BRAF-Inhibitor-Induced Metabolic Alterations in A375 Melanoma Cells
Abstract
:1. Introduction
2. Results
2.1. Vemurafenib Persister Cells Are Slow-Cycling Cells That Are Reversibly Drug Tolerant
2.2. VEM Treatment Affects the Expression Levels of Cancer Stem Cell Biomarkers
2.3. VEM-Induced Persister Cells Exhibit an Altered Metabolic Profile
2.4. TCA Cycle Activity of Persister Cells Is Not Significantly Different from That of Control Cells
2.5. Lactic Acid Consumption Is Significantly Upregulated in VEM Persister Cells
2.6. Persister Cells Have Increased Viability in a Minimal Medium
3. Discussion
4. Materials and Methods
4.1. Cell Culture Conditions
4.2. Generating Persister Kill Curves
4.3. Isolating Persister Cells
4.4. Assessing the Cell Viability with Microscopy
4.5. Apoptosis Assay
4.6. Clonogenic Survival Assay
4.7. Cell Count with a Flow Cytometer
4.8. Cancer Stem Cell Markers
4.9. ALDEFLUOR Assay
4.10. Cell Proliferation Assay
4.11. Metabolomics
4.12. Mitoplate Assays
4.13. PM-M1 Assays
4.14. Glucose and Lactate Consumption Assay
4.15. Viability Assay in a Minimal Medium
4.16. Statistical Analysis
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
- Jemal, A.; Siegel, R.; Miller, K.D. Home. American Cancer Society-Cancer Facts & Statistics. Available online: https://cancerstatisticscenter.cancer.org/?_ga=2.242081546.925338221.1599614578-1606375298.1599614578#!/ (accessed on 8 September 2020).
- SEER Melanoma of the Skin-Cancer Stat Facts. Available online: https://seer.cancer.gov/statfacts/html/melan.html (accessed on 26 July 2021).
- Feigelson, H.S.; Powers, J.D.; Kumar, M.; Carroll, N.M.; Pathy, A.; Ritzwoller, D.P. Melanoma incidence, recurrence, and mortality in an integrated healthcare system: A retrospective cohort study. Cancer Med. 2019, 8, 4508–4516. [Google Scholar] [CrossRef] [PubMed]
- Davies, H.; Bignell, G.R.; Cox, C.; Stephens, P.; Edkins, S.; Clegg, S.; Teague, J.; Woffendin, H.; Garnett, M.J.; Bottomley, W.; et al. Mutations of the BRAF gene in human cancer. Nature 2002, 417, 949–954. [Google Scholar] [CrossRef] [PubMed]
- Roberts, R. The extracellular signal-regulated kinase (ERK) pathway: A potential therapeutic target in hypertension. J. Exp. Pharmacol. 2012, 4, 77. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Daniotti, M.; Oggionni, M.; Ranzani, T.; Vallacchi, V.; Campi, V.; Di Stasi, D.; Della Torre, G.; Perrone, F.; Luoni, C.; Suardi, S.; et al. BRAF alterations are associated with complex mutational profiles in malignant melanoma. Oncogene 2004, 23, 5968–5977. [Google Scholar] [CrossRef] [Green Version]
- Hanahan, D.; Weinberg, R.A. Hallmarks of cancer: The next generation. Cell 2011, 144, 646–674. [Google Scholar] [CrossRef] [Green Version]
- Ratnikov, B.I.; Scott, D.A.; Osterman, A.L.; Smith, J.W.; Ronai, Z.A. Metabolic rewiring in melanoma. Oncogene 2017, 36, 147–157. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Kuphal, S.; Winklmeier, A.; Warnecke, C.; Bosserhoff, A.K. Constitutive HIF-1 activity in malignant melanoma. Eur. J. Cancer 2010, 46, 1159–1169. [Google Scholar] [CrossRef]
- Kaplon, J.; Zheng, L.; Meissl, K.; Chaneton, B.; Selivanov, V.A.; MacKay, G.; Van Der Burg, S.H.; Verdegaal, E.M.E.; Cascante, M.; Shlomi, T.; et al. A key role for mitochondrial gatekeeper pyruvate dehydrogenase in oncogene-induced senescence. Nature 2013, 498, 109–112. [Google Scholar] [CrossRef]
- Ho, J.; de Moura, M.B.; Lin, Y.; Vincent, G.; Thorne, S.; Duncan, L.M.; Hui-Min, L.; Kirkwood, J.M.; Becker, D.; Van Houten, B.; et al. Importance of glycolysis and oxidative phosphorylation in advanced melanoma. Mol. Cancer 2012, 11, 1–13. [Google Scholar] [CrossRef] [Green Version]
- Sharma, S.V.; Lee, D.Y.; Li, B.; Quinlan, M.P.; Takahashi, F.; Maheswaran, S.; McDermott, U.; Azizian, N.; Zou, L.; Fischbach, M.A.; et al. A Chromatin-mediated reversible drug-tolerant state in cancer cell subpopulations. Cell 2010, 141, 69–80. [Google Scholar] [CrossRef] [Green Version]
- Roesch, A.; Fukunaga-Kalabis, M.; Schmidt, E.C.; Zabierowski, S.E.; Brafford, P.A.; Vultur, A.; Basu, D.; Gimotty, P.; Vogt, T.; Herlyn, M. A temporarily distinct subpopulation of slow-cycling melanoma cells is required for continuous tumor growth. Cell 2010, 141, 583–594. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Ramirez, M.; Rajaram, S.; Steininger, R.J.; Osipchuk, D.; Roth, M.A.; Morinishi, L.S.; Evans, L.; Ji, W.; Hsu, C.H.; Thurley, K.; et al. Diverse drug-resistance mechanisms can emerge from drug-tolerant cancer persister cells. Nat. Commun. 2016, 7, 10690. [Google Scholar] [CrossRef] [PubMed]
- Hangauer, M.J.; Viswanathan, V.S.; Ryan, M.J.; Bole, D.; Eaton, J.K.; Matov, A.; Galeas, J.; Dhruv, H.D.; Berens, M.E.; Schreiber, S.L.; et al. Drug-tolerant persister cancer cells are vulnerable to GPX4 inhibition. Nature 2017, 551, 247–250. [Google Scholar] [CrossRef] [Green Version]
- Shen, S.; Faouzi, S.; Souquere, S.; Roy, S.; Routier, E.; Libenciuc, C.; André, F.; Pierron, G.; Scoazec, J.Y.; Robert, C. Melanoma persister cells are tolerant to BRAF/MEK inhibitors via ACOX1-mediated fatty acid oxidation. Cell Rep. 2020, 33, 108421. [Google Scholar] [CrossRef] [PubMed]
- Karki, P.; Angardi, V.; Mier, J.C.; Orman, M.A. A transient metabolic state in melanoma persister cells mediated by chemotherapeutic treatments. bioRxiv 2021. [Google Scholar] [CrossRef]
- Sharma, A.; Shah, S.R.; Illum, H.; Dowell, J. Vemurafenib: Targeted inhibition of mutated BRAF for treatment of advanced melanoma and its potential in other malignancies. Drugs 2012, 72, 2207–2222. [Google Scholar] [CrossRef]
- Shen, S.; Faouzi, S.; Bastide, A.; Martineau, S.; Malka-Mahieu, H.; Fu, Y.; Sun, X.; Mateus, C.; Routier, E.; Roy, S.; et al. An epitranscriptomic mechanism underlies selective mRNA translation remodelling in melanoma persister cells. Nat. Commun. 2019, 10, 5713. [Google Scholar] [CrossRef] [Green Version]
- Wang, Y.; Zhang, J.; Ren, S.; Sun, D.; Huang, H.Y.; Wang, H.; Jin, Y.; Li, F.; Zheng, C.; Yang, L.; et al. Branched-chain amino acid metabolic reprogramming orchestrates drug resistance to EGFR tyrosine kinase inhibitors. Cell Rep. 2019, 28, 512–525. [Google Scholar] [CrossRef]
- Wlodkowic, D.; Skommer, J.; Darzynkiewicz, Z. Flow cytometry-based apoptosis detection. Methods Mol. Biol. 2009, 559, 19–32. [Google Scholar] [CrossRef] [Green Version]
- Van Engeland, M.; Nieland, L.J.W.; Ramaekers, F.C.S.; Schutte, B.; Reutelingsperger, C.P.M. Annexin V-affinity assay: A review on an apoptosis detection system based on phosphatidylserine exposure. Cytometry 1998, 31, 1–9. [Google Scholar] [CrossRef]
- Lyons, A.B.; Blake, S.J.; Doherty, K.V. Flow cytometric analysis of cell division by dilution of CFSE and related dyes. Curr. Protoc. Cytom. 2013, 64, 9–11. [Google Scholar] [CrossRef] [PubMed]
- Parmiani, G. Melanoma cancer stem cells: Markers and functions. Cancers 2016, 8, 34. [Google Scholar] [CrossRef] [Green Version]
- Rambow, F.; Rogiers, A.; Marin-Bejar, O.; Aibar, S.; Femel, J.; Dewaele, M.; Karras, P.; Brown, D.; Chang, Y.H.; Debiec-Rychter, M.; et al. Toward minimal residual disease-directed therapy in melanoma. Cell 2018, 174, 843–855. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Restivo, G.; Diener, J.; Cheng, P.F.; Kiowski, G.; Bonalli, M.; Biedermann, T.; Reichmann, E.; Levesque, M.P.; Dummer, R.; Sommer, L. Low Neurotrophin receptor CD271 regulates phenotype switching in Melanoma. Nat. Commun. 2017, 8, 1–16. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Chong, J.; Wishart, D.S.; Xia, J. Using MetaboAnalyst 4.0 for comprehensive and integrative metabolomics data analysis. Curr. Protoc. Bioinform. 2019, 68, e86. [Google Scholar] [CrossRef] [PubMed]
- Roesch, A.; Vultur, A.; Bogeski, I.; Wang, H.; Zimmermann, K.M.; Speicher, D.; Körbel, C.; Laschke, M.W.; Gimotty, P.A.; Philipp, S.E.; et al. Overcoming intrinsic multidrug resistance in melanoma by blocking the mitochondrial respiratory chain of slow-cycling JARID1Bhigh cells. Cancer Cell 2013, 23, 811–825. [Google Scholar] [CrossRef] [Green Version]
- Viale, A.; Pettazzoni, P.; Lyssiotis, C.A.; Ying, H.; Sánchez, N.; Marchesini, M.; Carugo, A.; Green, T.; Seth, S.; Giuliani, V.; et al. Oncogene ablation-resistant pancreatic cancer cells depend on mitochondrial function. Nature 2014, 514, 628–632. [Google Scholar] [CrossRef] [Green Version]
- Merighi, S.; Mirandola, P.; Milani, D.; Varani, K.; Gessi, S.; Klotz, K.N.; Leung, E.; Baraldi, P.G.; Borea, P.A. Adenosine receptors as mediators of both cell proliferation and cell death of cultured human melanoma cells. J. Investig. Dermatol. 2002, 119, 923–933. [Google Scholar] [CrossRef] [Green Version]
- Schrimpe-Rutledge, A.C.; Codreanu, S.G.; Sherrod, S.D.; McLean, J.A. Untargeted metabolomics strategies—challenges and emerging directions. J. Am. Soc. Mass Spectrom. 2016, 27, 1897–1905. [Google Scholar] [CrossRef] [Green Version]
- Zhang, J.; Zhang, Q. Using seahorse machine to measure OCR and ECAR in cancer cells. In Methods in Molecular Biology; Humana Press: New York, NY, USA, 2019; Volume 1928, pp. 353–363. [Google Scholar]
- de la Cruz-López, K.G.; Castro-Muñoz, L.J.; Reyes-Hernández, D.O.; García-Carrancá, A.; Manzo-Merino, J. Lactate in the regulation of tumor microenvironment and therapeutic approaches. Front. Oncol. 2019, 9, 1143. [Google Scholar] [CrossRef] [Green Version]
- Chen, Y.J.; Mahieu, N.G.; Huang, X.; Singh, M.; Crawford, P.A.; Johnson, S.L.; Gross, R.W.; Schaefer, J.; Patti, G.J. Lactate metabolism is associated with mammalian mitochondria. Nat. Chem. Biol. 2016, 12, 937–943. [Google Scholar] [CrossRef] [Green Version]
- Germain, N.; Dhayer, M.; Boileau, M.; Fovez, Q.; Kluza, J.; Marchetti, P. Lipid metabolism and resistance to anticancer treatment. Biology 2020, 9, 474. [Google Scholar] [CrossRef]
- Kopecka, J.; Trouillas, P.; Gašparović, A.Č.; Gazzano, E.; Assaraf, Y.G.; Riganti, C. Phospholipids and cholesterol: Inducers of cancer multidrug resistance and therapeutic targets. Drug Resist. Updates 2020, 49, 100670. [Google Scholar] [CrossRef] [PubMed]
- Pellerin, L.; Carrié, L.; Dufau, C.; Nieto, L.; Ségui, B.; Levade, T.; Riond, J.; Andrieu-Abadie, N. Lipid metabolic reprogramming: Role in melanoma progression and therapeutic perspectives. Cancers 2020, 12, 3147. [Google Scholar] [CrossRef] [PubMed]
- Delgado-Goñi, T.; Galobart, T.C.; Wantuch, S.; Normantaite, D.; Leach, M.O.; Whittaker, S.R.; Beloueche-Babari, M. Increased inflammatory lipid metabolism and anaplerotic mitochondrial activation follow acquired resistance to vemurafenib in BRAF-mutant melanoma cells. Br. J. Cancer 2020, 122, 72–81. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Mashima, T.; Seimiya, H.; Tsuruo, T. De novo fatty-acid synthesis and related pathways as molecular targets for cancer therapy. Br. J. Cancer 2009, 100, 1369–1372. [Google Scholar] [CrossRef] [Green Version]
- Soares, A.S.; Costa, V.M.; Diniz, C.; Fresco, P. Inosine strongly enhances proliferation of human C32 melanoma cells through PLC-PKC-MEK1/2-ERK1/2 and PI3K pathways. Basic Clin. Pharmacol. Toxicol. 2015, 116, 25–36. [Google Scholar] [CrossRef]
- Shuvalov, O.; Petukhov, A.; Daks, A.; Fedorova, O.; Vasileva, E.; Barlev, N.A. One-carbon metabolism and nucleotide biosynthesis as attractive targets for anticancer therapy. Oncotarget 2017, 8, 23955–23977. [Google Scholar] [CrossRef] [Green Version]
- Newman, A.C.; Maddocks, O.D.K. One-carbon metabolism in cancer. Br. J. Cancer 2017, 116, 1499–1504. [Google Scholar] [CrossRef] [Green Version]
- Ananieva, E.A.; Wilkinson, A.C. Branched-chain amino acid metabolism in cancer. Curr. Opin. Clin. Nutr. Metab. Care 2018, 21, 64–70. [Google Scholar] [CrossRef] [Green Version]
- Sivanand, S.; Vander Heiden, M.G. Emerging roles for branched-chain amino acid metabolism in cancer. Cancer Cell 2020, 37, 147–156. [Google Scholar] [CrossRef]
- Xu, Y.; Yu, W.; Yang, T.; Zhang, M.; Liang, C.; Cai, X.; Shao, Q. Overexpression of BCAT1 is a prognostic marker in gastric cancer. Hum. Pathol. 2018, 75, 41–46. [Google Scholar] [CrossRef] [PubMed]
- Parmenter, T.J.; Kleinschmidt, M.; Kinross, K.M.; Bond, S.T.; Li, J.; Kaadige, M.R.; Rao, A.; Sheppard, K.E.; Hugo, W.; Pupo, G.M.; et al. Response of BRAF-mutant melanoma to BRAF inhibition is mediated by a network of transcriptional regulators of glycolysis. Cancer Discov. 2014, 4, 423–433. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Asati, V.; Mahapatra, D.K.; Bharti, S.K. PI3K/Akt/mTOR and Ras/Raf/MEK/ERK signaling pathways inhibitors as anticancer agents: Structural and pharmacological perspectives. Eur. J. Med. Chem. 2016, 109, 314–341. [Google Scholar] [CrossRef] [PubMed]
- Gough, D.J.; Koetz, L.; Levy, D.E. The MEK-ERK pathway is necessary for serine phosphorylation of mitochondrial STAT3 and ras-mediated transformation. PLoS ONE 2013, 8, e83395. [Google Scholar] [CrossRef]
- Ferber, E.C.; Peck, B.; Delpuech, O.; Bell, G.P.; East, P.; Schulze, A. FOXO3a regulates reactive oxygen metabolism by inhibiting mitochondrial gene expression. Cell Death Differ. 2012, 19, 968–979. [Google Scholar] [CrossRef] [PubMed]
- Kapitsinou, P.P.; Haase, V.H. The VHL tumor suppressor and HIF: Insights from genetic studies in mice. Cell Death Differ. 2008, 15, 650–659. [Google Scholar] [CrossRef] [PubMed]
- King, A.; Selak, M.A.; Gottlieb, E. Succinate dehydrogenase and fumarate hydratase: Linking mitochondrial dysfunction and cancer. Oncogene 2006, 25, 4675–4682. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Dang, C.V.; Kim, J.W.; Gao, P.; Yustein, J. The interplay between MYC and HIF in cancer. Nat. Rev. Cancer 2008, 8, 51–56. [Google Scholar] [CrossRef]
- Coller, H.A.; Grandori, C.; Tamayo, P.; Colbert, T.; Lander, E.S.; Eisenman, R.N.; Golub, T.R. Expression analysis with oligonucleotide microarrays reveals that MYC regulates genes involved in growth, cell cycle, signaling, and adhesion. Proc. Natl. Acad. Sci. USA 2000, 97, 3260–3265. [Google Scholar] [CrossRef] [Green Version]
- Theodosakis, N.; Held, M.A.; Marzuka-Alcala, A.; Meeth, K.M.; Micevic, G.; Long, G.V.; Scolyer, R.A.; Stern, D.F.; Bosenberg, M.W. BRAF inhibition decreases cellular glucose uptake in melanoma in Association with reduction in cell volume. Mol. Cancer Ther. 2015, 14, 1680–1692. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Corazao-Rozas, P.; Guerreschi, P.; André, F.; Gabert, P.E.; Lancel, S.; Dekiouk, S.; Fontaine, D.; Tardivel, M.; Savina, A.; Quesnel, B.; et al. Mitochondrial oxidative phosphorylation controls cancer cell’s life and death decisions upon exposure to MAPK inhibitors. Oncotarget 2016, 7, 39473–39485. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Hardeman, K.N.; Peng, C.; Paudel, B.B.; Meyer, C.T.; Luong, T.; Tyson, D.R.; Young, J.D.; Quaranta, V.; Fessel, J.P. Dependence on Glycolysis Sensitizes BRAF-mutated Melanomas for Increased Response to Targeted BRAF Inhibition. Sci. Rep. 2017, 7. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Delgado-Goni, T.; Miniotis, M.F.; Wantuch, S.; Parkes, H.G.; Marais, R.; Workman, P.; Leach, M.O.; Beloueche-Babari, M. The BRAF inhibitor vemurafenib activates mitochondrial metabolism and inhibits hyperpolarized pyruvate-lactate exchange in BRAF-mutant human melanoma cells. Mol. Cancer Ther. 2016, 15, 2987–2999. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Zhao, Y.; Li, M.; Yao, X.; Fei, Y.; Lin, Z.; Li, Z.; Cai, K.; Zhao, Y.; Luo, Z. HCAR1/MCT1 regulates tumor ferroptosis through the lactate-mediated AMPK-SCD1 activity and its therapeutic implications. Cell Rep. 2020, 33. [Google Scholar] [CrossRef] [PubMed]
- Louis, K.S.; Siegel, A.C. Cell viability analysis using trypan blue: Manual and automated methods. Methods Mol. Biol. 2011, 740, 7–12. [Google Scholar] [CrossRef] [PubMed]
- Evans, A.M.; DeHaven, C.D.; Barrett, T.; Mitchell, M.; Milgram, E. Integrated, nontargeted ultrahigh performance liquid chromatography/ electrospray ionization tandem mass spectrometry platform for the identification and relative quantification of the small-molecule complement of biological systems. Anal. Chem. 2009, 81, 6656–6667. [Google Scholar] [CrossRef] [PubMed]
- Xia, J.; Wishart, D.S. Web-based inference of biological patterns, functions and pathways from metabolomic data using metaboanalyst. Nat. Protoc. 2011, 6, 743–760. [Google Scholar] [CrossRef]
- Bochner, B.R.; Siri, M.; Huang, R.H.; Noble, S.; Lei, X.H.; Clemons, P.A.; Wagner, B.K. Assay of the multiple energy-producing pathways of mammalian cells. PLoS ONE 2011, 6, e18147. [Google Scholar] [CrossRef] [Green Version]
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. |
© 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Share and Cite
Karki, P.; Sensenbach, S.; Angardi, V.; Orman, M.A. BRAF-Inhibitor-Induced Metabolic Alterations in A375 Melanoma Cells. Metabolites 2021, 11, 777. https://0-doi-org.brum.beds.ac.uk/10.3390/metabo11110777
Karki P, Sensenbach S, Angardi V, Orman MA. BRAF-Inhibitor-Induced Metabolic Alterations in A375 Melanoma Cells. Metabolites. 2021; 11(11):777. https://0-doi-org.brum.beds.ac.uk/10.3390/metabo11110777
Chicago/Turabian StyleKarki, Prashant, Shayne Sensenbach, Vahideh Angardi, and Mehmet A. Orman. 2021. "BRAF-Inhibitor-Induced Metabolic Alterations in A375 Melanoma Cells" Metabolites 11, no. 11: 777. https://0-doi-org.brum.beds.ac.uk/10.3390/metabo11110777