bims-meluca 2019-05-26 papers

on Metabolism of non-small cell lung carcinoma

Issue of 2019–05–26
eight 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

Metabolic reprogramming and Notch activity distinguish between non-small cell lung cancer subtypes.
Cysteine dioxygenase 1 is a metabolic liability for non-small cell lung cancer.
Increased 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 activity in response to EGFR signaling contributes to non-small cell lung cancer cell survival.
Raman spectroscopy detects metabolic signatures of radiation response and hypoxic fluctuations in non-small cell lung cancer.
Antitumor activity of SR splicing-factor 5 knockdown by downregulating pyruvate kinase M2 in non-small cell lung cancer cells.
Metabolomics workflow for lung cancer: Discovery of biomarkers.
Genomic signatures defining responsiveness to allopurinol and combination therapy for lung cancer identified by systems therapeutics analyses.
Subcellular compartmentalization of PKM2 identifies anti-PKM2 therapy response in vitro and in vivo mouse model of human non-small-cell lung cancer.

Br J Cancer. 2019 May 22.

Metabolic reprogramming and Notch activity distinguish between non-small cell lung cancer subtypes.

Katherine Sellers, Thaddeus D Allen, Michael Bousamra, JinLian Tan, Andrés Méndez-Lucas, Wei Lin, Nourdine Bah, Yelena Chernyavskaya, James I MacRae, Richard M Higashi, Andrew N Lane, Teresa W-M Fan, Mariia O Yuneva.

BACKGROUND: Previous studies suggested that the metabolism is differently reprogrammed in the major subtypes of non-small cell lung cancer (NSCLC), squamous cell carcinomas (SCC) and adenocarcinomas (AdC). However, a comprehensive analysis of this differential metabolic reprogramming is lacking.
METHODS: Publicly available gene expression data from human lung cancer samples and cell lines were analysed. Stable isotope resolved metabolomics were performed on SCC and ADC tumours in human patients and in freshly resected tumour slices.
RESULTS: Analysis of multiple transcriptomics data from human samples identified a SCC-distinguishing enzyme gene signature. SCC tumours from patients infused with [U-13C]-glucose and SCC tissue slices incubated with stable isotope tracers demonstrated differential glucose and glutamine catabolism compared to AdCs or non-cancerous lung, confirming increased activity through pathways defined by the SCC metabolic gene signature. Furthermore, the upregulation of Notch target genes was a distinguishing feature of SCCs, which correlated with the metabolic signature. Notch and MYC-driven murine lung tumours recapitulated the SCC-distinguishing metabolic reprogramming. However, the differences between SCCs and AdCs disappear in established cell lines in 2D culture.
CONCLUSIONS: Our data emphasise the importance of studying lung cancer metabolism in vivo. They also highlight potential targets for therapeutic intervention in SCC patients including differentially expressed enzymes that catalyse reactions in glycolysis, glutamine catabolism, serine, nucleotide and glutathione biosynthesis.

DOI: https://doi.org/10.1038/s41416-019-0464-z
Elife. 2019 May 20. pii: e45572. [Epub ahead of print]8

Cysteine dioxygenase 1 is a metabolic liability for non-small cell lung cancer.

Yun Pyo Kang, Laura Torrente, Aimee Falzone, Cody M Elkins, Min Liu, John M Asara, Christian C Dibble, Gina DeNicola.

  NRF2 is emerging as a major regulator of cellular metabolism. However, most studies have been performed in cancer cells, where co-occurring mutations and tumor selective pressures complicate the influence of NRF2 on metabolism. Here we use genetically engineered, non-transformed primary murine cells to isolate the most immediate effects of NRF2 on cellular metabolism. We find that NRF2 promotes the accumulation of intracellular cysteine and engages the cysteine homeostatic control mechanism mediated by cysteine dioxygenase 1 (CDO1), which catalyzes the irreversible metabolism of cysteine to cysteine sulfinic acid (CSA). Notably, CDO1 is preferentially silenced by promoter methylation in human non-small cell lung cancers (NSCLC) harboring mutations in KEAP1, the negative regulator of NRF2. CDO1 silencing promotes proliferation of NSCLC by limiting the futile metabolism of cysteine to the wasteful and toxic byproducts CSA and sulfite (SO32-), and depletion of cellular NADPH. Thus, CDO1 is a metabolic liability for NSCLC cells with high intracellular cysteine, particularly NRF2/KEAP1 mutant cells.

Keywords:  biochemistry; cancer biology; chemical biology; human; mouse

DOI:  https://doi.org/10.7554/eLife.45572
J Biol Chem. 2019 May 24. pii: jbc.RA119.007784. [Epub ahead of print]

Increased 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 activity in response to EGFR signaling contributes to non-small cell lung cancer cell survival.

Nadiia Lypova, Sucheta Telang, Jason Chesney, Yoannis Imbert-Fernandez.

  Constitutive activation of the epidermal growth factor receptor (EGFR) due to somatic mutations of the EGFR gene is commonly observed in tumors of non-small cell lung cancer (NSCLC) patients. Consequently, tyrosine kinase inhibitors (TKI) targeting the EGFR are among the most effective therapies for patients with sensitizing EGFR mutations. Clinical responses to the EGFR-targeting TKIs are evaluated through 2-[18F]-fluoro-2-deoxy-glucose (18FDG-PET) uptake, which is decreased in patients responding favorably to therapy and is positively correlated with survival. Recent studies have reported that EGFR signaling drives glucose metabolism in NSCLC cells; however, the precise downstream effectors required for this EGFR-driven metabolic effect are largely unknown. 6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB3) is an essential glycolytic regulator that is consistently overexpressed in lung cancer. Here, we found that PFKFB3 is an essential target of EGFR signaling and that PFKFB3 activation is required for glycolysis stimulation upon EGFR activation. We demonstrate that exposing NSCLC cells harboring either wildtype or mutated EGFR to EGF rapidly increases PFKFB3 phosphorylation, expression, and activity, and that PFKFB3 inhibition markedly reduces the EGF-mediated increase in glycolysis. Furthermore, we found that prolonged NSCLC cell exposure to the TKI erlotinib drives PFKFB3 expression and that chemical PFKFB3 inhibition synergizes with erlotinib in increasing erlotinib's anti-proliferative activity in NSCLC cells. We conclude that PFKFB3 has a key role in mediating glucose metabolism and survival of NSCLC cells in response to EGFR signaling. These results support the potential clinical utility of using PFKFB3 inhibitors in combination with EGFR-TKIs to manage NSCLC.

Keywords:  6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase; PFKFB3; epidermal growth factor (EGF); epidermal growth factor receptor (EGFR); erlotinib; glycolysis; lung cancer; metabolism; tumorigenesis; tyrosine kinase inhibitor

DOI:  https://doi.org/10.1074/jbc.RA119.007784
BMC Cancer. 2019 May 20. 19(1): 474

Raman spectroscopy detects metabolic signatures of radiation response and hypoxic fluctuations in non-small cell lung cancer.

Samantha J Van Nest, Leah M Nicholson, Nils Pavey, Mathew N Hindi, Alexandre G Brolo, Andrew Jirasek, Julian J Lum.

   BACKGROUND: Radiation therapy is a standard form of treating non-small cell lung cancer, however, local recurrence is a major issue with this type of treatment. A better understanding of the metabolic response to radiation therapy may provide insight into improved approaches for local tumour control. Cyclic hypoxia is a well-established determinant that influences radiation response, though its impact on other metabolic pathways that control radiosensitivity remains unclear.
METHODS: We used an established Raman spectroscopic (RS) technique in combination with immunofluorescence staining to measure radiation-induced metabolic responses in human non-small cell lung cancer (NSCLC) tumour xenografts. Tumours were established in NOD.CB17-Prkdcscid/J mice, and were exposed to radiation doses of 15 Gy or left untreated. Tumours were harvested at 2 h, 1, 3 and 10 days post irradiation.
RESULTS: We report that xenografted NSCLC tumours demonstrate rapid and stable metabolic changes, following exposure to 15 Gy radiation doses, which can be measured by RS and are dictated by the extent of local tissue oxygenation. In particular, fluctuations in tissue glycogen content were observed as early as 2 h and as late as 10 days post irradiation. Metabolically, this signature was correlated to the extent of tumour regression. Immunofluorescence staining for γ-H2AX, pimonidazole and carbonic anhydrase IX (CAIX) correlated with RS-identified metabolic changes in hypoxia and reoxygenation following radiation exposure.
CONCLUSION: Our results indicate that RS can identify sequential changes in hypoxia and tumour reoxygenation in NSCLC, that play crucial roles in radiosensitivity.

Keywords:  Hypoxia; Immunofluorescence; Ionizing radiation; Non-small cell lung cancer; Raman spectroscopy; Reoxygenation

DOI:  https://doi.org/10.1186/s12885-019-5686-1
J Cell Biochem. 2019 May 20.

Antitumor activity of SR splicing-factor 5 knockdown by downregulating pyruvate kinase M2 in non-small cell lung cancer cells.

Jiliang Yan, Dongya Zhang, Yanjie Han, Zhongjie Wang, Chunyan Ma.

  SR splicing-factors (SRSFs) play a vital role in carcinogenesis. SRSF5 was demonstrated to be upregulated in lung cancer and identified as a novel prognostic indicator for small-cell lung cancer. However, the role of SRSF5 in the pathogenesis of non-small cell lung cancer (NSCLC) and the molecular mechanism involved are still undefined. The expression of SRSF5 in NSCLC cells was detected by quantitative real-time polymerase chain reaction and Western blot analysis. The proliferation of cells was evaluated by cell counting kit-8 and BrdU assays. Apoptosis was assessed by flow cytometry and Western blot analysis of apoptosis-associated proteins including B-cell lymphoma 2 (Bcl-2), Bax, and cytochrome C (Cyt C). Glycolysis was detected by determining glucose consumption, lactate production, and pyruvate kinase M2 (PKM2) expression. We found that SRSF5 messenger RNA and protein levels were elevated in NSCLC cells. SRSF5 knockdown inhibited the proliferation and Ki67 expression in NSCLC cells. SRSF5 silencing increased the apoptotic rate, upregulated Bax and Cyt C, and decreased Bcl-2 level in NSCLC cells. Moreover, Knockdown of SRSF5 repressed glycolysis in NSCLC cells via reducing PKM2 expression. Enhanced glycolysis by PKM2 overexpression attenuated the effects of SRSF5 silencing on NSCLC cell proliferation and apoptosis. Overall, knockdown of SRSF5 inhibited proliferative ability and induced apoptosis by suppressing PKM2 expression in NSCLC cells.

Keywords:  NSCLC; PKM2; SRSF5; apoptosis; glycolysis; proliferation

DOI:  https://doi.org/10.1002/jcb.28992
Clin Chim Acta. 2019 May 16. pii: S0009-8981(19)31863-7. [Epub ahead of print]

Metabolomics workflow for lung cancer: Discovery of biomarkers.

Yuqing Tang, Zhou Li, Lissy Lazar, Zhiling Fang, Chunlan Tang, Jinshun Zhao.

  Lung cancer is one of the most common cancers in the world. Due to the limitations of current diagnostic techniques and methods, most lung cancers are diagnosed at the advanced stage, which is not conducive to early treatment. The rise of metabolomics has provided new ideas for the early diagnosis of lung cancer. As a method for the comprehensive analysis of endogenous metabolites of the biological system, metabolomics has shown significant application potential for the early diagnosis and individualized treatment of various cancers including lung cancers. Via advanced analytical techniques and bioinformatics tools, the metabolome was excavated to find biomarkers related to cancer and its prognosis. In this review, the research methods and workflow of metabolomics are summarized, with an emphasis on the recent discovery of biomarkers and major metabolic pathways for lung cancers.

Keywords:  Biomarker; Lung cancer; Metabolic pathways; Metabolomics; Workflow

DOI:  https://doi.org/10.1016/j.cca.2019.05.012
Mol Oncol. 2019 May 22.

Genomic signatures defining responsiveness to allopurinol and combination therapy for lung cancer identified by systems therapeutics analyses.

Iman Tavassoly, Yuan Hu, Shan Zhao, Chiara Mariottini, Aislyn Boran, Yibang Chen, Lisa Li, Rosa E Tolentino, Gomathi Jayaraman, Joseph Goldfarb, James Gallo, Ravi Iyengar.

  The ability to predict responsiveness to drugs in individual patients is limited. We hypothesized that integrating molecular information from databases would yield predictions that could be experimentally tested to develop transcriptomic signatures for specific drugs. We analyzed lung adenocarcinoma patient data from The Cancer Genome Atlas (TCGA) and identified a subset of patients in which xanthine dehydrogenase expression correlated with decreased survival. We tested allopurinol, an FDA-approved drug that inhibits xanthine dehydrogenase, on human Non-Small Cell Lung Cancer (NSCLC) cell lines obtained from the Broad Institute Cancer Cell Line Encyclopedia (CCLE), and identified sensitive and resistant cell lines. We utilized the transcriptomic profiles of these cell lines to identify six-gene signatures for allopurinol-sensitive and -resistant cell lines. Transcriptomic networks identified JAK2 as an additional target in allopurinol-resistant lines. Treatment of resistant cell lines with allopurinol and CEP-33779 (a JAK2 inhibitor) resulted in cell death. The effectiveness of allopurinol alone or allopurinol and CEP-33779 were verified in vivo using tumor formation in NCR-nude mice. We utilized the six-gene signatures to predict five additional allopurinol-sensitive NSCLC cell lines, and four allopurinol-resistant cell lines susceptible to combination therapy. We searched the transcriptomic data from a library of patient-derived NSCLC tumors from Jackson Laboratory to identify tumors that would be predicted to be sensitive to allopurinol or allopurinol + CEP-33779 treatment. Patient-derived tumors showed the predicted drug sensitivity in vivo. These data indicate that we can use integrated molecular information from cancer databases to predict drug responsiveness in individual patients and thus enable precision medicine.

Keywords:   CCLE ; TCGA ; Cancer Genomics; Gene Signature; Lung cancer; Precision Oncology

DOI:  https://doi.org/10.1002/1878-0261.12521
PLoS One. 2019 ;14(5): e0217131

Subcellular compartmentalization of PKM2 identifies anti-PKM2 therapy response in vitro and in vivo mouse model of human non-small-cell lung cancer.

Akiko Suzuki, Sachin Puri, Pamela Leland, Ankit Puri, Tarsem Moudgil, Bernard A Fox, Raj K Puri, Bharat H Joshi.

Pyruvate kinase M2 (PKM2) is an alternatively spliced variant, which mediates the conversion of glucose to lactate in cancer cells under normoxic conditions, known as the Warburg effect. Previously, we demonstrated that PKM2 is one of 97 genes that are overexpressed in non-small-cell lung cancer (NSCLC) cell lines. Herein, we demonstrate a novel role of subcellular PKM2 expression as a biomarker of therapeutic response after targeting this gene by shRNA or small molecule inhibitor (SMI) of PKM2 enzyme activity in vitro and in vivo. We examined two established lung cancer cell lines, nine patients derived NSCLC and three normal lung fibroblast cell lines for PKM2 mRNA, protein and enzyme activity by RT-qPCR, immunocytochemistry (ICC), and Western blot analysis. All eleven NSCLC cell lines showed upregulated PKM2 enzymatic activity and protein expression mainly in their cytoplasm. Targeting PKM2 by shRNA or SMI, NSCLC cells showed significantly reduced mRNA, enzyme activity, cell viability, and colony formation, which also downregulated cytosolic PKM2 and upregulated nuclear enzyme activities. Normal lung fibroblast cell lines did not express PKM2, which served as negative controls. PKM2 targeting by SMI slowed tumor growth while gene-silencing significantly reduced growth of human NSCLC xenografts. Tumor sections from responding mice showed >70% reduction in cytoplasmic PKM2 with low or undetectable nuclear staining by immunohistochemistry (IHC). In sharp contrast, non-responding tumors showed a >38% increase in PKM2 nuclear staining with low or undetectable cytoplasmic staining. In conclusion, these results confirmed PKM2 as a target for cancer therapy and an unique function of subcellular PKM2, which may characterize therapeutic response to anti-PKM2 therapy in NSCLC.

DOI: https://doi.org/10.1371/journal.pone.0217131