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1.  Spatial Control of the TSC Complex Integrates Insulin and Nutrient Regulation of mTORC1 at the Lysosome 
Cell  2014;156(4):771-785.
SUMMARY
mTORC1 promotes cell growth in response to nutrients and growth factors. Insulin activates mTORC1 through the PI3K-Akt pathway, which inhibits the TSC1-TSC2-TBC1D7 complex (the TSC complex) to turn on Rheb, an essential activator of mTORC1. However, the mechanistic basis of how this pathway integrates with nutrient-sensing pathways is unknown. We demonstrate that insulin stimulates acute dissociation of the TSC complex from the lysosomal surface, where subpopulations of Rheb and mTORC1 reside. The TSC complex associates with the lysosome in a Rheb-dependent manner, and its dissociation in response to insulin requires Akt-mediated TSC2 phosphorylation. Loss of the PTEN tumor suppressor results in constitutive activation of mTORC1 through the Akt-dependent dissociation of the TSC complex from the lysosome. These findings provide a unifying mechanism by which independent pathways affecting the spatial recruitment of mTORC1 and the TSC complex to Rheb at the lysosomal surface serve to integrate diverse growth signals.
doi:10.1016/j.cell.2013.11.049
PMCID: PMC4030681  PMID: 24529379
2.  Vulnerabilities of PTEN-p53-deficient prostate cancers to compound PARP/PI3K inhibition 
Cancer discovery  2014;4(8):896-904.
Prostate cancer (CaP) is the most prevalent cancer in males and treatment options are limited for advanced forms of the disease. Loss of the PTEN and p53 tumor suppressor genes is commonly observed in CaP, while their compound loss is often observed in advanced CaP. Here we show, that PARP inhibition triggers a p53-dependent cellular senescence in a PTEN-deficient setting in the prostate. Surprisingly, we also find that PARP-induced cellular senescence is morphed into an apoptotic response upon compound loss of PTEN and p53. We further show that superactivation of the pro-survival signalling PI3K-AKT pathway limits the efficacy of a PARP-single-agent treatment, and that PARP and PI3K inhibitors effectively synergize to suppress tumorigenesis in human CaP cell lines and in a Pten/p53 deficient mouse model of advanced CaP. Our findings therefore identify a combinatorial treatment with PARP and PI3K inhibitors as an effective option for PTEN-deficient CaP.
doi:10.1158/2159-8290.CD-13-0230
PMCID: PMC4125493  PMID: 24866151
PTEN; Prostate; PARP; PI3K; Senescence
3.  Targeting metabolic scavenging in pancreatic cancer 
Pancreatic tumor metabolism is rewired to facilitate survival and growth in a nutrient-depleted environment. This leads to a unique dependence on metabolic recycling and scavenging pathways, including NAD salvage. Targeting this pathway in pancreatic cancer disrupts metabolic homeostasis and impairs tumor growth.
doi:10.1158/1078-0432.CCR-13-2570
PMCID: PMC3930347  PMID: 24166909
4.  What a Tangled Web We Weave: Emerging Resistance Mechanisms to Inhibition of the Phosphoinositide 3-kinase Pathway 
Cancer discovery  2013;3(12):10.1158/2159-8290.CD-13-0063.
The phosphoinositide 3-kinase (PI3K) pathway is one of the most frequently mutated pathways in cancer, and is actively being pursued as a therapeutic target. Despite the importance of the PI3K pathway in cancer, durable responses to PI3K-pathway targeted therapies are uncommon with monotherapy. Several in vitro and xenograft models have elucidated compensatory signaling and genomic changes which may limit the therapeutic effectiveness of PI3K inhibitors in the clinic. Future clinical trials with prospective evaluation of tumor signaling and genomic changes are likely to identify novel resistance mechanisms as well as subsets of patients who may derive maximal benefit from PI3K pathway inhibitors.
doi:10.1158/2159-8290.CD-13-0063
PMCID: PMC3864542  PMID: 24265156
phosphoinositide 3-kinase; resistance; mTOR; cancer; signaling
5.  Lin28 enhances tissue repair by reprogramming cellular metabolism 
Cell  2013;155(4):778-792.
SUMMARY
Regeneration capacity declines with age, but why juvenile organisms show enhanced tissue repair remains unexplained. Lin28a, a highly-conserved RNA binding protein expressed during embryogenesis, plays roles in development, pluripotency and metabolism. To determine if Lin28a might influence tissue repair in adults, we engineered the reactivation of Lin28a expression in several models of tissue injury. Lin28a reactivation improved hair regrowth by promoting anagen in hair follicles, and accelerated regrowth of cartilage, bone and mesenchyme after ear and digit injuries. Lin28a inhibits let-7 microRNA biogenesis; however let-7 repression was necessary but insufficient to enhance repair. Lin28a bound to and enhanced the translation of mRNAs for several metabolic enzymes, thereby increasing glycolysis and oxidative phosphorylation (OxPhos). Lin28a-mediated enhancement of tissue repair was negated by OxPhos inhibition, whereas a pharmacologically-induced increase in OxPhos enhanced repair. Thus, Lin28a enhances tissue repair in some adult tissues by reprogramming cellular bioenergetics.
doi:10.1016/j.cell.2013.09.059
PMCID: PMC3917449  PMID: 24209617
6.  Phosphorylation of BRAF by AMPK impairs BRAF-KSR1 association and cell proliferation 
Molecular cell  2013;52(2):161-172.
Summary
BRAF is an oncogenic protein kinase that drives cell growth and proliferation through the MEK-ERK signaling pathway. BRAF inhibitors have demonstrated anti-tumor efficacy in melanoma therapy, but have also found to be associated with the development of cutaneous squamous cell carcinomas (cSCC) in certain patients. Here, we report that BRAF is phosphorylated at Ser729 by AMP-activated protein kinase (AMPK), a critical energy sensor. This phosphorylation promotes the association of BRAF with 14-3-3 proteins and disrupts its interaction with the KSR1 scaffolding protein, leading to attenuation of the MEK-ERK signaling. We also show that phosphorylation of BRAF by AMPK impairs keratinocyte cell proliferation and cell cycle progression. Furthermore, AMPK activation attenuates BRAF inhibitor-induced ERK hyperactivation in keratinocytes and epidermal hyperplasia in mouse skin. Our findings reveal a mechanism for regulating BRAF signaling in response to energy stress and suggest a strategy for preventing the development of cSCC associated with BRAF-targeted therapy.
doi:10.1016/j.molcel.2013.08.044
PMCID: PMC3892895  PMID: 24095280
8.  Idelalisib — A PI3Kδ Inhibitor for B-Cell Cancers 
The New England journal of medicine  2014;370(11):1061-1062.
doi:10.1056/NEJMe1400055
PMCID: PMC4088325  PMID: 24620870
9.  Metabolic and Functional Genomic Studies Identify Deoxythymidylate Kinase as a target in LKB1 Mutant Lung Cancer 
Cancer discovery  2013;3(8):870-879.
The LKB1/STK11 tumor suppressor encodes a serine/threonine kinase which coordinates cell growth, polarity, motility, and metabolism. In non-small cell lung cancer, LKB1 is somatically inactivated in 25-30% of cases, often concurrently with activating KRAS mutation. Here, we employed an integrative approach to define novel therapeutic targets in KRAS-driven LKB1 mutant lung cancers. High-throughput RNAi screens in lung cancer cell lines from genetically engineered mouse models driven by activated KRAS with or without coincident Lkb1 deletion led to the identification of Dtymk, encoding deoxythymidylate kinase which catalyzes dTTP biosynthesis, as synthetically lethal with Lkb1 deficiency in mouse and human lung cancer lines. Global metabolite profiling demonstrated that Lkb1-null cells had striking decreases in multiple nucleotide metabolites as compared to the Lkb1-wt cells. Thus, LKB1 mutant lung cancers have deficits in nucleotide metabolism conferring hypersensitivity to DTYMK inhibition, suggesting that DTYMK is a potential therapeutic target in this aggressive subset of tumors.
doi:10.1158/2159-8290.CD-13-0015
PMCID: PMC3753578  PMID: 23715154
LKB1; KRAS; DTYMK; CHEK1; NSCLC; GEMM-derived cell line; genome wide RNAi screen; metabolic profiling
10.  microRNA-antagonism regulates breast cancer stemness and metastasis via TET family dependent chromatin remodeling 
Cell  2013;154(2):311-324.
SUMMARY
Tumor cells metastasize to distant organs through genetic and epigenetic alterations, including changes in microRNA (miR) expression. Here we find miR-22 triggers epithelial-mesenchymal transition (EMT), enhances invasiveness and promotes metastasis in mouse xenografts. In a conditional mammary gland-specific transgenic (TG) mouse model, we show that miR-22 enhances mammary gland side-branching, expands the stem cell compartment, and promotes tumor development. Critically, miR-22 promotes aggressive metastatic disease in MMTV-miR-22 TG mice, as well as compound MMTV-neu or -PyVT-miR-22 TG mice. We demonstrate that miR-22 exerts its metastatic potential by silencing anti-metastatic miR-200 through direct targeting of the TET (Ten eleven translocation) family of methylcytocine dioxygenases, thereby inhibiting demethylation of the mir-200 promoter. Finally, we show that miR-22 overexpression correlates with poor clinical outcomes and silencing of the TET-miR-200 axis in patients. Taken together, our findings implicate miR-22 as a crucial epigenetic modifier and promoter of EMT and breast cancer stemness towards metastasis.
doi:10.1016/j.cell.2013.06.026
PMCID: PMC3767157  PMID: 23830207
11.  Metformin decreases glucose oxidation and increases the dependency of prostate cancer cells on reductive glutamine metabolism 
Cancer research  2013;73(14):4429-4438.
Metformin inhibits cancer cell proliferation and epidemiology studies suggest an association with increased survival in cancer patients taking metformin, however, the mechanism by which metformin improves cancer outcomes remains controversial. To explore how metformin might directly affect cancer cells, we analyzed how metformin altered the metabolism of prostate cancer cells and tumors. We found that metformin decreased glucose oxidation and increased dependency on reductive glutamine metabolism in both cancer cell lines and in a mouse model of prostate cancer. Inhibition of glutamine anaplerosis in the presence of metformin further attenuated proliferation while increasing glutamine metabolism rescued the proliferative defect induced by metformin. These data suggest that interfering with glutamine may synergize with metformin to improve outcomes in patients with prostate cancer.
doi:10.1158/0008-5472.CAN-13-0080
PMCID: PMC3930683  PMID: 23687346
12.  Depletion of a Putatively Druggable Class of Phosphatidylinositol Kinases Inhibits Growth of p53-Null Tumors 
Cell  2013;155(4):844-857.
SUMMARY
Here, we show that a subset of breast cancers express high levels of the type 2 phosphatidylinositol-5-phosphate 4-kinases α and/or β (PI5P4Kα and β) and provide evidence that these kinases are essential for growth in the absence of p53. Knocking down PI5P4Kα and β in a breast cancer cell line bearing an amplification of the gene encoding PI5P4K β and deficient for p53 impaired growth on plastic and in xenografts. This growth phenotype was accompanied by enhanced levels of reactive oxygen species (ROS) leading to senescence. Mice with homozygous deletion of both TP53 and PIP4K2B were not viable, indicating a synthetic lethality for loss of these two genes. Importantly however, PIP4K2A−/−, PIP4K2B+/−, and TP53−/− mice were viable and had a dramatic reduction in tumor formation compared to TP53−/− littermates. These results indicate that inhibitors of PI5P4Ks could be effective in preventing or treating cancers with mutations in TP53.
doi:10.1016/j.cell.2013.09.057
PMCID: PMC4070383  PMID: 24209622
13.  Pancreatic cancers rely on a novel glutamine metabolism pathway to maintain redox balance 
Cell Cycle  2013;12(13):1987-1988.
doi:10.4161/cc.25307
PMCID: PMC3737294  PMID: 23759579
cancer metabolism; NADPH; aspartate aminotransferase; malic enzyme; glutamate dehydrogenase
15.  PI 3-kinase and disease 
Cancer & Metabolism  2014;2(Suppl 1):O30.
doi:10.1186/2049-3002-2-S1-O30
PMCID: PMC4073074
16.  The mTORC1 pathway stimulates glutamine metabolism and cell proliferation by repressing SIRT4 
Cell  2013;153(4):840-854.
Summary
Proliferating mammalian cells use glutamine as a source of nitrogen and as a key anaplerotic source to provide metabolites to the tricarboxylic acid cycle (TCA) for biosynthesis. Recently, mTORC1 activation has been correlated with increased nutrient uptake and metabolism, but no molecular connection to glutaminolysis has been reported. Here, we show that mTORC1 promotes glutamine anaplerosis by activating glutamate dehydrogenase (GDH). This regulation requires transcriptional repression of SIRT4, the mitochondrial-localized sirtuin that inhibits GDH. Mechanistically, mTORC1 represses SIRT4 by promoting the proteasome-mediated destabilization of cAMP response element binding-2 (CREB2). Thus, a relationship between mTORC1, SIRT4 and cancer is suggested by our findings. Indeed, SIRT4 expression is reduced in human cancer, and its overexpression reduces cell proliferation, transformation and tumor development. Finally, our data indicate that targeting nutrient metabolism in energy-addicted cancers with high mTORC1 signaling may be an effective therapeutic approach.
doi:10.1016/j.cell.2013.04.023
PMCID: PMC3684628  PMID: 23663782
17.  AMPK-dependent degradation of TXNIP upon energy stress leads to enhanced glucose uptake via GLUT1 
Molecular cell  2013;49(6):1167-1175.
Summary
TXNIP is an α-arrestin family protein that is induced in response to glucose elevation. It has been shown to provide a negative feedback loop to regulate glucose uptake into cells, though the biochemical mechanism of action has been obscure. Here, we report that TXNIP suppresses glucose uptake directly by binding to the glucose transporter, Glut1, inducing Glut1 internalization through clathrin coated pits, as well as indirectly by reducing the level of Glut1 mRNA. In addition, we show that energy stress results in phosphorylation of TXNIP by AMP-dependent protein kinase (AMPK), leading to its rapid degradation. This suppression of TXNIP results in an acute increase in Glut1 function and an increase in Glut1 mRNA (hence total protein levels) for long-term adaptation. The glucose influx through GLUT1 restores ATP/ADP ratios in the short run and ultimately induces TXNIP protein production to suppress glucose uptake once energy homeostasis is reestablished.
doi:10.1016/j.molcel.2013.01.035
PMCID: PMC3615143  PMID: 23453806
18.  Inhibition of pyruvate kinase M2 by reactive oxygen species contributes to cellular antioxidant responses 
Science (New York, N.Y.)  2011;334(6060):1278-1283.
Control of intracellular reactive oxygen species (ROS) concentrations is critical for cancer cell survival. We show that, in human lung cancer cells, acute increases in intracellular concentrations of ROS caused inhibition of the glycolytic enzyme pyruvate kinase M2 (PKM2) through oxidation of Cys358. This inhibition of PKM2 is required to divert glucose flux into the pentose phosphate pathway and thereby generate sufficient reducing potential for detoxification of ROS. Lung cancer cells in which endogenous PKM2 was replaced with the Cys358 to Ser358 oxidation-resistant mutant exhibited increased sensitivity to oxidative stress and impaired tumor formation in a xenograft model. Besides promoting metabolic changes required for proliferation, the regulatory properties of PKM2 may confer an additional advantage to cancer cells by allowing them to withstand oxidative stress.
doi:10.1126/science.1211485
PMCID: PMC3471535  PMID: 22052977
19.  Getting Knit-PI3Ky: PIK3CA Mutation Status to Direct Multimodality Therapy? 
Clinical Cancer Research  2009;15(22):6748-6750.
There is high morbidity associated with local recurrence of rectal cancer. However, the adjuvant therapies given to prevent such recurrences also have significant side effects and associated risks. The ability to select patients with the highest risk of recurrence and greatest therapeutic response will improve rectal cancer care.
doi:10.1158/1078-0432.CCR-09-2305
PMCID: PMC3400141  PMID: 19903790
20.  Fetal deficiency of Lin28 programs life-long aberrations in growth and glucose metabolism 
Stem cells (Dayton, Ohio)  2013;31(8):1563-1573.
LIN28A/B are RNA binding proteins implicated by genetic association studies in human growth and glucose metabolism. Mice with ectopic over-expression of Lin28a have shown related phenotypes. Here we describe the first comprehensive analysis of the physiologic consequences of Lin28a and Lin28b deficiency in knockout (KO) mice. Lin28a/b-deficiency led to dwarfism starting at different ages, and compound gene deletions showed a cumulative dosage effect on organismal growth. Conditional gene deletion at specific developmental stages revealed that fetal but neither neonatal nor adult deficiency resulted in growth defects and aberrations in glucose metabolism. Tissue-specific KO mice implicated skeletal muscle-deficiency in the abnormal programming of adult growth and metabolism. The effects of Lin28b KO can be rescued by Tsc1 haplo-insufficiency in skeletal muscles. Our data implicate fetal expression of Lin28a/b in the regulation of life-long effects on metabolism and growth, and demonstrate that fetal Lin28b acts at least in part via mTORC1 signaling.
doi:10.1002/stem.1423
PMCID: PMC3775935  PMID: 23666760
Lin28a; Lin28b; dwarfism; growth; glucose metabolism; diabetes; let-7; mTOR
21.  A Fluorescent Reporter of AMPK activity and Cellular Energy Stress 
Cell metabolism  2011;13(4):476-486.
SUMMARY
AMP-activated protein kinase (AMPK) is activated when the AMP/ATP ratio in cells is elevated due to energy stress. Here we describe a biosensor, AMPKAR, which exhibits enhanced fluorescence resonance energy transfer (FRET) in response to phosphorylation by AMPK, allowing spatio-temporal monitoring of AMPK activity in single cells. We show that this reporter responds to a variety of stimuli that are known to induce energy stress and that the response is dependent on AMPK α1 & α2 and on the upstream kinase, LKB1. Interestingly we found that AMPK activation is confined to the cytosol in response to energy stress but can be observed in both the cytosol and nucleus in response to calcium elevation. Finally, using this probe with U2OS cells in a microfluidics device, we observed a very high cell-to-cell variability in the amplitude and time course of AMPK activation and recovery in response to pulses of glucose deprivation.
doi:10.1016/j.cmet.2011.03.006
PMCID: PMC3070961  PMID: 21459332
22.  Metabolic stress controls mTORC1 lysosomal localization and dimerization by regulating the TTT-RUVBL1/2 complex 
Molecular cell  2012;49(1):172-185.
Summary
The metabolism of glucose and glutamine, primary carbon-sources utilized by mitochondria to generate energy and macromolecules for cell growth, is directly regulated by mTORC1. We show that glucose and glutamine, by supplying carbons to the TCA cycle to produce ATP, positively feed back to mTORC1 through an AMPK-, TSC1/2-, and Rag-independent mechanism by regulating mTORC1 assembly and its lysosomal localization. We discovered that ATP-dependent TTT-RUVBL1/2 complex was disassembled and repressed by energy depletion, resulting in its decreased interaction with mTOR. The TTT-RUVBL complex was necessary for the interaction between mTORC1 and Rag, and formation of mTORC1 obligate dimers. In cancer tissues, TTT-RUVBL complex mRNAs were elevated, and positively correlated with transcripts encoding proteins of anabolic metabolism and mitochondrial function - all mTORC1-regulated processes. Thus, the TTT-RUVBL1/2 complex responds to the cell’s metabolic state, directly regulating the functional assembly of mTORC1, and indirectly controlling the nutrient signal from Rags to mTORC1.
doi:10.1016/j.molcel.2012.10.003
PMCID: PMC3545014  PMID: 23142078
23.  A co-clinical approach identifies mechanisms and potential therapies for androgen deprivation resistance in prostate cancer 
Nature genetics  2013;45(7):747-755.
Here we report an integrated analysis that leverages data from treatment of genetic mouse models of prostate cancer along with clinical data from patients to elucidate new mechanisms of castration resistance. We show that castration counteracts tumor progression in a Pten-loss driven mouse model of prostate cancer through the induction of apoptosis and proliferation block. Conversely, this response is bypassed upon deletion of either Trp53 or Lrf together with Pten, leading to the development of castration resistant prostate cancer (CRPC). Mechanistically, the integrated acquisition of data from mouse models and patients identifies the expression patterns of XAF1-XIAP/SRD5A1 as a predictive and actionable signature for CRPC. Importantly, we show that combined inhibition of XIAP, SRD5A1, and AR pathways overcomes castration resistance. Thus, our co-clinical approach facilitates stratification of patients and the development of tailored and innovative therapeutic treatments.
doi:10.1038/ng.2650
PMCID: PMC3787876  PMID: 23727860
24.  Heterogeneity of tumor-induced gene expression changes in the human metabolic network 
Nature biotechnology  2013;31(6):522-529.
Reprogramming of cellular metabolism is an emerging hallmark of neoplastic transformation. However, it is not known how metabolic gene expression in tumors differs from that in normal tissues, or whether different tumor types exhibit similar metabolic changes. Here we compare expression patterns of metabolic genes across 22 diverse types of human tumors. Overall, the metabolic gene expression program in tumors is similar to that in the corresponding normal tissues. Although expression changes of some metabolic pathways (e.g., up-regulation of nucleotide biosynthesis and glycolysis) are frequently observed across tumors, expression changes of other pathways (e.g., oxidative phosphorylation and the tricarboxylic acid (TCA) cycle) are very heterogeneous. Our analysis also suggests that the expression changes of major metabolic processes across tumors can be rationalized in terms of several principal components. On the level of individual biochemical reactions, many hundreds of metabolic isoenzymes show significant and tumor-specific expression changes. These isoenzymes are potential targets for anticancer therapy.
doi:10.1038/nbt.2530
PMCID: PMC3681899  PMID: 23604282
25.  Glutamine supports pancreatic cancer growth through a Kras-regulated metabolic pathway 
Nature  2013;496(7443):101-105.
Cancer cells exhibit metabolic dependencies that distinguish them from their normal counterparts1. Among these addictions is an increased utilization of the amino acid glutamine (Gln) to fuel anabolic processes2. Indeed, the spectrum of Gln-dependent tumors and the mechanisms whereby Gln supports cancer metabolism remain areas of active investigation. Here we report the identification of a non-canonical pathway of Gln utilization in human pancreatic ductal adenocarcinoma (PDAC) cells that is required for tumor growth. While most cells utilize glutamate dehydrogenase (GLUD1) to convert Gln-derived glutamate (Glu) into α-ketoglutarate in the mitochondria to fuel the tricarboxylic acid (TCA) cycle, PDAC relies on a distinct pathway to fuel the TCA cycle such that Gln-derived aspartate is transported into the cytoplasm where it can be converted into oxaloacetate (OAA) by aspartate transaminase (GOT1). Subsequently, this OAA is converted into malate and then pyruvate, ostensibly increasing the NADPH/NADP+ ratio which can potentially maintain the cellular redox state. Importantly, PDAC cells are strongly dependent on this series of reactions, as Gln deprivation or genetic inhibition of any enzyme in this pathway leads to an increase in reactive oxygen species and a reduction in reduced glutathione. Moreover, knockdown of any component enzyme in this series of reactions also results in a pronounced suppression of PDAC growth in vitro and in vivo. Furthermore, we establish that the reprogramming of Gln metabolism is mediated by oncogenic Kras, the signature genetic alteration in PDAC, via the transcriptional upregulation and repression of key metabolic enzymes in this pathway. The essentiality of this pathway in PDAC and the fact that it is dispensable in normal cells may provide novel therapeutic approaches to treat these refractory tumors.
doi:10.1038/nature12040
PMCID: PMC3656466  PMID: 23535601

Results 1-25 (132)