RB1 Status in Triple Negative Breast Cancer Cells Dictates Response to Radiation Treatment and Selective Therapeutic Drugs Tyler J. W. Robinson 1 *, Jeff C. Liu 2 , Frederick Vizeacoumar 3 , Thomas Sun 3 , Neil Maclean 4 , Sean E. Egan 5 , Aaron D. Schimmer 4 , Alessandro Datti 3,6 , Eldad Zacksenhaus 1,2 * 1 Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada, 2 Division of Advanced Diagnostics, Toronto General Research Institute, University Health Network, Toronto, Ontario, Canada, 3 S.M.A.R.T. High Throughput Facility, Mount Sinai Hospital, Samuel Lunenfeld Research Institute, Toronto, Ontario, Canada, 4 Clinical Studies Resource Centre, OCI, University Health Network, Toronto, Ontario, Canada, 5 Program in Developmental and Stem Cell Biology, Hospital for Sick Children, Molecular Genetics, University of Toronto, Toronto, Ontario, Canada, 6 Department of Experimental Medicine and Biochemical Sciences, University of Perugia, Perugia, Italy Abstract Triple negative breast cancer (TNBC) includes basal-like and claudin-low subtypes for which only chemotherapy and radiation therapy are currently available. The retinoblastoma (RB1) tumor suppressor is frequently lost in human TNBC. Knockdown of RB1 in luminal BC cells was shown to affect response to endocrine, radiation and several antineoplastic drugs. However, the effect of RB1 status on radiation and chemo-sensitivity in TNBC cells and whether RB1 status affects response to divergent or specific treatment are unknown. Using multiple basal-like and claudin-low cell lines, we hereby demonstrate that RB-negative TNBC cell lines are highly sensitive to gamma-irradiation, and moderately more sensitive to doxorubicin and methotrexate compared to RB-positive TNBC cell lines. In contrast, RB1 status did not affect sensitivity of TNBC cells to multiple other drugs including cisplatin (CDDP), 5-fluorouracil, idarubicin, epirubicin, PRIMA-1 met , fludarabine and PD-0332991, some of which are used to treat TNBC patients. Moreover, a non-biased screen of ,3400 compounds, including FDA-approved drugs, revealed similar sensitivity of RB-proficient and -deficient TNBC cells. Finally, ESA + /CD24 2/ low /CD44 + cancer stem cells from RB-negative TNBC lines were consistently more sensitive to gamma-irradiation than RB- positive lines, whereas the effect of chemotherapy on the cancer stem cell fraction varied irrespective of RB1 expression. Our results suggest that patients carrying RB-deficient TNBCs would benefit from gamma-irradiation as well as doxorubicin and methotrexate therapy, but not necessarily from many other anti-neoplastic drugs. Citation: Robinson TJW, Liu JC, Vizeacoumar F, Sun T, Maclean N, et al. (2013) RB1 Status in Triple Negative Breast Cancer Cells Dictates Response to Radiation Treatment and Selective Therapeutic Drugs. PLoS ONE 8(11): e78641. doi:10.1371/journal.pone.0078641 Editor: Aamir Ahmad, Wayne State University School of Medicine, United States of America Received July 6, 2013; Accepted September 20, 2013; Published November 12, 2013 Copyright: ß 2013 Robinson 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 study was conducted with support of the Canadian BC Foundation, Canadian BC Research Alliance, Ontario Institute for Cancer Research through funding provided by the Government of Ontario to E. Zacksenhaus, as well as the Terry-Fox Foundation to S.E. Egan and E. Zacksenhaus. 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: tyler.robinson@mail.utoronto.ca (TJWR); eldad.zacksenhaus@utoronto.ca (EZ) Introduction Triple negative breast cancer (TNBC) represents a collection of tumors that lack expression of estrogen (ER) and progesterone (PR) receptors as well as the receptor tyrosine kinase HER2 [1]. These tumors can be further subdivided into basal-like, claudin- low and other subclasses. The former is characterized by expression of basal markers and elevated proliferation. The claudin-low subtype lacks basal markers but expresses low levels of tight junction proteins and cell adhesion proteins such as E- cadherin and certain claudins, as well as high levels of genes associated with epithelial-mesenchymal-transition (EMT) [2,3]. TNBC makes up 10–30% of all breast cancer cases. Compared to other subtypes, TN tumors are associated with poor prognosis, in part due to a lack of targeted treatment. Clinically, TNBCs respond more favorably to chemotherapy than other types, however prognosis still remains poor due to a greater risk of distal recurrence, with a rapid rise in relapse in the first 3 years post diagnosis [4–6]. Metastatic disease is extremely aggressive, and often arises in tissues that are difficult to treat, such as bone or brain. Therefore, it is pertinent to find more effective treatments for aggressive forms of TNBC. The tumor suppressor RB1 is often lost by mutation, deletion or transcriptional silencing as well as by hyper-phosphorylation of its gene product, pRb, in many human malignancies [7–9]. Indeed, it is deleted or rearranged in ,20–25% of BC cell lines [10–18]. It is primarily inactivated in TNBC [19]. Furthermore, recent genomic sequencing, transcriptome analysis, epigenetic and proteomic analysis identified RB1 loss in ,20% of TNBC [20]. Deletion of murine Rb in mammary epithelium induces basal-like and luminal tumors, whereas deletion of both Rb and p53 leads to claudin-low like tumors [21], hence demonstrating a causal role for RB1 in TNBC. Acute inactivation of RB1 in hormone-dependent luminal breast and colon cancer cells increases response to several antineoplastic drugs, suggesting that RB-deficiency affects thera- peutic outcome in certain tumor types including ER + breast PLOS ONE | www.plosone.org 1 November 2013 | Volume 8 | Issue 11 | e78641