YB-1 transforms human mammary epithelial cells through chromatin remodeling leading to the development of basal-like breast cancer - PubMed (original) (raw)

. 2014 Jun;32(6):1437-50.

doi: 10.1002/stem.1707.

Kristen M Reipas, Mary Rose Pambid, Rachel Berns, Anna L Stratford, Abbas Fotovati, Natalie Firmino, Arezoo Astanehe, Kaiji Hu, Christopher Maxwell, Gordon B Mills, Sandra E Dunn

Affiliations

YB-1 transforms human mammary epithelial cells through chromatin remodeling leading to the development of basal-like breast cancer

Alastair H Davies et al. Stem Cells. 2014 Jun.

Abstract

There is growing evidence that cancer-initiation could result from epigenetic changes. Y-box binding protein-1 (YB-1) is a transcription/translation factor that promotes the formation of tumors in transgenic mice; however, the underlying molecular events are not understood. To explore this in a human model system, YB-1 was expressed in mammary epithelial cells under the control of a tetracycline-inducible promoter. The induction of YB-1 promoted phenotypes associated with malignancy in three-dimensional breast acini cultures. This was attributed to YB-1 enhancing the expression and activity of the histone acetyltransferase p300 leading to chromatin remodeling. Specifically, this relaxation of chromatin allowed YB-1 to bind to the BMI1 promoter. The induction of BMI1 engaged the Polycomb complex resulting in histone H2A ubiquitylation and repression of the CDKN2A locus. These events manifested functionally as enhanced self-renewal capacity that occurred in a BMI1-dependent manner. Conversely, p300 inhibition with anacardic acid prevented YB-1 from binding to the BMI1 promoter and thereby subverted self-renewal. Despite these early changes, full malignant transformation was not achieved until RSK2 became overexpressed concomitant with elevated human telomerase reverse transcriptase (hTERT) activity. The YB-1/RSK2/hTERT expressing cells formed tumors in mice that were molecularly subtyped as basal-like breast cancer. We conclude that YB-1 cooperates with p300 to allow BMI1 to over-ride p16(INK4a) -mediated cell cycle arrest enabling self-renewal and the development of aggressive breast tumors.

Keywords: Breast cancer; Cancer stem cells; Epigenetics; Human mammary epithelial cells; Neoplastic cell transformation; Y-box binding protein-1.

© 2014 AlphaMed Press.

PubMed Disclaimer

Conflict of interest statement

Conflicts of Interest

No potential conflicts of interest.

Figures

Figure 1

Figure 1. YB-1 over-expression in HMECs drove migration and luminal translocation in breast acinar structures

(a) Immunoblot of YB-1 in the normal mammary epithelial MCF10A and 184hTERT cell lines compared to HTRZ and the YB-1-induced HTRY, HTRY-LT #1, and HTRY-LT #2 cell lines. (b) Immunoblot of YB-1 in HTRZ and HTRY cells compared to BLBC cell lines. (c) HTRY cells were grown as 3D acini on a reconstituted basement membrane. Following YB-1 induction, acinar structures were immunostained with ZO-1 (luminal), CD49f (basolateral), and CD44 antibodies. DAPI marked the nuclei. (d) Acinar structures (60 per time point) were evaluated for luminal filling and invasive outgrowths at the times indicated. (e) Acini were grown as above; however, YB-1 was induced along with the addition of DMSO or BI-D1870 (10 μM). At 96 hours luminal filling was evaluated. Scale bars represent 100 μm. UN, uninduced control. Data represented as mean ± SEM. P values were determined using t test. *, P <0.05; **, P < 0.01.

Figure 2

Figure 2. HMECs acquired TIC characteristics following short-term YB-1 over-expression

(a) qRT-PCR analysis of TIC-associated genes in HTRZ, HTRY, and HTRY-LT #1 cells. (b) Immunoblot of BMI1, ubiquitinated histone H2A (ubH2A), p16INK4a, and p14ARF, in addition to the TIC markers CD44 and CD49f, in HTRZ and HTRY cells. (c) HTRZ and HTRY cells grown in mammosphere cultures and serially passaged. Doxycycline was replenished at each generation. (d) Differentiation culture of cells from secondary mammospheres. Colonies (x = 50) were evaluated for markers of luminal (CK18) and basal (CK14) differentiation using immunofluorescence. (e) FACS histograms depicting two HTRY cell populations defined as CD44/CD49f double-positive and CD44/CD49f double-negative. (f) Mammosphere assay of sorted HTRY cells. Data represented as mean ± SEM. P values were determined using t test. *, P <0.05; **, P < 0.01.

Figure 3

Figure 3. Increased p300 HAT activity underlies the reprograming of HMECs into TICs

(a) Immunoblot analysis of p300 in normal mammary MCF10A and 184hTERT cell lines compared to HTRZ, HTRY, HTRY-LT #1, and HTRY-LT #2 cells and the BLBC cell line MDA-MB-231. (b) Immunoblot analysis of p300 and PI3K signaling components in HTRY cells treated for 24 hours with DMSO, LY294002 (LY, 20 μM), or LY in combination with MG132 (MG, 5 μM). (c) Immunofluorescence of p300 localization in HTRZ and HTRY cells. Arrows denote p300-positive nuclei (visualized by DAPI). Scale bar represents 100 μm. (d) Immunoblot analysis of cytoplasmic and nuclear fractions following siRNA-mediated p300 knockdown (5 nM) in HTRY cells. CREB and vinculin were used to assess the purity of the nuclear and cytoplasmic fractions, respectively. (e) HAT activity in nuclear lysate from HTRZ, HTRY, and HTRY cells treated for 96-hours with siRNA targeting EP300. 184hTERT and MDA-MB-231 were a negative and positive control for p300 activity, respectively. (f) Histone H3 (HH3) immunoprecipitation followed by immunoblotting to measure acetylated lysine residues. Protein levels were evaluated by densitometry (normalized to HTRZ). (g) ChIP targeting the promoters of BMI1, CD44, and CD49f in induced HTRZ and HTRY cells. DNA templates were pulled down with acetyl-histone H3 (Lys9) or nonimmune IgG antibody. GAPDH served as a control. (h) Immunoblot analysis of TIC markers in HTRY cells treated with EP300 siRNA for 96 hours. (i) qRT-PCR analysis of mRNA transcript in HTRY cells pre-treated with DMSO or anacardic acid (AA) for 4 hours prior to induction. (j) HTRY cells serially passaged as mammospheres in the presence of DMSO or AA. Primary mammospheres are shown. Scale bar represents 200 μm. Data represented as mean ± SEM. P values were determined using t test. *, P <0.05; **, P < 0.01.

Figure 4

Figure 4. YB-1 transcriptionally regulated BMI1 to enhance self-renewal capacity

(a) ChIP analysis of HTRY cells pre-treated with DMSO or anacardic acid (AA) for 4 hours prior to induction. DNA templates were pulled down with YB-1 or nonimmune IgG antibody and different promoter regions (ChIP a and ChIP b) were amplified using primers flanking YB-1 binding sites in the BMI1, CD44, and CD49f promoters. (b) Immunoblot analysis of HTRY cells treated with RSK1/2 siRNA for 96 hours or BI-D1870 for 24 hours. (c) The percentage of cells in each phase of the cell cycle at the indicated time points after plating was quantified by DNA content based on Hoechst 33342 intensity using an ArrayScan VTI. (d) Cell cycle profile of HTRY cells treated with scrambled control (scr) or BMI1 siRNA for 96 hours was measured using an ArrayScan VTI. Uninduced (UN) cells served as a control. Immunoblotting confirmed BMI1 knockdown. (e) Uninduced (UN) HTRY cells transfected with empty vector (EV) or BMI expression plasmid and induced HTRY cells treated with scrambled (scr) or BMI1 siRNA for 96 hours were grown in mammosphere cultures. BMI1 over-expression and knockdown was confirmed by immunoblotting. Data represented as mean ± SEM. P values were determined using t test. **, P < 0.01.

Figure 5

Figure 5. Synergism between YB-1, RSK2, and hTERT conferred complete transformation

(a) Quantification of HTRZ, HTRY, and HTRY-LT cell growth under anchorage-independent conditions. MDA-MB-231 and SUM149 cells acted as a positive control. (b) Immunoblot assessing RSK expression and activation. (c) Telomerase activity in HTRZ, HTRY, and HTRY-LT cell lysate. 184hTERT cells were used as a positive control. (d) Soft agar colony growth of HTRY cells expressing YB-1, RSK2, or YB-1 and RSK2. Uninduced HTRY cells served as the control. Immunoblotting confirmed transgene expression at 96 hours post-transfection. (e) Quantification of soft agar colony growth following treatment of HTRY-LT cells with scrambled (scr) or YB-1, RSK1, and RSK2 siRNA. Immunoblotting confirmed gene silencing. (f) The ability of HTRZ, HTRY-LT #1, and HTRY-LT #2 cells to form palpable tumors when transplanted into the mammary fat pad of NSG mice (6 mice/group). (g) qRT-PCR analysis of human YB-1 and BMI1 in tumors isolated from NSG mice inoculated with HTRZ and HTRY-LT #2 cells. Gene expression was normalized using eukaryotic 18S rRNA. (h) Hematoxylin-eosin (H&E) and immunoperoxidase staining with p300, BMI1, and YB-1 antibody in tumor tissue explanted 86 days and 142 days after implantation of HTRY-LT #1 cells into the mammary fat pad of NSG mice. The contralateral naïve gland served as a control. Scale bar represents 500 μm. Data represented as mean ± SEM. P values were determined using t test. **, P < 0.01.

Figure 6

Figure 6. HTRY-LT cells were molecularly classified as BLBC

(a) qRT-PCR and (b) immunoblot analysis of subtype biomarkers. MDA-MB-231 (TNBC), MCF-7 (ER+), and BT474 (PR+/HER2+) cells were used as controls. (c) qRT-PCR analysis of subtype biomarkers in HTRY-LT #2-derived xenografts from NSG mice. Data are reported relative to the appropriate control for each gene: MDA-MB-231 (EGFR+), T47D (ER+), and BT474 (ERBB2+ and PR+). (d) Box plot analyses of YB-1 expression among HU subtypes, Basal (n = 194), HER2 (n = 78), Luminal A (n = 221), Luminal B (n = 122), Normal-like (n = 121), and unclassified (n = 191). Data obtained using Gene expression based Outcome for Breast cancer Online (GOBO).

Figure 7

Figure 7. YB-1 transgenic mice form hormone-receptor negative tumors

(a) qRT-PCR analysis of h_YB-1_ mRNA transcript in wild-type (WT) and TG2 YB-1 transgenic mice normalized to 488 WT. (b) Gene expression analysis of YB-1 transgenic TG2 mice (n = 4) relative to the WT controls (n = 3) using the NanoString nCounter platform (red, high expression; green, low expression). (c) Depiction of the genetic and phenotypic features that define each step of YB-1-driven transformation of HMECs into a TNBC. The epigenetic alterations following YB-1 induction in the HTRY cells are shown in detail.

References

    1. Habibi G, Leung S, Law JH, et al. Redefining prognostic factors for breast cancer: YB-1 is a stronger predictor of relapse and disease-specific survival than estrogen receptor or HER-2 across all tumor subtypes. Breast Cancer Res. 2008;10:R86. - PMC - PubMed
    1. Wu J, Lee C, Yokom D, et al. Disruption of the Y-box binding protein-1 results in suppression of the epidermal growth factor receptor and HER-2. Cancer research. 2006;66:4872–4879. - PubMed
    1. Stratford AL, Fry CJ, Desilets C, et al. Y-box binding protein-1 serine 102 is a downstream target of p90 ribosomal S6 kinase in basal-like breast cancer cells. Breast Cancer Res. 2008;10:R99. - PMC - PubMed
    1. To K, Fotovati A, Reipas KM, et al. Y-box binding protein-1 induces the expression of CD44 and CD49f leading to enhanced self-renewal, mammosphere growth, and drug resistance. Cancer research. 2010;70:2840–2851. - PMC - PubMed
    1. Gupta PB, Chaffer CL, Weinberg RA. Cancer stem cells: mirage or reality? Nature medicine. 2009;15:1010–1012. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources