Search

Oncometabolites, such as D/L-2-hydroxyglutarate (2HG), have directly been implicated in carcinogenesis; however, the underlying molecular mechanisms remain poorly understood. Here, we showed that the levels of the L-enantiomer of 2HG (L2HG) were specifically increased in colorectal cancer (CRC) tissues and cell lines compared with the D-enantiomer of 2HG (D2HG). In addition, L2HG increased the expression of ATF4 and its target genes by activating the mTOR pathway, which subsequently provided amino acids and improved the survival of CRC cells under serum deprivation. Downregulating the expression of L-2-hydroxyglutarate dehydrogenase (L2HGDH) and oxoglutarate dehydrogenase (OGDH) increased L2HG levels in CRC, thereby activating mTOR-ATF4 signaling. Furthermore, L2HGDH overexpression reduced L2HG-mediated mTOR-ATF4 signaling under hypoxia, whereas L2HGDH knockdown promoted tumor growth and amino acid metabolism in vivo. Together, these results indicate that L2HG ameliorates nutritional stress by activating the mTOR-ATF4 axis and thus could be a potential therapeutic target for CRC.

Upregulation of nuclear factor κB (NFκB) signaling is a hallmark of aging and a major cause of age-related chronic inflammation. NFκB activity plays a critical role in transcriptional regulation and cell fate determination; however, its physiological function in inflammatory aging remains unclear. Here, we demonstrate that dysfunction of negative feedback regulators of NFκB, IκBα and A20, alters the NFκB nuclear dynamics from oscillatory to sustained, thereby promoting cellular senescence. Sustained NFκB activity by IκBα downregulation enhances inflammatory gene expression through increased NFκB-DNA binding, promotes purine catabolism by downregulating hypoxanthine phosphoribosyltransferase, and arrests the cell cycle. This regulatory mechanism was confirmed in aged mice heart tissues, suggesting that prolonged nuclear localization of NFκB drives chronic inflammation and metabolic rewiring associated with aging.

Notch signaling regulates cell-fate decisions during development and postnatal life. Little is known, however, about the role of Delta-like-4 (Dll4)-Notch signaling between cancer cells, or how this signaling affects cancer metastasis. We, therefore, assessed the role of Dll4-Notch signaling in cancer metastasis. We generated a soluble Dll4 fused to the IgG1 constant region (Dll4-Fc) that acts as a blocker of Dll4-Notch signaling and introduced it into human small cell lung cancer (SCLC) cell lines expressing either high levels (SBC-3 and H1048) or low levels (SBC-5) of Dll4. The effects of Dll4-Fc on metastasis of SCLC were evaluated using a mouse model. Although Dll4-Fc had no effect on the liver metastasis of SBC-5, the number of liver metastasis inoculated with SBC-3 and H1048 cells expressing Dll4-Fc was significantly lower than that injected with control cells. To study the molecular mechanisms of the effects of Dll4-Fc on liver metastasis, a PCR array analysis was conducted. Because the expression of NF-κB target genes was affected by Dll4-Fc, we conducted an electrophoretic mobility shift assay and observed that NF-κB activities, both with and without stimulation by TNF-α, were downregulated in Dll4-Fc–overexpressing SBC-3 and H1048 cells compared with control cells. Moreover, Dll4-Fc attenuates, at least in part, the classical and alternative NF-κB activation pathway by reducing Notch1 signaling. These results suggest that Dll4-Notch signaling in cancer cells plays a critical role in liver metastasis of SCLC by regulating NF-κB signaling. Mol Cancer Ther; 11(12); 2578–87. ©2012 AACR.

PDF file - 330K, Dll4-Fc reduced NF-κB/DNA binding in SBC-3 and H1048 cells

PDF file - 245K, Dll4-Fc promoted excessive angiogenesis of liver metastasis without affecting VEGFA expression and HUVECs proliferation

PDF file - 50K, The effect of recombinant Dll4-Fc on the NF-κB activity of SBC-3-vector cells

PDF file - 116K, Extracellular domain of murine Dll4-fused to Fc (Dll4-Fc) expressed in H1048 and SBC-5 cells

PDF file - 83K

PDF file - 174K, Dll4-Fc suppressed TNF-α induced NF-κB activity without attenuation of the cytoplasmic sequestration of NF-κB subunit

PDF file - 69K, Dll4-Fc suppresses genes-associated with metastasis in SCLC cells

PDF file - 47K, Taqman gene expression assays

Supplementary Table 1 from Molecular Basis for the Induction of an Angiogenesis Inhibitor, Thrombospondin-1, by 5-Fluorouracil

5-Fluorouracil (5-FU) is one of the most commonly used anticancer drugs in chemotherapy against various solid tumors. 5-FU dose-dependently increased the expression levels of intrinsic antiangiogenic factor thrombospondin-1 (TSP-1) in human colon carcinoma KM12C cells and human breast cancer MCF7 cells. We investigated the molecular basis for the induction of TSP-1 by 5-FU in KM12C cells. Promoter assays showed that the region with the Egr-1 binding site is critical for the induction of TSP-1 promoter activity by 5-FU. The binding of Egr-1 to the TSP-1 promoter was increased in KM12C cells treated with 5-FU. Immunofluorescence staining revealed that 5-FU significantly increased the level of Egr-1 in the nuclei of KM12C cells. The suppression of Egr-1 expression by small interfering RNA decreased the expression level of TSP-1. Furthermore, 5-FU induced the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and heat shock protein 27 (HSP27). Blockade of the p38 MAPK pathway by SB203580 remarkably inhibited the phosphorylation of HSP27 induced by 5-FU and decreased the induction of Egr-1 and TSP-1 by 5-FU in KM12C cells. These findings suggest that the p38 MAPK pathway plays a crucial role in the induction of Egr-1 by 5-FU and that induced Egr-1 augments TSP-1 promoter activity, with the subsequent production of TSP-1 mRNA and protein. [Cancer Res 2008;68(17):7035–41]

Our understanding of metabolic reprogramming in cancer has tremendously improved along with the technical progression of metabolomic analysis. Metabolic changes in cancer cells proved much more complicated than the classical Warburg effect. Previous studies have approached metabolic changes as therapeutic and/or chemopreventive targets. Recently, several clinical trials have reported anti-cancer agents associated with metabolism. However, whether cancer cells are dependent on metabolic reprogramming or favor suitable conditions remains nebulous. Both scenarios are possibly intertwined. Identification of downstream molecules and the understanding of mechanisms underlying reprogrammed metabolism can improve the effectiveness of cancer therapy. Here, we review several examples of the metabolic reprogramming of cancer cells and the therapies targeting the metabolism-related molecules as well as discuss practical approaches to improve the next generation of cancer therapies focused on the metabolic reprogramming of cancer.

Adapter proteins CRK and CRKL participate in a variety of signaling pathways, including cell adhesion, and fate regulation of mammalian cells. However, the molecular functions of CRK/CRKL in epigenetic regulation remain largely unknown. Here, we developed a pipeline to evaluate cell morphology using high-content image analysis combined with chemical screening of kinase and epigenetic modulators. We found that CRK/CRKL modulates gene regulatory networks associated with cell morphology through epigenetic alteration in mouse embryonic fibroblasts. Integrated epigenome and transcriptome analyses revealed that CRK/CRKL is involved in super-enhancer activity and upregulation of Cdt1, Rin1, and Spp1 expression for the regulation of cell morphology. Screening of a library of 80 epigenetic inhibitors showed that histone H3 modifiers, euchromatic histone methyltransferase 2 and mitogen- and stress-activated kinase 1, may be important for CRK/CRKL-mediated morphological changes. Taken together, our results indicate that CRK/CRKL plays a critical role in gene regulatory networks through epigenetic modification. DATABASES: Chromatin immunoprecipitation sequencing and RNA sequencing data were deposited in the DNA Data Bank of Japan under DRA011080 and DRA011081 accession numbers, respectively.

The spread of cancer to bone is invariably fatal, with complex cross-talk between tumor cells and the bone microenvironment responsible for driving disease progression. By combining in silico analysis of patient datasets with metabolomic profiling of prostate cancer cells cultured with bone cells, we demonstrate the changing energy requirements of prostate cancer cells in the bone microenvironment, identifying the pentose phosphate pathway (PPP) as elevated in prostate cancer bone metastasis, with increased expression of the PPP rate-limiting enzyme glucose-6-phosphate dehydrogenase (G6PD) associated with a reduction in progression-free survival. Genetic and pharmacologic manipulation demonstrates that G6PD inhibition reduces prostate cancer growth and migration, associated with changes in cellular redox state and increased chemosensitivity. Genetic blockade of G6PD in vivo results in reduction of tumor growth within bone. In summary, we demonstrate the metabolic plasticity of prostate cancer cells in the bone microenvironment, identifying the PPP and G6PD as metabolic targets for the treatment of prostate cancer bone metastasis.

Epithelial–mesenchymal transition (EMT)—a fundamental process in embryogenesis and wound healing—promotes tumor metastasis and resistance to chemotherapy. While studies have identified signaling components and transcriptional factors responsible in the TGF-β-dependent EMT, whether and how intracellular metabolism is integrated with EMT remains to be fully elucidated. Here, we showed that TGF-β induces reprogramming of intracellular amino acid metabolism, which is necessary to promote EMT in non-small cell lung cancer cells. Combined metabolome and transcriptome analysis identified prolyl 4-hydroxylase α3 (P4HA3), an enzyme implicated in cancer metabolism, to be upregulated during TGF-β stimulation. Further, knockdown of P4HA3 diminished TGF-β-dependent changes in amino acids, EMT, and tumor metastasis. Conversely, manipulation of extracellular amino acids induced EMT-like responses without TGF-β stimulation. These results suggest a previously unappreciated requirement for the reprogramming of amino acid metabolism via P4HA3 for TGF-β-dependent EMT and implicate a P4HA3 inhibitor as a potential therapeutic agent for cancer., Through metabolome and transcriptome analyses, Nakasuka et al find that TGF-β-induced epithelial–mesenchymal transition (EMT) in non-small cell lung cancer cells is associated with reprogramming of amino acid metabolism. They also identify P4HA3 as a key enzyme involved in these changes altogether providing insights into potential mechanisms of metastasis.

Extracellular vesicles (EVs) released from cancer cells contribute to various malignant phenotypes of cancer, including metastasis, cachexia, and angiogenesis. Although DNA, mRNAs, miRNAs, and proteins contained in EVs have been extensively studied, the function of metabolites in EVs remains unclear. In this study, we performed a comprehensive metabolomic analysis of pancreatic cancer cells, PANC-1, cultured under different oxygen concentrations, and small EVs (sEVs) released from them, considering the fact that hypoxia contributes to the malignant behavior of cells in pancreatic cancer, which is a poorly diagnosed cancer. sEVs were collected by ultracentrifugation, and hydrophilic metabolites were analyzed using capillary ion chromatography-mass spectrometry and liquid chromatography-mass spectrometry, and lipids were analyzed by supercritical fluid chromatography-tandem mass spectrometry. A total of 140 hydrophilic metabolites and 494 lipids were detected in sEVs, and their profiles were different from those in cells. In addition, the metabolomic profile of sEVs was observed to change under hypoxic stress, and an increase in metabolites involved in angiogenesis was also detected. We reveal the hallmark of the metabolites contained in sEVs and the effect of tumor hypoxia on their profiles, which may help in understanding EV-mediated cancer malignancy.

Background Cisplatin (CDDP) significantly prolongs survival in various cancers, but many patients also develop resistance that results in treatment failure. Thus, this study aimed to elucidate the underlying mechanisms by which ovarian cancer cells acquire CDDP resistance. Methods We evaluated the metabolic profiles in CDDP-sensitive ovarian cancer A2780 cells and CDDP-resistant A2780cis cells using capillary electrophoresis-time-of-flight mass spectrometry (CE-TOFMS). We further examined the expression of glutamine metabolism enzymes using real-time PCR and Western blot analyses. Cell viability was accessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Results The results showed that levels of glutamine, glutamate, and glutathione (GSH), a key drug resistance mediator synthesized from glutamate, were significantly elevated in A2780cis cells than those in A2780 cells. Furthermore, glutamine starvation decreased the GSH levels and CDDP resistance in A2780cis cells. Interestingly, the expression of glutamine synthetase (GS/GLUL), which synthesizes glutamine from glutamate and thereby negatively regulates GSH production, was almost completely suppressed in resistant A2780cis cells. In addition, treatment of A2780cis cells with 5-aza-2′-deoxycytidine, a DNA-demethylating agent, restored GS expression and reduced CDDP resistance. In contrast, GS knockdown in CDDP-sensitive A2780 cells induced CDDP resistance. Conclusions The results indicate that upregulation of GSH synthesis from glutamine via DNA methylation-mediated silencing of GS causes CDDP resistance in A2780cis cells. Therefore, glutamine metabolism could be a novel therapeutic target against CDDP resistance.

Thymidine phosphorylase (TP) is a rate-limiting enzyme in thymidine catabolism. TP has several important roles in biological and pharmacological mechanisms; importantly TP acts as an angiogenic factor and one of metabolic enzymes of fluoro-pyrimidine anticancer agents and modifies inflammation. Improving our understanding of the characteristics and functions of TP has led to the development of novel TP-based anticancer therapies. We recently reported that TP-dependent thymidine catabolism contributes to tumour survival in low nutrient conditions and the pathway from thymidine to the glycolysis cascade is affected in the context of physiological and metabolic conditions. In this review, we describe recent advancement in our understanding of TP, with a focus on cancer cell biology and the pharmacology of pyrimidine analogue anticancer agents. This review provides comprehensive understanding of the molecular mechanism of TP function in cancer.

The use of synthetic biomarkers is an emerging technique to improve disease diagnosis. Here, we report a novel design strategy that uses analyte-responsive acetaminophen (APAP) to expand the catalogue of analytes available for synthetic biomarker development. As proof-of-concept, we designed hydrogen peroxide (H2 O2 )-responsive APAP (HR-APAP) and succeeded in H2 O2 detection with cellular and animal experiments. In fact, for blood samples following HR-APAP injection, we demonstrated that the plasma concentration ratio [APAP+APAP conjugates]/[HR-APAP] accurately reflects in vivo differences in H2 O2 levels. We anticipate that our practical methodology will be broadly useful for the preparation of various synthetic biomarkers.

Oncogenes often promote cell death as well as proliferation. How oncogenes drive these diametrically opposed phenomena remains to be solved. A key question is whether cell death occurs as a response to aberrant proliferation signals or through a proliferation-independent mechanism. Here, we reveal that Src, the first identified oncogene, simultaneously drives cell proliferation and death in an obligatorily coupled manner through parallel MAPK pathways. The two MAPK pathways diverge from a lynchpin protein Slpr. A MAPK p38 drives proliferation whereas another MAPK JNK drives apoptosis independently of proliferation signals. Src-p38-induced proliferation is regulated by methionine-mediated Tor signaling. Reduction of dietary methionine uncouples the obligatory coupling of cell proliferation and death, suppressing tumorigenesis and tumor-induced lethality. Our findings provide an insight into how cells evolved to have a fail-safe mechanism that thwarts tumorigenesis by the oncogene Src. We also exemplify a diet-based approach to circumvent oncogenesis by exploiting the fail-safe mechanism.

Recently, ion chromatography coupled with mass spectrometry has been used for the determination of anionic metabolites. However, connection with a mass spectrometer in this method is not straightforward because backpressure produced by the addition of a make-up solution often affects the peak resolutions of the target metabolites. To overcome this problem, we developed a capillary ion chromatography-mass spectrometry method utilizing a double coaxial electrospray ionization sprayer. This method was not affected by backpressure and the number of theoretical plates was about three times that of a conventional sprayer. Under optimized conditions, 44 anionic metabolites, including organic acids, sugar phosphates, nucleotides, and cofactors, were successfully separated and selectively detected with a Q Exactive mass spectrometer. The calibration curves of the tested metabolites showed excellent linearity within the range of 1–100,000 nmol/L and the correlation coefficient was greater than 0.991. The detection limits for these metabolites were between 1 and 500 nmol/L (0.4 and 200 fmol). The developed method was applied to the quantitation of anionic metabolites in cultured cancer cell samples with tumor necrosis factor (TNF)-α stimulation. This allowed for the successful determination of 105 metabolites. The levels of tricarboxylic acid cycle intermediates changed significantly after TNF-α stimulation. These results demonstrate that the developed method is a promising new tool for comprehensive analysis of anionic metabolites.

Tumor biomarkers are highly desirable for the screening of patients with a risk of tumor development and progression. Here, we report a beta-galactosidase (β-gal)-responsive acetaminophen (β-GR-APAP) as a synthetic plasma biomarker for targeted tumor detection. Tumor β-gal labeling via the recognition of tumor-related antigen enabled the detection of a tumor using β-GR-APAP.

Thymidine phosphorylase (TP) is a rate-limiting enzyme in the thymidine catabolic pathway. TP is identical to platelet-derived endothelial cell growth factor and contributes to tumour angiogenesis. TP induces the generation of reactive oxygen species (ROS) and enhances the expression of oxidative stress-responsive genes, such as interleukin (IL)-8. However, the mechanism underlying ROS induction by TP remains unclear. In the present study, we demonstrated that TP promotes NADPH oxidase-derived ROS signalling in cancer cells. NADPH oxidase inhibition using apocynin or small interfering RNAs (siRNAs) abrogated the induction of IL-8 and ROS in TP-expressing cancer cells. Meanwhile, thymidine catabolism induced by TP increased the levels of NADPH and intermediates of the pentose phosphate pathway (PPP). Both siRNA knockdown of glucose 6-phosphate dehydrogenase (G6PD), a rate-limiting enzyme in PPP, and a G6PD inhibitor, dihydroepiandrosterone, reduced TP-induced ROS production. siRNA downregulation of 2-deoxy-D-ribose 5-phosphate (DR5P) aldolase, which is needed for DR5P to enter glycolysis, also suppressed the induction of NADPH and IL-8 in TP-expressing cells. These results suggested that TP-mediated thymidine catabolism increases the intracellular NADPH level via the PPP, which enhances the production of ROS by NADPH oxidase and activates its downstream signalling.

A sheath-flow interface is the most common ionization technique in CE-ESI-MS. However, this interface dilutes the analytes with the sheath liquid and decreases the sensitivity. In this study, we developed a sheathless CE-MS interface to improve sensitivity. The interface was fabricated by making a small crack approximately 2 cm from the end of a capillary column fixed on a plastic plate, and then covering the crack with a dialysis membrane to prevent metabolite loss during separation. A voltage for CE separation was applied between the capillary inlet and the buffer reservoir. Under optimum conditions, 52 cationic metabolite standards were separated and selectively detected using MS. With a pressure injection of 5 kPa for 15 s (ca. 1.4 nL), the detection limits for the tested compounds were between 0.06 and 1.7 μmol/L (S/N = 3). The method was applied to analysis of cationic metabolites extracted from a small number (12 000) of cancer cells, and the number of peaks detected was about 2.5 times higher than when using conventional sheath-flow CE-MS. Because the interface is easy to construct, it is cost-effective and can be adapted to any commercially available capillaries. This method is a powerful new tool for highly sensitive CE-MS-based metabolomic analysis.

Cancer cells alter their metabolism for the production of precursors of macromolecules. However, the control mechanisms underlying this reprogramming are poorly understood. Here we show that metabolic reprogramming of colorectal cancer is caused chiefly by aberrant MYC expression. Multiomics-based analyses of paired normal and tumor tissues from 275 patients with colorectal cancer revealed that metabolic alterations occur at the adenoma stage of carcinogenesis, in a manner not associated with specific gene mutations involved in colorectal carcinogenesis. MYC expression induced at least 215 metabolic reactions by changing the expression levels of 121 metabolic genes and 39 transporter genes. Further, MYC negatively regulated the expression of genes involved in mitochondrial biogenesis and maintenance but positively regulated genes involved in DNA and histone methylation. Knockdown of MYC in colorectal cancer cells reset the altered metabolism and suppressed cell growth. Moreover, inhibition of MYC target pyrimidine synthesis genes such as CAD, UMPS, and CTPS blocked cell growth, and thus are potential targets for colorectal cancer therapy.

Objective Thymidine phosphorylase (TP) in rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) is induced by tumor necrosis factor α (TNFα) and other cytokines that have been reported to be major inflammation mediators in RA. We previously demonstrated that TP plays an important role in angiogenesis and tumor growth, invasion, and metastasis. The aim of this study was to investigate whether the role of TP in the pathogenesis of RA is similar to its role in tumors. Methods In FLS obtained from 2 patients with RA, the expression of TP, interferon-γ (IFNγ)–inducible protein 10 (CXCL10), and other cytokines was measured by quantitative real-time polymerase chain reaction, immunoblotting, and enzyme-linked immunosorbent assays. Microarray analysis was performed using FLS transfected with TYMP complementary DNA and treated with a TP inhibitor. Results The expression of TP in FLS was up-regulated by TNFα, interleukin-1β (IL-1β), IL-17, IFNγ, and lipopolysaccharide. Microarray analysis of FLS overexpressing TP identified CXCL10 as a thymidine phosphorylase–related gene. The expression of CXCL10 was induced by TNFα, and this induction was suppressed by TYMP small interfering RNA and TP inhibitor. Furthermore, the combination of TNFα and IFNγ synergistically augmented the expression of TP and CXCL10. TP-induced CXCL10 expression was suppressed by the antioxidant EUK-8. In the synovial tissue of patients with RA, TP levels were significantly correlated with CXCL10 expression. Conclusion The combination of TNFα and IFNγ strongly induced the expression of thymidine phosphorylase in RA FLS. The induction of thymidine phosphorylase enhanced the expression of CXCL10, which may contribute to the Th1 phenotype and bone destruction observed in RA.

Despite recent advances in treatment, malignant pleural mesothelioma (MPM) remains a deadly disease. Targeted therapy generated broad interests and is highly expected for the treatment of MPM, yet promising preclinical results have not been translated into substantial clinical benefits for the patients. In this study, we tried to identify the genes which play functional roles in cell migration as well as to test whether they can be used as novel targets for molecular targeted therapy for MPM in preclinical model. In our study, pregnancy-associated plasma protein A (PAPPA) was identified as a gene whose expression level is correlated with MPM cell migration by correlation analysis combining MPM cell migration ability and their gene expression profiles. Highly migratory cells were selected from MPM cell lines, MSTO-211H, NCI-H290 and EHMES-1 in vitro and up-regulation of PAPPA in these cells were confirmed. In vitro, PAPPA was demonstrated to stimulate the MPM cell migration via cleavage of insulin-like growth factor-binding protein-4 and subsequent release of IGF-1. Gene silencing of PAPPA in MPM cells led to reduced migration, invasion and proliferation. Furthermore, PAPPA shRNA transfected NCI-H290 when orthotopically inoculated into pleural cavity of severe combined immunodeficiency recipient mice, failed to develop tumors and produce bloody pleural effusion as control shRNA transfected cells did. Our study suggests that PAPPA plays a functional role in promoting MPM cell migration and it might serve as a potential therapeutic target for the treatment of MPM.

Major vault protein (MVP) is identical to lung resistance-related protein (LRP), which is the major component of vaults. Vaults are considered to play a protective role against xenobiotics and other types of stress. In a previous study, we reported that the expression levels of MVP in SW620 human colon cancer cells were increased in hypertonic culture medium with sucrose. However, the molecular mechanism behind the induction of MVP expression by osmotic stress has not yet been elucidated. Therefore, in the present study, we investigated the mechanism behind the induction of MVP expression by osmotic stress. Under hyperosmotic stress conditions, the ubiquitination of specificity protein 1 (Sp1) decreased, Sp1 protein levels increased, its binding to the MVP promoter was enhanced, and small interfering RNA (siRNA) for Sp1 suppressed the induction of MVP expression. The inhibition of c-jun N-terminal kinase (JNK) by SP600125, a specific JNK inhibitor, decreased the expression of MVP and Sp1 under hyperosmotic conditions. Our data indicate that the stabilization and upregulation of Sp1 protein expression by JNK participate in the inhibition of the ubiquitination and degradation of Sp1, and thus in the induction of MVP expression under hyperosmotic conditions.

The angiogenic factor, platelet-derived endothelial cell growth factor/thymidine phosphorylase (PD-ECGF/TP), stimulates the chemotaxis of endothelial cells and confers resistance to apoptosis induced by hypoxia. 2-Deoxy-D-ribose, a degradation product of thymidine generated by TP enzymatic activity, inhibits the upregulation of hypoxia-inducible factor (HIF) 1α, BNIP3 and caspase-3 induced by hypoxia. In the present study, we investigated the molecular basis for the suppressive effect of 2-deoxy-D-ribose on the upregulation of HIF-1α. 2-Deoxy-D-ribose enhanced the interaction of HIF-1α and the von Hippel-Lindau (VHL) protein under hypoxic conditions. It did not affect the expression of HIF-1α, prolyl hydroxylase (PHD)1/2/3 and VHL mRNA under normoxic or hypoxic conditions, but enhanced the interaction of HIF-1α and PHD2 under hypoxic conditions. 2-Deoxy-D-ribose also increased the amount of hydroxy-HIF-1α in the presence of the proteasome inhibitor MG-132. The expression levels of TP are elevated in many types of malignant solid tumors and, thus, 2-deoxy-D-ribose generated by TP in these tumors may play an important role in tumor progression by preventing hypoxia-induced apoptosis.

Surfactant protein A (SP-A) is a large multimeric protein found in the lungs. In addition to its immunoregulatory function in infectious respiratory diseases, SP-A is also used as a marker of lung adenocarcinoma. Despite the finding that SP-A expression levels in cancer cells has a relationship with patient prognosis, the function of SP-A in lung cancer progression is unknown. We investigated the role of SP-A in lung cancer progression by introducing the SP-A gene into human lung adenocarcinoma cell lines. SP-A gene transduction suppressed the progression of tumor in subcutaneous xenograft or lung metastasis mouse models. Immunohistochemical analysis showed that the number of M1 antitumor tumor-associated macrophages (TAMs) was increased and the number of M2 tumor-promoting TAMs was not changed in the tumor tissue produced by SP-A-expressing cells. In addition, natural killer (NK) cells were also increased and activated in the SP-A-expressing tumor. Moreover, SP-A did not inhibit tumor progression in mice depleted of NK cells. Taking into account that SP-A did not directly activate NK cells, these results suggest that SP-A inhibited lung cancer progression by recruiting and activating NK cells via controlling the polarization of TAMs.

Notch signaling regulates cell-fate decisions during development and postnatal life. Little is known, however, about the role of Delta-like-4 (Dll4)-Notch signaling between cancer cells, or how this signaling affects cancer metastasis. We, therefore, assessed the role of Dll4-Notch signaling in cancer metastasis. We generated a soluble Dll4 fused to the IgG1 constant region (Dll4-Fc) that acts as a blocker of Dll4-Notch signaling and introduced it into human small cell lung cancer (SCLC) cell lines expressing either high levels (SBC-3 and H1048) or low levels (SBC-5) of Dll4. The effects of Dll4-Fc on metastasis of SCLC were evaluated using a mouse model. Although Dll4-Fc had no effect on the liver metastasis of SBC-5, the number of liver metastasis inoculated with SBC-3 and H1048 cells expressing Dll4-Fc was significantly lower than that injected with control cells. To study the molecular mechanisms of the effects of Dll4-Fc on liver metastasis, a PCR array analysis was conducted. Because the expression of NF-κB target genes was affected by Dll4-Fc, we conducted an electrophoretic mobility shift assay and observed that NF-κB activities, both with and without stimulation by TNF-α, were downregulated in Dll4-Fc–overexpressing SBC-3 and H1048 cells compared with control cells. Moreover, Dll4-Fc attenuates, at least in part, the classical and alternative NF-κB activation pathway by reducing Notch1 signaling. These results suggest that Dll4-Notch signaling in cancer cells plays a critical role in liver metastasis of SCLC by regulating NF-κB signaling. Mol Cancer Ther; 11(12); 2578–87. ©2012 AACR.

Cellular energy production processes are composed of many Mg2+ dependent enzymatic reactions. In fact, dysregulation of Mg2+ homeostasis is involved in various cellular malfunctions and diseases. Recently, mitochondria, energy-producing organelles, have been known as major intracellular Mg2+ stores. Several biological stimuli alter mitochondrial Mg2+ concentration by intracellular redistribution. However, in living cells, whether mitochondrial Mg2+ alteration affect cellular energy metabolism remains unclear. Mg2+ transporter of mitochondrial inner membrane MRS2 is an essential component of mitochondrial Mg2+ uptake system. Here, we comprehensively analyzed intracellular Mg2+ levels and energy metabolism in Mrs2 knockdown (KD) cells using fluorescence imaging and metabolome analysis. Dysregulation of mitochondrial Mg2+ homeostasis disrupted ATP production via shift of mitochondrial energy metabolism and morphology. Moreover, Mrs2 KD sensitized cellular tolerance against cellular stress. These results indicate regulation of mitochondrial Mg2+via MRS2 critically decides cellular energy status and cell vulnerability via regulation of mitochondrial Mg2+ level in response to physiological stimuli.

Bevacizumab exerts anti-angiogenic effects in cancer patients by inhibiting vascular endothelial growth factor (VEGF). However, its use is still limited due to the development of resistance to the treatment. Such resistance can be regulated by various factors, although the underlying mechanisms remain incompletely understood. Here we show that bone marrow-derived fibrocyte-like cells, defined as alpha-1 type I collagen-positive and CXCR4-positive cells, contribute to the acquired resistance to bevacizumab. In mouse models of malignant pleural mesothelioma and lung cancer, fibrocyte-like cells mediate the resistance to bevacizumab as the main producer of fibroblast growth factor 2. In clinical specimens of lung cancer, the number of fibrocyte-like cells is significantly increased in bevacizumab-treated tumours, and correlates with the number of treatment cycles, as well as CD31-positive vessels. Our results identify fibrocyte-like cells as a promising cell biomarker and a potential therapeutic target to overcome resistance to anti-VEGF therapy., Acquired resistance to anti-angiogenic drugs, including bevacizumab, may occur in cancer patients. In this study the authors identify in the tumour microenvironment, fibrocyte-like cells derived from the bone marrow that mediate the resistance to bevacizumab through the production of FGF2.

Thymidine phosphorylase (TP), a rate-limiting enzyme in the thymidine catabolic pathway, catalyzes the reversible conversion of thymidine to thymine and 2-deoxy-D-ribose 1-phosphate (DR1P). We previously demonstrated that TP is identical to an anigogenic factor, platelet-derived endothelial cell growth factor (PD-ECGF). Although many reports indicate that TP has angiogenic activity and plays a pivotal role in tumor progression, the mechanism of the TP functions is not fully understood. Here, we find that TP-mediated thymidine catabolism can supply the carbon source in the glycolytic pathway and thus contribute to cell survival under conditions of nutrient deprivation. In certain procaryotes, phosphopentosemutase converts thymidine-derived DR1P to 2-deoxy-D-ribose 5-phosphate (DR5P), which is then converted to glyceraldehyde 3-phosphate (GAP) and acetaldehyde by deoxyriboaldorase (DERA). Given that GAP is an intermediate in the glycolytic pathway and the pentose phosphate pathway (PPP), we hypothesized that the activation of thymidine catabolism by TP could supply the carbon source for those pathways. However, the thymidine catabolic pathway in mammalian cells remains unclarified. We used 13C-labeled thymidine (13C5-thymidine) as a tracer and examined whether 13C5-thymidine-derived metabolites enter the glycolytic pathway and the PPP in TP-expressing cancer cells using capillary electrophoresis time-of-flight mass spectrometry. Thymidine was converted to metabolites, including glucose 6-phosphate, lactate, 5-phospho-α-ribose 1-diphosphate, and serine, via the glycolytic pathway both in vitro and in vivo. These thymidine-derived metabolites were required for the survival of cells under low-glucose conditions. Furthermore, enhanced thymidine catabolism was observed in human gastric cancer. These findings suggest that the thymidine catabolism plays an important role in the survival and growth of cancer cells in a microenvironment where glucose is insufficient. Citation Format: Sho Tabata, Masatatsu Yamamoto, Hisatsugu Goto, Yasuhiko Nishioka, Masaru Tomita, Tomoyoshi Soga, Tatsuhiko Furukawa, Shin-ichi Akiyama. Novel linkage of thymidine catabolism and the glycolytic pathway in human cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1442.

5-FU is commonly used for treatment of various solid tumors including colon carcinoma. We have previously demonstrated that Egr-1 induced by 5-FU enhanced TSP-1 expression in human colon cancer KM12C cells. In this study, a Genechip analysis of KM12C cells treated with 5-FU revealed down-regulation of 924 genes and up-regulation of 460 genes. The decreased expression of c-Myc mRNA and phosphorylated c-Myc were detected and confirmed by RT-PCR and immunoblotting. Since 5-FU induced the expression of TSP-1, we examined the effect of c-Myc on the TSP-1 promoter. Deletion of the TSP-1 promoter region in which binding sites for c-Myc reside had no effect on the TSP-1 promoter activity induced by 5-FU. Meanwhile, 5-FU dose-dependently decreased the expression of miR-17-92 cluster. These findings suggest that 5-FU decreased the expression of c-Myc and consequently miR-17-92 cluster and increased the expression of TSP-1 mRNA.