Cancer Burden Is Controlled by Mural Cell-ß3-Integrin Regulated Crosstalk with Tumor Cells.

Enhanced blood vessel (BV) formation is thought to drive tumor growth through elevated nutrient delivery. However, this observation has overlooked potential roles for mural cells in directly affecting tumor growth independent of BV function. Here we provide clinical data correlating high percentages of mural-ß3-integrin-negative tumor BVs with increased tumor sizes but no effect on BV numbers. Mural-ß3-integrin loss also enhances tumor growth in implanted and autochthonous mouse tumor models with no detectable effects on BV numbers or function. At a molecular level, mural-cell ß3-integrin loss enhances signaling via FAK-p-HGFR-p-Akt-p-p65, driving CXCL1, CCL2, and TIMP-1 production. In particular, mural-cell-derived CCL2 stimulates tumor cell MEK1-ERK1/2-ROCK2-dependent signaling and enhances tumor cell survival and tumor growth. Overall, our data indicate that mural cells can control tumor growth via paracrine signals regulated by ß3-integrin, providing a previously unrecognized mechanism of cancer growth control.

Proteomic Characterization of Acute Myeloid Leukemia for Precision Medicine.

Acute myeloid leukemia (AML) is a highly heterogeneous cancer of the hematopoietic system with no cure for most patients. In addition to chemotherapy, treatment options for AML include recently approved therapies that target proteins with roles in AML pathobiology, such as FLT3, BLC2, and IDH1/2. However, due to disease complexity, these therapies produce very diverse responses, and survival rates are still low. Thus, despite considerable advances, there remains a need for therapies that target different aspects of leukemic biology and for associated biomarkers that define patient populations likely to respond to each available therapy. To meet this need, drugs that target different AML vulnerabilities are currently in advanced stages of clinical development. Here, we review proteomics and phosphoproteomics studies that aimed to provide insights into AML biology and clinical disease heterogeneity not attainable with genomic approaches. To place the discussion in context, we first provide an overview of genetic and clinical aspects of the disease, followed by a summary of proteins targeted by compounds that have been approved or are under clinical trials for AML treatment and, if available, the biomarkers that predict responses. We then discuss proteomics and phosphoproteomics studies that provided insights into AML pathogenesis, from which potential biomarkers and drug targets were identified, and studies that aimed to rationalize the use of synergistic drug combinations. When considered as a whole, the evidence summarized here suggests that proteomics and phosphoproteomics approaches can play a crucial role in the development and implementation of precision medicine for AML patients.

Systematic identification of biochemical networks in cancer cells by functional pathway inference analysis.

MOTIVATION: Pathway inference methods are important for annotating the genome, for providing insights into the mechanisms of biochemical processes and allow the discovery of signalling members and potential new drug targets. Here, we tested the hypothesis that genes with similar impact on cell viability across multiple cell lines belong to a common pathway, thus providing a conceptual basis for a pathway inference method based on correlated anti-proliferative gene properties. METHODS: To test this concept, we used recently available large-scale RNAi screens to develop a method, termed functional pathway inference analysis (FPIA), to systemically identify correlated gene dependencies. RESULTS: To assess FPIA, we initially focused on PI3K/AKT/MTOR signalling, a prototypic oncogenic pathway for which we have a good sense of ground truth. Dependencies for AKT1, MTOR and PDPK1 were among the most correlated with those for PIK3CA (encoding PI3Kα), as returned by FPIA, whereas negative regulators of PI3K/AKT/MTOR signalling, such as PTEN were anti-correlated. Following FPIA, MTOR, PIK3CA and PIK3CB produced significantly greater correlations for genes in the PI3K-Akt pathway versus other pathways. Application of FPIA to two additional pathways (p53 and MAPK) returned expected associations (e.g. MDM2 and TP53BP1 for p53 and MAPK1 and BRAF for MEK1). Over-representation analysis of FPIA-returned genes enriched the respective pathway, and FPIA restricted to specific tumour lineages uncovered cell type-specific networks. Overall, our study demonstrates the ability of FPIA to identify members of pro-survival biochemical pathways in cancer cells. AVAILABILITY AND IMPLEMENTATION: FPIA is implemented in a new R package named 'cordial' freely available from https://github.com/CutillasLab/cordial. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Community detection in empirical kinase networks identifies new potential members of signalling pathways.

Phosphoproteomics allows one to measure the activity of kinases that drive the fluxes of signal transduction pathways involved in biological processes such as immune function, senescence and cell growth. However, deriving knowledge of signalling network circuitry from these data is challenging due to a scarcity of phosphorylation sites that define kinase-kinase relationships. To address this issue, we previously identified around 6,000 phosphorylation sites as markers of kinase-kinase relationships (that may be conceptualised as network edges), from which empirical cell-model-specific weighted kinase networks may be reconstructed. Here, we assess whether the application of community detection algorithms to such networks can identify new components linked to canonical signalling pathways. Phosphoproteomics data from acute myeloid leukaemia (AML) cells treated separately with PI3K, AKT, MEK and ERK inhibitors were used to reconstruct individual kinase networks. We used modularity maximisation to detect communities in each network, and selected the community containing the main target of the inhibitor used to treat cells. These analyses returned communities that contained known canonical signalling components. Interestingly, in addition to canonical PI3K/AKT/mTOR members, the community assignments returned TTK (also known as MPS1) as a likely component of PI3K/AKT/mTOR signalling. We drew similar insights from an external phosphoproteomics dataset from breast cancer cells treated with rapamycin and oestrogen. We confirmed this observation with wet-lab laboratory experiments showing that TTK phosphorylation was decreased in AML cells treated with AKT and MTOR inhibitors. This study illustrates the application of community detection algorithms to the analysis of empirical kinase networks to uncover new members linked to canonical signalling pathways.

Causal Role for Neutrophil Elastase in Thoracic Aortic Dissection in Mice.

BACKGROUND: Thoracic aortic dissection (TAD) is a life-threatening aortic disease without effective medical treatment. Increasing evidence has suggested a role for NE (neutrophil elastase) in vascular diseases. In this study, we aimed at investigating a causal role for NE in TAD and exploring the molecular mechanisms involved. METHODS: ß-aminopropionitrile monofumarate was administrated in mice to induce TAD. NE deficiency mice, pharmacological inhibitor GW311616A, and adeno-associated virus-2-mediated in vivo gene transfer were applied to explore a causal role for NE and associated target gene in TAD formation. Multiple functional assays and biochemical analyses were conducted to unravel the underlying cellular and molecular mechanisms of NE in TAD. RESULTS: NE aortic gene expression and plasma activity was significantly increased during ß-aminopropionitrile monofumarate-induced TAD and in patients with acute TAD. NE deficiency prevents ß-aminopropionitrile monofumarate-induced TAD onset/development, and GW311616A administration ameliorated TAD formation/progression. Decreased levels of neutrophil extracellular traps, inflammatory cells, and MMP (matrix metalloproteinase)-2/9 were observed in NE-deficient mice. TBL1x (F-box-like/WD repeat-containing protein TBL1x) has been identified as a novel substrate and functional downstream target of NE in TAD. Loss-of-function studies revealed that NE mediated inflammatory cell transendothelial migration by modulating TBL1x-LTA4H (leukotriene A4 hydrolase) signaling and that NE regulated smooth muscle cell phenotype modulation under TAD pathological condition by regulating TBL1x-MECP2 (methyl CpG-binding protein 2) signal axis. Further mechanistic studies showed that TBL1x inhibition decreased the binding of TBL1x and HDAC3 (histone deacetylase 3) to MECP2 and LTA4H gene promoters, respectively. Finally, adeno-associated virus-2-mediated Tbl1x gene knockdown in aortic smooth muscle cells confirmed a regulatory role for TBL1x in NE-mediated TAD formation. CONCLUSIONS: We unravel a critical role of NE and its target TBL1x in regulating inflammatory cell migration and smooth muscle cell phenotype modulation in the context of TAD. Our findings suggest that the NE-TBL1x signal axis represents a valuable therapeutic for treating high-risk TAD patients.

eEF2K Activity Determines Synergy to Cotreatment of Cancer Cells With PI3K and MEK Inhibitors.

PI3K-mammalian target of rapamycin and MAPK/ERK kinase (MEK)/mitogen-activated protein kinase (MAPK) are the most frequently dysregulated signaling pathways in cancer. A problem that limits the success of therapies that target individual PI3K-MAPK members is that these pathways converge to regulate downstream functions and often compensate each other, leading to drug resistance and transient responses to therapy. In order to overcome resistance, therapies based on cotreatments with PI3K/AKT and MEK/MAPK inhibitors are now being investigated in clinical trials, but the mechanisms of sensitivity to cotreatment are not fully understood. Using LC-MS/MS-based phosphoproteomics, we found that eukaryotic elongation factor 2 kinase (eEF2K), a key convergence point downstream of MAPK and PI3K pathways, mediates synergism to cotreatment with trametinib plus pictilisib (which target MEK1/2 and PI3Kα/δ, respectively). Inhibition of eEF2K by siRNA or with a small molecule inhibitor reversed the antiproliferative effects of the cotreatment with PI3K plus MEK inhibitors in a cell model-specific manner. Systematic analysis in 12 acute myeloid leukemia cell lines revealed that eEF2K activity was increased in cells for which PI3K plus MEKi cotreatment is synergistic, while PKC potentially mediated resistance to such cotreatment. Together, our study uncovers eEF2K activity as a key mediator of responses to PI3Ki plus MEKi and as a potential biomarker to predict synergy to cotreatment in cancer cells.

Principles of phosphoproteomics and applications in cancer research.

Phosphorylation constitutes the most common and best-studied regulatory post-translational modification in biological systems and archetypal signalling pathways driven by protein and lipid kinases are disrupted in essentially all cancer types. Thus, the study of the phosphoproteome stands to provide unique biological information on signalling pathway activity and on kinase network circuitry that is not captured by genetic or transcriptomic technologies. Here, we discuss the methods and tools used in phosphoproteomics and highlight how this technique has been used, and can be used in the future, for cancer research. Challenges still exist in mass spectrometry phosphoproteomics and in the software required to provide biological information from these datasets. Nevertheless, improvements in mass spectrometers with enhanced scan rates, separation capabilities and sensitivity, in biochemical methods for sample preparation and in computational pipelines are enabling an increasingly deep analysis of the phosphoproteome, where previous bottlenecks in data acquisition, processing and interpretation are being relieved. These powerful hardware and algorithmic innovations are not only providing exciting new mechanistic insights into tumour biology, from where new drug targets may be derived, but are also leading to the discovery of phosphoproteins as mediators of drug sensitivity and resistance and as classifiers of disease subtypes. These studies are, therefore, uncovering phosphoproteins as a new generation of disruptive biomarkers to improve personalised anti-cancer therapies.

HDAC Inhibition Restores Response to HER2-Targeted Therapy in Breast Cancer via PHLDA1 Induction.

The downregulation of Pleckstrin Homology-Like Domain family A member 1 (PHLDA1) expression mediates resistance to targeted therapies in receptor tyrosine kinase-driven cancers. The restoration and maintenance of PHLDA1 levels in cancer cells thus constitutes a potential strategy to circumvent resistance to inhibitors of receptor tyrosine kinases. Through a pharmacological approach, we identify the inhibition of MAPK signalling as a crucial step in PHLDA1 downregulation. Further ChIP-qPCR analysis revealed that MEK1/2 inhibition produces significant epigenetic changes at the PHLDA1 locus, specifically a decrease in the activatory marks H3Kme3 and H3K27ac. In line with this, we show that treatment with the clinically relevant class I histone deacetylase (HDAC) inhibitor 4SC-202 restores PHLDA1 expression in lapatinib-resistant human epidermal growth factor receptor-2 (HER2)+ breast cancer cells. Critically, we show that when given in combination, 4SC-202 and lapatinib exert synergistic effects on 2D cell proliferation and colony formation capacity. We therefore propose that co-treatment with 4SC-202 may prolong the clinical efficacy of lapatinib in HER2+ breast cancer patients.

Bespoken Nanoceria: An Effective Treatment in Experimental Hepatocellular Carcinoma.

BACKGROUND AND AIMS: Despite the availability of new-generation drugs, hepatocellular carcinoma (HCC) is still the third most frequent cause of cancer-related deaths worldwide. Cerium oxide nanoparticles (CeO2 NPs) have emerged as an antioxidant agent in experimental liver disease because of their antioxidant, anti-inflammatory, and antisteatotic properties. In the present study, we aimed to elucidate the potential of CeO2 NPs as therapeutic agents in HCC. APPROACH AND RESULTS: HCC was induced in 110 Wistar rats by intraperitoneal administration of diethylnitrosamine for 16 weeks. Animals were treated with vehicle or CeO2 NPs at weeks 16 and 17. At the eighteenth week, nanoceria biodistribution was assessed by mass spectrometry (MS). The effect of CeO2 NPs on tumor progression and animal survival was investigated. Hepatic tissue MS-based phosphoproteomics as well as analysis of principal lipid components were performed. The intracellular uptake of CeO2 NPs by human ex vivo perfused livers and human hepatocytes was analyzed. Nanoceria was mainly accumulated in the liver, where it reduced macrophage infiltration and inflammatory gene expression. Nanoceria treatment increased liver apoptotic activity, while proliferation was attenuated. Phosphoproteomic analysis revealed that CeO2 NPs affected the phosphorylation of proteins mainly related to cell adhesion and RNA splicing. CeO2 NPs decreased phosphatidylcholine-derived arachidonic acid and reverted the HCC-induced increase of linoleic acid in several lipid components. Furthermore, CeO2 NPs reduced serum alpha-protein levels and improved the survival of HCC rats. Nanoceria uptake by ex vivo perfused human livers and in vitro human hepatocytes was also demonstrated. CONCLUSIONS: These data indicate that CeO2 NPs partially revert the cellular mechanisms involved in tumor progression and significantly increase survival in HCC rats, suggesting that they could be effective in patients with HCC.

PARP-1 activation after oxidative insult promotes energy stress-dependent phosphorylation of YAP1 and reduces cell viability.

Poly(ADP-ribose) polymerase 1 (PARP-1) is a nuclear enzyme that catalyze the transfer of ADP-ribose units from NAD+ to several target proteins involved in cellular stress responses. Using WRL68 (HeLa derivate) cells, we previously showed that PARP-1 activation induced by oxidative stress after H2O2 treatment lead to depletion of cellular NAD+ and ATP, which promoted cell death. In this work, LC-MS/MS-based phosphoproteomics in WRL68 cells showed that the oxidative damage induced by H2O2 increased the phosphorylation of YAP1, a transcriptional co-activator involved in cell survival, and modified the phosphorylation of other proteins involved in transcription. Genetic or pharmacological inhibition of PARP-1 in H2O2-treated cells reduced YAP1 phosphorylation and degradation and increased cell viability. YAP1 silencing abrogated the protective effect of PARP-1 inhibition, indicating that YAP1 is important for the survival of WRL68 cells exposed to oxidative damage. Supplementation of NAD+ also reduced YAP1 phosphorylation, suggesting that the loss of cellular NAD+ caused by PARP-1 activation after oxidative treatment is responsible for the phosphorylation of YAP1. Finally, PARP-1 silencing after oxidative treatment diminished the activation of the metabolic sensor AMPK. Since NAD+ supplementation reduced the phosphorylation of some AMPK substrates, we hypothesized that the loss of cellular NAD+ after PARP-1 activation may induce an energy stress that activates AMPK. In summary, we showed a new crucial role of PARP-1 in the response to oxidative stress in which PARP-1 activation reduced cell viability by promoting the phosphorylation and degradation of YAP1 through a mechanism that involves the depletion of NAD+.

A systematic molecular and pharmacologic evaluation of AKT inhibitors reveals new insight into their biological activity.

BACKGROUND: AKT, a critical effector of the phosphoinositide 3-kinase (PI3K) signalling cascade, is an intensely pursued therapeutic target in oncology. Two distinct classes of AKT inhibitors have been in clinical development, ATP-competitive and allosteric. Class-specific differences in drug activity are likely the result of differential structural and conformational requirements governing efficient target binding, which ultimately determine isoform-specific potency, selectivity profiles and activity against clinically relevant AKT mutant variants. METHODS: We have carried out a systematic evaluation of clinical AKT inhibitors using in vitro pharmacology, molecular profiling and biochemical assays together with structural modelling to better understand the context of drug-specific and drug-class-specific cell-killing activity. RESULTS: Our data demonstrate clear differences between ATP-competitive and allosteric AKT inhibitors, including differential effects on non-catalytic activity as measured by a novel functional readout. Surprisingly, we found that some mutations can cause drug resistance in an isoform-selective manner despite high structural conservation across AKT isoforms. Finally, we have derived drug-class-specific phosphoproteomic signatures and used them to identify effective drug combinations. CONCLUSIONS: These findings illustrate the utility of individual AKT inhibitors, both as drugs and as chemical probes, and the benefit of AKT inhibitor pharmacological diversity in providing a repertoire of context-specific therapeutic options.

Poly(ADP-Ribose) Polymerase-1 inhibition potentiates cell death and phosphorylation of DNA damage response proteins in oxidative stressed retinal cells.

Oxidative stress (OxS) is involved in the development of cell injures occurring in retinal diseases while Poly(ADP-ribose) Polymerase-1 (PARP-1) is a key protein involved in the repair of the DNA damage caused by OxS. Inhibition of PARP-1 activity with the pharmacological inhibitor PJ34 in mouse retinal explants subjected to H2O2-induced oxidative damage resulted in an increase of apoptotic cells. Reduction of cell growth was also observed in the mouse cone like cell line 661 W in the presence of PJ34 under OxS conditions. Mass spectrometry-based phosphoproteomics analysis performed in 661 W cells determined that OxS induced significant changes in the phosphorylation in 1807 of the 8131 peptides initially detected. Blockade of PARP-1 activity after the oxidative treatment additionally increased the phosphorylation of multiple proteins, many of them at SQ motifs and related to the DNA-damage response (DDR). These motifs are substrates of the kinases ATM/ATR, which play a central role in DDR. Western blot analysis confirmed that the ATM/ATR activity measured and the phosphorylation at SQ motifs of ATM/ATR substrates was augmented when PARP-1 activity was inhibited under OxS conditions, in 661 W cells. Phosphorylation of ATM/ATR substrates, including the phosphorylation of the histone H2AX were also induced in organotypic cultures of retinal explants subjected to PARP-1 inhibition during exposure to OxS. In conclusion, inhibition of PARP-1 increased the phosphorylation and hence the activation of several proteins involved in the response to DNA damage, like the ATM protein kinase. This finally resulted in an augmented injury in mouse retinal cells suffering from OxS. Therefore, the inhibition of PARP-1 activity may have a negative outcome in the treatment of retinal diseases in which OxS is involved.

Kinase activity ranking using phosphoproteomics data (KARP) quantifies the contribution of protein kinases to the regulation of cell viability.

Cell survival is regulated by a signaling network driven by the activity of protein kinases; however, determining the contribution that each kinase in the network makes to such regulation remains challenging. Here, we report a computational approach that uses mass spectrometry-based phosphoproteomics data to rank protein kinases based on their contribution to cell regulation. We found that the scores returned by this algorithm, which we have termed kinase activity ranking using phosphoproteomics data (KARP), were a quantitative measure of the contribution that individual kinases make to the signaling output. Application of KARP to the analysis of eight hematological cell lines revealed that cyclin-dependent kinase (CDK) 1/2, casein kinase (CK) 2, extracellular signal-related kinase (ERK), and p21-activated kinase (PAK) were the most frequently highly ranked kinases in these cell models. The patterns of kinase activation were cell-line specific yet showed a significant association with cell viability as a function of kinase inhibitor treatment. Thus, our study exemplifies KARP as an untargeted approach to empirically and systematically identify regulatory kinases within signaling networks.

Shedding of bevacizumab in tumour cells-derived extracellular vesicles as a new therapeutic escape mechanism in glioblastoma.

Glioblastoma (GBM) is the most aggressive type of primary brain tumours. Anti-angiogenic therapies (AAT), such as bevacizumab, have been developed to target the tumour blood supply. However, GBM presents mechanisms of escape from AAT activity, including a speculated direct effect of AAT on GBM cells. Furthermore, bevacizumab can alter the intercellular communication of GBM cells with their direct microenvironment. Extracellular vesicles (EVs) have been recently described as main acts in the GBM microenvironment, allowing tumour and stromal cells to exchange genetic and proteomic material. Herein, we examined and described the alterations in the EVs produced by GBM cells following bevacizumab treatment. Interestingly, bevacizumab that is able to neutralise GBM cells-derived VEGF-A, was found to be directly captured by GBM cells and eventually sorted at the surface of the respective EVs. We also identified early endosomes as potential pathways involved in the bevacizumab internalisation by GBM cells. Via MS analysis, we observed that treatment with bevacizumab induces changes in the EVs proteomic content, which are associated with tumour progression and therapeutic resistance. Accordingly, inhibition of EVs production by GBM cells improved the anti-tumour effect of bevacizumab. Together, this data suggests of a potential new mechanism of GBM escape from bevacizumab activity.

Impact of phosphoproteomics in the translation of kinase-targeted therapies.

Signaling pathways driven by protein and lipid kinases are altered in most human diseases. Therefore, pharmacological inhibitors of cell signaling are one of the most intensively pursued therapeutic approaches for the treatment of diseases such as cancer, neurodegeneration, and metabolic syndromes. Phosphoproteomics is a technique that measures the products of kinase activities and, with the appropriate bioinformatics techniques, the methodology can also provide measures of kinase pathway activation and network circuitry. Hence, due to recent technological advantages, LC-MS-based quantitative phosphoproteomics provides relevant information for the design and implementation of kinase inhibitor based therapies. Here, we review how phosphoproteome profiling is being used in translational research as a means to identify drug targets and biomarkers for personalizing therapies based on kinase inhibitors.

Characterization of the Extracellular Matrix of Normal and Diseased Tissues Using Proteomics.

The extracellular matrix (ECM) is a complex meshwork of insoluble fibrillar proteins and signaling factors interacting together to provide architectural and instructional cues to the surrounding cells. Alterations in ECM organization or composition and excessive ECM deposition have been observed in diseases such as fibrosis, cardiovascular diseases, and cancer. We provide here optimized protocols to solubilize ECM proteins from normal or tumor tissues, digest the proteins into peptides, analyze ECM peptides by mass spectrometry, and interpret the mass spectrometric data. In addition, we present here two novel R-script-based web tools allowing rapid annotation and relative quantification of ECM proteins, peptides, and intensity/abundance in mass spectrometric data output files. We illustrate this protocol with ECMs obtained from two pairs of tissues, which differ in ECM content and cellularity: triple-negative breast cancer and adjacent mammary tissue, and omental metastasis from high-grade serous ovarian cancer and normal omentum. The complete proteomics data set generated in this study has been deposited to the public repository ProteomeXchange with the data set identifier: PXD005554.

Integrating phosphoproteome and transcriptome reveals new determinants of macrophage multinucleation.

Macrophage multinucleation (MM) is essential for various biological processes such as osteoclast-mediated bone resorption and multinucleated giant cell-associated inflammatory reactions. Here we study the molecular pathways underlying multinucleation in the rat through an integrative approach combining MS-based quantitative phosphoproteomics (LC-MS/MS) and transcriptome (high-throughput RNA-sequencing) to identify new regulators of MM. We show that a strong metabolic shift toward HIF1-mediated glycolysis occurs at transcriptomic level during MM, together with modifications in phosphorylation of over 50 proteins including several ARF GTPase activators and polyphosphate inositol phosphatases. We use shortest-path analysis to link differential phosphorylation with the transcriptomic reprogramming of macrophages and identify LRRFIP1, SMARCA4, and DNMT1 as novel regulators of MM. We experimentally validate these predictions by showing that knock-down of these latter reduce macrophage multinucleation. These results provide a new framework for the combined analysis of transcriptional and post-translational changes during macrophage multinucleation, prioritizing essential genes, and revealing the sequential events leading to the multinucleation of macrophages.

Cross-species proteomics reveals specific modulation of signaling in cancer and stromal cells by phosphoinositide 3-kinase (PI3K) inhibitors.

The tumor microenvironment plays key roles in cancer biology, but its impact on the regulation of signaling pathway activity in cancer cells has not been systemically investigated. We designed an analytical strategy that allows differential analysis of signaling between cancer and stromal cells present in tumor xenografts. We used this approach to investigate how in vivo growth conditions and PI3K inhibitors regulate pathway activities in both cancer and stromal cell populations. We found that, despite inducing more modest changes in protein expression, in vivo growing conditions extensively rewired protein kinase networks in cancer cells. As a result, different sets of phosphorylation sites were modulated by PI3K inhibitors in cancer cells growing in tumors relative to when these cells were in culture. The p110δ PI3K-selective compound CAL-101 (Idelalisib) did not inhibit markers of PI3K activity in cancer or stromal cells; however, unexpectedly, it induced phosphorylation on SQ motifs in both subpopulations of tumor cells in vivo but not in vitro. Thus, the interaction between cancer cells and the stroma modulated the ability of PI3K inhibitors to induce the activation of apoptosis in solid tumors. Our study provides proof-of-principle of a proteomics workflow for measuring signaling specifically in cancer and stromal cells and for investigating how cancer biochemistry is modulated in vivo.

Environmental stress affects the activity of metabolic and growth factor signaling networks and induces autophagy markers in MCF7 breast cancer cells.

Phosphoproteomic techniques are contributing to our understanding of how signaling pathways interact and regulate biological processes. This technology is also being used to characterize how signaling networks are remodeled during disease progression and to identify biomarkers of signaling pathway activity and of responses to cancer therapy. A potential caveat in these studies is that phosphorylation is a very dynamic modification that can substantially change during the course of an experiment or the retrieval and processing of cellular samples. Here, we investigated how exposure of cells to ambient conditions modulates phosphorylation and signaling pathway activity in the MCF7 breast cancer cell line. About 1.5% of 3,500 sites measured showed a significant change in phosphorylation extent upon exposure of cells to ambient conditions for 15 min. The effects of this perturbation in modifying phosphorylation patterns did not involve random changes due to stochastic activation of kinases and phosphatases. Instead, exposure of cells to ambient conditions elicited an environmental stress reaction that involved a coordinated response to a metabolic stress situation, which included: (1) the activation of AMPK; (2) the inhibition of PI3K, AKT, and ERK; (3) an increase in markers of protein synthesis inhibition at the level of translation elongation; and (4) an increase in autophagy markers. We also observed that maintaining cells in ice modified but did not completely abolish this metabolic stress response. In summary, exposure of cells to ambient conditions affects the activity of signaling networks previously implicated in metabolic and growth factor signaling. Mass spectrometry data have been deposited to the ProteomeXchange with identifier PXD000472.

The urinary proteome and metabonome differ from normal in adults with mitochondrial disease.

We studied the extent and nature of renal involvement in a cohort of 117 adult patients with mitochondrial disease, by measuring urinary retinol-binding protein (RBP) and albumin; established markers of tubular and glomerular dysfunction, respectively. Seventy-five patients had the m.3243A>G mutation and the most frequent phenotypes within the entire cohort were 14 with MELAS, 33 with MIDD, and 17 with MERRF. Urinary RBP was increased in 29 of 75 of m.3243A>G patients, whereas albumin was increased in 23 of the 75. The corresponding numbers were 16 and 14, respectively, in the 42 non-m.3243A>G patients. RBP and albumin were higher in diabetic m.3243A>G patients than in nondiabetics, but there were no significant differences across the three major clinical phenotypes. The urine proteome (mass spectrometry) and metabonome (nuclear magnetic resonance) in a subset of the m.3243A>G patients were markedly different from controls, with the most significant alterations occurring in lysosomal proteins, calcium-binding proteins, and antioxidant defenses. Differences were also found between asymptomatic m.3243A>G carriers and controls. No patients had an elevated serum creatinine level, but 14% had hyponatremia, 10% had hypophosphatemia, and 14% had hypomagnesemia. Thus, abnormalities in kidney function are common in adults with mitochondrial disease, exist in the absence of elevated serum creatinine, and are not solely explained by diabetes.