Published OnlineFirst January 9, 2015; DOI: 10.1158/0008-5472.CAN-14-1851 Cancer Molecular and Cellular Pathobiology Research Interferon Regulatory Factor-1 Signaling Regulates the Switch between Autophagy and Apoptosis to Determine Breast Cancer Cell Fate Jessica L. Schwartz-Roberts1,2, Katherine L. Cook1,2, Chun Chen3,4, Ayesha N. Shajahan-Haq1,2, Margaret Axelrod1,2, Anni Warri€ 1,2, Rebecca B. Riggins1,2, Lu Jin1,2, Bassem R. Haddad1,2, Bhaskar V. Kallakury5, William T. Baumann3,4, and Robert Clarke1,2 Abstract Interferon regulatory factor-1 (IRF1) is a tumor suppressor that IGF1 receptor and mTOR survival signaling. Loss of IRF1 pro- regulates cell fate in several cell types. Here, we report an inverse moted resistance to antiestrogens, whereas combined silencing correlation in expression of nuclear IRF1 and the autophagy of ATG7 and IRF1 restored sensitivity to these agents. Using a regulator ATG7 in human breast cancer cells that directly affects mathematical model to prompt signaling hypotheses, we devel- their cell fate. In mice harboring mutant Atg7, nuclear IRF1 was oped evidence that ATG7 silencing could resensitize IRF1- increased in mammary tumors, spleen, and kidney. Mechanistic attenuated cells to apoptosis through mechanisms that involve investigations identified ATG7 and the cell death modulator other estrogen-regulated genes. Overall, our work shows how beclin-1 (BECN1) as negative regulators of IRF1. Silencing ATG7 inhibiting the autophagy proteins ATG7 and BECN1 can reg- or BECN1 caused estrogen receptor-a to exit the nucleus at the ulate IRF1-dependent and -independent signaling pathways in time when IRF1 nuclear localization occurred. Conversely, silenc- ways that engender a new therapeutic strategy to attack breast ing IRF1 promoted autophagy by increasing BECN1 and blunting cancer. Cancer Res; 75(6); 1–10. Ó2015 AACR. Introduction within breast cancer cells regulates the cell-fate decision in response to endocrine therapies remains largely unknown. Breast cancer remains one of the most prevalent cancers among Interferon regulatory factor-1 (IRF1) is a nuclear transcrip- American women (1). With more than 70% of these breast cancers þ tion factor that is activated by many immune effector mole- expressing the estrogen receptor-a (ER ), many tumors depend cules, including type I and type II interferons (IFNg), TNFa, on estrogen to drive cell proliferation and promote cell survival. retinoicacid,IL1,IL6,andinresponsetoviralinfection(5). The routine use of antiestrogens, such as tamoxifen and fulves- Upon activation, IRF1 alters the transcriptional activity of trant (Faslodex; ICI 182,780; ICI), and aromatase inhibitors, genes involved in immunomodulation, antiviral responses, including letrozole and anastrozole, are now established as effec- and tumor suppression (5, 6). Previous studies have estab- tive therapies for the treatment of breast cancer (2, 3). The lished a role for IRF1 in inhibiting breast cancer cell growth, widespread use of endocrine therapy is a major contributor including by opposing the actions of IRF2 and inducing apo- leading to the decline in breast cancer–related mortality in women þ ptosis (7, 8). Furthermore, gene-expression microarrays com- with ER disease (4). However, clinical efficacy of these therapies paring antiestrogen-responsive (MCF7/LCC1) and -resistant is often limited by aberrations in prosurvival and prodeath (MCF7/LCC9) human breast cancer cell lines report that IRF1 signaling leading to drug resistance. Precisely, how signaling is downregulated in resistant cells, suggesting that loss of IRF1 may contribute to endocrine resistance through its reduced ability to induce cell death (9–11). Indeed, IRF1 mRNA levels 1Department of Physiology and Biophysics, Lombardi Comprehensive Cancer Center, Georgetown University,Washington, DC. 2Department correlate with a pathologic complete response and reduced risk of Oncology, Lombardi Comprehensive Cancer Center, Georgetown of recurrence and death in some breast cancers (12, 13). Recent 3 University, Washington, DC. Department of Biological Sciences, Vir- evidence suggests that autophagy proteins have a major effect ginia Polytechnic Institute and State University, Blacksburg, Virginia. fl 4Department of Electrical and Computer Engineering, Virginia on the regulation of in ammatory transcriptional responses. Polytechnic Institute and State University, Blacksburg, Virginia. Embryonic mice lacking the autophagy-related 5 (Atg5)gene 5Department of Pathology, Georgetown University Medical Center, have increased inflammation in tissue with impaired clearance Washington, DC. of apoptotic cells, raising the possibility that autophagy has a Note: Supplementary data for this article are available at Cancer Research role in inflammation and autoimmunity (14). Online (http://cancerres.aacrjournals.org/). A critical decision point in the determination of cell fate in Corresponding Author: Robert Clarke, Georgetown University Medical Center— response to endocrine therapies appears to be affected by the Lombardi Comprehensive Cancer Center, Research Building W405A, 3970 balance between autophagy (prosurvival) and apoptosis (pro- Reservoir Road NW, Washington, DC 20853. Phone: 202-687-3755; Fax: 202- death; ref. 15). The activities of IRF1 in multiple cell types suggest 687-7505; E-mail: [email protected] that it may be a key component in regulating cell-fate decisions doi: 10.1158/0008-5472.CAN-14-1851 and perhaps a critical regulator of the switch between apoptosis Ó2015 American Association for Cancer Research. and autophagy. Thus, we investigated the biologic function of www.aacrjournals.org OF1 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2015 American Association for Cancer Research. Published OnlineFirst January 9, 2015; DOI: 10.1158/0008-5472.CAN-14-1851 Schwartz-Roberts et al. IRF1 in autophagy in the context of endocrine responsiveness in Cells were harvested 48 hours later and stained as described in the breast cancer. We established an important (inverse) link between Mitochondrial Permeability Detection Kit for flow cytometry ATG7 and IRF1 expression/function, which we confirmed using (Enzo). Accumulation of autophagic vesicles was measured using immunohistochemical studies in human breast carcinomas and a modified monodansylcadaverine according to the manufac- þ À tissue from Atg7 / mice. We also showed that inhibition of turer's instructions (Enzo Cyto-ID Autophagy detection kit). Total the autophagy proteins ATG7 and beclin-1 (BECN1) promoted reactive oxygen species (ROS) were stained according to the IRF1 signaling and induced apoptotic cell death. Conversely, Enzo's Total ROS Detection Kit instructions. Stained cells were inhibition of IRF1 prolonged cell survival and promoted autop- detected and appropriate signals measured by FACS (LCCC FACS hagy. Thus, our data suggest that IRF1 inhibits breast cancer cell Shared Resource). growth through its ability to regulate both autophagy and apo- ptosis. To better understand the molecular interactions, we con- Autophagosome maturation and localization studies structed an influence diagram of the novel signaling and then used Cells (1 Â 105) were reverse transfected with IRF1, ATG7, this as a guide to build mathematical models using our experi- BECN1, or Ctrl siRNA and seeded onto 18 Â 18-mm glass cover- mental data. Model simulations suggested additional experimen- slips. The following day, IRF1 siRNA cells were transfected with tation to distinguish whether ATG7 knockout promotes endo- LC3 tagged with a GFP. Twenty-four hours later, cells were treated crine responsiveness through either IRF1-dependent or -indepen- with 500 nmol/L ICI or vehicle, and then fixed and stained for dent signaling. Collectively, these data indicate a major role for IRF1 as previously described (11). IRF1 and ERa were also IRF1 in regulating cell-fate decisions in breast cancer and also measured in LCC1 cells following knockdown of ATG7, BECN1, establish an IRF1-independent path as a further signaling com- or IRF1 to determine subcellular localization. ponent in ATG7-mediated cell death. Mathematical modeling and data fitting Materials and Methods Matlab (version 7.9.0) was used to build the mathematical Cell culture, reagents, and siRNA treatments model and perform simulations. An ordinary differential equa- MCF7, T47D, BT-474, and MDA-MB-231 cells were maintained tion formalism (18–20) was used to model the outcomes in improved minimal essential media (IMEM) with phenol red observed in experiments. Experimental data on cell proliferation and supplemented with 5% FBS (Life Technologies). MCF7/LCC1 under different conditions were used to fit the model by a least- (LCC1) and MCF7/LCC9 (LCC9) cells were grown in phenol red- squares approach. Further details of the model are described in free IMEM supplemented with 5% charcoal-stripped calf serum. Supplementary Fig. S1. All cells were maintained in a humidified atmosphere at 37C and 95% air/5% CO2. ATG7 (SignalSilence; Cell Signaling Technol- In vivo experiments þ À ogy), BECN1 and STAT1 (three unique siRNAs for each target; Tumors from female Atg7 / and wild-type (WT) mice (21) OriGene), IRF1 (Silencer Select; consisting of three different were treated with medroxyprogesterone acetate (DepoProvera; siRNA for same target; Life Technologies), or control (Ctrl) siRNA Pfizer) and 7,12-dimethylbenz(a)anthracene (DMBA; Sigma) to were transfected in cells using Lipofectamine RNAiMAX (Life induce mammary tumors, as described earlier (22). Mice