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FOXO Transcription Factors Both Suppress and Support Breast Cancer Progression

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FOXO Transcription Factors Both Suppress and Support Breast Cancer Progression Author Manuscript Published OnlineFirst on February 12, 2018; DOI: 10.1158/0008-5472.CAN-17-2511 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. FOXO transcription factors both suppress and support breast cancer progression. Marten Hornsveld1,2,$, Lydia M.M. Smits1,2, Maaike Meerlo1,2, Miranda van Amersfoort3, Marian J.A. Groot Koerkamp4, Dik van Leenen2, David E.A. Kloet2, Frank C.P. Holstege4, Patrick W.B. Derksen3, Boudewijn M.T. Burgering1,2#, Tobias B. Dansen2# 1Oncode Institute and 2Center for Molecular Medicine, Molecular Cancer Research, University Medical Center Utrecht, Utrecht University, The Netherlands. 3Department of Pathology, University Medical Center Utrecht, Utrecht University, The Netherlands. 4Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands. #Equal contribution. $present address: Leiden University Medical Center, Cell and Chemical Biology department, Leiden, The Netherlands The authors declare that there is no conflict of interest Correspondence: University Medical Center Utrecht Center for molecular medicine, Molecular Cancer Research Universiteitsweg 100 3584CG Utrecht, The Netherlands Tel: +31 887568918 E-mail: TBD: [email protected] & BMTB: [email protected] 1 Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on February 12, 2018; DOI: 10.1158/0008-5472.CAN-17-2511 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Running title: FOXOs both suppress and support breast cancer progression. Abstract FOXO transcription factors are regulators of cellular homeostasis and putative tumor suppressors, yet the role of FOXO in cancer progression remains to be determined. The data on FOXO function, particularly for epithelial cancers, are fragmentary and come from studies that focused on isolated aspects of cancer. To clarify the role of FOXO in epithelial cancer progression, we characterized the effects of inducible FOXO activation and loss in a mouse model of metastatic invasive lobular carcinoma. Strikingly, either activation or loss of FOXO function suppressed tumor growth and metastasis. We show that the multitude of cellular processes critically affected by FOXO function include proliferation, survival, redox homeostasis, and PI3K-signaling, all of which must be carefully balanced for tumor cells to thrive. Significance FOXO proteins are not solely tumor suppressors but also support tumor growth and metastasis by regulating a multitude of cellular processes essential for tumorigenesis. 2 Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on February 12, 2018; DOI: 10.1158/0008-5472.CAN-17-2511 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Introduction The PI3K-AKT/PKB pathway is commonly hyper-activated in cancer and negatively regulates nuclear localization and thereby the activity of Forkhead Box O family of transcription factors (FOXO1, FOXO3, FOXO4). When active, FOXOs control a plethora of target genes involved in redox homeostasis, cell proliferation, differentiation, metabolism and apoptosis (1). FOXOs are putative tumor suppressors as combined loss of Foxo1, Foxo3 and Foxo4 in adult mice results in increased hematopoietic lineage specific tumors and FOXO activation arrests the cell cycle and induces apoptosis or anoikis in multiple types of tumor cells (2-5). Despite their tumor suppressive roles, mutational loss of all FOXO alleles is statistically unlikely and has not been reported in cancer. Apparently, AKT/PKB mediated inhibition of FOXO is either sufficient to avoid tumor suppression, or the maintenance of a certain level of FOXO activity is beneficial to tumor cells. It remains however to be established what level of activity and under what conditions FOXO activity then would be required. It has been suggested that FOXOs may also contribute to tumorigenesis, and a more active mutant form of FOXO1 has been reported in cancer (3,5-10) Additionally, FOXOs have been implicated in a drug resistance signaling feedback loop that re-establishes RTK-PI3K-AKT signaling after pharmacological inhibition of AKT/PKB (11-13). In line with these contrasting roles for FOXOs in cancer, studies aimed to use FOXO expression levels and localization as prognostic markers for cancer patient survival yielded diametrically opposing results (14). The current understanding of the role of FOXOs in cancer is clearly incomplete, scattered over many different studies, in various types of cancer and limited to 3 Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on February 12, 2018; DOI: 10.1158/0008-5472.CAN-17-2511 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. only the effects of FOXO loss or gain of function in isolated aspects of cancer (3). Studies designed to characterize the effects of both acute FOXO gain and loss of function within one cancer model covering the complete disease spectrum from tumor initiation to metastasis are lacking. To clarify the role of FOXOs in cancer we characterized inducible FOXO activation in a mouse model for metastatic invasive lobular carcinoma (mILC) that allows live monitoring of both primary tumor growth and metastasis (15,16). Our study reveals that FOXOs both support and suppress metastatic breast cancer progression and hence are not classical tumor suppressors. FOXO activity is actually required for efficient proliferation and dissemination, which suggests that FOXO inhibitors might be of use as cancer therapeutics. Materials and Methods Cell culture The Luciferase expressing Mouse invasive lobular carcinoma cell line (mILC1) has been described before and was originally derived from tumors that developed in female K14cre;Cdh1F/F;Trp53F/F mice and cultured as described before (15). The identity of these cells was regularly checked by Luciferase expression and PCR for CdhD/F and p53 and these cells behaved as expected in the transplantation studies. mILC1 cells used for the transplantation assays were in culture for two weeks prior to surgery. Cells virally transduced to express iFOXO3, iFOXO3.A3 or shFOXOs were cultured and selected for three weeks prior to freezing. MCF7, MDA-MB231 and SKBR3 cell lines were acquired from the ATCC and verified by STR analysis before freezing (2013-2014). Cells were passaged two times a week 4 Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on February 12, 2018; DOI: 10.1158/0008-5472.CAN-17-2511 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. for about two months after which fresh vials were thawed. All cells used tested negative for mycoplasma (tests were performed routinely every ~3 months). Cells were treated with 100nM Doxycline (Dox). 200µM Trolox, 20µM EUK-134 or 1mM N-acetyl cysteine (NAC) was used for daily antioxidant treatment. 3D spheroid cultures were established in 20 µL drops containing 40% matrigel, and 1,5mg/ml rat tail collagen I (Ibidi). For colony formation assays 2000 cells/well were plated, subsequently fixed with methanol and stained with 0.5% crystal violet. Fresh tumor tissue digestion into single cells was performed with collagenase and dispase. 50µg/mL neomycin and 100µg/mL primocin (Invivogen) were used to dispense of stromal cells and avoid infection. NADP+/NADPh ratio was determined using Amplite™ Fluorimetric NADP/NADPh Ratio Assay Kit (AAT Bioquest). Constructs, lentiviral transduction and transfections Third generation packaging vectors and hEK293T cells were used to generate lentiviral particles (17). Lentiviral cDNA expression vectors expressing FOXO3 and FOXO3.A3 were generated using Gateway cloning in the pINDUCER20 and selected with Neomycin (Addgene #44012)(18). Foxo1 and Foxo3 knockdown done using the lentiviral FH1tUTG Dox inducible vector (19,20). The Lentiviral constructs pLV-CMV-PIK3CA-IRES-Puromycin, pLV-CMV-PIK3CAH1047R-IRES- Puromycin, pCDh-myr-HA-PKB-puromycin (Addgene #46969) pLV-CMV- RICTOR-IRES-Hygromicin and pINDUCER20-GFP-FOXO3mt were used to express PI3K, PI3KH1047R, myrPKB, RICTOR and FOXO3mt respectively. 5 Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on February 12, 2018; DOI: 10.1158/0008-5472.CAN-17-2511 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Immunoblotting, immunofluorescence & antibodies For Western blot cells were lysed in sample buffer containing 0.2% m/v SDS, 10% v/v glycerol, 0.2% v/v b-mercaptoethanol, 60mM Tris ph6.8. Protein concentration was determined using Bradford (Biorad) and equal concentrations of protein were used for Western blot. Proteins were detected using 6-15% SDS- PAGE gels and subsequent Western-blot analysis with primary antibodies (Supplemental Table 1) detected by secondary hRP conjugated. Unless stated otherwise, non-specific bands were used as a loading control. For immunofluorescence, cells were fixed with 3,7% Paraformaldehyde in PBS, permeabilized with 0.1% v/v
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