bims-meluca 2019-04-28 papers

on Metabolism of non-small cell lung carcinoma

Issue of 2019–04–28
nine papers selected by
the Muñoz-Pinedo/Nadal (PReTT) lab, L’Institut d’Investigació Biomèdica de Bellvitge and Cristina Muñoz Pinedo, L’Institut d’Investigació Biomèdica de Bellvitge

A Novel Mechanism of High Dose Radiation Sensitization by Metformin.
Activity and molecular targets of pioglitazone via blockade of proliferation, invasiveness and bioenergetics in human NSCLC.
Cathepsin L Regulates Metabolic Networks Controlling Rapid Cell Growth and Proliferation.
Mammalian Eps15 homology domain 1 potentiates angiogenesis of non-small cell lung cancer by regulating β2AR signaling.
Screening common signaling pathways associated with drug resistance in non-small cell lung cancer via gene expression profile analysis.
Can Nutritional Status Predict Overall Survival in Patients with Advanced Non-Small Cell Lung Cancer?
[Study on the Metabolic Reprogramming of Lung Cancer Cells Regulated by Docetaxel Based on Metabolomics].
Inhibitor of ghrelin receptor reverses gefitinib resistance in lung cancer.
shRNA-WT1 Potentiates Anticancer Effects of Gemcitabine and Cisplatin Against B16F10 Lung Metastases In Vitro and In Vivo.

Front Oncol. 2019 ;9 247

A Novel Mechanism of High Dose Radiation Sensitization by Metformin.

Stephen L Brown, Andrew Kolozsvary, Derek M Isrow, Karine Al Feghali, Karen Lapanowski, Kenneth A Jenrow, Jae Ho Kim.

  Introduction: Metformin, the most widely used treatment for diabetes, is lethal to cancer cells and increases in toxicity when used in combination with radiation. In addition to various molecular and metabolic mechanisms that have been previously proposed, the studies presented provide evidence of an additional, novel mechanism of sensitization following high dose radiotherapy; the magnitude of sensitization depends on the microenvironmental levels of glucose and oxygen which are in turn affected by high dose radiation. Methods: Cancer cells (A549 and MCF7) were studied in vitro under various controlled conditions. Endpoints included clonogenic cell survival and ROS expression measured by DHE and DCFDA. CD1 nu/nu athymic mice implanted with A549 cells received metformin alone (200 mg/kg, i.p.), radiation alone (15 Gy) or a combination of metformin and radiation; the effect of treatment sequence on efficacy was assessed by tumor growth delay and histology. In a separate set of experiments, tumor blood flow was measured using a tracer clearance technique using SPECT after the administration of metformin alone, radiation alone and the combined treatment. Results: In vivo, metformin provided equally effective tumor growth delay when given 24 h after radiation as when given 1 h or 4 h before radiation, an observation not previously reported and, in fact, unexpected based on published scientific literature. When drug followed radiation, the tumors were histologically characterized by massive cellular necrosis. In vitro, cancer cells when glucose depleted and/or hypoxic were preferentially killed by metformin, in a drug dose dependent manner. A549 cells exposed to 5.0 mM of metformin was reduced seven fold in survival when in a glucose deprived as compared to a low-glucose medium (0 vs. 1.0 g/L). Finally, using a SPECT detector to follow the washout of a radioactive tracer, it was shown that a high single dose of radiosurgery (15 Gy) could dramatically inhibit blood flow and presumably diminish glucose and oxygen. Discussion: Insight into the best timing of drug and radiation administration is gained through an understanding of the mechanisms of interaction. A new mechanism of metformin sensitization by high dose radiation is proposed based on the blood flow, glucose and oxygen.

Keywords:  cancer; glucose; metabolism; metformin; radiation; tumor blood flow

DOI:  https://doi.org/10.3389/fonc.2019.00247
J Exp Clin Cancer Res. 2019 Apr 26. 38(1): 178

Activity and molecular targets of pioglitazone via blockade of proliferation, invasiveness and bioenergetics in human NSCLC.

Vincenza Ciaramella, Ferdinando Carlo Sasso, Raimondo Di Liello, Carminia Maria Della Corte, Giusi Barra, Giuseppe Viscardi, Giovanna Esposito, Francesca Sparano, Teresa Troiani, Erika Martinelli, Michele Orditura, Ferdinando De Vita, Fortunato Ciardiello, Floriana Morgillo.

   BACKGROUND: Pioglitazone, a synthetic peroxisome proliferator activated receptor (PPAR-γ) ligand, is known as an antidiabetic drug included in the thiazolidinediones (TZDs) class. It regulates the lipid and glucose cell metabolism and recently a role in the inhibition of numerous cancer cell processes has been described.
METHODS: In our work we investigate the anti-tumor effects of pioglitazone in in vitro models of non small cell lung cancer (NSCLC) and also, we generated ex-vivo three-dimensional (3D) cultures from human lung adenocarcinoma (ADK) as a model to test drug efficacy observed in vitro. The inhibitory effect of pioglitazone on cell proliferation, apoptosis and cell invasion in a panel of human NSCLC cell lines was evaluated by multiple assays.
RESULTS: Pioglitazone reduced proliferative and invasive abilities with an IC50 ranging between 5 and 10 μM and induced apoptosis of NSCLC cells. mRNA microarray expression profiling showed a down regulation of MAPK, Myc and Ras genes after treatment with pioglitazone; altered gene expression was confirmed by protein analysis in a dose-related reduction of survivin and phosphorylated proteins levels of MAPK pathway. Interestingly mRNA microarray analysis showed also that pioglitazone affects TGFβ pathway, which is important in the epithelial-to-mesenchimal transition (EMT) process, by down-regulating TGFβR1 and SMAD3 mRNA expression. In addition, extracellular acidification rate (ECAR) and a proportional reduction of markers of altered glucose metabolism in treated cells demonstrated also cell bioenergetics modulation by pioglitazone.
CONCLUSIONS: Data indicate that PPAR-γ agonists represent an attractive treatment tool and by suppression of cell growth (in vitro and ex vivo models) and of invasion via blockade of MAPK cascade and TGFβ/SMADs signaling, respectively, and its role in cancer bioenergetics and metabolism indicate that PPAR-γ agonists represent an attractive treatment tool for NSCLC.

Keywords:  Bioenergetics; EMT; Glitazones; Lung cancer; Metabolism

DOI:  https://doi.org/10.1186/s13046-019-1176-1
Mol Cell Proteomics. 2019 Apr 22. pii: mcp.RA119.001392. [Epub ahead of print]

Cathepsin L Regulates Metabolic Networks Controlling Rapid Cell Growth and Proliferation.

Tommy Weiss Sadan, Gal Itzhak, Farnusch Kaschani, Zhanru Yu, Mohamed Mahameed, Adi Anaki, Yael Ben-Nun, Emmanuelle Merquiol, Boaz Tirosh, Benedikt Kessler, Markus Kaiser, Galia Blum.

  Rapidly proliferating cells reshape their metabolism to satisfy their ever-lasting need for cellular building blocks. This phenomenon is exemplified in certain malignant conditions such as cancer but also during embryonic development when cells rely heavily on glycolytic metabolism to exploit its metabolic intermediates for biosynthetic processes. How cells reshape their metabolism is not fully understood. Here we report that loss of cathepsin L (Cts L) is associated with a fast proliferation rate and enhanced glycolytic metabolism that depend on lactate dehydrogenase A (LDHA) activity. Using mass spectrometry analysis of cells treated with a pan cathepsin inhibitor, we observed an increased abundance of proteins involved in central carbon metabolism. Further inspection of putative Cts L targets revealed an enrichment for glycolytic metabolism that was independently confirmed by metabolomic and biochemical analyses. Moreover, proteomic analysis of Cts L-knockout cells identified LDHA overexpression that was demonstrated to be a key metabolic junction in these cells. Lastly, we show that Cts L inhibition led to increased LDHA protein expression, suggesting a causal relationship between LDHA expression and function. In conclusion, we propose that Cts L regulates this metabolic circuit to keep cell division under control, suggesting the therapeutic potential of targeting this protein and its networks in cancer.

Keywords:  Cathepsin L; Gene Expression*; Glycolysis; Metabolomics; Pathway Analysis; Proliferation; Proteases*; Proteolysis*

DOI:  https://doi.org/10.1074/mcp.RA119.001392
J Exp Clin Cancer Res. 2019 Apr 25. 38(1): 174

Mammalian Eps15 homology domain 1 potentiates angiogenesis of non-small cell lung cancer by regulating β2AR signaling.

Ting Wang, Ying Xing, Qingwei Meng, Hailing Lu, Wei Liu, Shi Yan, Yang Song, Xinyuan Xu, Jian Huang, Yue Cui, Dexin Jia, Li Cai.

   BACKGROUND: Non-small cell lung cancer (NSCLC) is a devastating disease with a heterogeneous prognosis, and the molecular mechanisms underlying tumor progression remain elusive. Mammalian Eps15 homology domain 1 (EHD1) plays a promotive role in tumor progression, but its role in cancer angiogenesis remains unknown. This study thus explored the role of EHD1 in angiogenesis in NSCLC.
METHODS: The changes in angiogenesis were evaluated through human umbilical vein endothelial cell (HUVEC) proliferation, migration and tube formation assays. The impact of EHD1 on β2-adrenoceptor (β2AR) signaling was evaluated by Western blotting, quantitative real-time polymerase chain reaction (qRT-PCR) analysis, and enzyme-linked immunosorbent assay (ELISA). The interaction between EHD1 and β2AR was confirmed by immunofluorescence (IF) and coimmunoprecipitation (Co-IP) experiments, and confocal microscopy immunofluorescence studies revealed that β2AR colocalized with the recycling endosome marker Rab11, which indicated β2AR endocytosis. Xenograft tumor models were used to investigate the role of EHD1 in NSCLC tumor growth.
RESULTS: The microarray analysis revealed that EHD1 was significantly correlated with tumor angiogenesis, and loss- and gain-of-function experiments demonstrated that EHD1 potentiates HUVEC proliferation, migration and tube formation. EHD1 knockdown inhibited β2AR signaling activity, and EHD1 upregulation promoted vascular endothelial growth factor A (VEGFA) and β2AR expression. Interestingly, EHD1 interacted with β2AR and played a novel and critical role in β2AR endocytic recycling to prevent receptor degradation. Aberrant VEGFA or β2AR expression significantly affected EHD1-mediated tumor angiogenesis. The proangiogenic role of EHD1 was confirmed in xenograft tumor models, and immunohistochemistry (IHC) analysis confirmed that EHD1 expression was positively correlated with VEGFA expression, microvessel density (MVD) and β2AR expression in patient specimens.
CONCLUSION: Collectively, the data obtained in this study suggest that EHD1 plays a critical role in NSCLC angiogenesis via β2AR signaling and highlight a potential target for antiangiogenic therapy.

Keywords:  Angiogenesis; EHD1; Endocytosis; NSCLC; β2AR signaling

DOI:  https://doi.org/10.1186/s13046-019-1162-7
Cancer Med. 2019 Apr 25.

Screening common signaling pathways associated with drug resistance in non-small cell lung cancer via gene expression profile analysis.

Ting Sun, Qitai Zhao, Chaoqi Zhang, Ling Cao, Mengjia Song, Nomathamsanqa Resegofetse Maimela, Shasha Liu, Jinjin Wang, Qun Gao, Guohui Qin, Liping Wang, Yi Zhang.

  Lung cancer is the leading cause of cancer-related deaths worldwide. Although several therapeutic strategies have been employed to curb lung cancer, the survival rate is still poor owing to the development of drug resistance. The mechanisms underlying drug resistance development are incompletely understood. Here, we aimed to identify the common signaling pathways involved in drug resistance in non-small cell lung cancer (NSCLC). Three published transcriptome microarray data were downloaded from the Gene Expression Omnibus (GEO) database comprising different drug-resistant cell lines and their parental cell lines. Differentially expressed genes (DEGs) were identified and used to perform Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. An overlapping analysis was performed for KEGG pathways enriched from all the three datasets to identify the common signaling pathways. As a result, we found that metabolic pathways, ubiquitin-mediated proteolysis, and mitogen-activated protein kinase (MAPK) signaling were the most aberrantly expressed signaling pathways. The knockdown of nicotinamide phosphoribosyltransferase (NAMPT), the gene involved in metabolic pathways and known to be upregulated in drug-resistant tumor cells, was shown to increase the apoptosis of cisplatin-resistant A549 cells following cisplatin treatment. Thus, our results provide an in-depth analysis of the signaling pathways that are commonly altered in drug-resistant NSCLC cell lines and highlight the potential strategy that facilitates the development of interventions to interfere with upregulated signaling pathways as well as to boost downregulated signaling pathways in drug-resistant tumors for the elimination of multiple resistance of NSCLC.

Keywords:  common signaling pathways; drug resistance; non-small cell lung cancer; transcriptome microarray data

DOI:  https://doi.org/10.1002/cam4.2190
Nutr Cancer. 2019 Apr 22. 1-10

Can Nutritional Status Predict Overall Survival in Patients with Advanced Non-Small Cell Lung Cancer?

Youngkwang Seo, Wankyu Eo, Sehyun Kim, Bumsang Shim, Sookyung Lee.

Medical records of patients with advanced non-small cell lung cancer (NSCLC) were retrospectively reviewed to examine the prognostic impact of nutritional status on survival. Age, sex, body mass index (BMI), Eastern Cooperative Oncology Group Performance Status (ECOG-PS), histologic tumor type, pulmonary comorbidities, white blood cell (WBC) count, C-reactive protein (CRP) level, and prognostic nutritional index (PNI) were assessed. Overall survival was calculated using Kaplan-Meier analysis and compared using log-rank testing. Univariate and multivariate Cox regression model analyses were used to evaluate prognostic impact. Of the 183 enrolled patients, 166 had stage IV NSCLC; 70 had ECOG-PS scores of 2; and 129 had undergone prior anticancer therapy. Age ≥ 65 years, male sex, smoking, BMI < 21 kg/m2, ECOG-PS score of 2, WBC count > 11,000 cells/μL, CRP level > 1.0 mg/dL, and PNI ≤46.1 were associated with poor overall survival. Multivariate analysis revealed that BMI ≥ 21 kg/m2 (hazard ratio [HR], 0.64) and PNI > 46.1 (HR, 0.65) were associated with prolonged survival, while age ≥ 65 years (HR, 1.48) and CRP level > 1.0 mg/dL (HR, 1.82) were associated with poor survival. In conclusion, BMI and PNI, as indicators of nutritional status, were significant independent prognostic factors of survival.

DOI: https://doi.org/10.1080/01635581.2019.1598564
Zhongguo Fei Ai Za Zhi. 2019 Apr 20. 22(4): 208-215

[Study on the Metabolic Reprogramming of Lung Cancer Cells Regulated by Docetaxel Based on Metabolomics].

Haichao Sun, Hailong Piao, Huan Qi, Min Yan, Hongxu Liu.

   BACKGROUND: Docetaxel is a commonly used anti-tumor drug in clinic, especially as the first-line drug for advanced non-small cell lung cancer (NSCLC). However, the molecular mechanism of docetaxel against NSCLC is still unclear. Increasing studies have shown that metabolic reprogramming of tumor cells plays an important role in tumorigenesis. The aim of this study was to investigate the effects of docetaxel on the metabolic pathway of NSCLC cells based on metabolomics analysis and biological means.
METHODS: First, we performed CCK8 assay to analyze the effects of docetaxel on cell viability of NSCLC cells and also to screen the appropriate drug concentration. Then, the differential metabolites of docetaxel-treated and untreated NSCLC cells were analyzed by gas chromatography-mass spectrometry based metabolomics. Finally, the effects of docetaxel on the expression levels of key enzymes that regulate the relevant metabolic pathways were determined by Western blot.
RESULTS: Docetaxel inhibited cell viability of A549 and H1299 cells in a concentration- and time-dependent manner. With the prolonged treatment time of docetaxel, the apoptotic sensitive protein poly (ADP-ribose) polymerase (PARP) was gradually activated to form a P89 fragment. Metabolomics analysis showed that eight metabolites were significantly changed in both A549 and H1299 cells following docetaxel treatment, which were mainly in the tricarboxylic acid (TCA) cycle pathway. Moreover, after docetaxel treatment, the protein expression levels of isocitrate dehydrogenases, the key regulators of the TCA cycle, were obviously decreased in both A549 and H1299 cells.
CONCLUSIONS: Our findings suggest that the effect of docetaxel-induced proliferation inhibition and apoptosis in NSCLC might be associated with down-regulation of isocitrate dehydrogenases and suppression of the TCA cycle pathway.

Keywords:  Docetaxel; Gas chromatography-mass spectrometry; Lung neoplasms; Metabolomics; Tricarboxylic acid cycle

DOI:  https://doi.org/10.3779/j.issn.1009-3419.2019.04.02
Hum Cell. 2019 Apr 24.

Inhibitor of ghrelin receptor reverses gefitinib resistance in lung cancer.

Xiaoyou Li, Xia Zhao, Chenchen Li, Siwen Liu, Fei Yan, Yue Teng, Jifeng Feng, Dengshun Miao.

  Gefitinib is the first-generation EGFR tyrosine kinase inhibitor (EGFR-TKI), which is used in the treatment of NCSLC patients through interrupting EGFR signaling pathway. Although gefitinib prolongs patients' progression-free survival (PFS), acquired resistance occurs in advanced NSCLC patients. In this study, we mainly investigated the effects of antagonist for ghrelin-R (D-lys-3-GHRP-6) on conquering acquired gefitinib resistance in human lung cancer cells. We found that GHSR was overexpressed in our established HCC827/GR cells compared with parental cells, accompanied with increase of p-AKT and p-ERK1/2. Treatment of D-lys-3-GHRP-6 significantly decreased p-AKT and p-ERK1/2 expression in HCC827/GR cells. H1650 cells and HCC827/GR cells were treated with control, gefitinib, D-lys-3-GHRP-6 and D-lys-3-GHRP-6 + gefitinib, respectively. In H1650 and HCC827/GR cells, combination of D-lys-3-GHRP-6 and gefitinib significantly inhibited cell proliferation and Bcl2 protein level, induced the cell apoptosis and cleaved-caspase3 protein level compared with control group, while there was no significant difference between control and gefitinib group.

Keywords:  D-Lys-3-GHRP-6; Gefitinib-acquired resistance; Lung cancer

DOI:  https://doi.org/10.1007/s13577-019-00245-5
In Vivo. 2019 May-Jun;33(3):33(3): 777-785

shRNA-WT1 Potentiates Anticancer Effects of Gemcitabine and Cisplatin Against B16F10 Lung Metastases In Vitro and In Vivo.

Pablo Zapata-Benavides, Fernanda Guadalupe Thompson-Armendariz, Mariela Arellano-Rodríguez, Moisés Armides Franco-Molina, Edgar Mendoza-Gamboa, Santiago Saavedra-Alonso, José Luis Zacarias-Hernández, Laura María Trejo-Avila, Cristina Rodríguez-Padilla.

   BACKGROUND/AIM: High expression level of Wilm's tumor gene (WT1) in several types of tumors appears to confer disruption of apoptosis and resistance to chemotherapeutic drugs, and correlate with poor outcome. The aim of this work was to determine if down-regulation of WT1 expression results in decreased cell proliferation and the increased action of different types of drugs, both in vitro in B16F10 cells, and in vivo in C57BL/6 mice.
MATERIALS AND METHODS: Inhibition of cell proliferation by short hairpin RNA against WT1 (shRNA-WT1), cisplatin, and gemcitabine in B16F10 cells in vitro was determined by the MTT assay and analysis of clonogenic survival. The apoptosis rate was determined by flow cytometry for annexin-V- fluorescein isothiocyante and propidium iodide.
RESULTS: Compared to treatment with shRNA-WT1 alone, treatment with shRNA-WT1 in combination with drugs had a synergistic inhibitory effect on B16F10 cell proliferation, particularly for the combination of cisplatin and gemcitabine at their 25% cytotoxic concentrations in vitro. Furthermore, mice treated with shRNA-WT1 in combination with cisplatin and gemcitabine were protected in the same way as those treated with the drugs alone, but were in better physical condition.
CONCLUSION: Decreased WT1 expression induces cell death and potentiates the action of anticancer drugs by inducing synergistic effects both in vitro and in vivo, which may be an attractive strategy in lung cancer therapy.

Keywords:  WT1; cancer; cisplatin; gemcitabine; shRNA

DOI:  https://doi.org/10.21873/invivo.11539