamine uptake is regulated by SLC1A5 and loss of SLC1A5 function inhibits cell growth and activates autophagy. The molecular basis for L-glutamine sensitivity is due to SLC7A5/SLC3A2, a bidirectional transporter that regulates the simultaneous efflux of L-glutamine out of cells and transport of L-leucine/EAA into cells. Certain tumor cell lines with high basal cellular levels of L-glutamine bypass the need for L-glutamine uptake and are primed for mTOR activation. Thus, L-glutamine flux regulates mTOR, translation and autophagy to coordinate cell growth and proliferation.

LRRK2 deficiency impairs trans-Golgi to lysosome trafficking and endocytic cargo degradation in human renal proximal tubule epithelial cells.

Defects in vesicular trafficking underlie a wide variety of human diseases. Genetic disruption of leucine-rich repeat kinase 2 (LRRK2) in rodents results in epithelial vesicular trafficking errors that can also be induced by treatment of animals with LRRK2 kinase inhibitors. Here we demonstrate that defects in human renal cells lacking LRRK2 phenocopy those seen in the kidneys of Lrrk2 knockout mice, characterized by accumulation of intracellular waste vesicles and fragmentation of the Golgi apparatus. This phenotype can be recapitulated by knockdown of N-ethylmaleimide-sensitive factor, which physically associates with LRRK2 in renal cells. Deficiency in either protein leads to a defect in trans-Golgi to lysosome protein trafficking, which compromises the capacity of lysosomes to degrade endocytic and autophagic cargo. In contrast, neither bulk endocytosis nor autophagic flux are impaired when LRRK2 is acutely knocked down in normal immortalized human kidney (HK2) cells. These data collectively suggest that the primary renal defect caused by LRRK2 deficiency is in protein trafficking between the Golgi apparatus and late endosome/lysosome, which leads to progressive impairments in lysosomal function.

Activation of a metabolic gene regulatory network downstream of mTOR complex 1.

Aberrant activation of the mammalian target of rapamycin complex 1 (mTORC1) is a common molecular event in a variety of pathological settings, including genetic tumor syndromes, cancer, and obesity. However, the cell-intrinsic consequences of mTORC1 activation remain poorly defined. Through a combination of unbiased genomic, metabolomic, and bioinformatic approaches, we demonstrate that mTORC1 activation is sufficient to stimulate specific metabolic pathways, including glycolysis, the oxidative arm of the pentose phosphate pathway, and de novo lipid biosynthesis. This is achieved through the activation of a transcriptional program affecting metabolic gene targets of hypoxia-inducible factor (HIF1alpha) and sterol regulatory element-binding protein (SREBP1 and SREBP2). We find that SREBP1 and 2 promote proliferation downstream of mTORC1, and the activation of these transcription factors is mediated by S6K1. Therefore, in addition to promoting protein synthesis, mTORC1 activates specific bioenergetic and anabolic cellular processes that are likely to contribute to human physiology and disease.

Integrating virtual and biochemical screening for protein tyrosine phosphatase inhibitor discovery.

Protein tyrosine phosphatases (PTPs) represent an important class of enzymes that mediate signal transduction and control diverse aspects of cell behavior. The importance of their activity is exemplified by their significant contribution to disease etiology with over half of all human PTP genes implicated in at least one disease. Small molecule inhibitors targeting individual PTPs are important biological tools, and are needed to fully characterize the function of these enzymes. Moreover, potent and selective PTP inhibitors hold the promise to transform the treatment of many diseases. While numerous methods exist to develop PTP-directed small molecules, we have found that complimentary use of both virtual (in silico) and biochemical (in vitro) screening approaches expedite compound identification and drug development. Here, we summarize methods pertinent to our work and others. Focusing on specific challenges and successes we have experienced, we discuss the considerable caution that must be taken to avoid enrichment of inhibitors that function by non-selective oxidation. We also discuss the utility of using "open" PTP structures to identify active-site directed compounds, a rather unconventional choice for virtual screening. When integrated closely, virtual and biochemical screening can be used in a productive workflow to identify small molecules targeting PTPs.

Chromosomal amplification of leucine-rich repeat kinase-2 (LRRK2) is required for oncogenic MET signaling in papillary renal and thyroid carcinomas.

The receptor tyrosine kinase MET is frequently amplified in human tumors, resulting in high cell surface densities and constitutive activation even in the absence of growth factor stimulation by its endogenous ligand, hepatocyte growth factor (HGF). We sought to identify mechanisms of signaling crosstalk that promote MET activation by searching for kinases that are coordinately dysregulated with wild-type MET in human tumors. Our bioinformatic analysis identified leucine-rich repeat kinase-2 (LRRK2), which is amplified and overexpressed in papillary renal and thyroid carcinomas. Down-regulation of LRRK2 in cultured tumor cells compromises MET activation and selectively reduces downstream MET signaling to mTOR and STAT3. Loss of these critical mitogenic pathways induces cell cycle arrest and cell death due to loss of ATP production, indicating that MET and LRRK2 cooperate to promote efficient tumor cell growth and survival in these cancers.

Identification of PTPsigma as an autophagic phosphatase.

Macroautophagy is a dynamic process whereby portions of the cytosol are encapsulated in double-membrane vesicles and delivered to the lysosome for degradation. Phosphatidylinositol-3-phosphate (PtdIns3P) is concentrated on autophagic vesicles and recruits effector proteins that are crucial for this process. The production of PtdIns3P by the class III phosphatidylinositol 3-kinase Vps34, has been well established; however, protein phosphatases that antagonize this early step in autophagy remain to be identified. To identify such enzymes, we screened human phosphatase genes by RNA interference and found that loss of PTPσ, a dual-domain protein tyrosine phosphatase (PTP), increases levels of cellular PtdIns3P. The abundant PtdIns3P-positive vesicles conferred by loss of PTPσ strikingly phenocopied those observed in cells starved of amino acids. Accordingly, we discovered that loss of PTPσ hyperactivates both constitutive and induced autophagy. Finally, we found that PTPσ localizes to PtdIns3P-positive membranes in cells, and this vesicular localization is enhanced during autophagy. We therefore describe a novel role for PTPσ and provide insight into the regulation of autophagy. Mechanistic knowledge of this process is crucial for understanding and targeting therapies for several human diseases, including cancer and Alzheimer's disease, in which abnormal autophagy might be pathological.

Unique Metabolic Contexts Sensitize Cancer Cells and Discriminate between Glycolytic Tumor Types.

Cancer cells utilize variable metabolic programs in order to maintain homeostasis in response to environmental challenges. To interrogate cancer cell reliance on glycolytic programs under different nutrient availabilities, we analyzed a gene panel containing all glycolytic genes as well as pathways associated with glycolysis. Using this gene panel, we analyzed the impact of an siRNA library on cellular viability in cells containing only glucose or only pyruvate as the major bioenergetic nutrient source. From these panels, we aimed to identify genes that elicited conserved and glycolysis-dependent changes in cellular bioenergetics across glycolysis-promoting and OXPHOS-promoting conditions. To further characterize gene sets within this panel and identify similarities and differences amongst glycolytic tumor RNA-seq profiles across a pan-cancer cohort, we then used unsupervised statistical classification of RNA-seq profiles for glycolytic cancers and non-glycolytic cancer types. Here, Kidney renal clear cell carcinoma (KIRC); Head and Neck squamous cell carcinoma (HNSC); and Lung squamous cell carcinoma (LUSC) defined the glycolytic cancer group, while Prostate adenocarcinoma (PRAD), Thyroid carcinoma (THCA), and Thymoma (THYM) defined the non-glycolytic cancer group. These groups were defined based on glycolysis scoring from previous studies, where KIRC, HNSC, and LUSC had the highest glycolysis scores, meanwhile, PRAD, THCA, and THYM had the lowest. Collectively, these results aimed to identify multi-omic profiles across cancer types with demonstrated variably glycolytic rates. Our analyses provide further support for strategies aiming to classify tumors by metabolic phenotypes in order to therapeutically target tumor-specific vulnerabilities.

Molecular dynamics simulations provide insights into ULK-101 potency and selectivity toward autophagic kinases ULK1/2.

Kinase domains are highly conserved within protein kinases in both sequence and structure. Many factors, including phosphorylation, amino acid substitutions or mutations, and small molecule inhibitor binding, influence conformations of the kinase domain and enzymatic activity. The serine/threonine kinases ULK1 and ULK2 are highly conserved with N- and C-terminal domains, phosphate-binding P-loops, αC-helix, regulatory and catalytic spines, and activation loop DFG and APE motifs. Here, we performed molecular dynamics (MD) simulations to understand better the potency and selectivity of the ULK1/2 small molecule inhibitor, ULK-101. We observed stable bound states for ULK-101 to the adenosine triphosphate (ATP)-binding site of ULK2, coordinated by hydrogen bonding with the hinge backbone and the catalytic lysine sidechain. Notably, ULK-101 occupies a hydrophobic pocket associated with the N-terminus of the αC-helix. Large movements in the P-loop are also associated with ULK-101 inhibitor binding and exit from ULK2. Our data further suggests that ULK-101 could induce a folded P-loop conformation and hydrophobic pocket reflected in its nanomolar potency and kinome selectivity.

Quantitative Analysis of Autophagy in Single Cells: Differential Response to Amino Acid and Glucose Starvation.

Autophagy is a highly conserved, intracellular recycling process by which cytoplasmic contents are degraded in the lysosome. This process occurs at a low level constitutively; however, it is induced robustly in response to stressors, in particular, starvation of critical nutrients such as amino acids and glucose. That said, the relative contribution of these inputs is ambiguous and many starvation medias are poorly defined or devoid of multiple nutrients. Here, we sought to generate a quantitative catalog of autophagy across multiple stages and in single, living cells under normal growth conditions as well as in media starved specifically of amino acids or glucose. We found that autophagy is induced by starvation of amino acids, but not glucose, in U2OS cells, and that MTORC1-mediated ULK1 regulation and autophagy are tightly linked to amino acid levels. While autophagy is engaged immediately during amino acid starvation, a heightened response occurs during a period marked by transcriptional upregulation of autophagy genes during sustained starvation. Finally, we demonstrated that cells immediately return to their initial, low-autophagy state when nutrients are restored, highlighting the dynamic relationship between autophagy and environmental conditions. In addition to sharing our findings here, we provide our data as a high-quality resource for others interested in mathematical modeling or otherwise exploring autophagy in individual cells across a population.

Characterization of differential protein tethering at the plasma membrane in response to epidermal growth factor signaling.

Physical tethering of membrane proteins to the cortical actin cytoskeleton provides functional organization to the plasma membrane and contributes to diverse cellular processes including cell signaling, vesicular trafficking, endocytosis, and migration. For these processes to occur, membrane protein tethering must be dynamically regulated in response to environmental cues. In this study, we describe a novel biochemical scheme for isolating the complement of plasma membrane proteins that are physically tethered to the actin cytoskeleton. We utilized this method in combination with tandem liquid chromatography/mass spectrometry (LC-MS/MS) to demonstrate that cytoskeletal tethering of membrane proteins is acutely regulated by epidermal growth factor (EGF) in normal human kidney (HK2) cells. Our results indicate that several proteins known to be involved in EGF signaling, as well as other proteins not traditionally associated with this pathway, are tethered to the cytoskeleton in dynamic fashion. Further analysis of one hit from our proteomic survey, the receptor phosphotyrosine phosphatase PTPRS, revealed a correlation between cytoskeletal tethering and endosomal trafficking in response to EGF. This finding parallels previous indications that PTPRS is involved in the desensitization of EGFR and provides a potential mechanism to coordinate localization of these two membrane proteins in the same compartment upon EGFR activation.

Sensitized RNAi screen of human kinases and phosphatases identifies new regulators of apoptosis and chemoresistance.

Evasion from apoptosis is a hallmark of cancer, and recent success using targeted therapeutics underscores the importance of identifying anti-apoptotic survival pathways. Here we utilize RNA interference (RNAi) to systematically screen the kinase and phosphatase component of the human genome. In addition to known kinases, we identified several new survival kinases. Interestingly, numerous phosphatases and associated regulatory subunits contribute to cell survival, revealing a previously unrecognized general role for phosphatases as negative regulators of apoptosis. We also identified a subset of phosphatases with tumour-suppressor-like activity. Finally, RNAi targeting of specific protein kinases sensitizes resistant cells to chemotherapeutic agents. The development of inhibitors that target these kinases or phosphatases may lead to new anti-cancer strategies.

Chemical Biology Screening Identifies a Vulnerability to Checkpoint Kinase Inhibitors in TSC2-Deficient Renal Angiomyolipomas.

The tuberous sclerosis complex (TSC) is a rare genetic syndrome and multisystem disease resulting in tumor formation in major organs. A molecular hallmark of TSC is a dysregulation of the mammalian target of rapamycin (mTOR) through loss-of-function mutations in either tumor suppressor TSC1 or TSC2. Here, we sought to identify drug vulnerabilities conferred by TSC2 tumor-suppressor loss through cell-based chemical biology screening. Our small-molecule chemical screens reveal a sensitivity to inhibitors of checkpoint kinase 1/2 (CHK1/2), regulators of cell cycle, and DNA damage response, in both in vitro and in vivo models of TSC2-deficient renal angiomyolipoma (RA) tumors. Further, we performed transcriptional profiling on TSC2-deficient RA cell models and discovered that these recapitulate some of the features from TSC patient kidney tumors compared to normal kidneys. Taken together, our study provides a connection between mTOR-dependent tumor growth and CHK1/2, highlighting the importance of CHK1/2 inhibition as a potential antitumor strategy in TSC2-deficient tumors.

Spectrum of germline and somatic mitochondrial DNA variants in Tuberous Sclerosis Complex.

Tuberous Sclerosis Complex (TSC) is caused by loss of function variants in either TSC1 or TSC2 and is characterized by broad phenotypic heterogeneity. Currently, there is limited knowledge regarding the role of the mitochondrial genome (mtDNA) in TSC pathogenesis. In this study, we aimed to determine the prevalence and spectrum of germline and somatic mtDNA variants in TSC and identify potential disease modifiers. Analysis of mtDNA amplicon massively parallel sequencing (aMPS) data, off-target mtDNA from whole-exome sequencing (WES), and/or qPCR, revealed mtDNA alterations in 270 diverse tissues (139 TSC-associated tumors and 131 normal tissue samples) from 199 patients and six healthy individuals. Correlation of clinical features to mtDNA variants and haplogroup analysis was done in 102 buccal swabs (age: 20-71 years). No correlation was found between clinical features and either mtDNA variants or haplogroups. No pathogenic variants were identified in the buccal swab samples. Using in silico analysis, we identified three predicted pathogenic variants in tumor samples: MT-ND4 (m.11742G>A, p. Cys328Tyr, VAF: 43%, kidney angiomyolipoma), MT-CYB (m.14775T>C, p. Leu10Pro, VAF: 43%, LAM abdominal tumor) and MT-CYB (m.15555C>T, p. Pro270Leu, VAF: 7%, renal cell carcinoma). Large deletions of the mitochondrial genome were not detected. Analysis of tumors from 23 patients with corresponding normal tissue did not reveal any recurrent tumor-associated somatic variants. The mtDNA/gDNA ratio between tumors and corresponding normal tissue was also unchanged. Overall, our findings demonstrate that the mitochondrial genome is highly stable across tissues and within TSC-associated tumors.

Identification of SRC3/AIB1 as a preferred coactivator for hormone-activated androgen receptor.

Transcription activation by androgen receptor (AR), which depends on recruitment of coactivators, is required for the initiation and progression of prostate cancer, yet the mechanisms of how hormone-activated AR interacts with coactivators remain unclear. This is because AR, unlike any other nuclear receptor, prefers its own N-terminal FXXLF motif to the canonical LXXLL motifs of coactivators. Through biochemical and crystallographic studies, we identify that steroid receptor coactivator-3 (SRC3) (also named as amplified in breast cancer-1 or AIB1) interacts strongly with AR via synergistic binding of its first and third LXXLL motifs. Mutagenesis and functional studies confirm that SRC3 is a preferred coactivator for hormone-activated AR. Importantly, AR mutations found in prostate cancer patients correlate with their binding potency to SRC3, corroborating with the emerging role of SRC3 as a prostate cancer oncogene. These results provide a molecular mechanism for the selective utilization of SRC3 by hormone-activated AR, and they link the functional relationship between AR and SRC3 to the development and growth of prostate cancer.

Mitochondria in cancer: at the crossroads of life and death.

Mitochondrial processes play an important role in tumor initiation and progression. In this review, we focus on three critical processes by which mitochondrial function may contribute to cancer: through alterations in glucose metabolism, the production of reactive oxygen species (ROS) and compromise of intrinsic apoptotic function. Alterations in cancer glucose metabolism include the Warburg effect, leading to a shift in metabolism away from aerobic respiration toward glycolysis, even when sufficient oxygen is present to support respiration. Such alterations in cellular metabolism may favor tumor cell growth by increasing the availability of biosynthetic intermediates needed for cellular growth and proliferation. Mutations in specific metabolic enzymes, namely succinate dehydrogenase, fumarate hydratase and the isocitrate dehydrogenases, have been linked to human cancer. Mitochondrial ROS may contribute to cancer via DNA damage and the activation of aberrant signaling pathways. ROS-dependent stabilization of the transcription factor hypoxia-inducible factor (HIF) may be a particularly important event for tumorigenesis. Compromised function of intrinsic apoptosis removes an important cellular safeguard against cancer and has been implicated in tumorigenesis, tumor metastasis, and chemoresistance. Each of the major mitochondrial processes is linked. In this review, we outline the connections between them and address ways these mitochondrial pathways may be targeted for cancer therapy.

Kinase targets in renal-cell carcinomas: reassessing the old and discovering the new.

Renal-cell carcinoma is a heterogeneous group of tumours that arise in the adult kidneys. Irrespective of the type of renal tumour, traditional chemotherapeutic and radiation-based therapies have been largely ineffective at treating advanced tumours, with long-term survival being very low. Molecularly-targeted inhibitors of protein kinases are effective in delaying progression of advanced renal tumours. These therapies revolve around inhibition of the vascular endothelial growth factor receptor tyrosine kinase and the mammalian target of rapamycin serine or threonine kinase signalling pathways. The genetic complexity of renal tumours revealed by gene-expression profiling and other molecular-genetic technologies indicate that inhibition of additional kinase-associated pathways could also prevent renal tumour growth. In this review, we discuss the use of molecularly-targeted kinase inhibitors in the treatment of renal-cell carcinoma and identify the next generation of kinase inhibitors that show promise for treatment.

Down-regulation of class II phosphoinositide 3-kinase alpha expression below a critical threshold induces apoptotic cell death.

Members of the phosphoinositide 3-kinase (PI3K) family collectively control multiple cellular responses, including proliferation, growth, chemotaxis, and survival. These diverse effects can partly be attributed to the broad range of downstream effectors being regulated by the products of these lipid kinases, the 3'-phosphoinositides. However, an additional layer of complexity is introduced by the existence of multiple PI3K enzyme isoforms. Much has been learned over the last years on the roles of the classes I and III PI3K members in cellular signaling, but little is known about the isoform-specific tasks done by the class II PI3Ks (C2alpha, beta, and gamma). In this study, we used quantitative reverse transcription-PCR and RNA interference in mammalian cells to gain further insight into the function of these lesser studied PI3K enzymes. We find that PI3K-C2alpha, but not PI3K-C2beta, has an important role in controlling cell survival and by using a panel of RNA interference reagents, we were able to determine a critical threshold of PI3K-C2alpha mRNA levels, below which the apoptotic program is switched on, via the intrinsic cell death pathway. In addition, knockdown of PI3K-C2alpha to levels that by themselves do not induce apoptosis sensitize cells to the anticancer agent Taxol (paclitaxel). Lastly, we report that lowering the levels of PI3K-C2alpha in a number of cancer cell lines reduces their proliferation and cell viability, arguing that PI3K inhibitors targeting not only the class Ialpha isoform but also class IIalpha may contribute to an effective anticancer strategy.

Determining the Impact of Metabolic Nutrients on Autophagy.

Tumorigenesis relies on the ability of cancer cells to obtain necessary nutrients and fulfill increased energy demands associated with rapid proliferation. However, as a result of increased metabolite consumption and poor vascularization, most cancer cells must survive in a nutrient poor and high cellular stress microenvironment. Cancer cells undergo metabolic reprogramming to evade cell death and ensure proliferation; in particular, cancer cells utilize the catabolic process of autophagy. Autophagy creates an intracellular pool of metabolites by sequestering cytosolic macromolecules in double-membrane vesicles targeted for lysosomal degradation. During times of environmental stress and nutrient starvation, autophagy is upregulated through the dynamic interactions between two nutrient sensing proteins, AMP activated protein kinase (AMPK) and mechanistic target of rapamycin (mTOR), in cooperation with Unc-51 like autophagy activating kinase 1 (ULK1). In this way, a lack of metabolic nutrients plays a critical role in inducing autophagy, while the products of autophagy also serve as readily available fuel for the cell. In this chapter, we describe methods to visualize and quantify autophagy using a fluorescent sensor of autophagic membranes. Thus, the impact of specific nutrients on autophagy can be measured using live-cell fluorescent microscopy.

Identification of Kinases Responsible for p53-Dependent Autophagy.

In cancer, autophagy is upregulated to promote cell survival and tumor growth during times of nutrient stress and can confer resistance to drug treatments. Several major signaling networks control autophagy induction, including the p53 tumor suppressor pathway. In response to DNA damage and other cellular stresses, p53 is stabilized and activated, while HDM2 binds to and ubiquitinates p53 for proteasome degradation. Thus blocking the HDM2-p53 interaction is a promising therapeutic strategy in cancer; however, the potential survival advantage conferred by autophagy induction may limit therapeutic efficacy. In this study, we leveraged an HDM2 inhibitor to identify kinases required for p53-dependent autophagy. Interestingly, we discovered that p53-dependent autophagy requires several kinases, including the myotonic dystrophy protein kinase-like alpha (MRCKα). MRCKα is a CDC42 effector reported to activate actin-myosin cytoskeletal reorganization. Overall, this study provides evidence linking MRCKα to autophagy and reveals additional insights into the role of kinases in p53-dependent autophagy.

Spatially separate docking sites on ERK2 regulate distinct signaling events in vivo.

Inhibitors of the oncogenic Ras-MAPK pathway have been intensely pursued as therapeutics. Targeting this pathway, however, presents challenges due to the essential role of MAPK in homeostatic functions. The phosphorylation and activation of MAPK substrates is regulated by protein-protein interactions with MAPK docking sites. Active ERK1/2 (extracellular signal-regulated kinase 1/2)-MAPKs localize to effectors containing DEF (docking site for ERK, (F)/(Y) -X-(F)/(Y) -P)- or D-domain (docking domain) motifs. We have examined the in vivo activity of ERK2 mutants with impaired ability to signal via either docking site. Mutations in the DEF-domain binding pocket prevent activation of DEF-domain-containing effectors but not RSK (90 kDa ribosomal S6 kinase), which contains a D domain. Conversely, mutation of the ERK2 CD domain, which interacts with D domains, prevents RSK activation but not DEF-domain signaling. Uncoupling docking interactions does not compromise ERK2 phosphotransferase activity. ERK2 DEF mutants undergo regulated nuclear translocation but are defective for Elk-1/TCF transactivation and target gene induction. Thus, downstream branches of ERK2 signaling can be selectively inhibited without blocking total pathway activity. Significantly, several protooncogenes contain DEF domains and are regulated by ERK1/2. Therefore, disrupting ERK-DEF domain interactions could be an alternative to inhibiting oncogenic Ras-MAPK signaling.