Myc linked to dysregulation of cholesterol transport and storage in nonsmall cell lung cancer

Abstract

Nonsmall cell lung cancer (NSCLC) is a leading cause of cancer-related deaths. While mutations in Kras and overexpression of Myc are commonly found in patients, the role of altered lipid metabolism in lung cancer and its interplay with oncogenic Myc is poorly understood. Here we use a transgenic mouse model of Kras-driven lung adenocarcinoma with reversible activation of Myc combined with surface analysis lipid profiling of lung tumors and transcriptomics to study the effect of Myc activity on cholesterol homeostasis. Our findings reveal that the activation of Myc leads to the accumulation of cholesteryl esters (CEs) stored in lipid droplets. Subsequent Myc deactivation leads to further increases in CEs, in contrast to tumors in which Myc was never activated. Gene expression analysis linked cholesterol transport and storage pathways to Myc activity. Our results suggest that increased Myc activity is associated with increased cholesterol influx, reduced efflux, and accumulation of CE-rich lipid droplets in lung tumors. Targeting cholesterol homeostasis is proposed as a promising avenue to explore for novel treatments of lung cancer, with diagnostic and stratification potential in human NSCLC. Nonsmall cell lung cancer (NSCLC) is a leading cause of cancer-related deaths. While mutations in Kras and overexpression of Myc are commonly found in patients, the role of altered lipid metabolism in lung cancer and its interplay with oncogenic Myc is poorly understood. Here we use a transgenic mouse model of Kras-driven lung adenocarcinoma with reversible activation of Myc combined with surface analysis lipid profiling of lung tumors and transcriptomics to study the effect of Myc activity on cholesterol homeostasis. Our findings reveal that the activation of Myc leads to the accumulation of cholesteryl esters (CEs) stored in lipid droplets. Subsequent Myc deactivation leads to further increases in CEs, in contrast to tumors in which Myc was never activated. Gene expression analysis linked cholesterol transport and storage pathways to Myc activity. Our results suggest that increased Myc activity is associated with increased cholesterol influx, reduced efflux, and accumulation of CE-rich lipid droplets in lung tumors. Targeting cholesterol homeostasis is proposed as a promising avenue to explore for novel treatments of lung cancer, with diagnostic and stratification potential in human NSCLC. Lung cancer is the leading cause of cancer-related mortality, with nonsmall cell lung cancer (NSCLC) the most common subtype (1Dela Cruz C.S. Tanoue L.T. Matthay R.A. Lung cancer: epidemiology, etiology, and prevention.Clin. Chest Med. 2011; 32: 605-644Abstract Full Text Full Text PDF PubMed Scopus (912) Google Scholar). Mutations in Kras are found in more than 30% of NSCLC cases, while the RTK/RAS/RAF pathway is activated in 76% of cases (2The Cancer Genome Atlas Research Network Comprehensive molecular profiling of lung adenocarcinoma.Nature. 2014; 511: 543-550Crossref PubMed Scopus (3557) Google Scholar). In addition, Myc is frequently overexpressed, focal amplifications occurring in more than 30% of lung adenocarcinomas (3Schaub F.X. Dhankani V. Berger A.C. Trivedi M. Richardson A.B. Shaw R. Zhao W. Zhang X. Ventura A. Liuand Y. et al.Pan-cancer alterations of the MYC oncogene and its proximal network across the Cancer Genome Atlas.Cell Syst. 2018; 6: 282-300.e2Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). Myc is a transcription factor with numerous functions in healthy cellular processes, including regulation of cell cycle and cell growth. Deregulation of Myc leads to uncontrolled cell proliferation in many tissues and is implicated in tumorigenesis of some, perhaps all, tumors (4Murphy D.J. Junttila M.R. Pouyet L. Karnezis A. Shchors K. Bui D.A. Brown-Swigart L. Johnson L. Evan G.I. Distinct thresholds govern Myc's biological output in vivo.Cancer Cell. 2008; 14: 447-457Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar, 5Bywater M.J. Burkhart D.L. Straube J. Sabò A. Pendino V. Hudson J.E. Quaife-Ryan G.A. Porrello E.R. Rae J. Parton R.G. et al.Reactivation of Myc transcription in the mouse heart unlocks its proliferative capacity.Nat. Commun. 2020; 11: 1827Crossref PubMed Scopus (21) Google Scholar). Inhibition of Myc is therefore of interest as a cancer treatment (6Soucek L. Whitfield J.R. Sodir N.M. Masso-Valles D. Serrano E. Karnezis A.N. Swigart L.B. Evan G.I. Inhibition of Myc family proteins eradicates KRas-driven lung cancer in mice.Genes Dev. 2013; 27: 504-513Crossref PubMed Scopus (219) Google Scholar); however, its complex regulation of transcription factors and genes are not fully understood. Enhanced lipid synthesis is recognized as a signature of cancer (7Santos C.R. Schulze A. Lipid metabolism in cancer.FEBS J. 2012; 279: 2610-2623Crossref PubMed Scopus (918) Google Scholar, 8Hilvo M. Denkert C. Lehtinen L. Muller B. Brockmoller S. Seppanen-Laakso T. Budczies J. Bucher E. Yetukuri L. Castillo S. et al.Novel theranostic opportunities offered by characterization of altered membrane lipid metabolism in breast cancer progression.Cancer Res. 2011; 71: 3236-3245Crossref PubMed Scopus (378) Google Scholar, 9Beloribi-Djefaflia S. Vasseur S. Guillaumond F. Lipid metabolic reprogramming in cancer cells.Oncogenesis. 2016; 5: e189Crossref PubMed Google Scholar). Excess lipids are stored in lipid droplets, providing a source of energy for rapidly dividing cancer cells and structural components for building new membranes. Furthermore, lipids are increasingly being recognized as critical to signaling pathways in cancer. Cholesterol is a particularly important lipid messenger for signal transduction, control of membrane fluidity, and regulation of the innate immune response (10Gowdy K.M. Fessler M.B. Emerging roles for cholesterol and lipoproteins in lung disease.Pulm. Pharmacol. Ther. 2013; 26: 430-437Crossref PubMed Scopus (79) Google Scholar, 11Tall A.R. Yvan-Charvet L. Cholesterol, inflammation and innate immunity.Nat. Rev. Immunol. 2015; 15: 104-116Crossref PubMed Scopus (806) Google Scholar, 12Bozza P.T. Viola J.P. Lipid droplets in inflammation and cancer.Prostaglandins Leukot. Essent. Fatty Acids. 2010; 82: 243-250Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar), while cholesteryl ester (CE) accumulation has been implicated in prostate cancer aggressiveness (13Yue S. Li J. Lee S.Y. Lee H.J. Shao T. Song B. Cheng L. Masterson T.A. Liu X. Ratliff T.L. et al.Cholesteryl ester accumulation induced by PTEN loss and PI3K/AKT activation underlies human prostate cancer aggressiveness.Cell Metab. 2014; 19: 393-406Abstract Full Text Full Text PDF PubMed Scopus (529) Google Scholar). The link between oncogenic Myc and lipid metabolism is relatively underexplored. Previously, we showed that dysregulated Myc modulated the production of eicosanoids, critical for proliferation and cell survival, in lung adenocarcinoma (14Hall Z. Ament Z. Wilson C.H. Burkhart D.L. Ashmore T. Koulman A. Littlewood T. Evan G.I. Griffin J.L. Myc expression drives aberrant lipid metabolism in lung cancer.Cancer Res. 2016; 76: 4608-4618Crossref PubMed Scopus (48) Google Scholar). Other recent studies link Myc to increased lipogenesis in tumors (15Gouw A.M. Margulis K. Liu N.S. Raman S.J. Mancuso A. Toal G.G. Tong L. Mosley A. Hsieh A.L. Sullivan D.K. et al.The MYC oncogene cooperates with sterol-regulated element-binding protein to regulate lipogenesis essential for neoplastic growth.Cell Metab. 2019; 30: 556-572.e5Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 16Méndez-Lucas A. Lin W. Driscoll P.C. Legrave N. Novellasdemunt L. Xie C. Charles M. Wilson Z. Jones N.P. Rayport S. et al.Identifying strategies to target the metabolic flexibility of tumours.Nat. Metab. 2020; 2: 335-350Crossref PubMed Scopus (54) Google Scholar), while switching from a high- to low-fat diet attenuates the Myc transcriptional program in prostate cancer (17Labbé D.P. Zadra G. Yang M. Reyes J.M. Lin C.Y. Cacciatore S. Ebot E.M. Creech A.L. Giunchi F. Fiorentino M. et al.High-fat diet fuels prostate cancer progression by rewiring the metabolome and amplifying the MYC program.Nat. Commun. 2019; 10: 4358Crossref PubMed Scopus (61) Google Scholar). It is evident that lipids and their interaction with oncogenes, such as Myc, play a complex and elegant role in tumorigenesis and offer an underexploited therapeutic avenue. Here we use a transgenic mouse model of Kras-driven lung adenocarcinoma with reversible activation of Myc. We explore the effect of Myc activity on cholesterol homeostasis in lung tumors by integrating surface analysis MS-based lipidomics, transcriptomics, and quantitative gene expression analysis. Our results reveal that increased Myc activity favors cholesterol influx over efflux in tumors and leads to the accumulation of CEs stored in lipid droplets. Deactivation of Myc triggers the clearance of cholesterol through increasing efflux, decreasing influx and further increasing cholesterol esterification. No accumulation of cholesterol occurred in tumors in which Myc was never activated. These findings provide new insights into the role of oncogenic Myc and dysregulation of cholesterol homeostasis in lung cancer. Mice were maintained on a regular diet in a pathogen-free facility on a 12 h light/dark cycle with continuous access to food and water. Lung tumors were generated in adult Krastm4Tyj/+ (LSL-KrasG12D) or LSL-KrasG12D; R26LSL-CAG-c-MycER/LSL-CAG-c-MycER (R26LSL-CMER) mice as previously described (5Bywater M.J. Burkhart D.L. Straube J. Sabò A. Pendino V. Hudson J.E. Quaife-Ryan G.A. Porrello E.R. Rae J. Parton R.G. et al.Reactivation of Myc transcription in the mouse heart unlocks its proliferative capacity.Nat. Commun. 2020; 11: 1827Crossref PubMed Scopus (21) Google Scholar). Briefly, mice were anesthetized (isoflurane), and the intranasal instillation of adeno-Cre virus (7 × 108 – 3.5 × 109 plaque-forming units; University of Iowa Viral Vector Core) was performed by placing virus droplets on the nose of the mouse. This resulted in the expression of Cre-recombinase removing the stop element sporadically in the lung epithelium of both the Kras and Rosa26 alleles. Consequently, these lung epithelial cells express oncogenic KrasG12D, driving the formation of lung adenocarcinomas. Tamoxifen added to the diet of LSL-KrasG12D; R26LSL-CMER mice results in the activation of the c-MycERT2 protein, specifically within the Cre-deleted tumor tissues (18Wilson C.H. Gamper I. Perfetto A. Auw J. Littlewood T.D. Evan G.I. The kinetics of ER fusion protein activation in vivo.Oncogene. 2014; 33: 4877-4880Crossref PubMed Scopus (22) Google Scholar). The subsequent removal of tamoxifen results in the rapid deactivation of MycER. Twelve adeno-Cre-infected LSL-KrasG12D; R26LSL-CMER mice were maintained on a tamoxifen-containing diet for 1 month. Tamoxifen was removed from the diet for 24 h (n = 3) or 72 h (n = 3) to deactivate Myc before culling (“Myc inactive”). The remaining mice were continuously fed a tamoxifen-containing diet (“Myc activated”). LSL-KrasG12D were also fed a tamoxifen-containing diet; however, Myc expression was unaffected (“Kras only”; n = Lung were in and stored were by the and the University of for lipids was performed to an A. L. A. of lipids by for the metabolic in and 2013; PubMed Scopus Google Scholar). were with for to and in water. This was by with and a in water. were with and Lung cells from adeno-Cre-infected R26LSL-CMER and LSL-KrasG12D; R26LSL-CMER mice were as previously described S. I. S. D. 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Lee et to 2018; Full Text Full Text PDF PubMed Scopus Google Scholar). on the expression to were into expression gene of The between expression and were we found that expression was associated with a = we that pathways cholesterol transport and storage are modulated the gene expression in lung are linked to oncogenic Myc, and potential as diagnostic or stratification in human NSCLC. a of transcriptomics, lipidomics, and quantitative gene expression we that Myc activity in lung tumors is linked to a in cholesterol homeostasis. and pathway analysis the dysregulation of lipid metabolism in cells with the lipids from tumor we found a in CEs Myc was activated with Myc was we showed that the expression of genes to cholesterol influx, efflux, and were linked to Myc activity. was a between the expression of and Myc activity. These are in both epithelial cells and and regulate cholesterol clearance the cell to in their expression leads to increased cholesterol and inflammation in cells R. E. S. T. G. G. M. epithelial cells cholesterol induced cell 2015; PubMed Scopus Google Scholar, the for Lipid Res. Full Text Full Text PDF PubMed Google Scholar, S. critical role for in inflammation and lung Immunol. 2008; PubMed Scopus Google Scholar). It is therefore that the accumulation of cholesterol in tumors with Myc activity is a of the deactivation of that cholesterol efflux through the of The link between Myc and cholesterol is expression for genes to cholesterol was found in tumors with Myc activity. the a of cholesterol and was to in the analysis of oncogenic lung is that is by the of cholesterol in tumor which also for the of in tumors. that activation of Myc is a accumulation of cholesterol that results from the dysregulation of of cholesterol and storage in lipid droplets of cancer Myc is is a homeostasis. The influx of cholesterol and efflux and to further increases to cholesterol The accumulation of lipid droplets more has been associated with and linked to and P.T. Viola J.P. Lipid droplets in inflammation and cancer.Prostaglandins Leukot. Essent. Fatty Acids. 2010; 82: 243-250Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar, S.J. Zhang J. A.M. formation of lipid droplets as a response to Med. Cell. 2013; PubMed Scopus Google Scholar, A. G. L. K. M. K. J. C. et metabolic leading to accumulation of lipid droplets in tumor 2013; PubMed Scopus Google Scholar). These lipid droplets a of into and of these droplets and cholesterol that by cancer cells for membrane and cellular proliferation and cellular signaling B. Song C. Cholesterol metabolism in cancer: and therapeutic Metab. 2020; 2: PubMed Scopus Google Scholar, F. B. Schulze A. on the role of lipid synthesis in cancer metabolism and 2013; 6: PubMed Scopus Google Scholar). recent study has that Myc and in NSCLC to inflammation and immune Deregulation of Myc in lung triggers the of which These in and clearance of immune cells Sodir N.M. Wilson C.H. Burkhart D.L. L. Swigart L. Littlewood T.D. Evan G.I. Myc cooperates with by inflammation and immune Full Text Full Text PDF PubMed Scopus Google Scholar). cholesterol has been implicated as a to immune for cholesterol in signaling and activation of the A.R. Yvan-Charvet L. Cholesterol, inflammation and innate immunity.Nat. Rev. Immunol. 2015; 15: 104-116Crossref PubMed Scopus (806) Google Scholar). dysregulation of cholesterol homeostasis an important effect on many cellular including and immune The of provide a link to further these complex in These of cholesterol as for Y. a 2019; PubMed Scopus Google Scholar), and been linked to the of immune response and cancer and in immune cell Immunol. 2013; 14: PubMed Scopus Google Scholar). This study new potential for lung cancer with which cholesterol and are being in for a of also been to a of in which inflammation a as as for with NSCLC (10Gowdy K.M. Fessler M.B. Emerging roles for cholesterol and lipoproteins in lung disease.Pulm. Pharmacol. Ther. 2013; 26: 430-437Crossref PubMed Scopus (79) Google Scholar, B. Z. X. F. The between and breast cancer by and a Cancer Res. PubMed Scopus Google Scholar, N. K. G. J.P. The effect of on in with lung 2016; Full Text Full Text PDF PubMed Scopus Google Scholar). for NSCLC treatment is by the of This is being to cases of prostate cancer, while studies to and tumor and in mouse of J. D. Lee Song B. S. S. Ratliff T.L. Liu X. Xie J. et cholesterol and of 2016; PubMed Scopus Google Scholar, F. Cheng X. X. Cheng C. X. B. et of Cancer Res. 2016; PubMed Scopus Google Scholar, B. Schulze A. the of prostate Metab. 2014; 19: Full Text Full Text PDF PubMed Scopus Google Scholar). are in with a factor for a of in with a for B. Schulze A. Lipid metabolism the of diet and tumor 2019; 5: Full Text Full Text PDF PubMed Scopus Google Scholar). a model of lung adenocarcinoma was to link in cholesterol transport and storage to Myc to dysregulation of cholesterol homeostasis in NSCLC. The accumulation of cholesterol play a role in signaling or to cellular Targeting cholesterol metabolism is therefore proposed as a promising avenue for novel treatments of lung cancer. found in the and been in the were performed University of was performed by the The is generated by the Research Network with cholesteryl ester surface analysis nonsmall cell lung cancer

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