Ectopic YB-1 Expression in HMECs Promotes Luminal Filling of Breast Acini In Vitro
To profile the molecular events that govern breast cancer progression, we engineered a Tet-On YB-1 expression system into nonmalignant H16N2 HMECs. Our group has extensively characterized this system previously . Cells conditionally expressing YB-1 under control of the tetracycline-inducible promoter were termed HMEC Tet-repressed YB-1 (HTRY), while a LacZ-expressing control cell line was designated HMEC Tet-repressed LacZ (HTRZ). Moreover, we developed two variant HTRY cells lines, HTRY-LT #1 and HTRY-LT #2, through sustained, long-term YB-1 expression. These cell lines expressed more YB-1 compared to HTRZ cells, as well as the normal mammary 184hTERT and MCF10A cell lines based on immunoblotting and qRT-PCR (Fig. 1A and Supporting Information Fig. S1A). The level of YB-1 achieved in the Tet-On YB-1 over-expressing cell lines was 4–10-fold higher than in the HTRZ cells and was similar to the basal-like breast cancer (BLBC) cell lines SUM149, MDA-MB-231, and MDA-MB-435/LCC6 as measured by qRT-PCR (Fig. 1B and Supporting Information Fig. S1B).
Figure 1. YB-1 over-expression in human mammary epithelial cells 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 three-dimensional 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 < .05; **, p < .01. Abbreviations: BLBC, basal-like breast cancer; YB-1, Y-box binding protein-1.
Download figure to PowerPoint
To question whether the over-expression of YB-1 in HMECs could drive phenotypic changes associated with breast cancer progression, such as luminal filling, we cultured HTRY cells on a reconstituted basement membrane. These cells became organized as three-dimensional polarized acini structures with a hollow lumen (Fig. 1C). Subsequent induction of YB-1 led to luminal filling mirroring a ductal carcinoma in situ (DCIS). Notably, the earliest luminal outgrowths were found to express the TIC marker CD44, which, at later time points, was ubiquitously expressed by cells invading the luminal space (Fig. 1C). By 8 days post-YB-1 induction, cells began to invade through the basement membrane and into the surrounding microenvironment similar to an invasive ductal carcinoma (Fig. 1C, 1D). In agreement, YB-1 expression caused HTRY and HTRY-LT cells to become invasive through Matrigel-coated Transwell chambers (Supporting Information Fig. S2). As CD44 expression was previously found to be dependent upon YB-1 serine-102 phosphorylation by RSK , we questioned whether inhibiting this activation could prevent DCIS-like luminal outgrowths. Acini treated with the RSK inhibitor BI-D1870 failed to form luminal outgrowths following YB-1 induction and instead resembled the uninduced control (Fig. 1E).
HMECs Acquire Characteristics of Stem/Progenitor-Like TICs Following YB-1 Over-Expression
Genome-wide ChIP-on-chip analysis performed previously by our group identified a potential interaction between YB-1 and the BMI1 promoter . As expected, expression of BMI1 was elevated in HTRY cells, relative to HTRZ cells, at the mRNA transcript level (Fig. 2A), which correlated with increased protein abundance (Fig. 2B). This stimulated histone H2A ubiquitylation and as a result the CDKN2A locus (encoding p16INK4a and p14ARF) was repressed. Consistent with our previous findings , the YB-1 transcriptional targets CD44 and CD49f were also more highly expressed in the HTRY cells. Notably, there was not a significant increase in BMI1, CD44, and CD49f expression in the long-term YB-1 expressing HTRY-LT #1 and HTRY-LT #2 cell lines relative to HTRY cells suggesting that the acquisition of a stem cell phenotype is an early event in the genesis of breast cancer.
Figure 2. Human mammary epithelial cells acquired tumor-initiating cell (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): Fluorescence-activated cell sorting 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 < .05; **, p < .01. Abbreviation: YB-1, Y-box binding protein-1.
Download figure to PowerPoint
Accordingly, we questioned whether TIC marker expression correlated with stem cell properties. HTRZ and HTRY cells were plated into nonadherent mammosphere cultures as an in vitro surrogate assay for self-renewal capacity. In stark contrast to HTRZ cells, HTRY cells formed mammospheres that could subsequently be dissociated and single cells repassaged as new spheres for at least five generations (Fig. 2C). Moreover, we discovered that induction of YB-1 skewed cellular differentiation along the luminal lineage (Fig. 2D). To validate that these molecular changes were not influenced by the H16N2 genetic background, we transiently transfected a FLAG:YB-1 expression vector into 184hTERT and MCF10A HMECs. Concomitant with YB-1 over-expression, both cell lines exhibited an increase in the expression of TIC-associated genes (Supporting Information Fig. S3A), which translated into their ability to form mammospheres that could be serially passaged (Supporting Information Fig. S3B) and an increased propensity for luminal differentiation (Supporting Information Fig. S3C).
To address whether all HTRY cells or only a subpopulation acquired TIC properties, we analyzed the population by flow cytometry. Notably, we detected a fraction of cells enriched for CD44 and CD49f expression (Fig. 2E). Sorting HTRY cells into two populations defined by high/high and low/low marker expression revealed that CD44+/CD49f+ cells had an enhanced ability to form mammospheres relative to the double-negative population (Fig. 2F). These results suggest that YB-1 confers TIC properties to a small subpopulation of HTRY cells.
Chromatin Remodeling by p300 Underlies the Reprogramming of HMECs into TICs
Work from other groups has highlighted the importance of the HAT protein p300 in maintaining the stem cell compartment [27, 28]. Coupled with the fact that p300 is upregulated in CD44-positive breast cancer cells , we questioned whether its expression altered histone acetylation patterns to facilitate the reprogramming of HMECs into TICs. Compared to the normal mammary 184hTERT and HTRZ cells, p300 protein was strongly expressed in the YB-1 over-expressing cell lines as well as MDA-MB-231 cells (Fig. 3A), which corresponded to higher HAT activity (Supporting Information Fig. S4A). Anacardic acid (AA), a potent inhibitor of p300 , decreased the activity of recombinant p300 (Supporting Information Fig. S4B) as well as HAT activity in total cell lysate by more than 80% (Supporting Information Fig. S4C).
Figure 3. Increased p300 HAT activity underlies the reprograming of human mammary epithelial cells into tumor-initiating cells (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 basal-like breast cancer 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 pretreated with DMSO or 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 < .05; **, p < .01. Abbreviations: AA, anacardic acid; YB-1, Y-box binding protein-1.
Download figure to PowerPoint
To deduce the mechanism responsible for p300 upregulation, we discovered that the catalytic subunit of phosphoinositide 3-kinase (PI3K), p110α, was expressed in the HTRY cells and treatment with the PI3K inhibitor LY294002 suppressed AKT activation and decreased p300 expression (Fig. 3B). Furthermore, we noted that the deleterious effect of LY294002 on p300 expression could be partially rescued by concomitant treatment with the proteasome inhibitor MG132 (Fig. 3B). This is consistent with reports that phosphorylation by AKT protects p300 from proteasome-mediated degradation . We therefore attribute the upregulation of p300 to AKT-driven protein stabilization. The finding that YB-1 over-expression ensures p300 stability is further supported by a significant decrease in HAT activity following siRNA-mediated inhibition of YB-1 or its upstream kinases RSK1 and RSK2 (Supporting Information Fig. S4D).
We next questioned whether YB-1 over-expression in HMECs influenced p300 activity. The number of p300-positive nuclei and the intensity of staining were much greater in HTRY cells relative to HTRZ cells as visualized by immunofluorescence (Fig. 3C). In support, cytonuclear fractionation confirmed that both YB-1 and p300 were localized to the nuclear compartment in HTRY (Fig. 3D) and MDA-MB-231 cells (Supporting Information Fig. S4E). As a consequence of increased p300 expression HAT activity was nearly fourfold higher in the HTRY cells compared to the HTRZ cells (Fig. 3E). Silencing EP300 (encoding p300) in HTRY cells significantly decreased HAT activity suggesting that p300 is the predominant HAT enzyme present in these cells (Fig. 3E). As a control we report that 184hTERT cells have low p300 expression and low HAT activity compared to MDA-MB-231 cells, which express more p300 and have much higher HAT activity (Fig. 3E).
The p300 protein epigenetically regulates gene expression by acetylating lysine residues on histone proteins. Therefore, as a direct measure of p300 activity we assessed the extent of histone acetylation by immunoprecipitation of histone H3. Using an antiacetylated-lysine antibody we detected enrichment in the pool of acetylated histone H3 in HTRY cells relative to HTRZ cells (Fig. 3F). To refine this analysis, we next evaluated acetylation of histone H3 at lysine 9 (AcH3-K9) along promoter-centered chromatin of TIC-associated genes. The ninth lysine residue (H3K9) of histone H3 is a preferential substrate of p300 . DNA purified after immunoprecipitation with anti-AcH3-K9 antibody was evaluated by PCR using primers targeting the length of the BMI1, CD44, and CD49f promoters. Higher levels of promoter-associated histone H3-K9 acetylation were detected in HTRY cells compared to HTRZ cells (Fig. 3G).
To demonstrate that inhibition of p300 could prevent the reprogramming of HMECs into TICs, we silenced its expression and/or activity using an siRNA and pharmacologic approach. Silencing EP300 yielded a decrease in CD44 and CD49f expression in both HTRY cells (Fig. 3H) and MDA-MB-231 cells (Supporting Information Fig. S4F). We further demonstrate that inhibition of p300 activity using anacardic acid correlated with loss of BMI1, CD44, and CD49f mRNA expression (Fig. 3I). Quenching p300 activity resulted in fewer, and smaller, primary mammospheres that could not be serially passaged (Fig. 3J).
Chromatin Remodeling Permits YB-1 to Transcriptionally Regulate BMI1
We next questioned whether p300-mediated chromatin relaxation was a prerequisite for YB-1 to bind TIC-associated gene promoters. In addition to CD44 and CD49f, we now show that YB-1 binds to the BMI1 promoter in both HTRY and MDA-MB-231 breast cancer cell lines using conventional ChIP (Supporting Information Fig. S5A). Consistent with the role of BMI1 in the development of cancer its expression was inversely correlated with p16INK4a when normal mammary epithelial cells were compared to the BLBC cell lines SUM149 and MDA-MB-231 (Supporting Information Fig. S5B). Furthermore, in ChIP assays using an anti-YB-1 antibody, pretreating HTRY cells with AA to inhibit p300 activity prevented YB-1 binding to the BMI1 and CD49f promoters as well as one region on the CD44 promoter (Fig. 4A). This suggests that p300-mediated chromatin remodeling is upstream of YB-1 promoter binding and gene transcription.
Figure 4. YB-1 transcriptionally regulated BMI1 to enhance self-renewal capacity. (A): ChIP analysis of HTRY cells pretreated with DMSO or 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. UN cells served as a control. Immunoblotting confirmed BMI1 knockdown. (E): UN HTRY cells transfected with 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 were confirmed by immunoblotting. Data represented as mean ± SEM. p values were determined using t test. **, p < .01. Abbreviations: AA, anacardic acid; EV, empty vector; UN, uninduced; YB-1, Y-box binding protein-1.
Download figure to PowerPoint
Consistent with the novel finding that the induction of YB-1 increased BMI1 expression as described above, we also report that siRNA-mediated loss of YB-1 significantly decreased the expression of BMI1 in MDA-MB-231 as well as HTRY-LT #2 cells (Supporting Information Fig. S5C). As we have previously demonstrated that YB-1 Ser-102 phosphorylation by RSK is a prerequisite for its DNA binding and transcriptional activity [2, 32, 33], we inhibited the kinase using siRNA or the small molecule inhibitor BI-D1870. In HTRY cells, suppression of RSK1/2 expression and/or activity led to a decrease in activated pYB-1S102 resulting in loss of BMI1 and rescue of p16INK4a (Fig. 4B).
BMI1-mediated silencing of p16INK4a has been associated with a loss of G1/S checkpoint fidelity . In accordance, analysis of cell cycle kinetics revealed that the proportion of cells in G0/G1 was shifted in the YB-1 over-expressing cell lines compared to HTRZ cells (Fig. 4C). This could explain why cellular doubling time decreased from 118 hours in the HTRZ cells to 101-, 72-, and 88-hours in the HTRY, HTRY-LT #1, and HTRY-LT #2 cells, respectively (Supporting Information Fig. S6A). Consistent with this finding, HTRY cells had a higher proliferative rate relative to HTRZ cells based on EdU incorporation (Supporting Information Fig. S6B). We were able to partially restore G1/S checkpoint activity following knockdown of BMI1 in HTRY cells using siRNA (Fig. 4D). In support, loss of BMI1 suppressed the growth of MDA-MB-231 cells over 72 hours (Supporting Information Fig. S6C). This was due, in part, to an increase in nuclear p16INK4a accumulation (Supporting Information Fig. S7A, S7B). These observations suggest that YB-1 expression potentiated G1/S checkpoint slippage via a BMI1-dependent mechanism.
The above-mentioned findings prompted us to question whether the enhanced self-renewal potential of HTRY cells was a direct consequence of BMI1 expression. The mammosphere-forming capacity of uninduced HTRY cells was increased from 12 ± 2 to 73.7 ± 16.1 following transient transfection with a pMIN:BMI1 expression vector (Fig. 4E). Conversely, siRNA-mediated silencing of BMI1 in HTRY cells decreased their ability to form mammospheres by nearly 80% (from 104 ± 18.5 to 22.7 ± 2.8) (Fig. 4E). Notably, loss of BMI1 reduced the CD44+/CD24− stem cell population by ∼25% in MDA-MB-231 cells (Supporting Information Fig. S8). Perplexingly, no change was observed in CD44 or CD49f mRNA expression; however, CD44 protein level decreased (data not shown). Thus, we conclude that YB-1 cooperates with p300 and BMI1 to promote and sustain a population of cells with tumor-initiating potential.
Sustained Upregulation of YB-1 Leads to Full Transformation and Tumor Initiation
The ability of YB-1 to elicit phenotypes associated with malignant progression prompted us to investigate whether the oncogene could confer full transformation and ultimately tumor initiation. HTRY cells failed to form colonies in soft agar indicating that a single short-term pulse of YB-1 was insufficient to allow for anchorage-independent growth (Fig. 5A). However, long-term expression of YB-1 in the HTRY-LT #1 and HTRY-LT #2 cell lines led to colony formation at a level comparable to SUM149 and MDA-MB-231 cells (Fig. 5A). At the molecular level, HTRY-LT cells displayed elevated RSK1 and RSK2 protein expression relative to HTRZ cells (Fig. 5B). This was particularly interesting as previous work from our group and others has established that BLBCs are dependent on RSK2 signaling for growth and survival [35, 36]. To understand why these cells formed colonies in soft agar we asked whether this involved telomerase (hTERT) activity given its established role in neoplastic transformation. Notably, hTERT activity was detected in the HTRY-LT cell lines, but less so in HTRZ and HTRY cells (Fig. 5C).
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 (six mice/group). (G): qRT-PCR analysis of human YB-1 and BMI1 in a representative tumor isolated at Day 86 from NSG mice inoculated with HTRZ and HTRY-LT #2 cells. Gene expression was normalized using eukaryotic 18S rRNA. (H): 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 < .01. Abbreviations: H&E, hematoxylin-eosin; YB-1, Y-box binding protein-1.
Download figure to PowerPoint
We endeavored to uncover the minimal combination of genes necessary for the evolution of HTRY cells into fully transformed HTRY-LT cells. Uninduced HTRY cells acquired the ability to form soft agar colonies following YB-1/RSK2 double-transfection. Expression of YB-1 or RSK2 alone did not significantly enhance soft agar colony formation (Fig. 5D). In a reciprocal experiment, we silenced YB-1 and RSK2 in the HTRY-LT cell lines using siRNA. This led to a repression in the number of soft agar colonies. Knockdown of RSK1 had only a moderate effect highlighting the unique importance of the RSK2 isoform (Fig. 5E). Taken together, these results suggest that interplay between YB-1, RSK2, and hTERT is necessary for transformation. One cannot exclude that the H16N2 genetic background may have influenced the transformative potential of these cells; therefore, we introduced YB-1 into a second mammary epithelial cell line, 184hTERT. Following stable transfection, 184hTERT clones emerged that expressed YB-1, RSK1, and RSK2 to a similar level observed in MDA-MB-231 cells (Supporting Information Fig. S9A). Moreover, compared to cells transfected with empty vector, 184hTERT YB-1-expressing cells exhibited increased soft agar colony formation (Supporting Information Fig. S9B) and invasion through Matrigel-coated Transwell chambers (Supporting Information Fig. S9C). Together these data suggest that the addition of RSK2 enabled YB-1-expressing cells to become fully transformed through collaboration with hTERT.
Given the observation that RSK1 and RSK2 levels were increased in the HTRY-LT cell lines we asked whether they would be sensitive to pan-RSK inhibition. Treating the HTRY-LT cell lines with BI-D1870 suppressed YB-1 activation (Supporting Information Fig. S10A). This resulted in decreased growth; however, the drug had no effect on the HTRZ cells at low dose (≤2 µM) (Supporting Information Fig. S10B). This growth inhibitory effect translated into a significant reduction in the ability of BI-D1870-treated HTRY-LT cells to form soft agar colonies (Supporting Information Fig. S10C). Moreover, the capacity of these cells to grow as mammospheres was also abrogated (Supporting Information Fig. S10D). This strongly implies that BI-D1870 is not only eradicating bulk tumor cells but also the TIC subpopulation. Importantly, we demonstrate that treatment with BI-D1870 induced apoptosis in the HTRY-LT cell lines as measured by annexin V positivity (Supporting Information Fig. S10E). Thus, this model further demonstrates the potential for RSK inhibitors to control the growth of transformed cells that depend on YB-1.
To assess the tumorigenicity of HTRY-LT cells in vivo, we injected them into the mammary glands of NSG mice. HTRY-LT #1 and HTRY-LT #2 cells were characterized as expressing seven- and fourfold higher level of YB-1 mRNA, respectively, compared to HTRZ cells immediately prior to in vivo transplantation (Supporting Information Fig. S11A). RSK2 was also elevated (Supporting Information Fig. S11B), while p16INK4a was repressed (Supporting Information Fig. S11C), in the HTRY-LT cells relative to HTRZ cells. At 142 days post-transplantation, none of the HTRZ group grew tumors; however, all the mice injected with HTRY-LT #1 and HTRY-LT #2 cells developed mammary tumors (Fig. 5F and Supporting Information Fig. S11D) ranging in weight from 40 to 1,480 mg (Supporting Information Fig. S11E). The tumor expressed 16-fold higher human-YB-1 mRNA and 2.3-fold higher human-BMI1 mRNA relative to mammary glands injected with HTRZ cells (Fig. 5G). In addition, human EP300 was readily detectable in the tumors by qRT-PCR (Ct = 27) but undetectable in mammary glands from the HTRZ group (data not shown). Microscopic examination of mammary fat pads transplanted with HTRY-LT #1 cells showed the presence of solid masses of neoplastic cells (Fig. 5H) with a high proliferative index based on human Ki67 staining (Supporting Information Fig. S11F). The tumor cells also expressed high levels of p300 and BMI1 protein at 86 days postinjection and the levels were sustained out to 142 days (Fig. 5H). YB-1 protein was also expressed at both time points (Fig. 5H). To ascertain whether HTRY-LT cells could form tumors at limiting dilution, we injected 105, 104, and 102 HTRY-LT #1 and HTRZ cells bilaterally into the mammary gland of NOD/SCID mice. As few as 100 HTRY-LT cells were sufficient for tumor initiation (Supporting Information Fig. S12A). DCIS-like lesions and small palpable tumor masses were present in mammary glands transplanted with HTRY-LT cells (Supporting Information Fig. S12B, S12C). While tumors developed in both the NSG and NOD/SCID strains by 90 days postinjection, they were notably larger in the former. We suspect that this is a consequence of the lack of a functional immune system in NSG mice and the coinjection of cells with Matrigel and collagen I to recapitulate the human mammary gland microenvironment.
YB-1 Transforms HMECs into Cells with a BLBC Subtype
As breast cancer can be classified into distinct subtypes with differential aggressiveness and prognosis , we questioned which subtype the HTRY-LT cell line represented. During YB-1-mediated transformation, the HTRY-LT cells gained expression of epidermal growth factor receptor (EGFR) and lost expression of ESR1 (encoding ER) and PGR (encoding PR) as measured by qRT-PCR-based gene expression profiling (Fig. 6A) and immunoblotting (Fig. 6B). Negligible level of ERBB2 (HER2) was present in the HTRY-LT cell lines. Moreover, ex vivo analysis of a HTRY-LT tumor from NSG mice revealed that it expressed EGFR, but lacked ESR1, PGR, and ERBB2 (Fig. 6C). Therefore, upon YB-1 induction HMECs were transformed from hormone receptor positive to triple-negative (lacking ER, PR, and HER2). More specifically, the transformed cells represent the BLBC subtype, which is clinically defined as ER, PR, HER2-negative and EGFR and/or CK5/6-positive . The clinical relevance of our model can be appreciated by the fact that YB-1 transcript expression was significantly higher in high grade ER-negative tumors (p < .0001; Supporting Information Fig. S13A, S13B), with the highest expression observed in the basal-like subtype (p < .0001; Fig. 6D) when analyzed in a cohort of 1,881 breast cancer patients using the Gene expression-based Outcome for Breast cancer Online algorithm .
Figure 6. HTRY-LT cells were molecularly classified as basal-like breast cancer. (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.
Download figure to PowerPoint
To further support the observation that YB-1 is inversely correlated with the hormone receptors we characterized YB-1 transgenic mouse tissues . Tissues taken from 6- to 8-month-old TG2 mice, previously characterized as having preneoplastic changes by this time [4, 26], expressed high YB-1 as compared to age-matched wild-type (WT) mice (Fig. 7A). Notably, we detected an increase in both human and murine YB-1 transcript in the TG2 tissue relative to WT (Supporting Information Fig. S14A). This could be a consequence of human YB-1 inducing murine YB-1 via an autoregulatory mechanism . It is important to note that the human YB-1 qRT-PCR probe exhibited slight cross-reactivity with murine YB-1, which rationalizes why human YB-1 was detected in WT mouse tissue (Supporting Information Fig. S14B). NanoString gene expression profiling of the TG2 mammary glands showed a loss of ER1, ER2, PR, and CDKN2A, while a gain of the luminal lineage marker KRT18 and the TIC genes CD44 and ITGA6 (encoding CD49f) (Fig. 7B). These changes are consistent with the Tet-On expression model described above. However, in contrast to the human YB-1-driven system, EP300 and BMI1 levels were constant. This could be due, in part, to the timing in which the lesions were taken because by 8 months preneoplastic lesions were evident [4, 26].
Figure 7. YB-1 transgenic mice form hormone-receptor negative preneoplastic lesions in advance of tumor formation. (A): qRT-PCR analysis of hYB-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 human mammary epithelial cells into a TNBC. The epigenetic alterations following YB-1 induction in the HTRY cells are shown in detail. Abbreviations: BLBC, basal-like breast cancer; hTERT, human telomerase reverse transcriptase; YB-1, Y-box binding protein-1.
Download figure to PowerPoint
Using reverse phase protein arrays (RPPA) we validated the association between YB-1 and BLBC in a clinical cohort of 710 invasive breast cancers. By RPPA, YB-1 was inversely correlated with ER (r = −.132, p = .00041), PR (r = −.157, p = 2.7 × 10−5), and HER2 (r = −.163, p = 1.3 × 10−5) (Supporting Information Table S1). Moreover, we addressed which signaling pathways are associated with YB-1 in primary breast cancers using this platform. We discovered several members of the MAPK signaling cascade to be correlated with YB-1 expression including MEK, pMEK, pS6 ribosomal protein, and pRSK (Supporting Information Table S1). This is a key finding because the MAPK pathway is linked to BLBC . These clinical data support our finding that the induction of YB-1 in vitro converts hormone receptor-positive cells into a triple-negative cancer, which is consistent with its expression in primary BLBC.
In summary, our data convey that HMEC transformation by YB-1 occurs in a step-wise process. During an initial premalignant phase, YB-1-mediated activation of the HAT protein p300 alters the histone acetylation landscape. The relaxation of promoter-centered chromatin allows for YB-1 to bind and transcriptionally regulate BMI1 to instill stem/progenitor-like phenotypes, such as enhanced self-renewal and multipotent differentiation. Over time, pressures exerted by YB-1 leads to the emergence of tumorigenic cells with a BLBC subtype that express high levels of RSK2 and hTERT (Fig. 7C).