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Open Full Page Published OnlineFirst November 2, 2012; DOI: 10.1158/2159-8290.CD-12-0031 RESEARCH ARTICLE Targeting C4-Demethylating Genes in the Cholesterol Pathway Sensitizes Cancer Cells to EGF Receptor Inhibitors via Increased EGF Receptor Degradation Anna Sukhanova 1 , Andrey Gorin 1 , Ilya G. Serebriiskii 1 , Linara Gabitova 1 , Hui Zheng 1 , Diana Restifo 1 , Brian L. Egleston 2 , David Cunningham 3 , Tetyana Bagnyukova 1 , Hanqing Liu 1 , Anna Nikonova 1 , Gregory P. Adams 1 , Yan Zhou 2 , Dong-Hua Yang 1 , Ranee Mehra 1 , Barbara Burtness 1 , Kathy Q. Cai 1 , Andres Klein-Szanto 1 , Lisa E. Kratz 4 , Richard I. Kelley 4 , Louis M. Weiner 5 , Gail E. Herman 3 , Erica A. Golemis 1 , and Igor Astsaturov 1 Downloaded from cancerdiscovery.aacrjournals.org on September 24, 2021. © 2013 American Association for Cancer Research. Published OnlineFirst November 2, 2012; DOI: 10.1158/2159-8290.CD-12-0031 ABSTRACT Persistent signaling by the oncogenic EGF receptor (EGFR) is a major source of cancer resistance to EGFR targeting. We established that inactivation of 2 sterol biosynthesis pathway genes, SC4MOL (sterol C4-methyl oxidase–like) and its partner, NSDHL (NADP-dependent steroid dehydrogenase–like), sensitized tumor cells to EGFR inhibitors. Bioinfor- matics modeling of interactions for the sterol pathway genes in eukaryotes allowed us to hypothesize and then extensively validate an unexpected role for SC4MOL and NSDHL in controlling the signaling, vesicular traffi cking, and degradation of EGFR and its dimerization partners, ERBB2 and ERBB3. Meta- bolic block upstream of SC4MOL with ketoconazole or CYP51A1 siRNA rescued cancer cell viability and EGFR degradation. Inactivation of SC4MOL markedly sensitized A431 xenografts to cetuximab, a therapeutic anti-EGFR antibody. Analysis of Nsdhl -defi cient Bpa 1H / + mice confi rmed dramatic and selective loss of internalized platelet-derived growth factor receptor in fi broblasts, and reduced acti- vation of EGFR and its effectors in regions of skin lacking NSDHL. SIGNIFICANCE: This work identifi es a critical role for SC4MOL and NSDHL in the regulation of EGFR signaling and endocytic traffi cking and suggests novel strategies to increase the potency of EGFR antagonists in tumors. Cancer Discov; 3(1); 96–111. ©2012 AACR. INTRODUCTION sensitizes refractory tumor cells to EGFR inhibitors. The EGFR-focused library was designed on the basis of the systems The EGF receptor (EGFR) provides essential growth and biology prediction that genes engaged in synthetic lethal rela- prosurvival signals to epithelial cells and is often targeted ther- tionships involve those closely linked in signaling processes apeutically in epithelial malignancies. Unfortunately, EGFR- ( 8, 9 ). Among the validated hits that increased cell killing by antagonizing treatment strategies are often limited in their common EGFR antagonists, SC4MOL was consistently one effi cacy due to acquired or primary resistance in tumors. Recep- of the most effective modulators. SC4MOL, an intermediate tor switching or compensatory activation of EGFR coreceptors enzyme in the cholesterol biosynthetic pathway, was included such as the receptor tyrosine kinases (RTK) ERBB2 ( 1 ), ERBB3 in the library based on its representation in a high-confi dence (2 ), and insulin-like growth factor I receptor ( 3 ) are examples of Gene Expression Omnibus (GEO) transcriptional profi ling acquired resistance, whereas preexisting somatic mutations acti- dataset (GSE6521) as a transcript that rapidly undergoes sig- vating downstream effectors of the EGFR pathway, such as RAS nifi cant expression change in response to stimulation or inhi- and BRAF, commonly confer primary resistance to EGFR antag- bition of EGFR. Active sterol biosynthesis remains an essential onists in pancreatic or colon cancers ( 4 ). Persistent EGFR signal- metabolic component of cancer, and changes in the function ing is a basis for resistance to EGFR antagonists, as observed in of this pathway are thought to contribute to resistance to some PTEN-defi cient tumors ( 5 ), or when EGFR internalization and forms of cancer treatment ( 10 ). However, to date, no direct degradation are reduced ( 6 ). In many cases, the source of thera- functional connections between SC4MOL or other sterol path- peutic resistance remains unknown, limiting clinical efforts to way proteins and EGFR signaling have been reported. select patients for EGFR-targeting therapeutics or to generally In this study, we fi rst systematically explore the proteins enhance the potency of such therapeutics in all patients. of the distal cholesterol biosynthesis pathway to establish In a network-guided siRNA-based screen ( 7 ), we recently that depletion or loss of SC4MOL and its partner, NSDHL identifi ed SC4MOL (testis meiosis-activating sterol/sterol C4-methyl (NADP-dependent steroid dehydrogenase–like), specifi cally oxidase–like) among genes whose silencing signifi cantly synergize with common inhibitors of EGFR. We then model from the interaction networks of the evolutionarily con- Authors’ Affi liations: 1 Program in Developmental Therapeutics and served orthologs of these proteins in yeast to determine 2 Biostatistics and Bioinformatics Facility, Fox Chase Cancer Center, Phila- that SC4MOL and NSDHL directly regulate intracellular delphia, Pennsylvania; 3 The Research Institute at Nationwide Children’s vesicular traffi cking of EGFR, infl uencing its rate of degrada- Hospital and the Department of Pediatrics, The Ohio State University, tion. These results reveal a previously unidentifi ed means of Columbus, Ohio; 4 Kennedy Krieger Institute, Johns Hopkins University, Baltimore, Maryland; and 5 Lombardi Comprehensive Cancer Center, regulating RTK expression and activity, and suggest a more Georgetown University Medical Center, Washington, District of Columbia complex action for these cholesterol biosynthetic pathway A. Sukhanova and A. Gorin contributed equally to this work. enzymes than has been formerly appreciated. Note: Supplementary data for this article are available at Cancer Discovery Online (http://cancerdiscovery.aacrjournals.org/). RESULTS Corresponding Author: Igor Astsaturov, Fox Chase Cancer Center, 333 Probing the Sterol Biosynthesis Pathway for Cottman Avenue, Philadelphia, PA 19111. Phone: 215-728-3135; Fax: 215-728-3616; E-mail: [email protected] Regulation of Response to EGFR Inhibitors doi: 10.1158/2159-8290.CD-12-0031 To address the mechanism of SC4MOL regulation of © 2012 American Association for Cancer Research. response to the EGFR inhibitors erlotinib and cetuximab, we JANUARY 2013CANCER DISCOVERY | 97 Downloaded from cancerdiscovery.aacrjournals.org on September 24, 2021. © 2013 American Association for Cancer Research. Published OnlineFirst November 2, 2012; DOI: 10.1158/2159-8290.CD-12-0031 RESEARCH ARTICLE Sukhanova et al. A B % mRNA Remaining C A431 0 50 100 SCC61 Vehicle GL2 Vehicle Erlotinib, 0.5 μmol/L SC4MOL Erlotinib, 5 μmol/L Erlotinib, 2 μmol/L Erlotinib, 10 μmol/L Farnesyl-P 120 NSDHL 120 Lipid modification Geranylgeranyl-P HSD17B7 Ras, GTPases 100 100 AKT, GPCR C14orf Squalene 80 80 Squalene epoxidase * * (SQLE) 60 60 * * (S)-2,3-Epoxysqualene % Viability 40 40 * * Lanosterol synthase (LSS) % Viability * 20 20 * Lanosterol Lanosterol demethylase 0 0 (CYP51A1) Ketoconazole siRNA GL2 SC4 NSDHL HSD C14orf1 siRNA GL2SC4 NSDHL HSD C14orf1 MOL 17B7 MOL 17B7 4,4-Dimethyl-5α-cholesta-8,14,24-trien-3β-ol FF-MAS (follicular) D SCC68 E PC9 Δ14-reductases 120 120 Vehicle Vehicle (LBR, TM7SF2) 100 Erlotinib, 1 μmol/L 100 Erlotinib, 0.25 μmol/L 4,4-Dimethyl-5α-cholesta-8,24-dien-3β-ol Erlotinib, 5 μmol/L Erlotinib, 1 μmol/L 80 80 T-MAS (testicular) 60 60 * SC4MOL/Erg25 * * * * * NSDHL/Erg26 40 * * 40 % Viability HSD17B7/Erg27 % Viability C14Orf1/Erg28 20 20 Zymosterol 0 0 siRNA GL2 SC4MOL NSDHL siRNA GL2 SC4MOL NSDHL Δ7,8-isomerase (EBP) Lathosterol F A431 siRNA: GL2 Δ5-desaturase 120 SC4MOL (SC5DL) NSDHL 100 7-Dehydrodesmosterol * 80 * * * Δ7-reductase * * (DHCR7) 60 * Desmosterol * 40 Δ24-reductase % Viability 20 (DHCR24) Steroid hormones Cholesterol Bile acids 0 /L nib mol/L mol/L loti mol/L mol mol/L r μ nmol/L μ μ μ μ Vehicle U0126 CPT11 E MCP110 1 Dasatinib LY294022 20 200 nmol/L Cetuximab 2 Enzastaurin 10 Rapamycin 12.5 nmol/L 5 μ 5 g/mL 500 Figure 1. Effects of sterol pathway genes silencing on cancer cell drug sensitivity. A, schema of the cholesterol pathway. Relevant enzymes are itali- cized and biologic functions are shown in boxed text. B–F, silencing of SC4MOL and NSDHL increased cytotoxicity of erlotinib. A431 (B), SCC61 (C), PC9 (D), and SCC68 (E) cells were made defi cient in the indicated sterol pathway genes and, 24 hours later, were either mock treated or exposed to erlotinib for 72 hours. F, specifi city of activity of SC4MOL and NSDHL silencing. A431 cells made defi cient in SC4MOL and NSDHL were treated with indicated inhibitors at concentrations producing 20% to 30% decrement in viability. In B–F, viability data from 3 independent experiments were normalized to mock-treated GL2 control–transfected cells. Each column represents averaged results; bars, SDs. *, P < 0.001. fi rst considered whether this effect was general to proteins C4-demethylation step in the pathway. Similar results were operating in the cholesterol biosynthesis pathway ( Fig. 1A ) or obtained in the head and neck squamous carcinoma cell lines more specifi c. SC4MOL is highly conserved throughout evolu- SCC61 ( Fig. 1C ) and SCC68 ( Fig. 1D ) expressing moderate lev- tion, as are many genes operating upstream and downstream els of EGFR (Supplementary Fig. S2A), and in the lung adeno- in the sterol synthesis pathway ( 11 ). Three human catalytic carcinoma cell line PC9 ( Fig. 1E ), which expresses a mutated enzymes, SC4MOL, NSDHL, and HSD17B7, and a gene with form of EGFR, ΔE746-A750 ( 13 ), indicating the fi ndings were unknown function, C14ORF1 , are orthologous to a complex not specifi c to A431 cells. Sensitization was also observed of yeast C4-sterol demethylation genes that defi ne the “ergo- with 2 shRNA constructs targeting SC4MOL (Supplementary some” ( ERG25/SC4MOL , ERG26/NSDHL , ERG27/HSD17B7 , Fig. S1C) and was associated with marked enhancement of and ERG28/C14ORF1 ; ref. 12 ). apoptosis (Supplementary Fig.
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