Disruption of IFN-I Signaling Promotes HER2/Neu Tumor

Published OnlineFirst April 5, 2018; DOI: 10.1158/2326-6066.CIR-17-0675

Research Article Cancer Immunology Research Disruption of IFN-I Signaling Promotes HER2/Neu Tumor Progression and Breast Cancer Stem Cells Luciano Castiello1, Paola Sestili1, Giovanna Schiavoni1, Rosanna Dattilo2, Domenica M. Monque1, Fiorella Ciaffoni3, Manuela Iezzi4, Alessia Lamolinara4, Antonella Sistigu2,5, Federica Moschella1, Anna Maria Pacca1, Daniele Macchia1, Maria Ferrantini1, Ann Zeuner1, Mauro Biffoni1, Enrico Proietti1, Filippo Belardelli1, and Eleonora Arico1

Abstract

Type I interferon (IFN-I) is a class of antiviral immunomod- zation. IFNAR-neuT tumors exhibited deregulation of genes ulatory cytokines involved in many stages of tumor initiation having adverse prognostic value in breast cancer patients, includ- and progression. IFN-I acts directly on tumor cells to inhibit ing the breast cancer stem cell (BCSC) marker aldehyde dehy- cell growth and indirectly by activating immune cells to mount drogenase-1A1 (ALDH1A1). An increased number of BCSCs antitumor responses. To understand the role of endogenous were observed in IFNAR-neuT tumors, as assessed by ALDH1A1 IFN-I in spontaneous, oncogene-driven carcinogenesis, we enzymatic activity, clonogenic assay, and tumorigenic capacity. þ characterized tumors arising in HER2/neu transgenic (neuT) In vitro exposure of neuT mammospheres and cell lines to þ mice carrying a nonfunctional mutation in the IFNI receptor antibodies to IFN-I resulted in increased frequency of ALDH (IFNAR1). Such mice are unresponsive to this family of cyto- cells, suggesting that IFN-I controls stemness in tumor cells. þ kines. Compared with parental neu / mice (neuT mice), Altogether, these results reveal a role of IFN-I in neuT-driven þ IFNAR1 / neu / mice (IFNAR-neuT mice) showed earlier spontaneous carcinogenesis through intrinsic control of BCSCs. onset and increased tumor multiplicity with marked vasculari- Cancer Immunol Res; 6(6); 658–70. 2018 AACR.

Introduction in enhanced transplantable tumor growth, suggesting that endogenous IFN-I controls tumor development and progres- Type I interferon (IFN-I) are pleiotropic cytokines sion in immunocompetent mice (3). Endogenous IFN-I that exert multiple biological effects in viral infections and functions in cancer immune surveillance (4) and in the innate cancer (1, 2). Injection of IFN-I–blocking antibody resulted recognition of tumors by several immune cells (5). Studies in mouse models show that IFN-I inhibits growth of transplantable tumors and exerts biological effects on immune 1Department of Oncology and Molecular Medicine, Istituto Superiore di Sanita, 2 cells, including NK cells, dendritic cells (DCs), T, and B cells (2). Rome, Italy. Unit of Tumor Immunology and Immunotherapy, Department of Thus IFN-I links innate immunity and development of the anti- Research, Advanced Diagnostics and Technological Innovation Regina Elena National Cancer Institute, Rome, Italy. 3Department of Cell Biology and tumor immune response (5). Neurosciences, Istituto Superiore di Sanita, Rome, Italy. 4Department of Med- HER2 is an oncogene overexpressed in 15% to 30% breast þ icine and Aging Science, Center of Excellence on Aging and Translational cancers (6). HER2 breast cancers are characterized by a Medicine (CeSi-Met), G. D'Annunzio University, Chieti-Pescara, Italy. 5Depart- molecular signature distinguishing these cancers from other ment of General Pathology and Physiopathology, Universita Cattolica del Sacro breast cancer types (7). HER2 overexpression is a negative Cuore, Rome, Italy. prognostic factor, associated with poorly differentiated, high- Note: Supplementary data for this article are available at Cancer Immunology grade tumors, shorter disease-free and overall survival (6). Research Online (http://cancerimmunolres.aacrjournals.org/). Mice engineered to express an activated rat neu gene (Erbb2, L. Castiello and P. Sestili contributed equally to this article. ortholog of HER2) mimic most of the features observed in þ fi Current affiliation for L. Castiello: Istituto Pasteur Italia—Fondazione Cenci human HER2 breast cancers (8, 9). In fact, during the rst Bolognetti, Rome, Italy; current affiliation for F. Belardelli: Institute of Transla- postnatal weeks, foci of atypical hyperplasia can be observed tional Pharmacology, CNR, Rome, Italy; and current affiliation for E. Arico: in mammary glands of neu transgenicmice.Gradually,these FaBioCell, Core Facilities, Istituto Superiore di Sanita, Rome, Italy. foci coalesce to form large carcinomas in situ,whichprogress Corresponding Authors: Filippo Belardelli, Istituto Superiore di Sanita, to invasive carcinomas (10). Viale Regina Elena, 288, Rome 00161, Italy. Phone: þ390649902414; Fax: Despite the extensive characterization of IFN signaling in þ3949902140; E-mail: fi[email protected]; and Eleonora Arico, FaBio- tumor biology and studies on the mechanisms behind neu- Cell, Core Facilities, Istituto Superiore di Sanita, Viale Regina Elena 299, driven tumorigenesis, little is known about endogenous IFN-I 00161, Rome, Italy. Phone: þ390649902414; E-mail: [email protected] signaling in neu transformation and progression and in spon- doi: 10.1158/2326-6066.CIR-17-0675 taneous carcinogenesis. Here, we studied neu-driven tumori- 2018 American Association for Cancer Research. genesis in mice unresponsive to IFN-I by generating a mouse

658 Cancer Immunol Res; 6(6) June 2018

IFN-I Signaling Regulates HER2 Carcinogenesis and Stemness

strain transgenic for HER2/neu and lacking the IFN-I receptor 30 minutes. After incubation, 5 mL RPMI 10% FCS EDTA IFNAR1 (IFNAR-neuT mice). 2 mmol/L was added, and cells were then filtered in cell strainers and washed twice in PBS. For mammosphere culture, cells recovered from transgenic Materials and Methods mice (13) tumors were cultured at clonal density in serum-free Mice medium supplemented with 20 ng/mL epidermal growth factor NeuT and IFNAR-neuT transgenic mice were generated on (EGF) and 10 ng/mL basic fibroblast growth factor (bFGF). 129sv background and bred in the animal facility of the Depart- Ultralow attachment cell culture flasks were used. The medium ment of Oncology and Molecular Medicine at the Istituto was replaced or supplemented with fresh growth factors once a Superiore di Sanita (Rome, Italy). Pure neuT and IFNAR-neuT week until cells started forming floating aggregates. Cultures were mice were obtained by at least 12 backcrosses of BALB-neuT expanded by mechanical dissociation of spheres, followed by transgenic males (kindly provided by Dr. Guido Forni, Univer- replating of both single cells and residual small aggregates in sity of Torino, Torino, Italy) with 129Sv IFNAR1 / female mice complete fresh medium. (A129, originally purchased from B&K Universal Ltd, now þ þ Marshall BioResources; ref. 11) or the IFNAR1 / counterpart Immunophenotypic analysis (Charles River Laboratories). The BALB-neuT strain originated Immunophenotypic analysis of cells isolated from spleens, from a transgenic CD1 random-bred breeder male mouse lymph nodes, bone marrow, blood, and mammary carcinomas (no. 1330) carrying the mutated rat HER2/neu oncogene driven of mice was carried out as follows: cell suspensions were treated by the MMTV promoter (9). Mice were maintained under strict inbreeding conditions. The presence of the rat HER2 transgene with lysis buffer (140 mmol/L NH4Cl, 17 mmol/L Tris HCl, pH 7.2) to eliminate red blood cells and then stained with the was routinely checked by polymerase chain reaction (PCR) on fl tail DNA using primers hybridizing to vector (5-ATCGGT- following uorescently labeled mAbs: anti-CD45 (30-F11), anti- CD11c (N418), anti-CD3 (145-2C11), anti-CD8a (53-6.7), anti- GATG