Reversal by RARα agonist Am580 of c-Myc-induced imbalance in RARα/RARγ expression during MMTV-Myc tumorigenesis

Antibody sources were as follows: anti-p27 and anti-E-cadherin (BD Transduction Labs, Hoboken, NJ, USA); anti-CYP26A1 and anti-CRBP1 (Santa Cruz Biotechnology, CA); anti-RARα, -RARβ and -RARγ (Abcam, Cambridge, MA, USA), anti-Akt, anti-pAkt anti-pErk, anti-Erk and anti-pRB (Cell Signaling, Beverly, MA, USA); anti-GAPDH (Calbiochem, Gibbstown, NJ, USA); anti-tubulin (Sigma Diagnostics, St. Louis, MO, USA).

Tumor samples were mechanically homogenized in radioimmunoprecipitation assay (RIPA) buffer (1% Triton X-100, 10 mM Tris, pH 8, 140 mM NaCl, and 0.1% SDS). Primary cultures derived from wild type FVB mice mammary gland epithelium were washed in PBS, pH 7.4, and lysed with RIPA buffer. Immunoblotting was performed following standard procedures as described previously [39].

Three-month-old uniparous MMTV-Myc female mice (NCI Frederick Mouse Repository, Frederick, MD, USA) (30 mice/group) were fed with 0.3 mg/kg/day of the RARa agonist Am580 (4-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthyl)carboxamido]benzoic acid), which was kindly provided by Dr K Shudo (Research Foundation Itsuu Laboratory, Tokyo, Japan). Am580 was mixed into their regular diet by the vendor (Purina 5053, Richmond, IN, USA). Food consumption was measured to calculate the amount of Am580 to be added to the diet to achieve the daily dose as described previously [39]. Regular diet was used as the control. Because the objective was to study the effect of Am580 on tumor initiation and development, mice that developed tumors within the first month were removed from the study on the assumption that their tumors had developed before treatment began. Mice were palpated twice weekly and the onset of tumor development was recorded. Once palpable, the tumor sizes were measured weekly in two dimensions and volumes calculated using the equation Vol = Dxd²/2 (where D = major diameter and d = minor diameter). Tumor-free survival was calculated from Kaplan-Meier curves, and statistical significance was determined using the log-rank test for survival and the t-test for tumor growth. Metastasis dissemination was evaluated by dissecting the lungs from euthanized mice and inspecting the Bouin-fixed (Sigma, St. Louis, MO, USA) lung surface for lesions using a stereoscope (Nikon SMZ800 stereoscope X3 to X5). For xenograft experiments, 8-week-old syngenic FVB mice were used (NCI Frederick Mouse Repository). Cells or tumor fragments were inoculated into the mammary fat pad of the inguinal mammary glands (gland numbers 4 and 8) under soft anesthesia and analgesia in accordance with the Institutional Animal Care and Use Committee (IACUC) guidelines.

Tumor samples were fixed in 10% buffered formalin for 24 h, transferred to 70% ethanol and kept at 4ºC until use. Sections were prepared from eight tumors per group, subjected to standard antigen retrieval and incubated with primary antibody overnight at 4ºC. Sections were processed using the VectaStain ABC Elite Kit (Vector Laboratories, Burlingame, CA, USA), signals were detected using the Metal Enhanced DAB Substrate Kit (Pierce Laboratories, Rockford, IL, USA) and sections counterstained with Harris Hematoxylin Solution (Sigma Diagnostics).

After euthanasia by CO₂ overdose, tumors from untreated female MMTV-Myc mice and mammary glands from wild-type-FVB female mice were removed by dissection and minced into fragments, which were then digested with 5 ml of 1.5 mg/ml collagenase (Sigma, St. Louis, MO, USA) in PBS containing 25 mg/ml BSA (Sigma), 100 mM Ca²⁺ and 100 mM Mg²⁺ per approximate 500 mg of tissue at 37ºC for 30 to 45 minutes with gentle agitation. Cells were maintained in DMEM-F12 (Cellgro, Manassas, VA, USA) containing 5% (MMTV-Myc cells) or 10% (wt-FVB cells) fetal bovine serum (FBS) and 4 μg/ml insulin (Sigma Diagnostics). Immortalized nontumorigenic human MCF-10A breast epithelial cells, which were a generous gift from Dr J Brugge (Department of Cell Biology, Harvard Medical School, Boston, MA, USA), were maintained as described by Debnath et al. [50]. Human breast cancer MCF-7 (ER⁺) and MDA-MB-231 (ER^-) cells were obtained from the American Tissue Culture Collection (ATCC, Manassas, VA, USA). Both cell lines were propagated using ATCC protocols. FVB or Myc cells were transfected with either pSG5-empty, pSG5-RARγ, pSG5-mCRBP1 (kindly provided by Dr Chambon, IGBMC, Strasbourg, France), pcDNA3, or pcDNA3-h-c-Myc (Addgene, Ricci et al. [51]) vectors using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) in Opti-MEM (Gibco, Carlsbad, CA, USA). At 24 h after transfection, cells were treated for 16 h with 1 μM ATRA (Sigma, St. Louis, MO, USA) in dimethyl sulfoxide (DMSO) (0.01% final concentration; Sigma Diagnostics).

Primary Myc cell suspensions obtained after a 45-minute collagenase digestion (1.5 mg/ml collagenase and 25 mg/ml BSA in PBS plus 100 mM Ca²⁺ and 100 mM Mg²⁺) of MMTV-Myc tumor fragments were grown in DMEM/F12 medium supplemented with 5% horse serum, 100 ng/ml cholera toxin, 5 mg/ml insulin, 0.5 mg/ml hydrocortisone, 1% Pen/Strep, 1% glutamine (Gibco), 1% non-essential amino acids (Invitrogen) and 20 ng/ml EGF (PeproTech, Rock Hill, NJ, USA). Cells were transfected with shRARγ(2 μg) in 35-mm dishes, following the manufacturer's instructions (Open Biosystems, Huntsville, AL, USA) and seeded (3 × 10³/well) in quadruplicate onto Matrigel^® (BD Bioscience, San Jose, CA, USA) beds in 8-well culture slides (BD Bioscience, Bedford, MA, USA) to prepare three-dimensional cultures as described by Debnath et al. [50]. The media was changed every 48 h for 8 consecutive days. An additional shRARγ transfection was done at day 4 to maintain RARγ knockdown. Colony morphology was determined by phase-contrast microscopy. RARs were silenced using shRNAs from Open Biosystems (Open Biosystems, Huntsville, AL, USA) shRARα (Oligo ID: V2MM_6881) , shRARβ (Oligo ID: V2HS_239292) or shRARγ (Oligo ID: V2MM_62330). Scrambled shRNA (Open Biosystems, Huntsville, AL, USA) was used as the control.

Primary Myc cells (2 × 10⁴) were seeded in triplicate in 6-well culture dishes and allowed to attach overnight. They were then washed with culture medium and treated with the RARγ antagonist SR11253 (2-(4-carboxyphenyl)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthyl)-1,3-dithiolane) at increasing doses (10, 50 and 250 nM) or with DMSO (0.001% final concentration) alone, and then detached with 0.05% trypsin (Gibco) and counted every 24 h for 4 days. Statistical significance was determined by t-test. Following the same protocol described above for Myc, MCF-10A, MCF-7 and MDA-MB-231 cells were treated with 1 μM ATRA (RAR pan-agonist), 200 nM Am580 (RARα agonist), 100 nM CD437 (the RARγ/β agonist 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalenecarboxylic acid, AHPN) (Sigma), 30 nM BMS961 (the RARγ agonist 3-fluoro-4-[[2-hydroxy-2-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-2-naphthalenyl)acetyl]amino]benzoic acid) (Tocris, Bristol, UK) and SR11253 or DMSO (0.001% final concentration) alone as the control.

Total RNA from FVB mammary gland epithelial cells was isolated using the RNeasy mini kit (Qiagen, Valencia, CA, USA) and reverse-transcribed using the iScript cDNA synthesis kit (Bio-Rad, Philadelphia, PA, USA). cDNA was amplified by real-time PCR using an iQ5 Real-Time PCR detection systems kit (Bio-Rad) and SYBR green PCR master mix (Applied Biosystems, Carlsbad, CA, USA). Primer sequences were: RARγ1-sense: 5'-TGG GGC CTG GAT CTG GTT AC-3', RARγ1-antisense: 5'-TTC ACA GGA GCT GAC CCC AT; RARγ2-sense: 5'-GCC GGG TCG CGA TGT ACG AC and RARγ2-antisense: 5'- TTC ACA GGA GCT GAC CC CAT; RARβ2-sense: 5'-ATG GAG TTC GTG GAC TTT TCT GTG-3' and RARβ2-antisense: 5'-CTC GCA GGC ACT GAC GCC AT-3'; CRBP1-sense: 5'-ACG GGT ACT GGA AGA TGC TG-3' and CRBP1-antisense: 5'-CCA TCC TGC ACG ATC TCT TT-3'; Hmq-c-Myc-sense: 5'-AGC GAC TCT GAG GAG GAA CA-3' and Hmq-c-Myc-antisense: 5'-AGT GGG CTG TGA GGA GGT TT-3'; GAPDH-sense: 5'-CGT AGA CAA AAT GGT GAA GG-3' and GAPDH-antisense: 5'-GAC TCC ACG ACA TAC TCA GC-3'.

Cells from FVB mammary gland epithelium were isolated as described above and seeded in 140-mm plates pre-coated with Matrigel^® (BD Bioscience, San Jose, CA, USA). At 70% confluency, cells were transfected with pSG5 or pSG5-RARγ (10 μg) using Lipofectamine 2000 in Opti-MEM, 24 h after transfection, cells were treated with 1 μM ATRA for 16 h, and then crosslinked using 1% formaldehyde. Cell nuclei were isolated using hypertonic buffer A (150 mM NaCl, 50 mM Tris-HCl, pH 8, 1% NP40, 1% sodium deoxycholate, 0.5% SDS, and 2 mM EDTA), centrifuged, lysed in SDS lysis buffer (50 mM Tris, pH 8,10 mM EDTA, 1% SDS), and then sonicated. Chromatin from the nuclear fraction obtained was pre-cleared with protein G agarose/sperm salmon (Millipore, Billerica, MA, USA) and irrelevant immunoglobulin G (IgG). RARγ was immunoprecipitated overnight at 4°C, and immunoprecipitates were incubated with protein G agarose/sperm salmon for 2 h at 4°C. Immunoprecipitates were washed and eluted with elution buffer (100 mM Na₂CO₃ and 1% SDS) from agarose beads and incubated in 5 M NaCl at 65°C overnight. DNA was isolated by digestion with proteinase K, treatment with RNAse A, and purification using the QIAquick DNA purification kit (Qiagen, Valencia, CA, USA). Primers for the detection of the RA response element (RARE) sequence of CRBP1 were designed based on Smith et al. [52]: CRBP1-sense 5'-CTT GCC TAC CCT GAT GGT GT-3' and CRBP1-antisense: 5'-CCC TTC TCA CCT GCT ACC TG-3'. As a control, nonspecific primers were designed using 9000-bp upstream of the CRBP1 promoter; nonspecific-sense: 5'-GCA AGA CTG CTT GCT CTC CT-3' and nonspecific-antisense: 5'-AAC ACA TCG TGG GTG GTC TT-3'.

We hypothesized that over-expression of c-Myc in the mammary epithelium of MMTV-Myc transgenic mice leads to up-regulation of RARγ and determine if this up-regulation happens before the development of palpable tumors. To test this hypothesis, we compared RARγ expression in mammary gland epithelium isolated from 15-week-old virgin MMTV-Myc female mice, at which point the mammary epithelium is already hyperplastic, to that of primary mammary epithelium from wt-FVB female mice of the same age. Quantitative (Q)-PCR results (Figure 1A) revealed that compared to normal epithelium, the MMTV-Myc mouse hyperplastic epithelium had elevated levels of RARγ1 and γ2 mRNA and reduced RARβ2, which is commonly silenced in cancer [53]. Western blot analysis confirmed that compared to primary cells cultured from normal FVB mammary epithelium, Myc tumors had elevated RARγ but reduced RARβ protein levels, whereas RARα levels remained unchanged (Figure 1B). Moreover, the elevation in the level of RARγ protein in c-Myc over-expressing cells compared to that in normal FVB cells, appeared to correlate with the reduction in the level of CRBP1 protein (Figure 1C).

To determine whether modulation of RARγ and CRBP1 were functionally linked to c-Myc expression, primary cultures of FVB mammary epithelial cells were transfected with c-Myc or a control vector (c-Myc expression is shown in Figure S1A in Additional file 1) and for the transfected cells were evaluated for the expression of RARγ and that of the RARα target genes CRBP1 and RARβ [5‐8]. Forced expression of c-Myc specifically elevates RARγ1 mRNA relative to the vector control, but not that of RARγ2 (Figure 1D). Knockdown of RARγ1 in primary Myc cells using shRARγ1 (Figure S1B in Additional file 1) followed by Am580 treatment resulted in an even higher level of CRBP1 expression (Figure 1E), showing that in these cells RARγ has a repressive effect on the RARα target gene CRBP1.

These results suggest that when the RARγ ligand is absent or present only at low physiologic concentration, RARγ over-expression represses CRBP1 expression, but this effect is independent of its canonical activation.

Our results suggest that RARγ is involved in the repression of RARβ and CRBP1 expression, both of which have a tumor-suppressive function [54]. To determine whether RARγ has a pro-tumorigenic function in the context of c-Myc-driven transformation, RARγ was either stably knocked down or over-expressed in primary Myc cells and inoculated orthotopically into the mammary fat pad of syngeneic FVB mice. After 20 days, the tumors formed by RARγ null myc cells displayed a 60% reduction in size in comparison to those formed by Myc-control cells. The Myc cells in which RARγ was over-expressed produced approximately 40% larger tumors (Figure 2A and 2B). These results allude to the fact that RARγ is likely to confer pro-tumorigenic activity.

Immunohistological analysis of tumor sections isolated from mice implanted with shRARγ-transfected Myc cells showed increased expression of p27 (Figure 2C), which is a marker of cell-cycle arrest [55], and membrane-associated E-cadherin (Figure 2D arrow), which is a marker of epithelial cell differentiation [56].

Given the pro-tumorigenic activity of RARγ, we examined the effect of RARγ knockdown on tumor invasiveness by generating three-dimensional cultures. In this approach, Myc cells were oligofected with siRARγ both before being plated in Matrigel^® as described by Debnath et al. [50] and then again on day 4. The colonies were then cultured for 8 days before the size and morphology of the resulting colonies were determined. Colonies formed by control cells transfected by vector alone were large (>200 um) and invasive, with long matrix-penetrating spikes. In contrast, the siRARγ-transfected cells developed only small (around 100 μm), smooth, rounded acinar-like colonies. The difference in invasiveness between control and siRARγ-transfected cells was quantified by counting 100 colonies per group (n = 4 wells/group) and was found to be highly statistically significant (Figure 2E).

In addition to knocking down RARγ, we also explored the effect of pharmacologic inhibition of its activity using the RARγ antagonist SR11253 [57, 58] in Myc cells in vitro. SR11253 treatment reduced Myc cell proliferation in a dose-dependent manner (IC₅₀ value = 50 nM after 96 h) (Figure 2F). The anti-proliferative effect achieved by reducing the expression of RARγ coincided with up-regulation of p27 and down-regulation of pRb, as shown by confocal immunofluorescence (Figure 2G).

In order to determine if the pro-proliferative effect of RARγ in the Myc-expressing mouse mammary tumor cells is also present in human mammary cells, RARγ was knocked-down with siRNAs in immortalized non-tumorigenic human MCF-10A cells, estrogen receptor (ER⁺) MCF-7 cells and triple negative (ER^-/progesterone receptor^-/HER2^-) MDA-MB-231 breast cancer cells. As shown in Figure 3A-C, the proliferation of all three cell lines was significantly impaired by RARγ knockdown. Treatment with 200 nM Am580 enhanced the anti-proliferative effect exhibited by RARγ knockdown in the MCF-10A and MCF-7 cell lines but not in the MDA-MB-231 cells. The lack of an effect in MDA-MB-231 is most likely due to the low expression level of RARα in these cells [59]. Although, c-Myc expression was low in MCF-10A cells and high in MCF-7 and MDA-MB-231 cells) (Figure 3D), this difference did not impinge on the changes in the rate of proliferation induced by modulation of RARγ expression.

We treated the three human cell lines and Myc-expressing mouse mammary cells with RARγ agonist BMS961, RARγ/β agonist CD347 and RARγ antagonist SR11253 alone, or in combination with ATRA or Am580. Similarly to the results obtained using RARγ knockdown (Figure 3), co-treatment of these cell lines, including the mouse Myc cells, with the RARγ antagonist SR11253 and RARα agonist Am580, resulted in strong growth inhibition (Figure 4). As expected, Am580-inhibited MCF-10A- and MCF-7-cell growth (Figure 4, upper panels) were growth-inhibited by Am580, whereas RARγ agonist BMS961 increased their proliferation. Antagonist SR11253 inhibited the proliferation of MDA-MB-231 cells (Figure 4, lower left panel), which was not further inhibited by combination with Am580, as was expected due to their low expression of RARα. The general pattern of responses of the Myc mouse cells to treatment was similar to the human cells, except that the CD437/AHPN (RARγ/β agonist) and its combination with Am580 impaired cell proliferation most effectively (Figure 4, lower right panel). These results show that the pro-proliferative effect of RARγ is ligand-dependent and more importantly, that it can be targeted by RARγ-specific antagonists such as SR11253, alone or in combination with Am580.

To further examine the specific roles of the RAR isotypes, each one (α, β or γ) was individually knocked down in MCF-10A cells using specific shRNAs (Figure 5C). Down-regulation of RARα or RARβ altered the cellular morphology from a more cobblestone-like structure to that of a more spindle-like shape (Figure 5A), suggesting that both isotypes could participate in control of epithelial to mesenchymal transition (EMT). In contrast, down-regulation of RARγ impaired cell survival, with cells showing a progressively increasing unhealthy star-like morphology (Figure 5A, right panel). Confocal immunofluorescence analysis using pRb (green) as a surrogate marker for proliferation and p27 (red) for cell-cycle arrest, indicated that knocking down RARα or RARβ increased proliferation (increased pRb expression), whereas knocking down RARγ promoted cell-cycle arrest (increased p27 expression). The p27 levels increased further when shRARγ-transfected cells were treated with 1 uM ATRA for 48 h (Figure 5B), suggesting that changing the ratio of RARα or β to RARγ affects the cellular response to ATRA.

Based on our prior work [39] and the above results, we reasoned that use of a RARα-selective agonist might circumvent the pro-proliferative and pRb repressive effects induced by activation of RARγ [11] and thereby prevent tumor development. To accomplish this goal, we selected the RARα agonist Am580 [60], which was reported to have 10-fold higher binding affinity for RARα than RARβ and no detectable affinity for RARγ [40]. Uniparous, 15-week-old MMTV-Myc female mice, 30 per group, were fed standard diet containing Am580 or the standard diet alone (Myc-Ctrl group) and palpated twice weekly for the appearance of tumors. Tumors were first noted at week 16 in both experimental groups (control diet and Am580 diet). At week 50, no significant difference in tumor development was observed between the two groups analyzed by Kaplan-Meier plot. At that time, 80% of the Am580-treated mice and 100% of the control mice had tumors (Figure 6A, Myc-Ctrl vs. Am580-treated; NR+R, where NR are Am580 non-responders, and R are Am580 responders). Analysis of the tumor growth rates after initial tumor detection revealed two distinct sub-populations in the Am580-treatment group, those with fast-growing tumors (Figure 6B, Am580 non-responders denoted by squares) and those with slow-growing tumors (Figure 6B, Am580-responders denoted by triangles). Of 27 treated mice available for evaluation at week 50, 17 (63%) responded to Am580 treatment with 90% reduction in tumor size relative to the untreated controls, while 10 (37%) did not show a tumor size reduction (non-responders) (Figure 6B). Comparison of the Am580 responders to the Myc-Ctrl group by Kaplan-Meier analysis showed that tumor latency in the responders was significantly extended (P = 0.0465, log-rank test, Am580 R vs. Myc-Ctrl), with 35% of the treated Am580-responding mice not developing tumors at 50 weeks (Figure 6B).

Metastatic dissemination of tumor cells is the ultimate cause of death in breast cancer with the lungs being one of the major target organs for metastasis to occur [61]. Analysis of lung metastasis incidence and number of metastatic lesions per mouse revealed that, compared to untreated mice (Myc-Ctrl) of which 66.6% developed lung metastases, only 16.6% of the Am580-responsive mice had metastases. The incidence of metastases in Am580 non-responsive mice was 71.4%, whereas compared to this group, Am580 responders had 36.8% metastasis incidence. The number of metastases per responder mouse was also reduced by Am580 (Figure 6C). Together these results demonstrate the ability of Am580 to reduce tumor growth and aggressiveness in the MMTV-Myc tumor model.

Because the MMTV-Myc mouse is an inbred population, we did not expect the difference in response to Am580 to be host-dependent. However, to rule it out formally, two tumors from each Am580 treatment group were excised, minced and then inoculated into the mammary fat pads of separate groups of syngenic FVB females, which have the same genetic background of the MMTV-Myc transgenic model. Inoculated mice were fed the diet containing Am580 for 20 days. The transplanted tumors recapitulated the individual patterns of response and non-response of the original tumor (Additional file 2, Figure S2) indicating that the response to Am580 was independent of the host and intrinsic to the tumor.

We, therefore, tested whether an imbalance in RAR isotype expression defined the response to Am580 treatment. Analysis of RAR isotype protein levels showed that Am580 NR tumors expressed higher RARγ protein levels than Am580 R tumors (Figure 7A). To confirm that high expression of RARγ correlated with lack of responsiveness to Am580, sections from untreated (control) and treated responsive and nonresponsive tumors were examined for protein levels of RARα target genes by western blotting and immunohistochemistry. As shown in Figure 7A, tumor lysates of Am580-responsive mice treated with Am580 exhibited increased levels of growth arrest (p27) and differentiation (E-cadherin) markers compared to the Am580-nonresponsive tumors. Tumor lysates of Am580-treated nonresponsive mice also had increased levels of p-Erk1/2 (Figure 7A), which also suggests an increased proliferative response. RARα protein levels were low in both Am580 treatment groups, whereas RARγ was much higher in the Am580 non-responsive tumors. Immunohistochemistry of tumor sections (Figure 7B) confirmed the western blot results by showing enhanced levels of E-cadherin and p27 in the Am580 responsive tumors and also revealed, that the RARα target gene involved in ATRA catabolism, cytosolic Cyp26A1, was strongly induced in these tumors compared to the Am580 nonresponsive and Myc-Ctrl tumors. Both immunohistochemistry (Figure 7C) and western blotting (Figure 8A) showed that Am580 treatment induced robust CRBP1 expression exclusively in the responsive tumors. The only CRBP1-positive cells detected in the untreated control and nonresponsive tumors were host stromal cells (black arrows in Figure 7C and data not shown). Interestingly, the responsive tumors showed focal outgrowths negative for CRBP1 staining, which may correspond to nonresponsive precursors (white arrows in Figure 7C). Despite induction of several differentiation markers, no overt morphological signs of tissue differentiation, such as the acinar-like structures observed in the three-dimensional cultures (Figure 1E), were detected in these tumor sections. However, scattered necrotic areas in both nonresponsive and responsive tumor sections were observed and found to be bigger and more prevalent in the responsive sections (Figure 7D, white arrows).

Since RARγ and CRBP1 expression levels were inversely regulated in c-Myc-expressing tumors (Figure 1B), as shown by up-regulation of CRBP1 protein by RARγ inhibition [39] and (Figure 1D), we explored the possibility that ligand-free RARγ could transcriptionally repress CRBP1. We ectopically expressed RARγ in primary mammary epithelial cells isolated from FVB mice and analyzed its effect on CRBP1 expression (mRNA) and protein levels (Figure 8B and 8C, respectively) and found a slight down-regulation in CRBP1 levels. ATRA treatment strongly induced CRBP1 expression at mRNA (Figure 8B) and, to a lesser degree, protein levels (Figure 8C). It is possible that the CRBP1 protein level continues to accumulate beyond the 24 h shown here, as was shown in rat Sertoli cells in which the half-life of CRBP1 was calculated to be 48 h [62].

To test the regulation of CRBP1 in mammary epithelial cells by retinoids, primary mammary epithelium from three 9-week-old FVB females, was treated for 3 days with 100 nM Am580, 1 uM ATRA, 100 nM SR11253, 60 nM BMS961, or their combination and CRBP1 levels were evaluated by western blot analysis (Figure 8D). CRBP1 protein was increased by RARα agonist Am580 alone or with RARγ agonist BMS961 (Figure 2 second and eighth lanes) and by RAR pan-agonist ATRA plus BMS961 (Figure 2 ninth lane) but reduced by RARγ antagonist SR11253. No major changes in CRBP1 expression level were observed in cells treated with ATRA, Am580 plus SR11253, ATRA plus SR11253, or BMS961. These results show that in contrast to Myc-expressing cells, CRBP1 protein levels are reduced in normal mammary epithelium when RARγ transcriptional activation is blocked, suggesting that oncogenic stress changes the RARγ function, possibly through changes in the RARγ-associated transcriptional complex, additional studies are required to elucidate these differences.

Although we did not examine the composition of the transcriptional complex, using ChiP assays we showed that over-expression of RARγ in normal mammary cells led to an enrichment of RARγ promoter (Figure 8E); whereas treatment of these cells with ATRA led to a slight CRBP1 reduction in CRBP1 promoter-associated RARγ and re-expression of CRBP1 (Figure 8B-D). These results suggest a repressive role for RARγ. Because ATRA activates all three RAR isotypes, we propose that in normal cells, activated RARα competes with RARγ for binding to the RARE in the CRBP1 promoter. Under the influence of over-expressed c-Myc, the ratio of RARα/RARγ changes profoundly in favor of RARγ, thus limiting the ability of RARα to displace RARγ from this promoter.