bims-meluca 2019-02-10 papers

on Metabolism of non-small cell lung carcinoma

Issue of 2019–02–10
ten 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

Metformin enhances the radiosensitizing effect of cisplatin in non-small cell lung cancer cell lines with different cisplatin sensitivities.
Suppression of Myeloid Cell Arginase Activity leads to Therapeutic Response in a NSCLC Mouse Model by Activating Anti-Tumor Immunity.
The discovery and characterization of K-563, a novel inhibitor of the Keap1/Nrf2 pathway produced by Streptomyces sp.
Methioninase Gene Therapy.
Identification of a novel glycolysis-related gene signature that can predict the survival of patients with lung adenocarcinoma.
An Integrative Systems Biology and Experimental Approach Identifies Convergence of Epithelial Plasticity, Metabolism, and Autophagy to Promote Chemoresistance.
Cyclopamine tartrate, a modulator of hedgehog signaling and mitochondrial respiration, effectively arrests lung tumor growth and progression.
Evidence for an alternative fatty acid desaturation pathway increasing cancer plasticity.
Repeatability of [18F]FDG PET/CT total metabolic active tumour volume and total tumour burden in NSCLC patients.
Extracellular vesicles secreted by hypoxia pre-challenged mesenchymal stem cells promote non-small cell lung cancer cell growth and mobility as well as macrophage M2 polarization via miR-21-5p delivery.

Sci Rep. 2019 Feb 04. 9(1): 1282

Metformin enhances the radiosensitizing effect of cisplatin in non-small cell lung cancer cell lines with different cisplatin sensitivities.

Muhammad Assad Riaz, Ali Sak, Yasin Bahadir Erol, Michael Groneberg, Jürgen Thomale, Martin Stuschke.

Cisplatin is an extensively used chemotherapeutic drug for lung cancer, but the development of resistance decreases its effectiveness in the treatments of non-small cell lung cancer (NSCLC). In this study, we examined the effects of metformin, a widely used antidiabetic drug, on cisplatin radiosensitization in NSCLC cell lines. Human NSCLC cell lines, A549 (cisplatin-resistant) and H460 (cisplatin-sensitive), were treated with metformin, cisplatin or a combination of both drugs before ionizing radiation. Cell proliferation, clonogenic assays, western blotting, cisplatin-DNA adduct formation and immunocytochemistry were used to characterize the treatments effects. Metformin increased the radiosensitivity of NSCLC cells. Metformin showed additive and over-additive effects in combination with cisplatin and the radiation response in the clonogenic assay in H460 and A549 cell lines (p = 0.018 for the interaction effect between cisplatin and metformin), respectively. At the molecular level, metformin led to a significant increase in cisplatin-DNA adduct formation compared with cisplatin alone (p < 0.01, ANOVA-F test). This was accompanied by a decreased expression of the excision repair cross-complementation 1 expression (ERCC1), a key enzyme in nucleotide excision repair pathway. Furthermore, compared with each treatment alone metformin in combination with cisplatin yielded the lowest level of radiation-induced Rad51 foci, an essential protein of homologous recombination repair. Ionizing radiation-induced γ-H2AX and 53BP1 foci persisted longer in both cell lines in the presence of metformin. Pharmacological inhibition of AMP-activated protein kinase (AMPK) demonstrated that metformin enhances the radiosensitizing effect of cisplatin through an AMPK-dependent pathway only in H460 but not in A549 cells. Our results suggest that metformin can enhance the effect of combined cisplatin and radiotherapy in NSCLC and can sensitize these cells to radiation that are not sensitized by cisplatin alone.

DOI: https://doi.org/10.1038/s41598-018-38004-5
J Immunother Cancer. 2019 Feb 06. 7(1): 32

Suppression of Myeloid Cell Arginase Activity leads to Therapeutic Response in a NSCLC Mouse Model by Activating Anti-Tumor Immunity.

Juan J Miret, Paul Kirschmeier, Shohei Koyama, Mingrui Zhu, Yvonne Y Li, Yujiro Naito, Min Wu, Venkat S Malladi, Wei Huang, William Walker, Sangeetha Palakurthi, Glenn Dranoff, Peter S Hammerman, Chad V Pecot, Kwok-Kin Wong, Esra A Akbay.

   BACKGROUND: Tumor orchestrated metabolic changes in the microenvironment limit generation of anti-tumor immune responses. Availability of arginine, a semi-essential amino acid, is critical for lymphocyte proliferation and function. Levels of arginine are regulated by the enzymes arginase 1,2 and nitric oxide synthase (NOS). However, the role of arginase activity in lung tumor maintenance has not been investigated in clinically relevant orthotopic tumor models.
METHODS: RNA sequencing (RNA-seq) of sorted cell populations from mouse lung adenocarcinomas derived from immunocompetent genetically engineered mouse models (GEMM)s was performed. To complement mouse studies, a patient tissue microarray consisting of 150 lung adenocarcinomas, 103 squamous tumors, and 54 matched normal tissue were stained for arginase, CD3, and CD66b by multiplex immunohistochemistry. Efficacy of a novel arginase inhibitor compound 9 in reversing arginase mediated T cell suppression was determined in splenocyte ex vivo assays. Additionally, the anti-tumor activity of this compound was determined in vitro and in an autochthonous immunocompetent KrasG12D GEMM of lung adenocarcinoma model.
RESULTS: Analysis of RNA-seq of sorted myeloid cells suggested that arginase expression is elevated in myeloid cells in the tumor as compared to the normal lung tissue. Accordingly, in the patient samples arginase 1 expression was mainly localized in the granulocytic myeloid cells and significantly elevated in both lung adenocarcinoma and squamous tumors as compared to the controls. Our ex vivo analysis demonstrated that myeloid derived suppressor cell (MDSC)s cause T cell suppression by arginine depletion, and suppression of arginase activity by a novel ARG1/2 inhibitor, compound 9, led to restoration of T cell function by increasing arginine. Treatment of KrasG12D GEMM of lung cancer model with compound 9 led to a significant tumor regression associated with increased T cell numbers and function, while it had no activity across several murine and human non-small cell (NSCLC) lung cancer lines in vitro.
CONCLUSIONS: We show that arginase expression is elevated in mouse and patient lung tumors. In a KRASG12D GEMM arginase inhibition diminished growth of established tumors. Our data suggest arginase as an immunomodulatory target that should further be investigated in lung tumors with high arginase activity.

Keywords:  Aminoacid; Arginase; Arginine; Autochthonous; Immunocompetent; MDSC; Metabolic checkpoint

DOI:  https://doi.org/10.1186/s40425-019-0504-5
Cancer Med. 2019 Feb 08.

The discovery and characterization of K-563, a novel inhibitor of the Keap1/Nrf2 pathway produced by Streptomyces sp.

Ran Hori, Kozo Yamaguchi, Hidetaka Sato, Miwa Watanabe, Kyoko Tsutsumi, Susumu Iwamoto, Masayuki Abe, Hideyuki Onodera, Satoshi Nakamura, Ryuichiro Nakai.

  Keap1/Nrf2 pathway regulates the antioxidant stress response, detoxification response, and energy metabolism. Previous reports found that aberrant Keap1/Nrf2 pathway activation due to Kelch-like ECH-associated protein 1 (Keap1) mutations or Nuclear factor E2-related factor 2 (Nrf2) mutations induced resistance of cancer cells to chemotherapy and accelerated cell growth via the supply of nutrients. Therefore, Keap1/Nrf2 pathway activation is associated with a poor prognosis in many cancers. These previous findings suggested that inhibition of Keap1/Nrf2 pathway could be a target for anti-cancer therapies. To discover a small-molecule Keap1/Nrf2 pathway inhibitor, we conducted high-throughput screening in Keap1 mutant human lung cancer A549 cells using a transcriptional reporter assay. Through this screening, we identified the novel Keap1/Nrf2 pathway inhibitor K-563, which was isolated from actinomycete Streptomyces sp. K-563 suppressed the expression of Keap1/Nrf2 pathway downstream target genes or the downstream target protein, which induced suppression of GSH production, and activated reactive oxygen species production in A549 cells. K-563 also inhibited the expression of downstream target genes in other Keap1- or Nrf2-mutated cancer cells. Furthermore, K-563 exerted anti-proliferative activities in these mutated cancer cells. These in vitro analyses showed that K-563 was able to inhibit cell growth in Keap1- or Nrf2-mutated cancer cells by Keap1/Nrf2 pathway inhibition. K-563 also exerted synergistic combinational effects with lung cancer chemotherapeutic agents. An in vivo study in mice xenotransplanted with A549 cells to further explore the therapeutic potential of K-563 revealed that it also inhibited Keap1/Nrf2 pathway in lung cancer tumors. K-563, a novel Keap1/Nrf2 pathway inhibitor, may be a lead compound for development as an anti-cancer agent.

Keywords:   NSCLC ; Keap1/Nrf2 pathway; Streptomyces sp; anti-cancer agent; drug resistance

DOI:  https://doi.org/10.1002/cam4.1949
Methods Mol Biol. 2019 ;1866 173-197

Methioninase Gene Therapy.

Robert M Hoffman, Kenji Miki, Waddah Al-Refaie, Mingxu Xu, Yuying Tan.

  Recombinant methioninase (rMETase) derived from Pseudomonas putida targets the elevated methionine (MET) requirement of cancer cells (methionine dependence) and has shown efficacy against a variety of cancer types in mouse models. To enhance the efficacy of rMETase, we constructed the pLGFP-METSN retrovirus encoding the P. putida methioninase (METase) gene fused with the green fluorescent protein (GFP) gene. pLGFP-METSN or control vector pLGFPSN was introduced into the human lung cancer cell line H460. The retrovirus-mediated METase gene transfer decreased the intracellular MET level of the cancer cells and consequently enhanced the efficacy of treatment with the rMETase protein. The rMETase gene was introduced into an adenovirus. rAd-METase transduction of human OVACAR-8 ovarian cancer cells and human fibrosarcoma HT1080 cells in vitro and in vivo resulted in high levels of METase expression up to 10% or more of the total protein of the cells, depending on the multiplicity of infection. The combination of rAd-METase and rMETase was synergistic to kill these cells. Normal fibroblasts, on the other hand, appeared relatively resistant to the METase gene in the presence of rMETase. Adenoviral METase-transduced cancer cells were used in combination with selenomethionine, releasing highly toxic methylselenol, which killed both the cancer cells containing the METase gene and bystanders. Methylselenol damaged the mitochondria via oxidative stress and caused cytochrome c release into the cytosol, thereby activating the caspase cascade and cancer-cell apoptosis. Adenoviral METase-gene/SeMET treatment also inhibited tumor growth in rodents and significantly prolonged their survival. AdMETase/SeMET therapy was effective against Bcl-2-overproducing A549 lung cancer cells, which were resistant to staurosporine-induced apoptosis, with a strong bystander effect. The combination of Ad-METase/SeMET and doxorubicin (DOX) delayed the growth of the H460 human lung cancer, growing subcutaneously in nude mice. These results demonstrate the potential of methionine restriction (MR) for cancer treatment.

Keywords:  Adenovirus; By-stander effect; Cancer; GFP; Gene cloning; Gene therapy; Methioninase; Methionine dependence; Methylselenol; Prodrug; Selenomethionine

DOI:  https://doi.org/10.1007/978-1-4939-8796-2_14
Cell Cycle. 2019 Feb 06.

Identification of a novel glycolysis-related gene signature that can predict the survival of patients with lung adenocarcinoma.

Chang Liu, YinYan Li, Minjie Wei, Lin Zhao, Yangyang Yu, Guang Li.

  Lung cancer is one of the most malignant cancers worldwide, and lung adenocarcinoma (LUAD) is the most common histologic subtype. Thousands of biomarkers related to the survival and prognosis of patients with this cancer type have been investigated through database mining; however, the prediction effect of a single gene biomarker is not satisfactorily specific or sensitive. Thus, the present study aimed to develop a novel gene signature of prognostic values for patients with LUAD. Using a data-mining method, we performed expression profiling of 1145 mRNAs in large cohorts with LUAD (n=511) from The Cancer Genome Atlas database. Using the Gene Set Enrichment Analysis, we selected 198 genes related to GLYCOLYSIS, which is the most important enrichment gene set. Moreover, these genes were identified using Cox proportional regression modeling. We established a risk score staging system to predict the outcome of patients with LUAD and subsequently identified four genes (AGRN, AKR1A1, DDIT4, and HMMR) that were closely related to the prognosis of patients with LUAD. The identified genes allowed us to classify patients into the high-risk group (with poor outcome) and low-risk group (with better outcome). Compared with other clinical factors, the risk score has a better performance in predicting the outcome of patients with LUAD, particularly in the early stage of LUAD. In conclusion, we developed a four-gene signature related to glycolysis by utilizing the Cox regression model and a risk staging model for LUAD, which might prove valuable for the clinical management of patients with LUAD.

Keywords:  glycolysis; lung cancer; mRNAs; prognostic; survival

DOI:  https://doi.org/10.1080/15384101.2019.1578146
J Clin Med. 2019 Feb 07. pii: E205. [Epub ahead of print]8(2):

An Integrative Systems Biology and Experimental Approach Identifies Convergence of Epithelial Plasticity, Metabolism, and Autophagy to Promote Chemoresistance.

Shengnan Xu, Kathryn E Ware, Yuantong Ding, So Young Kim, Maya U Sheth, Sneha Rao, Wesley Chan, Andrew J Armstrong, William C Eward, Mohit Kumar Jolly, Jason A Somarelli.

  The evolution of therapeutic resistance is a major cause of death for cancer patients. The development of therapy resistance is shaped by the ecological dynamics within the tumor microenvironment and the selective pressure of the host immune system. These selective forces often lead to evolutionary convergence on pathways or hallmarks that drive progression. Thus, a deeper understanding of the evolutionary convergences that occur could reveal vulnerabilities to treat therapy-resistant cancer. To this end, we combined phylogenetic clustering, systems biology analyses, and molecular experimentation to identify convergences in gene expression data onto common signaling pathways. We applied these methods to derive new insights about the networks at play during transforming growth factor-β (TGF-β)-mediated epithelial⁻mesenchymal transition in lung cancer. Phylogenetic analyses of gene expression data from TGF-β-treated cells revealed convergence of cells toward amine metabolic pathways and autophagy during TGF-β treatment. Knockdown of the autophagy regulatory, ATG16L1, re-sensitized lung cancer cells to cancer therapies following TGF-β-induced resistance, implicating autophagy as a TGF-β-mediated chemoresistance mechanism. In addition, high ATG16L expression was found to be a poor prognostic marker in multiple cancer types. These analyses reveal the usefulness of combining evolutionary and systems biology methods with experimental validation to illuminate new therapeutic vulnerabilities for cancer.

Keywords:  autophagy; epithelial–mesenchymal transition; evolution; lung cancer; metabolism; systems biology; tumor invasiveness

DOI:  https://doi.org/10.3390/jcm8020205
Sci Rep. 2019 Feb 05. 9(1): 1405

Cyclopamine tartrate, a modulator of hedgehog signaling and mitochondrial respiration, effectively arrests lung tumor growth and progression.

Sarada Preeta Kalainayakan, Poorva Ghosh, Sanchareeka Dey, Keely E Fitzgerald, Sagar Sohoni, Purna Chaitanya Konduri, Massoud Garrossian, Li Liu, Li Zhang.

Lung cancer remains the leading cause of cancer-related death, despite the advent of targeted therapies and immunotherapies. Therefore, it is crucial to identify novel molecular features unique to lung tumors. Here, we show that cyclopamine tartrate (CycT) strongly suppresses the growth of subcutaneously implanted non-small cell lung cancer (NSCLC) xenografts and nearly eradicated orthotopically implanted NSCLC xenografts. CycT reduces heme synthesis and degradation in NSCLC cells and suppresses oxygen consumption in purified mitochondria. In orthotopic tumors, CycT decreases the levels of proteins and enzymes crucial for heme synthesis, uptake, and oxidative phosphorylation (OXPHOS). CycT also decreases the levels of two regulators promoting OXPHOS, MYC and MCL1, and effectively alleviates tumor hypoxia. Evidently, CycT acts via multiple modes to suppress OXPHOS. One mode is to directly inhibit mitochondrial respiration/OXPHOS. Another mode is to inhibit heme synthesis and degradation. Both modes appear to be independent of hedgehog signaling. Addition of heme to NSCLC cells partially reverses the effect of CycT on oxygen consumption, proliferation, and tumorigenic functions. Together, our results strongly suggest that CycT suppress tumor growth in the lung by inhibiting heme metabolism and OXPHOS. Targeting heme metabolism and OXPHOS may be an effective strategy to combat lung cancer.

DOI: https://doi.org/10.1038/s41598-018-38345-1
Nature. 2019 Feb 06.

Evidence for an alternative fatty acid desaturation pathway increasing cancer plasticity.

Kim Vriens, Stefan Christen, Sweta Parik, Dorien Broekaert, Kazuaki Yoshinaga, Ali Talebi, Jonas Dehairs, Carmen Escalona-Noguero, Roberta Schmieder, Thomas Cornfield, Catriona Charlton, Laura Romero-Pérez, Matteo Rossi, Gianmarco Rinaldi, Martin F Orth, Ruben Boon, Axelle Kerstens, Suet Ying Kwan, Brandon Faubert, Andrés Méndez-Lucas, Charlotte C Kopitz, Ting Chen, Juan Fernandez-Garcia, João A G Duarte, Arndt A Schmitz, Patrick Steigemann, Mustapha Najimi, Andrea Hägebarth, Jo A Van Ginderachter, Etienne Sokal, Naohiro Gotoh, Kwok-Kin Wong, Catherine Verfaillie, Rita Derua, Sebastian Munck, Mariia Yuneva, Laura Beretta, Ralph J DeBerardinis, Johannes V Swinnen, Leanne Hodson, David Cassiman, Chris Verslype, Sven Christian, Sylvia Grünewald, Thomas G P Grünewald, Sarah-Maria Fendt.

Most tumours have an aberrantly activated lipid metabolism1,2 that enables them to synthesize, elongate and desaturate fatty acids to support proliferation. However, only particular subsets of cancer cells are sensitive to approaches that target fatty acid metabolism and, in particular, fatty acid desaturation3. This suggests that many cancer cells contain an unexplored plasticity in their fatty acid metabolism. Here we show that some cancer cells can exploit an alternative fatty acid desaturation pathway. We identify various cancer cell lines, mouse hepatocellular carcinomas, and primary human liver and lung carcinomas that desaturate palmitate to the unusual fatty acid sapienate to support membrane biosynthesis during proliferation. Accordingly, we found that sapienate biosynthesis enables cancer cells to bypass the known fatty acid desaturation pathway that is dependent on stearoyl-CoA desaturase. Thus, only by targeting both desaturation pathways is the in vitro and in vivo proliferation of cancer cells that synthesize sapienate impaired. Our discovery explains metabolic plasticity in fatty acid desaturation and constitutes an unexplored metabolic rewiring in cancers.

DOI: https://doi.org/10.1038/s41586-019-0904-1
EJNMMI Res. 2019 Feb 07. 9(1): 14

Repeatability of [18F]FDG PET/CT total metabolic active tumour volume and total tumour burden in NSCLC patients.

Guilherme D Kolinger, David Vállez García, Gerbrand M Kramer, Virginie Frings, Egbert F Smit, Adrianus J de Langen, Rudi A J O Dierckx, Otto S Hoekstra, Ronald Boellaard.

   BACKGROUND: Total metabolic active tumour volume (TMATV) and total tumour burden (TTB) are increasingly studied as prognostic and predictive factors in non-small cell lung cancer (NSCLC) patients. In this study, we investigated the repeatability of TMATV and TTB as function of uptake interval, positron emission tomography/computed tomography (PET/CT) image reconstruction settings, and lesion delineation method. We used six lesion delineation methods, four direct PET image-derived delineations and two based on a majority vote approach, i.e. intersection between two or more delineations (MV2) and between three or more delineations (MV3). To evaluate the accuracy of those methods, they were compared with a reference delineation obtained from the consensus of the segmentations performed by three experienced observers. Ten NSCLC patients underwent two baseline whole-body [18F]2-Fluoro-2-deoxy-2-D-glucose ([18F]FDG) PET/CT studies on separate days, within 3 days. Two scans were obtained on each day at 60 and 90 min post-injection to assess the influence of tracer uptake interval. PET/CT images were reconstructed following the European Association of Nuclear Medicine Research Ltd. (EARL) compliant settings and with point-spread-function (PSF) modelling. Repeatability between the measurements of each day was determined and the influence of uptake interval, reconstruction settings, and lesion delineation method was assessed using the generalized estimating equations model.
RESULTS: Based on the Jaccard index with the reference delineation, the MV2 lesion delineation method was the most successful method for automated lesion segmentation. The best overall repeatability (lowest repeatability coefficient, RC) was found for TTB from 90 min of tracer uptake scans reconstructed with EARL compliant settings and delineated with 41% of lesion's maximum SUV method (RC = 11%). In most cases, TMATV and TTB repeatability were not significantly affected by changes in tracer uptake time or reconstruction settings. However, some lesion delineation methods had significantly different repeatability when applied to the same images.
CONCLUSIONS: This study suggests that under some circumstances TMATV and TTB repeatability are significantly affected by the lesion delineation method used. Performing the delineation with a majority vote approach improves reliability and does not hamper repeatability, regardless of acquisition and reconstruction settings. It is therefore concluded that by using a majority vote based tumour segmentation approach, TMATV and TTB in NSCLC patients can be measured with high reliability and precision.

Keywords:  FDG PET/CT; Majority vote; Metabolic active tumour volume; NSCLC; Repeatability; Total tumour burden; Tracer uptake interval; Tumour delineation

DOI:  https://doi.org/10.1186/s13550-019-0481-1
J Exp Clin Cancer Res. 2019 Feb 08. 38(1): 62

Extracellular vesicles secreted by hypoxia pre-challenged mesenchymal stem cells promote non-small cell lung cancer cell growth and mobility as well as macrophage M2 polarization via miR-21-5p delivery.

Weihua Ren, Jianfeng Hou, Chenguang Yang, Hanjun Wang, Shuangting Wu, Yabin Wu, Xingpeng Zhao, Chao Lu.

   OBJECTIVE: To investigate the lung cancer-promoting mechanism of mesenchymal stem cell-secreted extracellular vesicles (MSC-EV).
METHODS: EV were isolated from culture media of human bone marrow-derived MSCs that were pre-challenged with or without hypoxia (referred to as H-EV and N-EV, respectively). After treatment with N-EV or H-EV, A549 and H23 cell proliferation, apoptosis, trans-well invasion and epithelial-to-mesenchymal transition (EMT) were examined. Polarization of human primary monocytes-derived macrophages with or without N-EV or H-EV induction were analyzed by flow cytometry and ELISA. PTEN, PDCD4 or RECK gene was overexpressed in A549 cells, while miR-21-5p was knocked down in MSCs, A549 or H23 lung cancer cells or primary monocytes by miR-21-5p inhibitor transfection. Protein level of PTEN, PDCD4, RECK, AKT or STAT3 as well as phosphorylation level of AKT or STAT3 protein were assayed by western blot. Tumorigenicity of A549 and H23 cells with or without MSC-EV co-injection was assayed on immunocompromised mice. The xenograft tumor were examined for cell proliferation, angiogenesis, apoptosis and intra-tumoral M1/M2 macrophage polarization.
RESULTS: Comparing to N-EV, H-EV treatment significantly increased A549 and H23 cell proliferation, survival, invasiveness and EMT as well as macrophage M2 polarization. MiR-21-5p knocked down significantly abrogated the cancer-promoting and macrophage M2 polarizing effects of H-EV treatment. H-EV treatment downregulated PTEN, PDCD4 and RECK gene expression largely through miR-21-5p. Overexpressing PTEN, PDCD4 and RECK in A549 cells significantly reduced the miR-21-5p-mediated anti-apoptotic and pro-metastatic effect of H-EV, while overexpressing PTEN in monocytes significantly reduced macrophage M2 polarization after induction with the presence of H-EV. H-EV co-injection significantly increased tumor growth, cancer cell proliferation, intra-tumoral angiogenesis and M2 polarization of macrophages in vivo partially through miR-21-5p.
CONCLUSIONS: Increased miR-21-5p delivery by MSC-EV after hypoxia pre-challenge can promote lung cancer development by reducing apoptosis and promoting macrophage M2 polarization.

Keywords:  Extracellular vesicles; Hypoxia; Lung cancer; Macrophage polarization; Mesenchymal stem cells; miR-21-5p

DOI:  https://doi.org/10.1186/s13046-019-1027-0