Search

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.

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.

DBF4-dependent kinase (DDK) is a two-subunit kinase required for initiating DNA replication at individual origins and is composed of CDC7 kinase and its regulatory subunit DBF4. Both subunits are highly expressed in many diverse tumor cell lines and primary tumors, and this is correlated with poor prognosis. Inhibiting DDK causes apoptosis of tumor cells, but not normal cells, through a largely unknown mechanism. Firstly, to understand why DDK is often overexpressed in tumors, we identified gene expression signatures that correlate with DDK high- and DDK low-expressing lung adenocarcinomas. We found that increased DDK expression is highly correlated with inactivation of RB1-E2F and p53 tumor suppressor pathways. Both CDC7 and DBF4 promoters bind E2F, suggesting that increased E2F activity in RB1 mutant cancers promotes increased DDK expression. Surprisingly, increased DDK expression levels are also correlated with both increased chemoresistance and genome-wide mutation frequencies. Our data further suggest that high DDK levels directly promote elevated mutation frequencies. Secondly, we performed an RNAi screen to investigate how DDK inhibition causes apoptosis of tumor cells. We identified 23 kinases and phosphatases required for apoptosis when DDK is inhibited. These hits include checkpoint genes, G2/M cell cycle regulators, and known tumor suppressors leading to the hypothesis that inhibiting mitotic progression can protect against DDKi-induced apoptosis. Characterization of one novel hit, the LATS2 tumor suppressor, suggests that it promotes apoptosis independently of the upstream MST1/2 kinases in the Hippo signaling pathway.

Background Among breast cancers, the triple-negative breast cancer (TNBC) subtype has the worst prognosis with no approved targeted therapies and only standard chemotherapy as the backbone of systemic therapy. Unique metabolic changes in cancer progression provide innovative therapeutic opportunities. The receptor tyrosine kinases (RTKs) epidermal growth factor receptor (EGFR), and MET receptor are highly expressed in TNBC, making both promising therapeutic targets. RTK signaling profoundly alters cellular metabolism by increasing glucose consumption and subsequently diverting glucose carbon sources into metabolic pathways necessary to support the tumorigenesis. Therefore, detailed metabolic profiles of TNBC subtypes and their response to tyrosine kinase inhibitors may identify therapeutic sensitivities. Methods We quantified the metabolic profiles of TNBC cell lines representing multiple TNBC subtypes using gas chromatography mass spectrometry. In addition, we subjected MDA-MB-231, MDA-MB-468, Hs578T, and HCC70 cell lines to metabolic flux analysis of basal and maximal glycolytic and mitochondrial oxidative rates. Metabolic pool size and flux measurements were performed in the presence and absence of the MET inhibitor, INC280/capmatinib, and the EGFR inhibitor, erlotinib. Further, the sensitivities of these cells to modulators of core metabolic pathways were determined. In addition, we annotated a rate-limiting metabolic enzymes library and performed a siRNA screen in combination with MET or EGFR inhibitors to validate synergistic effects. Results TNBC cell line models displayed significant metabolic heterogeneity with respect to basal and maximal metabolic rates and responses to RTK and metabolic pathway inhibitors. Comprehensive systems biology analysis of metabolic perturbations, combined siRNA and tyrosine kinase inhibitor screens identified a core set of TCA cycle and fatty acid pathways whose perturbation sensitizes TNBC cells to small molecule targeting of receptor tyrosine kinases. Conclusions Similar to the genomic heterogeneity observed in TNBC, our results reveal metabolic heterogeneity among TNBC subtypes and demonstrate that understanding metabolic profiles and drug responses may prove valuable in targeting TNBC subtypes and identifying therapeutic susceptibilities in TNBC patients. Perturbation of metabolic pathways sensitizes TNBC to inhibition of receptor tyrosine kinases. Such metabolic vulnerabilities offer promise for effective therapeutic targeting for TNBC patients. Electronic supplementary material The online version of this article (doi:10.1186/s40170-017-0168-x) contains supplementary material, which is available to authorized users.

The adaptor protein SH2B1β participates in regulation of the actin cytoskeleton during processes such as cell migration and differentiation. Here, we identify SH2B1β as a new focal adhesion protein. We provide evidence that SH2B1β is phosphorylated in response to phorbol 12-myristate 13-acetate (PMA)-induced protein kinase C (PKC) activation and show that PMA induces a rapid redistribution of SH2B1β out of focal adhesions. We also show that growth hormone (GH) increases cycling of SH2B1β into and out of focal adhesions. Ser161 and Ser165 in SH2B1β fall within consensus PKC substrate motifs. Mutating these two serine residues into alanine residues abrogates PMA-induced redistribution of SH2B1β out of focal adhesions, decreases SH2B1β cycling into and out of focal adhesions in control and GH-stimulated cells, and increases the size of focal adhesions. By contrast, mutating Ser165 into a glutamate residue decreases the amount of SH2B1β in focal adhesions and increases the number of focal adhesions per cell. These results suggest that activation of PKC regulates SH2B1β focal adhesion localization through phosphorylation of Ser161 and/or Ser165. The finding that phosphorylation of SH2B1β increases the number of focal adhesions suggests a mechanism for the stimulatory effect on cell motility of SH2B1β.

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.

We previously performed an RNA interference (RNAi) screen and found that the knockdown of the catalytically inactive phosphatase, MK-STYX [MAPK (mitogen-activated protein kinase) phospho-serine/threonine/tyrosine-binding protein], resulted in potent chemoresistance. Our follow-up studies demonstrated that knockdown of MK-STYX prevents cells from undergoing apoptosis through a block in cytochrome c release, but that MK-STYX does not localize proximal to the molecular machinery currently known to control this process. In an effort to define its molecular mechanism, we utilized an unbiased proteomics approach to identify proteins that interact with MK-STYX. We identified the mitochondrial phosphatase, PTPMT1 (PTP localized to mitochondrion 1), as the most significant and unique interaction partner of MK-STYX. We previously reported that knockdown of PTPMT1, an important component of the cardiolipin biosynthetic pathway, is sufficient to induce apoptosis and increase chemosensitivity. Accordingly, we hypothesized that MK-STYX and PTPMT1 interact and serve opposing functions in mitochondrial-dependent cell death. We confirmed that MK-STYX and PTPMT1 interact in cells and, importantly, found that MK-STYX suppresses PTPMT1 catalytic activity. Furthermore, we found that knockdown of PTPMT1 resensitizes MK-STYX knockdown cells to chemotherapeutics and restores the ability to release cytochrome c. Taken together, our data support a model in which MK-STYX controls apoptosis by negatively regulating PTPMT1. Given the important role of PTPMT1 in the production of cardiolipin and other phospholipids, this raises the possibility that dysregulated mitochondrial lipid metabolism may facilitate chemoresistance.

Previous studies have shown that growth hormone (GH) recruits the adapter protein SH2B1β to the GH-activated, GH receptor-associated tyrosine kinase JAK2, implicating SH2B1β in GH-dependent actin cytoskeleton remodeling, and suggesting that phosphorylation at serines 161 and 165 in SH2B1β releases SH2B1β from the plasma membrane. Here, we examined the role of SH2B1β in GH regulation of macrophage migration. We show that GH stimulates migration of cultured RAW264.7 macrophages, and primary cultures of peritoneal and bone marrow-derived macrophages. SH2B1β overexpression enhances, whereas SH2B1 knockdown inhibits, GH-dependent motility of RAW macrophages. At least two independent mechanisms regulate the SH2B1β-mediated changes in motility. In response to GH, tyrosines 439 and 494 in SH2B1β are phosphorylated. Mutating these tyrosines in SH2B1β decreases both basal and GH-stimulated macrophage migration. In addition, mutating the polybasic nuclear localization sequence (NLS) in SH2B1β or creating the phosphomimetics SH2B1β(S161E) or SH2B1β(S165E), all of which release SH2B1β from the plasma membrane, enhances macrophage motility. Conversely, SH2B1β(S161/165A) exhibits increased localization at the plasma membrane and decreased macrophage migration. Mutating the NLS or the nearby serine residues does not alter GH-dependent phosphorylation on tyrosines 439 and 494 in SH2B1β. Mutating tyrosines 439 and 494 does not affect localization of SH2B1β at the plasma membrane or movement of SH2B1β into focal adhesions. Taken together, these results suggest that SH2B1β enhances GH-stimulated macrophage motility via mechanisms involving phosphorylation of SH2B1β on tyrosines 439 and 494 and movement of SH2B1β out of the plasma membrane (e.g. as a result of phosphorylation of serines 161 and 165).

Protein Tyrosine Phosphatase localized to the Mitochondrion 1 (PTPMT1) is a dual specificity phosphatase exclusively localized to the mitochondria, and has recently been shown to be a critical component in the cardiolipin biosynthetic pathway. The downregulation of PTPMT1 in pancreatic beta cells has been shown to increase cellular ATP levels and insulin production, however, the generalized role of PTPMT1 in cancer cells has not been characterized. Here we report that downregulation of PTPMT1 activity is sufficient to induce apoptosis of cancer cells. Additionally, the silencing of PTPMT1 decreases cardiolipin levels in cancer cells, while selectively increasing ATP levels in glycolytic media. Additionally, sublethal downregulation of PTPMT1 synergizes with low doses of paclitaxel to promote cancer cell death. Our data suggest that inhibition of PTPMT1 causes a metabolic crisis in cancer cells that induces cell death, and may be a mechanism by which cancer cells can be sensitized to currently available therapies.

Evasion of apoptosis is a significant problem affecting an array of cancers. In order to identify novel regulators of apoptosis, we performed an RNA interference (RNAi) screen against all kinases and phosphatases in the human genome. We identified MK-STYX (STYXL1), a catalytically inactive phosphatase with homology to the mitogen-activated protein kinase (MAPK) phosphatases. Despite this homology, MK-STYX knockdown does not significantly regulate MAPK signaling in response to growth factors or apoptotic stimuli. Rather, RNAi-mediated knockdown of MK-STYX inhibits cells from undergoing apoptosis induced by cellular stressors activating mitochondrion-dependent apoptosis. This MK-STYX phenotype mimics the loss of Bax and Bak, two potent guardians of mitochondrial apoptotic potential. Similar to loss of both Bax and Bak, cells without MK-STYX expression are unable to release cytochrome c. Proapoptotic members of the BCL-2 family (Bax, Bid, and Bim) are unable to trigger cytochrome c release in MK-STYX-depleted cells, placing the apoptotic deficiency at the level of mitochondrial outer membrane permeabilization (MOMP). MK-STYX was found to localize to the mitochondria but is neither released from the mitochondria upon apoptotic stress nor proximal to the machinery currently known to control MOMP, indicating that MK-STYX regulates MOMP using a distinct mechanism.

Regulation of c-Fos transcription by GH is mediated by CCAAT/enhancer binding protein (C/ EBP ). This study examines the role of C/EBP in mediating GH activation of other early response genes, including Cyr61, Btg2, Socs3, Zfp36, and Socs1. C/EBP depletion using short hairpin RNA impaired responsiveness of these genes to GH, as seen for c-Fos. Rescue with wild-type C/EBP led to GH-dependent recruitment of the coactivator p300 to the c-Fos promoter. In contrast, rescue with C/EBP mutated at the ERK phosphorylation site at T188 failed to induce GH-dependent recruitment of p300, indicating that ERK-mediated phosphorylation of C/EBP at T188 is required for GH-induced recruitment of p300 to c-Fos. GH also induced the occupancy of phosphorylated C/EBP and p300 on Cyr61, Btg2, and Socs3 at predicted C/EBP-cAMP response element-binding protein motifs in their promoters. Consistent with a role for ERKs in GH-induced expression of these genes, treatment with U0126 to block ERK phosphorylation inhibited their GH-induced expression. In contrast, GH-dependent expression of Zfp36 and Socs1 was not inhibited by U0126. Thus, induction of multiple early response genes by GH in 3T3-F442A cells is mediated by C/EBP . A subset of these genes is regulated similarly to c-Fos, through a mechanism involving GH-stimulated ERK 1/2 activation, phosphorylation of C/EBP , and recruitment of p300. Overall, these studies suggest that C/EBP , like the signal transducer and activator of transcription proteins, regulates multiple genes in response to GH. Fil: Cui, Tracy X.. University Of Michigan; Estados Unidos Fil: Lin, Grace. University Of Michigan; Estados Unidos Fil: Lapensee, Christopher R.. University Of Michigan; Estados Unidos Fil: Calinescu, Anda Alexandra. University Of Michigan; Estados Unidos Fil: Rathore, Maanjot. University Of Michigan; Estados Unidos Fil: Streeter, Cale. University Of Michigan; Estados Unidos Fil: Piwien Pilipuk, Graciela. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina. University Of Michigan; Estados Unidos Fil: Lanning, Nathan. University Of Michigan; Estados Unidos Fil: Jin, Hui. University Of Michigan; Estados Unidos Fil: Carter Su, Christin. University Of Michigan; Estados Unidos Fil: Qin, Zhaohui S.. University Of Michigan; Estados Unidos Fil: Schwartz, Jessica . University Of Michigan; Estados Unidos

Janus kinase 2 (JAK2), a tyrosine kinase that associates with the GH receptor and is activated by GH, has been implicated as a key mediator of GH signaling. Several published reports suggest that members of the Src family of tyrosine kinases may also participate in GH signaling. We therefore investigated the extent to which JAK2 and Src family kinases mediate GH activation of signal transducers and activators of transcription (STATs) 1, 3, and 5a/b, ERKs 1 and 2, and Akt, in the highly GH-responsive cell lines 3T3-F442A preadipocytes and H4IIE hepatoma cells. GH activation of Src family kinases was not detected in either cell line. Further, blocking basal activity of Src kinases with the Src inhibitors PP1 and PP2 did not inhibit GH activation of STATs 1, 3, or 5a/b, or ERKs 1 and 2. When levels of JAK2 were depressed by short hairpin RNA in 3T3-F442A and H4IIE cells, GH-stimulated activation of STATs 1, 3, and 5a/b, ERKs 1 and 2, and Akt were significantly reduced; however, basal activity of Src family kinases was unaffected. These results were supported genetically by experiments showing that GH robustly activates JAK2, STATs 3 and 5a/b, ERKs 1 and 2, and Akt in murine embryonic fibroblasts derived from Src/Yes/ Fyn triple-knockout embryos that lack known Src kinases. These results strongly suggest that JAK2, but not Src family kinases, is critical for transducing these GH signals in 3T3-F442A and H4IIE cells.

Growth hormone (GH) is a major regulatory factor for overall body growth as evidenced by the height extremes in people with abnormal circulating GH levels or GH receptor (GHR) disruptions. GH also affects metabolism, cardiac and immune function, mental agility and aging. Currently, GH is being used therapeutically for a variety of clinical conditions including promotion of growth in short statured children, treatment of adults with GH deficiency and HIV-associated wasting. To help reveal previous unrecognized functions of GH, better understand the known functions of GH, and avoid adverse consequences that are often associated with exogenous GH administration, careful delineation of the molecular mechanisms whereby GH induces its diverse effects is needed. GH is a peptide hormone that is secreted into the circulation by the anterior pituitary and acts upon various target tissues expressing GHR. GH binding of GHR activates the tyrosine kinase Janus kinase 2 (JAK2), thus initiating a multitude of signaling cascades that result in a variety of biological responses including cellular proliferation, differentiation and migration, prevention of apoptosis, cytoskeletal reorganization and regulation of metabolic pathways. A number of signaling proteins and pathways activated by GH have been identified, including JAKs, signal transducers and activators of transcription (Stats), the mitogen activated protein kinase (MAPK) pathway, and the phosphatidylinositol 3′-kinase (PI3K) pathway. Although these signal transduction pathways have been well characterized, the manner by which GH activates these pathways, the downstream signals induced by these pathways, and the cross-talk with other pathways are not completely understood. Recent findings have added vital information to our understanding of these downstream signals induced by GH and mechanisms that terminate GH signaling, and identified new GH signaling proteins and pathways. This review will highlight some of these findings, many of which are unexpected and some of which challenge previously held beliefs about the mechanisms of GH signaling.

The notoriously high glucose uptake by metastatic melanoma (MM) lesions is commonly exploited clinically using PET/CT imaging to evaluate disease progression and treatment. The purpose of this study was to use patient-derived xenograft (PDX) MM models and a PDX derived (MM8.1) cell line, to determine how driver oncogene mutations and drugs targeting the RAS-RAF-MEK signaling pathways impact glucose uptake, ATP production and mitochondrial plasticity of MM cells both in vivo and in vitro and the implications on drug resistance. MM BRAF V600E+ patients are routinely treated targeting either mutant BRAF kinase (vemurafenib), or the MAPK downstream signaling intermediate; MEK. To begin exploring links between oncogenic BRAF and metabolism, we first conducted differential gene expression (DGE) profiling of 30 different MM lesions (n=15 patients: pre- and post-engraftment). Focusing on PGC1α; a master mitochondrial regulator promoting oxidative phosphorylation (OXPHOS) revealed lower expression levels in BRAF V600E+ tumors compared to BRAF V600V+ tumors. Further analysis of a BRAF V600E+ MM PDX model revealed strong glucose uptake by PET/CT imaging at baseline. However, 48 hrs. after treatment with vemurafenib triggered growth arrest, accompanied by increased PGC1α; mRNA levels and significant reduction in glucose uptake without change in tumor volume. Upon acquisition of acquired vemurafenib resistance in the PDX model (day 70), rapidly growing tumors displayed reversal to high glucose uptake; which was again diminished by targeting MEK (PD0325901). DGE analysis of vehicle vs. 48 hrs. day 50 and day 70 vemurafenib treated tumors revealed oscillation in the expression of genes regulating glycolysis and oxidative phosphorylation (OXPHOS) that is reflected in the PET/CT imaging. To gain deeper insight, a cell line derived from a vehicle treated tumor, was examined using the Seahorse metabolic analyzer. Vehicle treated MM8.1 cells display high glycolytic capacity and predominantly glycolytic-derived ATP production, which is reflected by only minor inhibition of ATP production with oligomycin (an inhibitor of ATP synthase, a component of the OXHPOS system). Upon treatment with PLX4032, there was rapid reduction in glycolytic capacity along with a concurrent increase in maximum oxygen consumption capacity. PLX4032 treatment additionally enhanced MM8.1 sensitivity to oligomycin, but only transiently (24 hrs. pre-treatment), as later exposure times did not induce oligomycin sensitivity despite DGE changes to indicate otherwise. In conclusion, caution is warranted in evaluating drug responses in patients using PET/CT depending on duration of treatment. Cross-talk involving MAPK signaling and energy metabolism is a dynamic process, with more of a rheostat than switch-type properties, requiring further evaluation to identify therapeutic approaches exploiting metabolic events within MM cells. Citation Format: Nathan J. Lanning, Pimiento Jose, Noel R. Monks, Paula J. Davidson, Andrew S. Borgman, Ting-Tung Chang, Anderson S. Peck, David J. Monsma, Chelsea A. Peterson, Mackeigan P. Jeff, Brian J. Nickoloff. Complex crosstalk between MAPK signaling and energy metabolism in melanoma. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr A37. doi: 10.1158/1557-3125.RASONC14-A37

Degenerative joint disease, also known as osteoarthritis, is the most common joint disorder in human beings. The molecular mechanism underlying this disease is not fully understood. Here, we report that disruption of mitogen-inducible gene 6 ( Mig-6 ) in mice by homologous recombination leads to early onset degenerative joint disease, which is revealed by simultaneous enlargement and deformity of multiple joints, degradation of articular cartilage, and the development of bony outgrowths or osteophyte formation within joint space. The osteophyte formation appears to be derived from proliferation of mesenchymal progenitor cells followed by differentiation into chondrocytes. Absence of the Rag2 gene does not rescue the joint phenotype, excluding a role for the acquired immune system in the development of this disease. Our results provide insight into the mechanism of osteoarthritis by showing that loss of Mig-6 leads to early onset of this disease, implying that this gene or its pathway is important in normal joint maintenance. Because of the striking similarity of osteoarthritis in humans and mice, the Mig-6 mutant mouse should provide a useful animal model for studying the mechanism of this disease and for testing drugs or therapies for treating osteoarthritis.

Downstream signaling that results from the interaction of hepatocyte growth factor/scatter factor (HGF/SF) with the receptor tyrosine kinase Met plays critical roles in tumor development, progression, and metastasis. This ligand-receptor pair is an attractive target for new diagnostic and therapeutic agents, preclinical development of which requires suitable animal models. The growth of heterotopic and orthotopic Met-expressing human tumor xenografts in conventional strains of immunocompromised mice inadequately replicates the paracrine stimulation by human HGF/SF (hHGF/SF) that occurs in humans with cancer. We have therefore generated a mouse strain transgenic for hHGF/SF (designated hHGF-Tg) on a severe combined immunodeficiency (SCID) background. We report here that the presence of ectopically expressed hHGF/SF ligand significantly enhances growth of heterotopic subcutaneous xenografts derived from human Met-expressing cancer cells, including the lines SK-LMS-1 (human leiomyosarcoma), U118 (human glioblastoma), and DU145 (human prostate carcinoma), but not that of M14-Mel xenografts (human melanoma that expresses insignificant levels of Met). Our results indicate that ectopic hHGF/SF can specifically activate Met in human tumor xenografts. This new hHGF-Tg strain of mice should provide a powerful tool for evaluating drugs and diagnostic agents that target the various pathways influenced by Met activity.

Summary Altered cellular bioenergetics and mitochondrial function are major features of several diseases, including cancer, diabetes, and neurodegenerative disorders. Given this important link to human health, we sought to define proteins within mitochondria that are critical for maintaining homeostatic ATP levels. We screened an RNAi library targeting >1,000 nuclear-encoded genes whose protein products localize to the mitochondria in multiple metabolic conditions in order to examine their effects on cellular ATP levels. We identified a mechanism by which electron transport chain (ETC) perturbation under glycolytic conditions increased ATP production through enhanced glycolytic flux, thereby highlighting the cellular potential for metabolic plasticity. Additionally, we identified a mitochondrial adenylate kinase (AK4) that regulates cellular ATP levels and AMPK signaling and whose expression significantly correlates with glioma patient survival. This study maps the bioenergetic landscape of >1,000 mitochondrial proteins in the context of varied metabolic substrates and begins to link key metabolic genes with clinical outcome.