The Ability to Generate Senescent Progeny as a Mechanism Underlying Breast Cancer Cell Heterogeneity Mine Mumcuoglu1, Sevgi Bagislar1,2, Haluk Yuzugullu1,2, Hani Alotaibi1, Serif Senturk1, Pelin Telkoparan 1 , Bala Gur-Dedeoglu1, Burcu Cingoz1, Betul Bozkurt3, Uygar H. Tazebay1, Isik G. Yulug 1 , K. Can Akcali1, Mehmet Ozturk1,2* 1 BilGen Genetics and Biotechnology Center, Department of Molecular Biology and Genetics, Bilkent University, Ankara, Turkey, 2 INSERM - Universite´ Joseph Fourrier, CRI U823, Grenoble, France, 3 Department of Surgery, Ankara Numune Research and Teaching Hospital, Ankara, Turkey Abstract Background: Breast cancer is a remarkably heterogeneous disease. Luminal, basal-like, ‘‘normal-like’’, and ERBB2+ subgroups were identified and were shown to have different prognoses. The mechanisms underlying this heterogeneity are poorly understood. In our study, we explored the role of cellular differentiation and senescence as a potential cause of heterogeneity. Methodology/Principal Findings: A panel of breast cancer cell lines, isogenic clones, and breast tumors were used. Based on their ability to generate senescent progeny under low-density clonogenic conditions, we classified breast cancer cell lines as senescent cell progenitor (SCP) and immortal cell progenitor (ICP) subtypes. All SCP cell lines expressed estrogen receptor (ER). Loss of ER expression combined with the accumulation of p21Cip1 correlated with senescence in these cell lines. p21Cip1 knockdown, estrogen-mediated ER activation or ectopic ER overexpression protected cells against senescence. In contrast, tamoxifen triggered a robust senescence response. As ER expression has been linked to luminal differentiation, we compared the differentiation status of SCP and ICP cell lines using stem/progenitor, luminal, and myoepithelial markers. The SCP cells produced CD24+ or ER+ luminal-like and ASMA+ myoepithelial-like progeny, in addition to CD44+ stem/progenitor-like cells. In contrast, ICP cell lines acted as differentiation-defective stem/progenitor cells. Some ICP cell lines generated only CD44+/ CD24-/ER-/ASMA- progenitor/stem-like cells, and others also produced CD24+/ER- luminal-like, but not ASMA+ myoepithelial- like cells. Furthermore, gene expression profiles clustered SCP cell lines with luminal A and ‘‘normal-like’’ tumors, and ICP cell lines with luminal B and basal-like tumors. The ICP cells displayed higher tumorigenicity in immunodeficient mice. Conclusions/Significance: Luminal A and ‘‘normal-like’’ breast cancer cell lines were able to generate luminal-like and myoepithelial-like progeny undergoing senescence arrest. In contrast, luminal B/basal-like cell lines acted as stem/ progenitor cells with defective differentiation capacities. Our findings suggest that the malignancy of breast tumors is directly correlated with stem/progenitor phenotypes and poor differentiation potential. Citation: Mumcuoglu M, Bagislar S, Yuzugullu H, Alotaibi H, Senturk S, et al. (2010) The Ability to Generate Senescent Progeny as a Mechanism Underlying Breast Cancer Cell Heterogeneity. PLoS ONE 5(6): e11288. doi:10.1371/journal.pone.0011288 Editor: Syed A. Aziz, Health Canada, Canada Received March 16, 2010; Accepted June 4, 2010; Published June 24, 2010 Copyright: 2010 Mumcuoglu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by TUBITAK (The Scientific and Technological Research Council of Turkey), DPT (State Planning Office of Turkey) and TUBA (Turkish Academy of Sciences). Additional funding was from Institut National de Cancer and INSERM of France. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: ozturkm@ujf-grenoble.fr Introduction Human breast tumors are heterogeneous, both in their pathology and in their molecular profiles. Gene expression analyses classify breast tumors into distinct subtypes, such as luminal A, luminal B, ERBB2-positive (ERBB2+) and basal-like [1,2,3]. The prognosis and therapeutic response of each subtype is different. Luminal A cancers are mostly estrogen receptor-a- positive (ER+) and sensitive to anti-estrogen therapy, with the best metastasis-free and overall survival rates. Luminal B tumors have an incomplete anti-estrogen response and lower survival rates. Basal-like and ERBB2+ tumors are ER- and display the worst survival rates [2,3]. The patterns of genetic changes such as chromosomal aberrations and gene mutations observed in breast tumors indicate that breast tumorigenesis does not follow a stepwise linear progression from well-differentiated to poorly differentiated tumors with cumulative genetic aberrations [4]. This suggests that different breast tumor subtypes do not represent different stages of tumor progression, but rather represent the cells from which they initiate [4]. The mammary gland is composed of differentiated luminal and myoepithelial cells that are generated from multi-lineage, luminal-restricted, and myoepithelial-restrict- ed progenitors originating from a hypothetical breast epithelial stem cell. Thus, different types of breast cancers might originate from such stem or progenitor cells at a given stage of commitment and differentiation, as observed in hematological malignancies [4,5]. Without compromising the author’s hypothesis, it is also possible that the molecular heterogeneity of breast cancer is due to subtle differences in the ability of tumor-initiating cells to generate differentiated progeny. PLoS ONE | www.plosone.org 1 June 2010 | Volume 5 | Issue 6 | e11288