Author Manuscript Published OnlineFirst on May 13, 2020; DOI: 10.1158/1535-7163.MCT-19-1172 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 1 TITLE PAGE 2 Title: Autocrine CCL5 effect mediates trastuzumab resistance by ERK pathway 3 activation in HER2-positive breast cancer 4 Authors: Sandra Zazo1, Paula González-Alonso1, Ester Martín-Aparicio1, Cristina 5 Chamizo1, Melani Luque1, Marta Sanz-Álvarez1, Pablo Mínguez2, Gonzalo Gómez- 6 López3, Ion Cristóbal4, Cristina Caramés4, Jesús García-Foncillas4, Pilar Eroles5, Ana 7 Lluch5,6, Oriol Arpí7, Ana Rovira7,8, Joan Albanell7,8,9, Juan Madoz-Gúrpide1§, Federico 8 Rojo1§ 9 Affiliations: 1Pathology, Fundación Jiménez Díaz University Hospital Health Research 10 Institute (IIS—FJD, UAM)—CIBERONC, Madrid 28040, Spain; 2Genetics 11 Department, Health Research Institute-Fundación Jiménez Díaz (IIS-FJD, UAM), 12 Center for Biomedical Network Research on Rare Diseases (CIBERER), ISCIII, Madrid 13 28040, Spain; 3Bioinformatics Unit, Spanish National Cancer Research Centre (CNIO), 14 Madrid 28029, Spain; 4Translational Oncology Division, OncoHealth Institute, Health 15 Research Institute-Fundación Jiménez Díaz (IIS-FJD, UAM), Madrid 28040, Spain; 16 5Institute of Health Research INCLIVA-CIBERONC, Valencia 46010, Spain; 17 6Medicine Department, University of Valencia, Valencia 46010, Spain; 7Cancer 18 Research Program, IMIM (Hospital del Mar Research Institute), Barcelona 08003, 19 Spain; 8Medical Oncology Department, Hospital del Mar-CIBERONC, Barcelona 20 08003, Spain; 9Universitat Pompeu Fabra, Barcelona 08002, Spain; §these authors 21 contributed equally to this work. 22 Running title: Trastuzumab resistance by CCL5/ERK axis activation 23 Keywords: breast cancer, HER2-positive, anti-receptor therapy, trastuzumab, 24 resistance, cytokines, CCL5, cell lines 1 Downloaded from mct.aacrjournals.org on October 2, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on May 13, 2020; DOI: 10.1158/1535-7163.MCT-19-1172 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 1 Financial support: The present work was supported by grants from the Spanish 2 Ministry of Health, Consumer Affairs and Social Welfare (AES Program, grants 3 PI15/00934; PI18/00382 and PI18/00006); the Biomedical Research Networking Centre 4 for Cancer (CIBERONC); the Biobanks Platform, PT13/0010/0012; the Community of 5 Madrid (S2010/BMD-2344); and ProteoRed (PRB2-ISCIII, PT13/0001). PGA was 6 supported by a Fundación Conchita Rábago de Jiménez Díaz grant. PM was supported 7 by the ISCIII Miguel Servet Program (CP16/00116). 8 Corresponding authors: §Dr. Juan Madoz-Gúrpide, Ph.D., Pathology Department, IIS- 9 Fundación Jiménez Díaz, UAM, Avda. Reyes Católicos 2, E-28040 Madrid, Spain. E- 10 mail: [email protected]. Phone: +34-915504800. 11 §Dr. Federico Rojo, M.D. Ph.D., Pathology Department, University Hospital Fundación 12 Jiménez Díaz, Avda. Reyes Católicos 2, E-28040 Madrid, Spain. E-mail: [email protected]. 13 Phone: +34-915504800. 14 Conflict of interest: AL has a consulting or advisory role in Novartis, Pfizer, 15 Roche/Genentech, Eisai, Celgene (recipient herself) and research funding from Roche 16 Pharma AG, AstraZeneca, Merck, PharmaMar, Boehringer Ingelheim, Amgen, 17 GlaxoSmithKline, Novartis, Pfizer, Eisai, Celgene, Pierre Fabre. The rest of the authors 18 declare no competing financial interests. 2 Downloaded from mct.aacrjournals.org on October 2, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on May 13, 2020; DOI: 10.1158/1535-7163.MCT-19-1172 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 1 ABSTRACT 2 HER2-positive breast cancer is currently managed with chemotherapy in combination 3 with specific anti-HER2 therapies, including trastuzumab. However, a high percentage 4 of patients with HER2-positive tumors do not respond to trastuzumab (primary 5 resistance) or either recur (acquired resistance), mostly due to molecular alterations in 6 the tumor that are either unknown or undetermined in clinical practice. Those alterations 7 may cause the tumor to be refractory to treatment with trastuzumab, promoting tumor 8 proliferation and metastasis. 9 Using continued exposure of a HER2-positive cell line to trastuzumab we 10 generated a model of acquired resistance characterized by increased expression of 11 several cytokines. Differential gene expression analysis indicated an overexpression of 12 15 genes, including 5 different chemokines, and highlighting CCL5/RANTES as the 13 most overexpressed one. Functional studies, either by in vitro gene silencing or by in 14 vitro and in vivo pharmacological inhibition of the CCL5/CCR5 interaction with 15 maraviroc, confirmed that CCL5 overexpression was implicated in acquired resistance 16 to trastuzumab, which was mediated by ERK activation. In patient samples, increased 17 CCL5 expression significantly correlated with lower rates of complete response after 18 neoadjuvant therapy, confirmed by detection of high serum CCL5 levels by ELISA. 19 Overexpression of CCL5 correlated with ERK phosphorylation in tumor cells and was 20 statistically associated with worse disease-free survival and overall cancer survival in 21 patients with early HER2-positive breast cancer. 3 Downloaded from mct.aacrjournals.org on October 2, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on May 13, 2020; DOI: 10.1158/1535-7163.MCT-19-1172 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 1 INTRODUCTION 2 Breast cancer accounts for 20-25% of all cancer cases worldwide and is the most 3 prevalent in women(1). Breast cancer comprises many biologically different diseases 4 with distinct pathological features and clinical implications, thus making accurate 5 grouping of clinically relevant subtypes of importance(2). Among these subtypes, 6 HER2-positive breast cancer, which accounts for 20% of all breast cancers(3), is 7 characterized by gene overexpression, high rates of cell proliferation and metastasis, 8 poor prognosis, low overall survival (OS), and variable chemotherapy response with 9 poor outcome(4). Together with this prognostic value, the HER2 receptor is currently 10 considered part of the standard assessment protocol as a predictor of response to 11 treatment(5). 12 During the past decade, systemic therapeutic management of breast cancer has 13 undergone a significant transformation, leading to the emergence of targeted therapy. 14 For HER2-positive breast cancer patients, targeting HER2 has become an attractive 15 therapeutic approach. Trastuzumab (Herceptin), a humanized IgG1 monoclonal 16 antibody that selectively targets the HER2 receptor, became the first FDA-approved 17 targeted therapy for metastatic breast cancer in 1998(6). Since then, therapies such as 18 trastuzumab combined with chemotherapy have been considered the standard of care for 19 HER2-positive breast cancer patients(7). However, about 25% of HER2-positive breast 20 cancers do not respond initially to trastuzumab(8), and 70% of the trastuzumab- 21 responsive metastatic cancers progress to therapy within the first year due to acquisition 22 of trastuzumab resistance(9). Several potential resistance mechanisms to trastuzumab 23 have been reported during the last decade and their details have been described in 24 numerous reviews(10-12). 4 Downloaded from mct.aacrjournals.org on October 2, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on May 13, 2020; DOI: 10.1158/1535-7163.MCT-19-1172 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 1 Nowadays, it is widely accepted that many pathways may be involved in the 2 development of resistance to antibody-based treatments. Specifically, there is a 3 widespread belief that a tumor must be examined in the context of its 4 microenvironment, and therefore should be considered as an entity with a heterogeneous 5 cellular origin in continuous interaction with the stroma, non-tumor cells, and the 6 immune system(13), highly modulated by the inflammatory cells found in the 7 tumor(14). This interaction of the tumor with the microenvironment is mainly regulated 8 through cytokines, which signal the participation of distinct pathways in processes of 9 cell proliferation and differentiation(15). 10 Many chemokines and their receptors are expressed by tumor cells. The 11 chemokine CCL5/ RANTES, is well-recognized for its activities in the immune context, 12 where it induces leukocyte-directed motility. CCL5 has affinity for the G protein- 13 coupled receptors (GPCR) CCR1, CCR3, and, especially, CCR5; and a lesser binding 14 capacity with other receptors. Recently, CCL5/CCR5 have been implicated in 15 proliferation and metastasis in breast cancer(16, 17) and have been recognized as 16 potential therapeutic targets. Moreover, a recent study showed that CCL5 signaling 17 promotes breast cancer recurrence following HER2 inhibition through the recruitment 18 of macrophages(18). In the context of HIV/AIDS studies, potent antagonist inhibitors 19 have developed, which prevent binding of ligands to the receptors; the only CCR5 20 antagonist currently approved by the FDA and the EMA for the treatment of infected 21 patients is maraviroc (Pfizer, USA)(19, 20). The