The Lipogenic Regulator SREBP2 Induces Transferrin in Circulating Melanoma Cells and Suppresses Ferroptosis
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Published OnlineFirst November 17, 2020; DOI: 10.1158/2159-8290.CD-19-1500 RESEARCH ARTICLE The Lipogenic Regulator SREBP2 Induces Transferrin in Circulating Melanoma Cells and Suppresses Ferroptosis Xin Hong1, Whijae Roh2, Ryan J. Sullivan1,3, Keith H.K. Wong4,5, Ben S. Wittner1, Hongshan Guo1, Taronish D. Dubash1, Moshe Sade-Feldman1,2, Benjamin Wesley1, Elad Horwitz1, Genevieve M. Boland1,6, Dieuwke L. Marvin1, Todd Bonesteel1, Chenyue Lu1, François Aguet2, Risa Burr1, Samuel S. Freeman2, Laxmi Parida9, Katherine Calhoun1,4, Michelle K. Jewett1,4, Linda T. Nieman1, Nir Hacohen1,2, Anders M. Näär1, David T. Ting1,3, Mehmet Toner4,5,6, Shannon L. Stott1,3, Gad Getz1,2,7, Shyamala Maheswaran1,6, and Daniel A. Haber1,3,8 ABSTRACT Circulating tumor cells (CTC) are shed by cancer into the bloodstream, where a viable subset overcomes oxidative stress to initiate metastasis. We show that Bianca Dunn by Illustration single CTCs from patients with melanoma coordinately upregulate lipogenesis and iron homeostasis pathways. These are correlated with both intrinsic and acquired resistance to BRAF inhibitors across clonal cultures of BRAF-mutant CTCs. The lipogenesis regulator SREBP2 directly induces transcription of the iron carrier Transferrin (TF), reducing intracellular iron pools, reactive oxygen species, and lipid peroxidation, thereby conferring resistance to inducers of ferroptosis. Knockdown of endogenous TF impairs tumor formation by melanoma CTCs, and their tumorigenic defects are partially rescued by the lipophilic antioxidants ferrostatin-1 and vitamin E. In a prospective melanoma cohort, presence of CTCs with high lipogenic and iron metabolic RNA signatures is correlated with adverse clinical outcome, irre- spective of treatment regimen. Thus, SREBP2-driven iron homeostatic pathways contribute to cancer progression, drug resistance, and metastasis. SIGNIFICANCE: Through single-cell analysis of primary and cultured melanoma CTCs, we have uncov- ered intrinsic cancer cell heterogeneity within lipogenic and iron homeostatic pathways that modulates resistance to BRAF inhibitors and to ferroptosis inducers. Activation of these pathways within CTCs is correlated with adverse clinical outcome, pointing to therapeutic opportunities. 1Massachusetts General Hospital Cancer Center and Harvard Medical X. Hong and W. Roh contributed equally to this article. 2 School, Boston, Massachusetts. Broad Institute of Harvard and MIT, Cam- Current address for X. Hong: School of Medicine, Southern University of 3 bridge, Massachusetts. Department of Medicine, Massachusetts General Science and Technology, Shen Zhen, China; and current address for A.M. 4 Hospital and Harvard Medical School, Boston, Massachusetts. Center for Näär, Department of Nutritional Sciences and Toxicology, University of Engineering in Medicine, Department of Surgery, Massachusetts General California, Berkeley, California. Hospital and Harvard Medical School, Boston, Massachusetts. 5Shriners Hospitals for Children, Boston, Massachusetts. 6Department of Surgery, Corresponding Authors: Daniel A. Haber, MGH Cancer Center, Building Massachusetts General Hospital and Harvard Medical School, Boston, Mas- 149, 13th Street, Charlestown, MA 02129. Phone: 617-726-7805; E-mail: sachusetts. 7Department of Pathology, Massachusetts General Hospital and [email protected]; and Shyamala Maheswaran, maheswaran@ Harvard Medical School, Boston, Massachusetts. 8Howard Hughes Medical helix.mgh.harvard.edu Institute, Bethesda, Maryland. 9IBM Research, Yorktown Heights, New York. Cancer Discov 2021;11:678–95 Note: Supplementary data for this article are available at Cancer Discovery doi: 10.1158/2159-8290.CD-19-1500 Online (http://cancerdiscovery.aacrjournals.org/). ©2020 American Association for Cancer Research. 678 | CANCER DISCOVERY MARCH 2021 AACRJournals.org Downloaded from cancerdiscovery.aacrjournals.org on September 30, 2021. © 2021 American Association for Cancer Research. Published OnlineFirst November 17, 2020; DOI: 10.1158/2159-8290.CD-19-1500 Illustration by Bianca Dunn by Illustration INTRODUCTION lating tumor cells (CTC) to diverse metastatic sites, as is the progression from oligometastatic to widespread metastatic Metastatic melanoma remains a highly lethal malignancy, disease. Although melanoma cells are notoriously invasive despite breakthroughs in the targeted treatment of BRAF- and resistant to any therapeutic interventions other than mutant cases (1–3) and the application of immune check- immunologic and BRAF-targeted treatments, the exceptional point therapies irrespective of genotype (4–8). Melanomas with environment in the bloodstream may provide new therapeu- BRAFV600E and associated oncogenic variants typically undergo tic opportunities to suppress metastasis. rapid tumor shrinkage following treatment with combina- Specifically, the high oxygen content of blood has been tion BRAF and MEK inhibitors, although there is consider- shown to render primary melanoma cells vulnerable to reac- able variation in the degree of initial response, and virtually tive oxygen species (ROS; ref. 17). Identifying innate mecha- all cases recur with drug-resistant disease. The effectiveness nisms that confer resistance to ROS, and thereby enable a of immune-targeted therapies appears to be determined in subset of melanoma cells to remain viable as they circulate in part by total mutational burden in the tumor and the associ- the blood, may thus provide new therapeutic targets to sup- ated presentation of neoantigens, which are typically high in press metastatic precursors. melanoma (9–14), but only a third of patients have sustained The small number of CTCs present within a blood speci- responses (15, 16). For both targeted and immune-based thera- men, admixed to billions of normal blood cells, presents pies, innate and acquired resistance is compounded by tumor significant technical challenges to their isolation and func- heterogeneity in signaling pathways, expression of cell surface tional analysis (18). Neural crest–derived melanoma cells epitopes, and intrinsic cell survival pathways. do not express the epithelial cell surface protein epithelial The transition from primary melanoma to metastatic dis- cell adhesion molecule EPCAM, which is traditionally used ease is characterized by blood-based dissemination of circu- to capture CTCs using magnetically conjugated antibody March 2021 CANCER DISCOVERY | 679 Downloaded from cancerdiscovery.aacrjournals.org on September 30, 2021. © 2021 American Association for Cancer Research. Published OnlineFirst November 17, 2020; DOI: 10.1158/2159-8290.CD-19-1500 RESEARCH ARTICLE Hong et al. enrichment (19, 20). Furthermore, melanoma cells display a independent media, under hypoxic conditions, and in the high degree of heterogeneity in cell surface epitope expres- presence of heparin and RHO kinase inhibitor (refs. 22, 23, 25; sion, which complicates their direct capture using lineage- or see Methods). To confirm their identity as melanoma cells, tumor-specific antibody panels (21). In designing a tumor- the cultured CTCs were tested for expression of characteristic agnostic strategy for isolating CTCs within blood specimens, melanoma markers (ref. 24; Fig. 1A; Supplementary Fig. S1A we developed a platform (CTC-iChip) for efficient depletion and S1B) and shown to have shared mutational profiles with of normal blood cells, thereby enriching for cancer cells irre- their respective parental tumors, including the canonical spective of cell surface markers (22, 23). Taking advantage drug-sensitizing BRAFV600E or NRASQ61K mutations, as well of microfluidic flow kinetics, this CTC-iChip achieves initial as displaying predicted BRAF and MEK inhibitor sensitivity size-based exclusion of red blood cells and platelets, followed patterns (Fig. 1A; Supplementary Fig. S1C and S1D). The by inertial focusing of all nucleated blood cells into a single melanoma CTC lines were tested for tumorigenesis potential streamline, through which antibody-tagged leukocytes are in immunosuppressed NOD/SCID gamma (NSG) mice. All magnetically separated from unlabeled CTCs. Across multi- five cultured lines are highly tumorigenic following sub- ple tumor types, this platform achieves 104 enrichment for cutaneous inoculation, and four of them (PEM-78 being CTCs, and we have applied a digital RNA-based readout to the exception) generated metastases upon direct intravascu- allow highly sensitive measurement of melanoma tumor cell lar injection, either by tail-vein or intracardiac inoculation burden in the blood (24). The enrichment of untagged and (Fig. 1B and C; Supplementary Fig. S1E–S1J). Both pri- unmanipulated CTCs not only enhances their RNA quality mary tumors and metastases generated from GFP-luciferase– for such diagnostic purposes, but also helps to preserve their tagged CTCs show preservation of characteristic melanosome cell viability for functional studies. markers (Fig. 1C; Supplementary Fig. S1F, S1H, and S1K). We We have previously reported the establishment of long- selected two of these CTC lines, Mel-167 and Mel-182-2, for term CTC cultures from blood specimens of women with detailed functional studies. hormone receptor–positive breast cancer, which are often Having established robust tumorigenic and metastatic oligoclonal in origin and which recapitulate somatic muta- competent CTC cultures, we compared their transcriptomic tions acquired during the course of therapy, and their epige- profiles with those of standard melanoma tumor–derived cell netic cell states