American Association for Cancer Research
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Supplementary Information_clean version from Activating Transcription Factor 4 Modulates TGFβ-Induced Aggressiveness in Triple-Negative Breast Cancer via SMAD2/3/4 and mTORC2 Signaling

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journal contribution
posted on 2023-03-31, 20:51 authored by Adrián González-González, Esperanza Muñoz-Muela, Juan A. Marchal, Francisca E. Cara, Maria P. Molina, Marina Cruz-Lozano, Gema Jiménez, Akanksha Verma, Alberto Ramírez, Wei Qian, Wen Chen, Anthony J. Kozielski, Olivier Elemento, María D. Martín-Salvago, Rafael J. Luque, Carmen Rosa-Garrido, David Landeira, María Quintana-Romero, Roberto R. Rosato, Maria A. García, Cesar L. Ramirez-Tortosa, Hanna Kim, Cristian Rodriguez-Aguayo, Gabriel Lopez-Berestein, Anil K. Sood, Jose A. Lorente, Pedro Sánchez-Rovira, Jenny C. Chang, Sergio Granados-Principal

Non-tracked version of the supplementary information document that includes supplementary materials and methods, supplementary references and supplementary figure legends


Institute of Health Carlos III

Ministry of Economy and Competitiveness

Ministry of Health, Andalusian Regional Government

Andalusian Regional Government and University of Granada



Purpose: On the basis of the identified stress-independent cellular functions of activating transcription factor 4 (ATF4), we reported enhanced ATF4 levels in MCF10A cells treated with TGFβ1. ATF4 is overexpressed in patients with triple-negative breast cancer (TNBC), but its impact on patient survival and the underlying mechanisms remain unknown. We aimed to determine ATF4 effects on patients with breast cancer survival and TNBC aggressiveness, and the relationships between TGFβ and ATF4. Defining the signaling pathways may help us identify a cell signaling–tailored gene signature.Experimental Design: Patient survival data were determined by Kaplan–Meier analysis. Relationship between TGFβ and ATF4, their effects on aggressiveness (tumor proliferation, metastasis, and stemness), and the underlying pathways were analyzed in three TNBC cell lines and in vivo using patient-derived xenografts (PDX).Results: ATF4 overexpression correlated with TNBC patient survival decrease and a SMAD-dependent crosstalk between ATF4 and TGFβ was identified. ATF4 expression inhibition reduced migration, invasiveness, mammosphere-forming efficiency, proliferation, epithelial–mesenchymal transition, and antiapoptotic and stemness marker levels. In PDX models, ATF4 silencing decreased metastases, tumor growth, and relapse after chemotherapy. ATF4 was shown to be active downstream of SMAD2/3/4 and mTORC2, regulating TGFβ/SMAD and mTOR/RAC1–RHOA pathways independently of stress. We defined an eight-gene signature with prognostic potential, altered in 45% of 2,509 patients with breast cancer.Conclusions: ATF4 may represent a valuable prognostic biomarker and therapeutic target in patients with TNBC, and we identified a cell signaling pathway–based gene signature that may contribute to the development of combinatorial targeted therapies for breast cancer. Clin Cancer Res; 24(22); 5697–709. ©2018 AACR.