Y-box binding protein-1 (YB-1) is an oncogenic transcription/translation factor expressed in >40% of breast cancers, where it is associated with poor prognosis, disease recurrence, and drug resistance. We questioned whether this may be linked to the ability of YB-1 to induce the expression of genes linked to cancer stem cells such as CD44 and CD49f. Herein, we report that YB-1 binds the CD44 and CD49f promoters to transcriptionally upregulate their expressions. The introduction of wild-type (WT) YB-1 or activated P-YB-1S102 stimulated the production of CD44 and CD49f in MDA-MB-231 and SUM 149 breast cancer cell lines. YB-1–transfected cells also bound to the CD44 ligand hyaluronan more than the control cells. Similarly, YB-1 was induced in immortalized breast epithelial cells and upregulated CD44. Conversely, silencing YB-1 decreased CD44 expression as well as reporter activity in SUM 149 cells. In mice, expression of YB-1 in the mammary gland induces CD44 and CD49f with associated hyperplasia. Further, activated mutant YB-1S102D enhances self-renewal, primary and secondary mammosphere growth, and soft-agar colony growth, which were reversible via loss of CD44 or CD49f. We next addressed the consequence of this system on therapeutic responsiveness. Here, we show that paclitaxel induces P-YB-1S102 expression, nuclear localization of activated YB-1, and CD44 expression. The overexpression of WT YB-1 promotes mammosphere growth in the presence of paclitaxel. Importantly, targeting YB-1 sensitized the CD44High/CD24Low cells to paclitaxel. In conclusion, YB-1 promotes cancer cell growth and drug resistance through its induction of CD44 and CD49f. Cancer Res; 70(7); 2840–51
- breast cancer
- tumor-initiating cells
- drug resistance
- cancer recurrence
Breast cancer relapse confers a poor prognosis, decreasing survival rates from 80% to 60% when the recurrence is local, and from 80% to 10% for metastatic disease. Relapse is postulated to be mediated by the persistence of cancer stem cells that have survived an initial treatment regimen with radiation (1) and/or chemotherapy (2–4) possibly due to a selective resistance of the cancer stem cells to these agents. Human breast cancer stem cells have been identified as a phenotypically restricted subset of CD44+/CD24−/low cells that form tumors in immunocompromised mice in limiting dilution transplant assays (5). The same CD44+/CD24−/low subset has also been associated with breast tumor mammosphere generation in vitro (3). However, the nature of the essential molecular properties of breast cancer stem cells has remained poorly defined.
Y-box binding protein-1 (YB-1) is a transcription/translation factor that is commonly overexpressed in many cancers, including human breast cancer (40%; refs. 6–8). This elevated expression of YB-1 in human breast cancer correlates with high rates of relapse (6). Targeted overexpression of YB-1 in the mouse mammary gland leads to the development of mammary tumors (9), confirming a role of YB-1 as an oncogene in that tissue. YB-1 is directly phosphorylated, and therefore activated, on its serine 102 site by Akt (10) and even more potently by ribosomal S6 kinase (RSK; ref. 11), a major component of the mitogen-activated protein kinase (MAPK) pathway. Thus, YB-1 is positioned as a key player in the phosphoinositide 3-kinase (PI3K)/Akt and MAPK pathways. YB-1 regulates genes that promote breast cancer cell growth and survival, including EGFR (12), Her-2 (erbB2; ref. 7), PIK3CA (13), and the MET (c-Met) receptor (14), and is biologically essential for breast cancer cell growth in vitro (7, 10–12) and in vivo (15). YB-1 expression has also been shown to be associated with drug resistance via the induction of genes such as MDR-1 (16, 17). Consistent with this, inhibition of YB-1 was found to sensitize breast cancer cells to paclitaxel (15), a chemotherapeutic agent commonly used in the clinic to treat advanced breast cancer. To elucidate the transcriptional programming activity of YB-1, we did chromatin immunoprecipitation-on-chip (ChIP-on-chip) assays. This screen revealed a subset of genes known to be active in and important to a number of stem cell populations, including c-KIT, BMI-1, members of the WNT and NOTCH signaling pathways, as well as CD44 and CD49f (also known as α6 integrin; ref. 14). We also found that YB-1 transcripts were present in purified CD44+CD49f+ subpopulations of primitive human mammary progenitor cells isolated from normal reduction mammoplasties (14). Taken together, this led us to test the hypothesis that YB-1 plays a key role as an oncogene by transactivating genes associated with a cancer stem cell phenotype.
Materials and Methods
Cell lines and culturing
The human breast cancer cell lines MDA-MB-231, MDA-MB-468, and SUM 149 were purchased and maintained as previously described (13, 14). The cell lines were characterized for CD44 and CD24 expression by flow cytometry (Supplementary Fig. S1A–E). Cell autofluorescence was taken into account.
ChIP using a polyclonal chicken antibody to precipitate endogenous YB-1 was done as previously described (7). Three primer sets were designed to flank seven putative YB-1 binding sites (ATTG) in the first 2 kb of the CD44 promoter, and similarly, two primer sets flanking eight putative YB-1 binding sites in the first 2 kb of the CD49f promoter were also designed. Details are described in Supplementary Materials and Methods.
Immunofluorescence assays were done as previously described (13, 14). Antibodies used were rat anti-human and anti-mouse FITC-conjugated anti-CD49f (1:25, clone GoH3, BD Pharmingen), rat anti-human phycoerythrin (PE)-conjugated anti-CD44 (1:100, clone 515, BD Pharmingen), unconjugated rabbit anti-human phospho-YB-1S102 (P-YB-1S102; 1:100, clone C34A2, Cell Signaling Technology), and secondary anti-rabbit FITC (1:200; Jackson ImmunoResearch Laboratories, Inc.). Details are described in Supplementary Materials and Methods.
Fluorescence-activated cell sorting and analysis
A single-cell suspension was achieved by first scraping the adherent cells and digesting with a dispase solution (Stem Cell Technologies) and DNase (Sigma-Aldrich) at 37°C for 10 min. Cells were diluted in a 2% fetal bovine serum (FBS)-PBS buffer and passed through a 40-μm cell strainer to achieve a single-cell suspension. The single-cell suspension was stained with anti-CD44 conjugated to PE (BD Pharmingen). 7-Aminoactinomycin D (7-AAD) viability dye (BD Pharmingen) was added to the 2% FBS-PBS resuspension buffer. For cell sorting, the top and bottom 10% of cells with the highest and lowest CD44-PE fluorescence were collected into 50% FBS-PBS buffer and seeded subsequently into a 96-well plate for immunofluorescence staining or growth assays. Sorted cells were seeded at 1 × 104 per well in 96-well plates. Details are given in Supplementary Materials and Methods.
A single-cell suspension of MDA-MB-231 or SUM 149 cells was seeded into nonadherent mammosphere culturing conditions and counted after 7 d. The single cells were seeded into secondary cultures under the same conditions and counted after 7 d. Cells were dissociated enzymatically into a single-cell suspension with dispase digestion and mechanical dissociation by repeated pipetting. Cells were seeded at 5 × 103 (MDA-MB-231) and 2 × 104 (SUM 149) per well into Ultra-Low Attachment–coated six-well culture plates (Corning) in a 1:1 DMEM/F12 (Invitrogen) basal medium freshly supplemented with 20 ng/mL human basic fibroblast growth factor (Invitrogen), 20 ng/mL epidermal growth factor (Invitrogen), 10 μg/mL heparin (Sigma-Aldrich), and 1:50 B27 supplement without vitamin A (Sigma-Aldrich). Spheres containing approximately >15 cells were counted 7 d after seeding. In the self-renewal serial passaging experiments, all cells from primary mammosphere cultures were collected, centrifuged at 350 × g for 5 min, and dissociated into a single-cell suspension with 0.25% trypsin for 5 min at 37°C and counted.
Soft-agar anchorage-independent growth assay
Small interfering RNA transfection
Small interfering RNAs (siRNA) were transfected into cells using the Lipofectamine RNAiMAX transfection reagent (Invitrogen) according to the manufacturer's protocol. Unless otherwise specified, in all experiments, 20 nmol/L of siRNAs were incubated with the cells for 96 h before assessment of biochemical/biological effects. Details are described in Supplementary Materials and Methods.
Quantitative real-time PCR
Immunoblotting analyses were done as described previously (7). Antibodies used were as follows: anti–total YB-1 (1:2,000, #ab12148, Abcam; 1:1,000, #2749, Cell Signaling Technology; 1:2,000, a polyclonal antibody designed against the COOH terminus of YB-1, produced by and a generous gift from Dr. Colleen Nelson, University of British Columbia, Vancouver, BC, Canada), anti–P-YB-1S102 (1:1,000, clone C34A2, Cell Signaling Technology), anti-CD44 (1:500, clone EPR1013Y, Abcam), anti-CD49f (1:1,000, #3750, Cell Signaling Technology), anti-Flag (1:2,000, clone M2, Sigma-Aldrich), anti–total Akt (T-Akt; 1:1000, #9272, Cell Signaling Technology), anti–pan-actin (1:1,000, #4968, Cell Signaling Technology), and anti-vinculin (1:1,000, clone hVIN-1, Sigma-Aldrich).
Luciferase reporter assay
SUM 149 EV and shYB-1 cells were seeded in a six-well plate (4 × 105 cells per well) and allowed to adhere. The cells were then transfected with pGL3b (1 μg) or pGL3b-CD44P (1 μg, Addgene, deposited by Dr. Robert Weinberg, Department of Biology, Massachusetts Institute of Technology, Cambridge, MA; ref. 2) in triplicate using Lipofectamine 2000 (Invitrogen). Cells were lysed 24 h after transfection in passive lysis buffer (Promega) and luciferase activity was measured.
Matrigel semi-fluid basement membrane growth assay
Matrigel basement membranes (BD Biosciences) were added at 40 μL/well in a 96-well plate and incubated briefly at 37°C for solidification. MDA-MB-231 and SUM 149 cells in single-cell suspension were added in their respective media at 2 × 103 and 5 × 103 cells per well. Growth was assessed in photographed fields 7 d after seeding.
DNA plasmid transfection
In a six-well plate format, 2 or 4 μg of a Flag:EV, Flag:YB-1WT, or Flag:YB-1S102D plasmid were transfected with 5 or 10 μL of Lipofectamine 2000 (Invitrogen), respectively, as specified by the manufacturer's protocol. Cells were harvested 96 h after transfection.
Stable transfectant cell lines
Stable EV, shYB-1, Flag:EV, Flag:YB-1WT, and Flag:YB-1S102D cell lines were established by transfecting 4 μg of DNA constructs as described. G418 (400 μg/mL; Calbiochem, EMD Chemicals) was added to the culture media and replaced every 3 to 4 d. Cells were continually split at low density to allow for optimal selection of transfectants with acquired G418 resistance.
YB-1 transgenic mouse and mammary gland sectioning
YB-1 transgenic (Tg 2 line) and wild-type (WT) mice were revived from cryopreserved embryos generated and characterized previously (9), where expression of a human hemagglutinin (HA)-tagged YB-1 cDNA was controlled by the ovine β-lactoglobulin promoter. For our study, histologic sections were obtained from the mammary glands of 6- to 8-mo-old female mice post-lactation after two cycles of mating and nursing. The mice were mated twice and their pups were nursed for 3 wk each time. The YB-1 transgenic and control mice were euthanized at the end of their second lactation (day 20 to 22 after the birth of the pups) and mammary gland tissues were collected. The mammary glands were dipped into Tissue-Tek optimum cutting temperature (OCT) compound (Sakura Finetek USA), frozen, and kept at −80°C. OCT compound–embedded tissues were sectioned (6 μm) using a cryostat and placed on Fisherbrand Superfrost Plus glass slides (Thermo Fisher Scientific) and kept at −80°C. The sections were defrosted at room temperature and incubated with PBS for 5 min, fixed with 2% formaldehyde for 20 min, and rinsed twice with PBS. Subsequent immunofluorescence staining and imaging protocol were done as described above. The only exception is that a primary purified rat anti-mouse CD44 antibody (1:100, clone IM7, BD Pharmingen) and a secondary antirat Alexa 546 antibody (Molecular Probes, Invitrogen) were used. A HA-specific antibody (Santa Cruz Biotechnology) was used with immunohistochemistry methods. Details are described in Supplementary Materials and Methods.
Anticancer drug screen and further evaluation of paclitaxel treatment
SUM 149 cells were treated with commonly used chemotherapeutic agents at 10 μmol/L and cell viability was assessed as previously described (18). The cells were also stained with anti-CD44-PE (1:5, clone G44-26, BD Pharmingen) and assessed as previously described (18). SUM 149 cells were also treated with paclitaxel (Sigma-Aldrich) dissolved in DMSO at 10 nmol/L for 48 or 72 h before doing immunoblotting and ChIP as described. MDA-MB-231 Flag:EV and Flag:YB-1WT cells were treated in monolayer or in the mammosphere assay in the presence of 0 nmol/L or 10 nmol/L of paclitaxel with DMSO as vehicle control for 7 d. MDA-MB-231 cells grown in monolayer had the media changed at day 4 and cells were harvested for immunoblotting on day 7.
All quantitative data are represented as mean ± SE of three independent experiments. P values were generated using paired Student's t test, where * denotes P < 0.05, ** denotes P < 0.01, and *** denotes P < 0.001.
YB-1 binds to upstream regulatory regions of the genes encoding CD44 and CD49f and regulates their expression
Several putative YB-1 binding sites were located 2 kb upstream of the transcriptional start site of the CD44 and CD49f genes, initially identified in a ChIP-on-chip screen (ref. 14; Fig. 1A, top). To validate these results, we did conventional ChIP analyses and provided direct evidence of the ability of YB-1 to bind to the promoters of CD44 and CD49f genes in two human breast cancer cell lines (MDA-MB-468 and SUM 149; Fig. 1A, bottom). Immunohistochemical analysis of SUM 149 cells grown either in monolayer cultures or as xenografts illustrated coincident staining of P-YB-1S102 and CD44 protein in individual cells (Fig. 1B). Therefore, we first asked whether the expression of CD44 would fluctuate concordantly with changes in activated YB-1 (P-YB-1S102). Quantification of this association of CD44 expression with the presence of P-YB-1S102 was shown by isolating the highest and lowest 10% of CD44-expressing viable SUM 149 cells by fluorescence-activated cell sorting (FACS) and costaining with CD44 and P-YB-1S102 antibodies. There, we observed a >2-fold difference in P-YB-1S102 levels between the subsets expressing the highest and lowest levels of CD44 (Fig. 1C, top), corresponding to enhanced growth in mammosphere cultures and soft agar (Fig. 1C, bottom).
We next asked whether YB-1 is a determining regulator of CD44 and CD49f expression in breast cancer cells by knocking down YB-1 expression in MDA-MB-231 and SUM 149 cells using three siRNA oligonucleotides designed to target YB-1 transcripts specifically. The result was a decrease in the transcript and protein levels of CD44 and CD49f (Fig. 2A and B). To obtain stable inhibition of YB-1 expression, SUM 149 cells were transfected with anti-YB-1 shRNAs. This produced sustained decreases in CD44 and CD49f transcript and protein levels as compared with control cells transfected with an empty vector (EV; Fig. 2C). Stable knockdown of YB-1 transcripts also resulted in a decreased expression of CD104 (β4 integrin), a β-chain partner of CD49f (Fig. 2B and C). Further, YB-1 shRNA–expressing cells showed a 50% decrease in CD44 promoter activity (Fig. 2D), suggesting YB-1 as a significant regulator of CD44.
Conversely, when we forced a transient elevation in the expression of either Flag:YB-1WT or a constitutively active Flag:YB-1S102D, increases in CD44 and CD49f transcript levels were obtained in both cases, with the largest increases resulting from the constitutively active YB-1 construct (Fig. 3A). A similar result was seen when these constructs were stably expressed in the same cells (Fig. 3B). Importantly, we confirmed that a heightened expression of CD44 and CD49f was concomitantly induced in the same transfected YB-1–overexpressing cells (Fig. 3C). Likewise, expression of YB-1 in immortalized breast epithelial cells, which typically have very little of this protein, caused a measurable increase in CD44 mRNA and protein (Supplementary Fig. S2). To functionalize the effect in breast cancer cells, we determined that MDA-MB-231 cells that stably express Flag:YB-1WT or Flag:YB-1S102D have a greater affinity for the CD44 ligand hyaluronan (Supplementary Fig. S3).
To determine whether elevated YB-1 expression would have a similar effect on the expression of CD44 and CD49f in primary mammary epithelial cells in vivo, we evaluated the mammary glands of 6- to 8-month-old transgenic mice expressing human HA-tagged YB-1 cDNA under the control of the ovine β-lactoglobulin promoter (9). To confirm transgene expression, we stained the tissues for HA and showed an increase in expression with an associated elevation in P-YB-1S102 (Fig. 3D, i–ii). These mice had higher levels of CD49f and CD44 protein (Fig. 3D, iii–iv; Supplementary Fig. S4A and B) as compared with mammary cells from WT animals (n = 4 per group). The increased expression of CD49f in the YB-1 transgenic mice was further validated with a second antibody (Supplementary Fig. S4B). The specificity of the CD44 antibody staining was confirmed by staining B6/129 CD44−/− mouse kidney cells (Supplementary Fig. S4C). We also noted that the cells in the mammary glands from the YB-1 transgenic mice were hyperplastic and displayed atypical nuclei in comparison with those in the WT controls when stained with H&E (Supplementary Fig. S4D). Taken together, these findings indicate that YB-1 regulates the expression of CD44 and CD49f both in human transformed mammary cells in vitro and in primary mouse mammary cells generated in vivo.
YB-1–regulated expression of CD44 and CD49f controls the anchorage-independent growth of transformed breast cells
To test whether the ability of YB-1 to regulate CD44 and/or CD49f expression was important for YB-1–dependent tumor cell growth (15), we next examined the effect of treating MDA-MB-231 cells with anti-CD44 and anti-CD49f siRNAs on mammosphere formation (Fig. 4A). SiRNAs directed against transcripts for YB-1 as well as CD44 and CD49f all inhibited primary mammosphere formation by >50%. Conversely, forced increased expression of Flag:YB-1WT or Flag:YB-1S102D increased the yield of mammospheres by up to 8-fold (Fig. 4B). Moreover, this effect was inhibited if the Flag:YB-1S102D–expressing cells were treated with either CD44 or CD49f siRNAs (Fig. 4B). Further, this effect is perpetuated over time through passaging as SUM 149 cells transduced to obtain stable high expression of either Flag:YB-1WT or Flag:YB-1S102D also produced a higher number of cells in mammospheres in secondary nonadherent cultures than control cells transduced with an empty vector plasmid (Flag:EV; Fig. 4B), an indicator of cell self-renewal (19, 20). Consistent with the mammosphere assays, analogous experiments with siRNA-treated MDA-MB-231 and SUM 149 cells showed both CD44 and CD49f to be required for the clonal anchorage-independent growth of both of these cell lines in soft agar (Fig. 4C). CD49f was needed for SUM 149 cells, but not for MDA-MB-231 cells, to form colonies (Fig. 4C). Conversely, forced expression of Flag:YB-1S102D increased the ability of transfected cells to grow in soft agar and this was again blocked when the expression of either CD44 or CD49f was silenced (Fig. 4C). The ability of MDA-MB-231 cells to proliferate and form extensive structures in Matrigel cultures was also inhibited when the cells were pretreated with CD44, CD49f, or YB-1 siRNAs (Fig. 4D). These findings show a critical role of YB-1 in enabling breast cancer cells to grow in three-dimensional assays in a manner that is dependent on the downstream upregulation of CD44 and CD49f. Notably, the expressions of CD44 and CD49f seem to be interdependent in these cell lines, suggesting a functional association.
Paclitaxel-treated SUM 149 cells show increased expression of YB-1 and CD44
The preceding findings intimate that the increased expression of YB-1 associated with breast cancer relapse in patients might be due to a selection of cells showing an increased expression of CD44. To investigate this possibility, we screened a panel of commonly used anticancer drugs (all at 10 μmol/L) for their ability to kill SUM 149 cells in vitro in parallel with an assessment of the proportion of CD44High cells present 72 hours later. All compounds killed >80% of the input cells and the percentage of CD44High cells among the survivors was consistently higher than that in the control cells (Supplementary Table S1). The most marked effect in this regard was obtained with paclitaxel treatment, which produced an almost 10-fold increase in the proportion of viable CD44High cells (Supplementary Table S1). Next, we determined that paclitaxel at a much lower and more clinically relevant concentration of 10 nmol/L was able to promote nuclear expression of P-YB-1S102 (Supplementary Fig. S5). Likewise, the chemotherapeutic agent cisplatin had the same effect (Supplementary Fig. S5). Related to these findings, paclitaxel increased YB-1 binding to the CD44 promoter after 48 hours (Fig. 5A), which also induced P-YB-1S102, correlating with activation of the RSK pathway based on higher levels of P-RSKS380 and a second substrate, P-GSKβS9 (Fig. 5A). The induction of this pathway then led to increased CD44 after 72 hours (Fig. 5B). In MDA-MB-231 cells stably expressing Flag:YB-1WT, after 10 nmol/L paclitaxel treatment, there was a further increase in CD44 expression and a greater amount of Flag:YB-1WT detected per cell on the whole (Fig. 5C). Further, the Flag:YB-1WT–transfected MDA-MB-231 cells formed more mammospheres in the presence of paclitaxel (10 nmol/L) as compared with control transduced cells (Fig. 5D). Finally, we sorted the SUM 149 cells for CD44High/CD24Low by FACS (Fig. 6A) and confirmed enrichment by immunofluorescence, comparing CD44High sorted cells to unsorted cells for CD44 expression (Fig. 6B). The CD44High/CD24Low cells were then transfected with YB-1 siRNAs for 48 hours and treated with paclitaxel (10 nmol/L) for 24 hours. Notably, the CD44High/CD24Low cells treated with the scrambled control were resistant to paclitaxel; however, if YB-1 was silenced, the growth of this tumor-initiating cell population was significantly attenuated based on crystal violet staining (Fig. 6C) and quantification of cell viability by Hoechst33342 staining (Fig. 6D).
Here, we present YB-1 as the first oncogene identified that induces both CD44 and CD49f. YB-1 uses CD44 and CD49f to promote self-renewal, mammosphere growth, soft-agar colony formation, and drug resistance in breast cancer cells. The YB-1/CD44/CD49f relationship was also evident in the mammary glands of YB-1 transgenic mice as well as in immortalized normal breast epithelial cells. These findings have broad implications given the important role for CD44 in mediating tumor initiation and cancer relapse. It also gives us some insight into why YB-1 may be so highly associated with breast cancer recurrence as previously reported (6). Consistent with this idea, we showed that CD44 cells were resistant to chemotherapy whereby the agents stimulate YB-1 to bind to the CD44 promoter. Furthermore, we report that eliminating YB-1 in the CD44High/CD24Low population can improve cellular response to paclitaxel.
We question whether YB-1 somehow hijacks normal breast stem cells during neoplastic progression. This extends our initial finding that YB-1 is detectable in primary mammary progenitor cells from women who have undergone reduction mammoplasties (14). In the study presented herein, we show that inducing YB-1 in immortalized breast epithelial cells (HTRY) causes an increase in CD44 mRNA and protein. Consistent with this, both CD44 and CD49f are selectively expressed on normal stem cells in the breast and other tissues (21–24). We therefore propose that early activation of YB-1 during mammary tumorigenesis involves a primitive cell population.
CD44 is a widely recognized marker of many cells with cancer-initiating (5, 25–27) and, in some cases, metastatic activities (28–30). As such, understanding its regulation has implications in tumor cell invasion, escape from apoptosis, and drug resistance. CD44 couples to a number of receptor tyrosine kinases such as epidermal growth factor receptor (EGFR), c-Met receptor, and CD49f to stimulate signal transduction (14, 31). It also partners with the oncogene RHAMM to promote signaling through the extracellular signal–regulated kinase-1/2 pathway whereby CD44 stimulates tumor cell invasion (32, 33). Importantly, CD44 expression is thought to confer drug resistance by directly inducing downstream stem cell–associated and/or drug-resistant genes, including Nanog and MDR-1, through its interaction with hyaluronan (34–36). This is attributable to activation of the PKC and signal transducer and activator of transcription-3 (STAT3) signaling pathways (34–36). Notably, as CD44 expression was increased, cells were less sensitive to paclitaxel due to STAT3 activation and consequent upregulation of MDR-1. YB-1 expression has already been reported to be associated with chemoresistance perhaps through the regulation of MDR-1 (16, 17). Bridging these studies is the evidence for the role of YB-1 in invasion (13) that may also be attributed to CD44 and CD49f. It is foreseeable that it induces other genes associated with cancer stem cells as well. We reported that YB-1 also regulates the stem cell–associated Met receptor (c-Met), EGFR, and Her-2 (erbB2; refs. 7, 14), signaling partners of CD44 and CD49f (37–40), suggesting a cooperative complex on the surface of tumor-initiating cells that activates downstream pathways such as the PI3K-Akt (40–42) and MAPK pathways (refs. 33, 43; Supplementary Fig. S6). The crosstalk between these receptors and their downstream effectors provide interesting opportunities for future therapeutic possibilities. We conclude that YB-1 inhibition may be a new approach to eliminating tumor-initiating cells and, by extension, possibly cancer recurrence.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
We thank Lisa Xu of the Child and Family Research Institute (CFRI) Core FACS Facility, as well as Lucy Yang Zhao and Peter Eirew (UBC, Vancouver, BC, Canada) for technical assistance. We would also like to acknowledge Michel Roberge (UBC, Vancouver, BC, Canada) for his assistance in accessing the Chemical Biology Network.
Grant Support: Canadian Breast Cancer Research Alliance (S.E. Dunn and C.J. Eaves), NIH grant R01-CA114017 (S.E. Dunn and I.M. Berquin), the Canadian Stem Cell Network (A. Raouf and C.J. Eaves) and the Canadian Breast Cancer Foundation (CBCF) British Columbia and Yukon Division (C.J. Eaves). K. To was a recipient of a CIHR Canada Graduate Scholarship Master's Award and A. Astanehe, a CIHR M.D./Ph.D. Award. K. To, A. Astanehe, and A.H. Davies were recipients of Michael Smith Foundation for Health Research Graduate Studentships. A.L. Stratford was awarded the CFRI and CBCF Postdoctoral Fellowships. A. Raouf held a CBCF British Columbia and Yukon Division Fellowship and a CIHR Fellowship.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).
- Received August 24, 2009.
- Revision received January 19, 2010.
- Accepted January 21, 2010.
- ©2010 American Association for Cancer Research.