Oncogenic K-RasG12V cannot overcome proliferation failure caused by loss of Ppp6c in mouse embryonic fibroblasts.

Protein phosphatase 6 is a Ser/Thr protein phosphatase and its catalytic subunit is Ppp6c. Ppp6c is thought to be indispensable for proper growth of normal cells. On the other hand, loss of Ppp6c accelerates growth of oncogenic Ras-expressing cells. Although it has been studied in multiple contexts, the role(s) of Ppp6c in cell proliferation remains controversial. It is unclear how oncogenic K-Ras overcomes cell proliferation failure induced by Ppp6c deficiency; therefore, in this study, we attempted to shed light on how oncogenic K-Ras modulates tumor cell growth. Contrary to our expectations, loss of Ppp6c decreased proliferation, anchorage-independent growth in soft agar, and tumor formation of oncogenic Ras-expressing mouse embryonic fibroblasts (MEFs). These findings show that oncogenic K-RasG12V cannot overcome proliferation failure caused by loss of Ppp6c in MEFs.

Niacin restriction with NAMPT-inhibition is synthetic lethal to neuroendocrine carcinoma.

Nicotinamide phosphoribosyltransferase (NAMPT) plays a major role in NAD biosynthesis in many cancers and is an attractive potential cancer target. However, factors dictating therapeutic efficacy of NAMPT inhibitors (NAMPTi) are unclear. We report that neuroendocrine phenotypes predict lung and prostate carcinoma vulnerability to NAMPTi, and that NAMPTi therapy against those cancers is enhanced by dietary modification. Neuroendocrine differentiation of tumor cells is associated with down-regulation of genes relevant to quinolinate phosphoribosyltransferase-dependent de novo NAD synthesis, promoting NAMPTi susceptibility in vitro. We also report that circulating nicotinic acid riboside (NAR), a non-canonical niacin absent in culture media, antagonizes NAMPTi efficacy as it fuels NAMPT-independent but nicotinamide riboside kinase 1-dependent NAD synthesis in tumors. In mouse transplantation models, depleting blood NAR by nutritional or genetic manipulations is synthetic lethal to tumors when combined with NAMPTi. Our findings provide a rationale for simultaneous targeting of NAR metabolism and NAMPT therapeutically in neuroendocrine carcinoma.

PP6 deficiency in mice with KRAS mutation and Trp53 loss promotes early death by PDAC with cachexia-like features.

To examine effects of PP6 gene (Ppp6c) deficiency on pancreatic tumor development, we developed pancreas-specific, tamoxifen-inducible Cre-mediated KP (KRAS(G12D) plus Trp53-deficient) mice (cKP mice) and crossed them with Ppp6cflox / flox mice. cKP mice with the homozygous Ppp6c deletion developed pancreatic tumors, became emaciated and required euthanasia within 150 days of mutation induction, phenotypes that were not seen in heterozygous or wild-type (WT) mice. At 30 days, a comparative analysis of genes commonly altered in homozygous versus WT Ppp6c cKP mice revealed enhanced activation of Erk and NFκB pathways in homozygotes. By 80 days, the number and size of tumors and number of precancerous lesions had significantly increased in the pancreas of Ppp6c homozygous relative to heterozygous or WT cKP mice. Ppp6c-/- tumors were pathologically diagnosed as pancreatic ductal adenocarcinoma (PDAC) undergoing the epithelial-mesenchymal transition (EMT), and cancer cells had invaded surrounding tissues in three out of six cases. Transcriptome and metabolome analyses indicated an enhanced cancer-specific glycolytic metabolism in Ppp6c-deficient cKP mice and the increased expression of inflammatory cytokines. Individual Ppp6c-/- cKP mice showed weight loss, decreased skeletal muscle and adipose tissue, and increased circulating tumor necrosis factor (TNF)-α and IL-6 levels, suggestive of systemic inflammation. Overall, Ppp6c deficiency in the presence of K-ras mutations and Trp53 gene deficiency promoted pancreatic tumorigenesis with generalized cachexia and early death. This study provided the first evidence that Ppp6c suppresses mouse pancreatic carcinogenesis and supports the use of Ppp6c-deficient cKP mice as a model for developing treatments for cachexia associated with pancreatic cancer.

Ppp6c deficiency accelerates K-rasG12D -induced tongue carcinogenesis.

BACKGROUND: Effective treatments for cancer harboring mutant RAS are lacking. In Drosophila, it was reported that PP6 suppresses tumorigenicity of mutant RAS. However, the information how PP6 regulates oncogenic RAS in mammals is limited. METHODS: We examined the effects of PP6 gene (Ppp6c) deficiency on tongue tumor development in K (K-rasG12D)- and KP (K-rasG12D + Trp53-deficient)-inducible mice. RESULTS: Mice of K and KP genotypes developed squamous cell carcinoma in situ in the tongue approximately 2 weeks after the induction of Ppp6c deficiency and was euthanized due to 20% loss of body weight. Transcriptome analysis revealed significantly different gene expressions between tissues of Ppp6c-deficient tongues and those of Ppp6c wild type, while Trp53 deficiency had a relatively smaller effect. We then analyzed genes commonly altered by Ppp6c deficiency, with or without Trp53 deficiency, and identified a group concentrated in KEGG database pathways defined as 'Pathways in Cancer' and 'Cytokine-cytokine receptor interaction'. We then evaluated signals downstream of oncogenic RAS and those regulated by PP6 substrates and found that in the presence of K-rasG12D, Ppp6c deletion enhanced the activation of the ERK-ELK1-FOS, AKT-4EBP1, and AKT-FOXO-CyclinD1 axes. Ppp6c deletion combined with K-rasG12D also enhanced DNA double-strand break (DSB) accumulation and activated NFκB signaling, upregulating IL-1ß, COX2, and TNF.

Robustness of a Cancer Profiling Test Using Formalin-fixed Paraffin Embedded Tumor Specimens.

BACKGROUND/AIM: Cancer profiling tests using formalin-fixed paraffin-embedded (FFPE) specimens with various conditions have become an essential tool for cancer treatment. The robustness of these tests needs to be addressed. MATERIALS AND METHODS: A cancer profiling test, NCC oncopanel, was tested with FFPE specimens from various tissues with different storage conditions and fixation lengths. Next generation sequencing was performed with Miseq and the data were assembled using the human reference genome hg19. RESULTS: Duration of storage and fixation affected the mapping statistics. Prolonged storage increased outward read paring and longer fixation rates caused increased singletons and unmapped reads. CONCLUSION: Our results indicate that a cancer profiling test with target capturing method, NCC oncopanel, shows robustness for FFPE cancer specimens with various storage conditions.

Ppp6c haploinsufficiency accelerates UV-induced BRAF(V600E)-initiated melanomagenesis.

According to TCGA database, mutations in PPP6C (encoding phosphatase PP6) are found in c. 10% of tumors from melanoma patients, in which they coexist with BRAF and NRAS mutations. To assess PP6 function in melanoma carcinogenesis, we generated mice in which we could specifically induce BRAF(V600E) expression and delete Ppp6c in melanocytes. In these mice, melanoma susceptibility following UVB irradiation exhibited the following pattern: Ppp6c semi-deficient (heterozygous) > Ppp6c wild-type > Ppp6c-deficient (homozygous) tumor types. Next-generation sequencing of Ppp6c heterozygous and wild-type melanoma tumors revealed that all harbored Trp53 mutations. However, Ppp6c heterozygous tumors showed a higher Signature 1 (mitotic/mitotic clock) mutation index compared with Ppp6c wild-type tumors, suggesting increased cell division. Analysis of cell lines derived from either Ppp6c heterozygous or wild-type melanoma tissues showed that both formed tumors in nude mice, but Ppp6c heterozygous tumors grew faster compared with those from the wild-type line. Ppp6c knockdown via siRNA in the Ppp6c heterozygous line promoted the accumulation of genomic damage and enhanced apoptosis relative to siRNA controls. We conclude that in the presence of BRAF(V600E) expression and UV-induced Trp53 mutation, Ppp6c haploinsufficiency promotes tumorigenesis.

Dietary intervention as a therapeutic for cancer.

Cancer metabolism is influenced by availability of nutrients in the microenvironment and can to some extent be manipulated by dietary modifications that target oncogenic metabolism. As yet, few dietary interventions have been scientifically proven to mitigate disease progression or enhance any other kind of therapy in human cancer. However, recent advances in the understanding of cancer metabolism enable us to predict or devise effective dietary interventions that might antagonize tumor growth. In fact, evidence emerging from preclinical models suggests that appropriate combinations of specific cancer therapies with dietary interventions could critically impact therapeutic efficacy. Here, we review the potential benefits of precision nutrition approaches in augmenting the efficacy of cancer treatment.

Comparison of the ischemic and non-ischemic lung cancer metabolome reveals hyper activity of the TCA cycle and autophagy.

Recent advances in cancer biology reveal the importance of metabolic changes in cancer; however, less is known about how metabolic pathways in tumors are regulated in vivo. Here, we report analysis of the lung cancer metabolism based on different surgical procedures, namely lobectomy and partial resection. In lobectomy, but not in partial resection, pulmonary arteries and veins are ligated prior to removal of tissues, rendering tissues ischemic. We show that tumors indeed undergo ischemia upon lobectomy and that the tumor metabolome differs markedly from that of tumors removed by partial resection. Comparison of the responses to ischemia in tumor and normal lung tissues revealed that lung cancer tissue exhibits greater TCA cycle and autophagic activity than do normal lung tissues in vivo in patients. Finally, we report that deleting ATG7, which encodes a protein essential for autophagy, antagonizes growth of tumors derived from lung cancer cell lines, suggesting that autophagy confers metabolic advantages to lung cancer. Our findings shed light on divergent metabolic responses to ischemia seen in tumors and normal tissues.

Structural Basis of Beneficial Design for Effective Nicotinamide Phosphoribosyltransferase Inhibitors.

Inhibition of nicotinamide phosphoribosyltransferase (NAMPT) is an attractive therapeutic strategy for targeting cancer metabolism. So far, many potent NAMPT inhibitors have been developed and shown to bind to two unique tunnel-shaped cavities existing adjacent to each active site of a NAMPT homodimer. However, cytotoxicities and resistances to NAMPT inhibitors have become apparent. Therefore, there remains an urgent need to develop effective and safe NAMPT inhibitors. Thus, we designed and synthesized two close structural analogues of NAMPT inhibitors, azaindole-piperidine (3a)- and azaindole-piperazine (3b)-motif compounds, which were modified from the well-known NAMPT inhibitor FK866 (1). Notably, 3a displayed considerably stronger enzyme inhibitory activity and cellular potency than did 3b and 1. The main reason for this phenomenon was revealed to be due to apparent electronic repulsion between the replaced nitrogen atom (N1) of piperazine in 3b and the Nδ atom of His191 in NAMPT by our in silico binding mode analyses. Indeed, 3b had a lower binding affinity score than did 3a and 1, although these inhibitors took similar stable chair conformations in the tunnel region. Taken together, these observations indicate that the electrostatic enthalpy potential rather than entropy effects inside the tunnel cavity has a significant impact on the different binding affinity of 3a from that of 3b in the disparate enzymatic and cellular potencies. Thus, it is better to avoid or minimize interactions with His191 in designing further effective NAMPT inhibitors.

Divergent metabolic responses dictate vulnerability to NAMPT inhibition in ovarian cancer.

It is of current interest to target cancer metabolism as treatment for many malignancies, including ovarian cancer (OVC), in which few druggable driver mutations have been identified. Nicotinamide phosphoribosyltransferase (NAMPT), a rate-limiting enzyme in the NAD salvage pathway, is a potential therapeutic target in OVC. However, factors that determine responsiveness to NAMPT inhibition are not fully understood. Here, we report that OVC cell lines can be divided into subgroups exhibiting NAMPT-dependent or NAMPT-independent glycolysis, and these metabolic differences correlate with vulnerability to NAMPT inhibition. Interestingly, cells showing NAMPT-dependent glycolysis were enriched in a group of cells lacking BRCA1/2 gene mutations. Our findings suggest the importance of selecting appropriate patients for NAMPT-targeting therapy in OVC.

Novel activating KRAS mutation candidates in lung adenocarcinoma.

Lung adenocarcinoma (LUAC) is a unique lung cancer subtype that is responsive to several therapeutic agents. The KRAS gene is the second most frequently mutated gene in LUAC and the majority of KRAS mutations are one of three classical activating mutations (G12, G13, and Q61). Recently, other types of "minor" KRAS mutation have been identified among LUAC patients and some may have similar transforming activities to those of the classical KRAS mutations. Here we describe minor KRAS mutations in LUAC patients, some of which (A66T, A66V, and G75E) may have tumor-forming activity in mouse embryonic fibroblasts in an allograft model. RNA-Seq analysis revealed that mouse embryonic fibroblasts overexpressing these three minor KRAS mutations have distinct expression profiles compared with overexpression of the wild type but similar expression profiles compared with overexpression of the classical KRAS mutants. Our results indicate that some of the minor KRAS mutations cause varying tumor formation activity and are important targets for developing anti-RAS agents as chemotherapeutic agents.

Revisiting glucose metabolism in cancer: lessons from a PKM knock-in model.

Isoform selection of pyruvate kinase M (PKM), a glycolytic enzyme, influences fates of glucose-derived carbons in cellular metabolic networks. We recently developed novel mouse lines to study PKM isoform function and identified PKM1 as a potential target in a subset of human lung cancers. This work provides new insight into cancer metabolism.

Loss of protein phosphatase 6 in mouse keratinocytes enhances K-rasG12D -driven tumor promotion.

Here, we address the function of protein phosphatase 6 (PP6) loss on K-ras-initiated tumorigenesis in keratinocytes. To do so, we developed tamoxifen-inducible double mutant (K-rasG12D -expressing and Ppp6c-deficient) mice in which K-rasG12D expression is driven by the cytokeratin 14 (K14) promoter. Doubly-mutant mice showed early onset tumor formation in lips, nipples, external genitalia, anus and palms, and had to be killed by 3 weeks after induction by tamoxifen, while comparably-treated K-rasG12D -expressing mice did not. H&E-staining of lip tumors before euthanasia revealed that all were papillomas, some containing focal squamous cell carcinomas. Immunohistochemical analysis of lips of doubly-mutant vs K-rasG12D mice revealed that cell proliferation and cell size increased approximately 2-fold relative to K-rasG12D -expressing mutants, and epidermal thickness of lip tissue greatly increased relative to that seen in K-rasG12D -only mice. Moreover, AKT phosphorylation increased in K-rasG12D -expressing/Ppp6c-deficient cells, as did phosphorylation of the downstream effectors 4EBP1, S6 and GSK3, suggesting that protein synthesis and survival signals are enhanced in lip tissues of doubly-mutant mice. Finally, increased numbers of K14-positive cells were present in the suprabasal layer of doubly-mutant mice, indicating abnormal keratinocyte differentiation, and γH2AX-positive cells accumulated, indicating perturbed DNA repair. Taken together, Ppp6c deficiency enhances K-rasG12D -dependent tumor promotion.

PKM1 Confers Metabolic Advantages and Promotes Cell-Autonomous Tumor Cell Growth.

Expression of PKM2, which diverts glucose-derived carbon from catabolic to biosynthetic pathways, is a hallmark of cancer. However, PKM2 function in tumorigenesis remains controversial. Here, we show that, when expressed rather than PKM2, the PKM isoform PKM1 exhibits a tumor-promoting function in KRASG12D-induced or carcinogen-initiated mouse models or in some human cancers. Analysis of Pkm mutant mouse lines expressing specific PKM isoforms established that PKM1 boosts tumor growth cell intrinsically. PKM1 activated glucose catabolism and stimulated autophagy/mitophagy, favoring malignancy. Importantly, we observed that pulmonary neuroendocrine tumors (NETs), including small-cell lung cancer (SCLC), express PKM1, and that PKM1 expression is required for SCLC cell proliferation. Our findings provide a rationale for targeting PKM1 therapeutically in certain cancer subtypes, including pulmonary NETs.

Pyruvate kinase type M2 contributes to the development of pancreatic ductal adenocarcinoma by regulating the production of metabolites and reactive oxygen species.

The majority of cancer cells maintain a high glycolytic activity and an increased lactate production, even in a well oxygenated environment. This phenomenon is known as the Warburg effect. Previous studies have revealed that various types of cancer selectively express the pyruvate kinase M2 isoform (PKM2), and that PKM2 plays a pivotal role in the Warburg effect. Although elevated PKM2 levels have been observed in pancreatic cancer and other types of cancer, little is known about the biological function of PKM2. In this study, in order to examine the expression and role of PKM2 in pancreatic ductal adenocarcinoma (PDAC), we knocked down PKM2 in PDAC cells by introducing small interfering and short hairpin RNAs, and examined the gene expression profiles in the cells by microarray analysis. We analyzed the energy-producing pathways in the cells by XFe Extracellular Flux Analyzers, and detected intracellular metabolites by capillary electrophoresis time-of-flight mass spectrometry. We found that the RNAi-mediated knockdown of PKM2 diminished the proliferative, migratory and tumorigenic ability of the PDAC cell-lines. PKM2 knockdown also resulted in lower glycolytic activities and decreased levels of some intracellular metabolites, such as pyruvate and polyamine; however, it led to elevated levels of reactive oxygen species. Microarray analysis revealed the functional association between PKM2 and the expression of genes that drive the cell cycle. On the whole, the findings of this study demonstrate that PKM2 plays an important role in metabolic activities, as well as in the malignancy of PDAC cells.

Cysteinyl-tRNA synthetase governs cysteine polysulfidation and mitochondrial bioenergetics.

Cysteine hydropersulfide (CysSSH) occurs in abundant quantities in various organisms, yet little is known about its biosynthesis and physiological functions. Extensive persulfide formation is apparent in cysteine-containing proteins in Escherichia coli and mammalian cells and is believed to result from post-translational processes involving hydrogen sulfide-related chemistry. Here we demonstrate effective CysSSH synthesis from the substrate L-cysteine, a reaction catalyzed by prokaryotic and mammalian cysteinyl-tRNA synthetases (CARSs). Targeted disruption of the genes encoding mitochondrial CARSs in mice and human cells shows that CARSs have a crucial role in endogenous CysSSH production and suggests that these enzymes serve as the principal cysteine persulfide synthases in vivo. CARSs also catalyze co-translational cysteine polysulfidation and are involved in the regulation of mitochondrial biogenesis and bioenergetics. Investigating CARS-dependent persulfide production may thus clarify aberrant redox signaling in physiological and pathophysiological conditions, and suggest therapeutic targets based on oxidative stress and mitochondrial dysfunction.

Enhanced expression of the M2 isoform of pyruvate kinase is involved in gastric cancer development by regulating cancer-specific metabolism.

Recent studies have indicated that increased expression of the M2 isoform of pyruvate kinase (PKM2) is involved in glycolysis and tumor development. However, little is known about the role of PKM2 in gastric cancer (GC). Therefore, we examined the expression and function of PKM2 in human GC. We evaluated PKM1 and PKM2 expression by quantitative RT-PCR in gastric tissues from 10 patients who underwent gastric endoscopic submucosal dissection, 80 patients who underwent gastrectomy, and seven healthy volunteers, and analyzed the correlation with clinicopathological variables. To assess the function of PKM2, we generated PKM2-knockdown GC cells, and investigated the phenotypic changes. Furthermore, we examined the induction of PKM2 expression by cytotoxin-associated gene A (CagA), a pathogenic factor of Helicobacter pylori, using CagA-inducible GC cells. We found that PKM2 was predominantly expressed not only in GC lesions but also in the normal gastric regions of GC patients and in the gastric mucosa of healthy volunteers. The PKM2 expression was significantly higher in carcinoma compared to non-cancerous tissue and was associated with venous invasion. Knockdown of PKM2 in GC cells caused significant decreases in cellular proliferation, migration, anchorage-independent growth, and sphere formation in vitro, and in tumor growth and liver metastasis in vivo. The serine concentration-dependent cell proliferation was also inhibited by PKM2 silencing. Furthermore, we found that PKM2 expression was upregulated by CagA by way of the Erk pathway. These results suggested that enhanced PKM2 expression plays a pivotal role in the carcinogenesis and development of GC in part by regulating cancer-specific metabolism.

Ex vivo model of non-small cell lung cancer using mouse lung epithelial cells.

Lung cancer is the most common cause of cancer mortality, however, efficient methods to culture, expand and transform lung epithelial (LE) cells have not been established. In the present study, an efficient ex vivo method was applied to recapitulate lung carcinogenesis using mouse LE cells. A Matrigel-assisted three-dimensional culture was used to isolate and selectively expand LE cells from mouse lungs. Purified LE cells were passaged and expanded for at least 2 to 3 months while maintaining epidermal growth factor-dependence. LE cells were also easily transformed by genetic manipulations using retroviral vectors. A SV40 large-T antigen, suppressing p53 and pRB, plus an activated oncogene, such as KrasG12V or EGFRex19del, were required to transform LE cells. Transformed cells formed tumors resembling non-small cell lung cancer (NSCLC) in allograft models and exhibited strong oncogene addiction. This experimental system provided a unique model system to study lung tumorigenesis and develop novel therapeutics against NSCLC.

The protein phosphatase 6 catalytic subunit (Ppp6c) is indispensable for proper post-implantation embryogenesis.

Ppp6c, which encodes the catalytic subunit of phosphoprotein phosphatase 6 (PP6), is conserved among eukaryotes from yeast to humans. In mammalian cells, PP6 targets IκBε for degradation, activates DNA-dependent protein kinase to trigger DNA repair, and is reportedly required for normal mitosis. Recently, Ppp6c mutations were identified as candidate drivers of melanoma and skin cancer. Nonetheless, little is known about the physiological role of Ppp6c. To investigate this function in vivo, we established mice lacking the Ppp6c phosphatase domain by crossing heterozygous mutants. No viable homozygous pups were born, indicative of a lethal mutation. Ppp6c homozygous mutant embryos were identified among blastocysts, which exhibited a normal appearance, but embryos degenerated by E7.5 and showed clear developmental defects at E8.5, suggesting that mutant embryos die after implantation. Accordingly, homozygous blastocysts showed significant growth failure of the inner cell mass (ICM) in in vitro blastocyst culture, and primary Ppp6c exon4-deficient MEFs showed greatly reduced proliferation. These results establish for the first time that the Ppp6c phosphatase domain is indispensable for mouse embryogenesis after implantation.