Liu3, Li Chai1* Abstract SALL4, a Stem Cell Factor, Affects the Side Population by Regulation of the ATP-Binding Cassette Drug Transport Genes Ha-Won Jeong1., Wei Cui2., Youyang Yang1, Jiayun Lu1, Jie He1, Ailing Li1, David Song1, Ye Guo2, Bee H. 1 The Department of Pathology, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts, United States of America, 2 Department of Clinical Laboratory, Chinese Academy of Medical Sciences, Peking Union Medical College Hospital, Beijing, China, 3 Centre for Life Sciences, Cancer Science Institute of Singapore, National University of Singapore, Singapore, Singapore Our previous work shows that the stem cell factor SALL4 plays a central role in embryonic and leukemic stem cells. In this study, we report that SALL4 expression was higher in drug resistant primary acute myeloid leukemic patients than those from drug-responsive cases. In addition, while overexpression of SALL4 led to drug resistance in cell lines, cells with decreased SALL4 expression were more sensitive to drug treatments than the parental cells. This led to our investigation of the implication of SALL4 in drug resistance and its role in side population (SP) cancer stem cells. SALL4 expression was higher in SP cells compared to non-SP cells by 2–4 fold in various malignant hematopoietic cell lines. Knocking down of SALL4 in isolated SP cells resulted in a reduction of SP cells, indicating that SALL4 is required for their self-renewal. The SP phenotype is known to be mediated by members of the ATP-binding cassette (ABC) drug transport protein family, such as ABCG2 and ABCA3. Using chromatin-immunoprecipitation (ChIP), quantitative reverse transcription polymerase chain reaction (qRT-PCR) and electrophoretic mobility shift assay(EMSA), we demonstrated that SALL4 was able to bind to the promoter region of ABCA3 and activate its expression while regulating the expression of ABCG2 indirectly. Furthermore, SALL4 expression was positively correlated to those of ABCG2 and ABCA3 in primary leukemic patient samples. Taken together, our results suggest a novel role for SALL4 in drug sensitivity, at least in part through the maintenance of SP cells, and therefore may be responsible for drug-resistance in leukemia. We are the first to demonstrate a direct link between stem cell factor SALL4, SP and drug resistance in leukemia. Citation: Jeong H-W, Cui W, Yang Y, Lu J, He J, et al. (2011) SALL4, a Stem Cell Factor, Affects the Side Population by Regulation of the ATP-Binding Cassette Drug Transport Genes. PLoS ONE 6(4): e18372. doi:10.1371/journal.pone.0018372 Editor: Alfons Navarro, University of Barcelona, Spain Received August 8, 2010; Accepted March 5, 2011; Published April 19, 2011 Copyright: 2011 Jeong 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 in part through National Institutes of Health grants RO1HL092437, RO1HL092437-A1S1, PO1 DK080665 and PO1HL095489 (to Dr. Li Chai). 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: lchai@partners.org . These authors contributed equally to this work. Introduction Leukemic stem cells (LSCs) are cells that can give rise to leukemia in transplant murine models, and therefore have self- renewal property. It is hypothesized that these cells are not targeted under current chemotherapy regimens and therefore may account for drug resistance and leukemia relapse. Identifying genes or signaling pathways involved in self-renewal of LSCs will likely promote the development of more effective treatments for leukemia and other cancers [1–4]. Several methods are currently being used to isolate and study hematopoietic stem cells (HSCs) or LSCs, including the use of HSCs or LSCs cell surface markers [5–10] and the use of Hoechst 33342 dye efflux to identify side population (SP) [11–16] . There are advantages and disadvantages to using either approach. Isolation of HSCs or LSCs by cell surface markers is better suited for subsequent functional studies such as in vivo transplantation since the Hoechst dye is toxic to the cells and therefore might impair their physiological functions. On the other hand, studying SP cells might be a better approach to identify LSCs with potential resistance to chemotherapeutic agents that may account for leukemic recurrence. As characterized by Goodell et al [17], SP from primary AML patients not only has the ability to transfer leukemic phenotypes in NOD-SCID mouse model, but more importantly, these cells have the capacity for rapid efflux of anti-leukemic drugs such as daunorubicin and mitoxantrone. Therefore, SP cells may be a major target for leukemic remission. In addition to blood cells, the SP approach has been used for enrichment for other cancer stem cells, such as breast cancer stem cells [15]. Several ATP binding cassette (ABC) transporter genes have been reported to be responsible for the SP phenotype. Currently, the ABC family has 48 members, which is further divided into 7 subfamilies based on similarities in protein structure, (ABCA, B, C, D, E, F and G) [18] . Among these transporters are the breast cancer resistance protein (BCRP or ABCG2) and ABCA3 [19,12,13,20–22]. ABCG2 is one of the earliest ABC transporters to be identified and characterized. It is expressed in normal hematopoietic CD34+ cells and has been associated with a subpopulation of tumor cells that PLoS ONE | www.plosone.org 1 April 2011 | Volume 6 | Issue 4 | e18372