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DNA Damage Induces a Secretory Program in the Quiescent TME That Published OnlineFirst March 29, 2017; DOI: 10.1158/1541-7786.MCR-16-0387 DNA Damage and Repair Molecular Cancer Research DNA Damage Induces a Secretory Program in the Quiescent TME that Fosters Adverse Cancer Phenotypes Luis Gomez-Sarosi1, Yu Sun2, Ilsa Coleman1, Daniella Bianchi-Frias1, and Peter S. Nelson1,3 Abstract Carcinomas develop in complex environments that include a using gene expression profiling. The secretory damage response diverse spectrum of cell types that influence tumor cell behavior. in quiescent cells was highly concordant with that of actively These microenvironments represent dynamic systems that con- dividing cells. Quiescent human prostate stroma exposed to tribute to pathologic processes. Damage to DNA is a notable genotoxic agents (e.g., mitoxantrone) in vivo resulted in signi- inducer of both transient and permanent alterations in cellular ficant upregulation (2.7- to 5.7-fold; P 0.01) of growth factors phenotypes. Induction of a DNA damage secretory program is and cytokines including IL1b, MMP3, IL6, and IL8. The para- known to promote adverse tumor cell behaviors such as pro- crine effects of damaged quiescent cells consistently increased liferation, invasion, metastasis, and treatment resistance. How- the proliferation and invasion of prostate cancer cells and ever, prior studies designed to identify genotoxic stress–induced promoted cell survival and resistance to apoptosis following factors evaluated actively proliferating in vitro cultures of cells exposure to chemotherapy. such as fibroblasts as experimental models. Conversely, the vast majority of benign cells in a typical tumor microenvironment Implications: Benign quiescent cells in the TME respond to (TME) are not proliferating but rather exist in quiescent (i.e., genotoxic stress by inducing a secretory program capable of G0) or in terminally differentiated states. In this study, the promoting therapy resistance. Developing approaches to suppress diversity and magnitude of transcriptional responses to geno- the secretory program may improve treatment responses. toxic damage in quiescent prostate fibroblasts were assessed Mol Cancer Res; 15(7); 842–51. Ó2017 AACR. Introduction signaling from other juxtaposed cell types, concentrations of hormones and systemic growth factors, pathogens, nutrients, Malignant neoplasms arise in complex biophysical environ- oxygen, pH, and a spectrum of other influences that can either ments composed of a diverse spectrum of cell types, structural reversibly or irreversibly alter cellular functions (6). components, and biochemical constituents that have profound Damage to DNA is a notable inducer of both transient and influences on tumor cell behavior (1–3). Of importance, organ permanent alterations in cellular phenotype. Genotoxic stress can and tissue microenvironments represent dynamic systems with result from a variety of events that include exposure to free shifts in the numbers and types of benign resident cells—such as radicals, telomere shortening, oncogenes, errors in DNA replica- fibroblasts and endothelium, and immigrating cells—including tion, and treatment with cancer therapeutics. Cell-cycle arrest is a those of immune lineage, in the context of normal development well-described consequence of DNA damage, with subsequent and pathologic processes (4, 5). The phenotypes of these cells also proliferation if damage is repaired, or if severe, irreversible growth vary depending on responses to extrinsic factors such as paracrine arrest manifest as senescence or programmed cell death (7–9). DNA damage response (DDR) programs provide mechanisms to avoid propagating oncogenic mutations and also activate a secre- 1Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, tory program that comprises a diverse spectrum of proteases, Washington. 2Shanghai Institutes for Biology Sciences, Chinese Academy of 3 growth factors, and cytokines, collectively and somewhat synon- Sciences, Shanghai, China. Division of Clinical Research, Fred Hutchinson ymously termed a senescence-associated secretory phenotype Cancer Research Center, Seattle, Washington. (SASP), senescence messaging secretome, acute stress-associated Note: Supplementary data for this article are available at Molecular Cancer secretome, and DNA damage secretory program (DDSP; refs. Research Online (http://mcr.aacrjournals.org/). 10–13). The composite effects of these programs have been L. Gomez-Sarosi and Y. Sun contributed equally to this study. shown to contribute to wound healing, aging phenotypes, altered Corresponding Author: Peter S. Nelson, Division of Human Biology, Fred immune responses and are also capable of promoting adverse Hutchinson Cancer Research Center, Mailstop D4-100, 1100 Fairview Ave N, tumor cell behaviors such as proliferation, invasion, metastasis, Seattle, WA 98109. Phone: 206-667-3377; Fax: 206-667-2917; E-mail: and treatment resistance (13–17). [email protected] Large-scale discovery-driven efforts designed to define the doi: 10.1158/1541-7786.MCR-16-0387 spectrum of secreted proteins induced by genotoxic damage have Ó2017 American Association for Cancer Research. identified several hundred growth factors, cytokines, enzymes, 842 Mol Cancer Res; 15(7) July 2017 Downloaded from mcr.aacrjournals.org on September 28, 2021. © 2017 American Association for Cancer Research. Published OnlineFirst March 29, 2017; DOI: 10.1158/1541-7786.MCR-16-0387 A DNA Damage Secretory Program in Quiescent Cells and matricellular proteins that are altered in benign cells my specimens into PAP membrane slides. Approximately, 1,000 following genotoxic stress or in the context of cellular senescence cells were separately microdissected for prostate cancer epitheli- (13, 18, 19). However, to date, the majority of these profiling um (CPE), benign prostate epithelium (BPE), and stroma adja- studies have used actively proliferating in vitro cultures of cells cent to cancer (CAS). The corresponding benign cells for each case such as fibroblasts as experimental models (18–20). Conversely, were microdissected from separate blocks identified as containing the vast majority of benign cells in a typical tumor microenvi- no adenocarcinoma cells (first choice) or from non-neoplastic ronment, including fibroblasts, endothelium, smooth muscle, tissues at a distance >1 mm from the cancer. Digital images of and inflammatory cells, are not proliferating but rather exist in tissue sections before, during, and after laser capture microdis- quiescent, G0, or terminally differentiated states. As the cell-cycle section (LCM) were taken and assessed to confirm the cell type phase has been shown to influence cellular responses to genotoxic specificity of the captured cells. exposures and other stresses (21, 22), it is unclear to what extent damage to proliferating cells reflects that of nondividing cells in Growth arrest conditions and cell treatments tissue microenvironments. In this study, we sought to assess the PSC27 fibroblasts were plated at a density of 2 Â 104 cells per diversity and magnitude of transcriptional responses to genotoxic cm2 in PSC medium and allowed to attach to the tissue culture damage in quiescent fibroblasts, compare the secretory damage dishes. To induce quiescence by growth factor starvation, the response to that of actively dividing cells, and determine whether medium was changed to DMEM with 0.1% serum and cultured the paracrine-acting factors derived from quiescent cells promote for 4 days before analysis. These cells were designated PSC27- adverse cancer cell phenotypes such as proliferation, invasion, QSS. To arrest cells by contact inhibition, cells were plated at a and resistance to cancer treatment–induced cell death. density of 2 Â 104 cells per cm2 in stromal medium and allowedtogrowtoconfluence, usually reaching complete fl Materials and Methods con uency in about 7 to 10 days. These cells were designated PSC27-QCI. Proliferating or quiescent cells were treated with Biospecimens, cell lines, and culture conditions 1 mmol/L mitoxantrone in PSC medium or ionizing radiation Tissue samples were obtained under IRB-approved biospeci- by a 137Cs source at 743 rad/min. Media under each condition men collection and handling protocols. The primary human were changed every 3 days for 10 days until cells were lysed for prostate fibroblast cell line, designated PSC27, was a gift from analysis. For quiescent cells allowed to resume proliferation, Dr. Beatrice Knudsen. PSC27 cells were cultured in prostate quiescent cells were trypsinized, replated to cell culture vessels stromal cell (PSC) complete medium as described previously of larger growth area, or divided into multiple vessels, with the (23). The human prostatic epithelial cell line BPH1 was a gift same media applied for each subculture. For each condition, 3 from Dr. Simon Hayward and was derived from nonmalignant independent replicates were performed. prostatic tissue with benign hyperplasia, immortalized by SV40- LT antigen, and cultured as previously described (24). The HeLa, Immunofluorescent analysis and quantitation of DNA PC3, VCaP, LNCaP, and DU145 cell lines were obtained from damage foci ATCC and routinely subcultured as per ATCC recommendations. Cells grown on coverslips were rinsed in PBS, subjected to Cells were either used within 4 passages after receipt from ATCC or fixation in 4% paraformaldehyde, and permeabilized with 0.1% authenticated before initiating the studies by genotyping at DNA Triton-X 100 before immunostaining. Primary mouse monoclo- Diagnostics Center (Fairfield,
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