Although most oncogenic phenotypes of PTEN loss are attributed to AKT activation, AKT alone is not sufficient to induce all of the biological activities associated with PTEN inactivation. We searched for additional PTEN-regulated pathways through gene set enrichment analysis (GSEA) and identified genes associated with JNK activation. PTEN null cells exhibit higher JNK activity, and genetic studies demonstrate that JNK functions parallel to and independently of AKT. Furthermore, PTEN deficiency sensitizes cells to JNK inhibition and negative feedback regulation of PI3K was impaired in PTEN null cells. Akt and JNK activation are highly correlated in human prostate cancer. These findings implicate JNK in PI3K-driven cancers and demonstrate the utility of GSEA to identify functional pathways using genetically defined systems.

Cancer cells may overcome growth factor dependence by deregulating oncogenic and/or tumor suppressor pathways that affect their metabolism, or by activating metabolic pathways de novo with targeted mutations in critical metabolic enzymes. It is unknown whether human prostate tumors develop a similar metabolic response to different oncogenic drivers or a particular oncogenic event results in its own metabolic reprogramming. Akt and Myc are arguably the most prevalent driving oncogenes in prostate cancer. Mass spectrometry-based metabolite profiling was performed on immortalized human prostate epithelial cells transformed by AKT1 or MYC, transgenic mice driven by the same oncogenes under the control of a prostate-specific promoter, and human prostate specimens characterized for the expression and activation of these oncoproteins. Integrative analysis of these metabolomic datasets revealed that AKT1 activation was associated with accumulation of aerobic glycolysis metabolites, whereas MYC overexpression was associated with dysregulated lipid metabolism. Selected metabolites that differentially accumulated in the MYC-high vs. AKT1-high tumors, or in normal vs. tumor prostate tissue by untargeted metabolomics, were validated using absolute quantitation assays. Importantly, the AKT1/MYC status was independent of Gleason grade and pathologic staging. Our findings show how prostate tumors undergo a metabolic reprogramming which reflects their molecular phenotypes, with implications for the development of metabolic diagnostics and targeted therapeutics.

S tringent response is the main strategy used by bacteria to cope with fluctuating nutrient supplies and metabolic and oxidative stresses 1,2 . This process rapidly redirects energy from cell proliferation toward stress survival by reduction of biosynthesis, conservation of ATP and blockage of GTP production 3 . The stringent response is triggered by the accumulation of the bacterial 'alarmone' (p)ppGpp (guanosine tetra-or penta-phosphate, shortened as ppGpp below) through the regulation of ppGpp synthetases and hydrolases in the RelA and SpoT homologue family 2 .Recent studies suggest that the stringent response may also function in metazoans, as metazoan genomes encode a homologue of bacterial SpoT-MESH1 (Metazoan SpoT Homologue 1, encoded by HDDC3)-that can hydrolyse ppGpp in vitro and functionally complement SpoT in Escherichia coli 4 . Furthermore, Mesh1 deletion in Drosophila displays impaired starvation resistance and extensive transcriptional reprogramming 4 . Despite these supporting lines of evidence, neither ppGpp nor its synthetase has been discovered in metazoans, thus obscuring the genuine function and the relevant substrate(s) of MESH1 in mammalian cells. Here, we have identified NADPH as an efficient substrate of MESH1. MESH1 is a cytosolic NADPH phosphatase that is induced under stress conditions, leading to the NADPH depletion and ferroptosis-a novel form of iron-dependent regulated cell death characterized by lipid peroxidation 5 . Accordingly, MESH1 removal preserves the NADPH level in stressed cells and promotes their ferroptotic survival.Critical to the bacterial stringent response is the rapid relocation of resources from proliferation toward stress survival through the respective accumulation and degradation of (p)ppGpp by RelA and SpoT homologues. While mammalian genomes encode MESH1, a homologue of the bacterial (p)ppGpp hydrolase SpoT, neither (p)ppGpp nor its synthetase has been identified in mammalian cells. Here, we show that human MESH1 is an efficient cytosolic NADPH phosphatase that facilitates ferroptosis. Visualization of the MESH1-NADPH crystal structure revealed a bona fide affinity for the NADPH substrate. Ferroptosisinducing erastin or cystine deprivation elevates MESH1, whose overexpression depletes NADPH and sensitizes cells to ferroptosis, whereas MESH1 depletion promotes ferroptosis survival by sustaining the levels of NADPH and GSH and by reducing lipid peroxidation. The ferroptotic protection by MESH1 depletion is ablated by suppression of the cytosolic NAD(H) kinase, NADK, but not its mitochondrial counterpart NADK2. Collectively, these data shed light on the importance of cytosolic NADPH levels and their regulation under ferroptosis-inducing conditions in mammalian cells.

Histidine kinases are key regulators in the bacterial two-component systems that mediate the cellular response to environmental changes. The vast majority of the sensor histidine kinases belong to the bifunctional HisKA family, displaying both kinase and phosphatase activities toward their substrates. The molecular mechanisms regulating the opposing activities of these enzymes are not well understood. Through a combined NMR and crystallographic study on the histidine kinase HK853 and its response regulator RR468 from Thermotoga maritima, here we report a pH-mediated conformational switch of HK853 that shuts off its phosphatase activity under acidic conditions. Such a pH-sensing mechanism is further demonstrated in the EnvZ-OmpR two-component system from Salmonella enterica in vitro and in vivo, which directly contributes to the bacterial infectivity. Our finding reveals a broadly conserved mechanism that regulates the phosphatase activity of the largest family of bifunctional histidine kinases in response to the change of environmental pH.

Gene expression profiling has identified several potentially useful gene signatures for predicting outcome or for selecting targeted therapy. However, these signatures have been developed in fresh or frozen tissue, and there is a need to apply them to routinely processed samples. Here, we demonstrate the feasibility of a potentially high-throughput methodology combining automated in situ hybridization with quantum dot-labeled oligonucleotide probes followed by spectral imaging for the detection and subsequent deconvolution of multiple signals. This method is semiautomated and quantitative and can be applied to formalin-fixed, paraffin-embedded tissues. We have combined dual in situ hybridization with immunohistochemistry, enabling simultaneous measurement of gene expression and cell lineage determination. The technique achieves levels of sensitivity and specificity sufficient for the potential application of known expression signatures to biopsy specimens in a semiquantitative way, and the semiautomated nature of the method enables application to highthroughput studies.

15 16 17 18 19 2 Nutrient deprivation triggers stringent response in bacteria, allowing rapid 20 reallocation of resources from proliferation toward stress survival. Critical to this process 21 is the accumulation/degradation of (p)ppGpp regulated by the RelA/SpoT homologues. 22 While mammalian genomes encode MESH1, a homologue of the bacterial (p)ppGpp 23 hydrolase SpoT, neither (p)ppGpp nor its synthetase has been identified in mammalian 24 cells. Therefore, the function of MESH1 remains a mystery. Here, we report that human 25 MESH1 is an efficient cytosolic NADPH phosphatase, an unexpected enzymatic activity 26 that is captured by the crystal structure of the MESH1-NADPH complex. MESH1 27 depletion promotes cell survival under ferroptosis-inducing conditions by sustaining the 28 level of NADPH, an effect that is reversed by the simultaneous depletion of the cytosolic 29 NAD(H) kinase, NADK, but not its mitochondrial counterpart NADK2. Importantly, 30 MESH1 depletion also triggers extensive transcriptional changes that are distinct from the 31 canonical integrated stress response but resemble the bacterial stringent response, 32implicating MESH1 in a previously uncharacterized stress response in mammalian cells. 33 Stringent response is the main strategy for bacteria to cope with fluctuating nutrient 34 supplies and metabolic stresses 1,2 . During this process, alterations in transcriptional and 35 metabolic profiles rapidly redirect energy from cell proliferation toward stress survival by 36 reduction of biosynthesis, conservation of ATP, and blockage of GTP production 3 . The stringent 37 response is triggered by the accumulation of the bacterial "alarmone" (p)ppGpp (guanosine tetra-38 or penta-phosphate, shortened as ppGpp below) through the regulation of ppGpp synthetases and 39 hydrolases in the RelA/SpoT Homologue family 2 . Recent studies suggest that the stringent 40 response may also function in metazoans, as metazoan genomes encode a homologue of bacterial 41 SpoT, MESH1 (Metazoan SpoT Homolog 1) that hydrolyzes ppGpp in vitro and functionally 42 3 complements SpoT in E. coli 4 . Furthermore, Mesh1 deletion in Drosophila displays impaired 43 starvation resistance, a susceptibility that is fully rescued by Mesh1 expression 4 . Despite these 44 supporting lines of evidence, the existence of a ppGpp-mediated stringent response pathway in 45 metazoans has serious impediments, as neither ppGpp nor its synthetase has been discovered in 46 metazoans, and no physiological substrate for MESH1 has been reported. 47 48 MESH1 is an efficient NADPH phosphatase 49 We reasoned that MESH1 may function through alternate metabolic substrate(s) from 50 ppGpp in mammalian cells. Consequently, we tested purified-recombinant human MESH1 51 (hMESH1) against a set of common metabolites, such as UDP-glucose (uridine diphosphate 52 glucose), GDP (guanosine diphosphate), CDP (cytidine diphosphate), pyrophosphate, creatine 53 phosphate, GDP-fucose, thiamine pyrophosphate and various form of inositol phosphates, but 54 ...

Collagen I, the most abundant protein in humans, is ubiquitous in solid tumors where it provides a rich source of exploitable metabolic fuel for cancer cells. While tumor cells were unable to exploit collagen directly, here we show they can usurp metabolic byproducts of collagen-consuming tumor-associated stroma. Using genetically engineered mouse models, we discovered that solid tumor growth depends upon collagen binding and uptake mediated by the TEM8/ANTXR1 cell surface protein in tumor-associated stroma. Tumor-associated stromal cells processed collagen into glutamine, which was then released and internalized by cancer cells. Under chronic nutrient starvation, a condition driven by the high metabolic demand of tumors, cancer cells exploited glutamine to survive, an effect that could be reversed by blocking collagen uptake with TEM8 neutralizing antibodies. These studies reveal that cancer cells exploit collagen-consuming stromal cells for survival, exposing an important vulnerability across solid tumors with implications for developing improved anticancer therapy.

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