Cell Cycle and Senescence The Role of Versican in Modulating Breast Cancer Cell Self-renewal

Versican is highly expressed during the early stages of tissuedevelopment and its expression is elevated duringwound repair and tumor growth. There is little literature on the potential role of breast cancer stem cells on the cellular– extracellular matrix interactions involving versican. An anti-versican short hairpin RNA (shRNA) was used to observe the effect of reduction of versican on breast cancer self-renewal. A versicanG3 construct was exogenously expressed in breast cancer cell lines. Colony formation and mammosphere formation assays were conducted; flow cytometry was applied to analyze the prevalence of side population cells. The versican G3and vector-transfected 66c14 cells were injected transdermally intoBALB/cmice as a 10-fold dilution series from1 10 to 1 10 cells permouse. Versican G3 domain enhanced breast cancer self-renewal in both experimental in vitro and in vivo models. Versican G3–transfected cells contained high levels of side population cells, formed more mammospheres when cultured in the serum-free medium, and formed a greater number and larger colonies. Reduction of versican's functionality through anti-versican shRNA or knocking out the EGF-like motifs reduced the effect of versican on enhancing mammosphere and colony formation. Versican-enhanced self-renewal played a role in enhanced chemotherapeutic drug resistance, relating partly to the upregulated expression of EGF receptor (EGFR) signaling. Versican is highly expressed in breast cancer progenitor cells and was maintained at high levels before cell differentiation. Overexpression of versican enhanced breast cancer self-renewal through EGFR/AKT/GSK-3b (S9P) signaling and conferred resistant to chemotherapeutic drugs tested. Mol Cancer Res; 11(5); 1–13. 2013 AACR. Introduction Experimental and clinical evidence support the understanding that tumorigenesis is sustained by a small population of cells that function as tumor stem/progenitor cells (1, 2). In breast cancer, these cells arise from mutated mammary stem/progenitor cells, which have been characterized by the expression of key cell surface markers (e.g., CD24, CD44, CD29, or Sca-1) as well as dye efflux assays based on the differential release of incorporated Hoechst dyes or rhodamine-121 through overexpressed or overactivated drug transporters in the cell membrane (3–5). These tumor progenitor cells are characterized by their ability to form new serially transplantable tumors in mice and to display stem/progenitor cell properties such as competence for self-renewal and the capacity to reestablish tumor heterogeneity (6, 7). In addition to flawed regulation of the selfrenewal pathways, the number of cells within a tumor that have the ability to self-renew is constantly expanding, resulting in a continuous expansion of tumorigenic cancer cells (8). Thus, the identification of mechanisms in which cancer cells regenerate through self-renewal is critical to our understanding of tumorigenesis (9). A number of developmental signaling pathways, such asWnt,Notch, andHedgehog, have been observed to play pivotal roles in governing both self-renewal and the process of malignant transformation (10, 11). Tumor stem cells, as well as other cells in the tissue, do not function autonomously as independent self decision-making entities. Increasing evidence shows the influence of the cellular microenvironment on tumor development and progression (2, 12). Cellular activity is closely regulated through interactions with adjacent cells that create well-defined microenvironments in which tumor stem cells reside. However, tumor cells also possess the ability to interact with and influence their surrounding environment. Examples of these properties include neo-angiogenesis, recruitment of epithelial cells, and modification of tissue architecture. Interestingly, certain extracellular matrix (ECM) and cell-surface Authors' Affiliations: Sunnybrook Research Institute; Department of Laboratory Medicine and Pathobiology; Centre for the Study of Bone Metastasis, Odette Cancer Centre, and HollandMusculoskeletal Program, Sunnybrook Health Sciences Centre, Division of Orthopaedic Surgery, Department of Surgery, University of Toronto, Toronto, Ontario, Canada; and Division of Life Science, Graduate School at Shenzhen, Tsinghua University, Shenzhen, China Note: Supplementary data for this article are available at Molecular Cancer Research Online (http://mcr.aacrjournals.org/). Corresponding Author: Albert J. Yee, Sunnybrook Health Sciences Centre and Centre for the Study of Bone Metastasis, Odette Cancer Centre, Department of Surgery, University of Toronto, 2075 Bayview Avenue, Rm. MG 371-B, Toronto, Ontario M4N 3M5, Canada. Phone: 416-480-6815; Fax: 416-480-5886; E-mail: [email protected] doi: 10.1158/1541-7786.MCR-12-0461 2013 American Association for Cancer Research. Molecular Cancer Research www.aacrjournals.org OF1 on June 27, 2017. © 2013 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from Published OnlineFirst February 28, 2013; DOI: 10.1158/1541-7786.MCR-12-0461