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. 2010;12(5):R79.
doi: 10.1186/bcr2724. Epub 2010 Oct 5.

RARα1 control of mammary gland ductal morphogenesis and wnt1-tumorigenesis

Ellen Cohn  1 , Liliana Ossowski, Silvina Bertran, Christine Marzan, Eduardo F Farias
Affiliations

RARα1 control of mammary gland ductal morphogenesis and wnt1-tumorigenesis

Ellen Cohn et al. Breast Cancer Res. 2010.

Abstract

Introduction: Retinoic acid signaling pathways are disabled in human breast cancer suggesting a controlling role in normal mammary growth that might be lost in tumorigenesis. We tested a single receptor isotype, RARα1, for its role in mouse mammary gland morphogenesis and MMTV-wnt1-induced oncogenesis.

Methods: The role of RARα1 in mammary morphogenesis was tested in RARα1-knockout (KO) mice and in mammary tumorigenesis in bi-genic (RARα1/KO crossed with MMTV-wnt1) mice. We used whole mounts analysis, stem cells/progenitor quantification, mammary gland repopulation, Q-PCR, test of tumor-free survival, tumor fragments and cell transplantation.

Results: In 2 genetic backgrounds (129/Bl-6 and FVB) the neo-natal RARα1/KO-mammary epithelial tree was 2-fold larger and the pubertal tree had 2-fold more branch points and 5-fold more mature end buds, a phenotype that was predominantly epithelial cell autonomous. The stem/progenitor compartment of the RARα1/KO mammary, defined as CD24(low)/ALDH(high activity) was increased by a median 1.7 fold, but the mammary stem cell (MaSC)-containing compartment, (CD24(low)/CD29(high)), was larger (~1.5 fold) in the wt-glands, and the mammary repopulating ability of the wt-gland epithelium was ~2-fold greater. In MMTV-wnt1 transgenic glands the progenitor (CD24(low)/ALDH(high activity)) content was 2.6-fold greater than in the wt and was further increased in the RARα1/KO-wnt1 glands. The tumor-free survival of RARα1/KO-wnt1 mice was significantly (p=0.0002, Kaplan Meier) longer, the in vivo growth of RARα1/KO-wnt1 transplanted tumor fragments was significantly (p=0.01) slower and RARα1/KO-wnt1 tumors cell suspension produced tumors after much longer latency.

Conclusions: In vitamin A-replete mice, RARα1 is required to maintain normal mammary morphogenesis, but paradoxically, also efficient tumorigenesis. While its loss increases the density of the mammary epithelial tree and the content of luminal mammary progenitors, it appears to reduce the size of the MaSC-containing compartment, the mammary repopulating activity, and to delay significantly the MMTV-wnt1-mammary tumorigenesis. Whether the delay in tumorigenesis is solely due to a reduction in wnt1 target cells or due to additional mechanisms remains to be determined. These results reveal the intricate nature of the retinoid signaling pathways in mammary development and carcinogenesis and suggest that a better understanding will be needed before retinoids can join the armament of effective anti- breast cancer therapies.

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Figures

Figure 1
Figure 1
The effect of RARα1/KO on neonatal and pubertal gland development. (a) Effect on the size of the neonatal glands. Number 4 glands were dissected from five to six days old wt and RARα1/KO C57Bl/6 or FVB, four mice per group, and whole mounts were prepared as described in Materials and methods. The area taken up by the mammary tree was analyzed using the Image J software. Bars are mean and SD of four glands per group. (b-e) Effect on pubertal mammary tree branching morphogenesis and terminal end buds. Number 4 mammary glands from seven to eight weeks old wt or RARα1/KO C57Bl/6 (b-d) and FVB pubertal mice (e) were dissected and processed for whole mounts and paraffin sections. Branching points were counted along the entire length of the three longest ducts. Bars show mean and SD of seven pairs (14 total) of number 4 glands per group (P = 0.0001, t-test). The same glands were used to count the peripheral terminal end buds (TEBs) (P = 0.00001, t-test).
Figure 2
Figure 2
Mammary gland transplantation. Fragments of epithelium containing mammary glands of eight to ten weeks old virgin mice (wt or RARα1/KO) were transplanted into number 4 glands pre-cleared of epithelium of three weeks old wild type or RARα1/KO animals (see Materials and methods). At week 11 (eight weeks after transplantation) the transplant-recipient glands and intact glands number 3 from the same mouse, as control, were processed for whole mounts. >90% of wt-FVB transplants repopulated the recipient gland while <50% of the FVB-RARα1/KO did (see Table 1).
Figure 3
Figure 3
Growth, composition and regulation of primary mammospheres. (a) Comparison of mammosphere growth. Primary cells obtained from wt and RARα1/KO mammary glands, were inoculated at 2.5 to 5 × 104 under conditions of ultra-low adhesion and serum-free medium and eight days later, the mammospheres were disassociated and the cells counted (see Materials and methods). The bars show mean (4,750 and 8,833 cells respectively, and SD of three individual experiments and three to four samples per group; P = 0.04 by unpaired t-test. (b) Bi-potential cells in mammospheres. Primary mammary epithelial cells were incubated for eight days until most single cells died; mammospheres were dissociated, inoculated into 96 wells, at 1cell/well (see Materials and methods) and when colonies formed stained for CK18 and CK14. (Red - CK18; green - CK14; blue - DAPI). Total number of colonies stained = 21. Scale bar = 10 um. (c) Regulation of mammosphere growth by retinoids. Cells isolated from mammary glands of seven- to eight-week old FVB mice were inoculated under conditions of mammosphere formation and eight to ten days later were treated with 10 nM of Am580, 200 nM of atRA, and/or 10-fold excess (100 nm and 2 μM, respectively) of RARα antagonist, Ro41-5253 for seven to eleven days. Each symbol represents an individual well; there were two to four wells per experiment, and a total of four experiments. Kruskal-Wallis test P = 0.0002; *indicates significance at P < 0.05 by Dunn's Multiple Comparison Test. (d) RARγ1 expression. RNA was extracted from freshly isolated and partially purified mammary epithelial cells and wt and RARα1/KO, and subjected to Q-PCR analysis as described in Materials and methods. The bars show mean of three experiments, two samples in each.
Figure 4
Figure 4
Characterization of the stem/progenitor cell compartment of wt-FVB and RARα1/KO. (a) Representative histograms of flow cytometry analysis of CD24low/ALDHhigh compartment in mammary epithelium. Primary mammary epithelial cells (approximately1 × 106) were incubated with ALDH substrate with inhibitor (first and third from left upper and lower panels) or without inhibitor (second and fourth from left upper and lower panels) followed by incubation with anti-CD24 Ab and detected with secondary Ab (APC-conjugated) as described in Materials and methods. Inclusion of the DEAB inhibitor reduced the ALDH activity by 82 and 87%, in FVB and RARα1/KO cells, respectively. Gated: CD24low/ALDHhigh (b) Summary of seven individual experiments. Experiments were performed as in A, shown are median, 3.9% and 6.7% respectively, (mean 4.4% and 6.6%, respectively, P = 0.04, unpaired t-test).
Figure 5
Figure 5
Characterization of MaSC-containing and luminal progenitor containing compartments. Mammary epithelial cells isolated as in Figure 4A, were incubated with PE-conjugated anti CD24 Ab, or APC-conjugated anti CD29 Ab and analyzed as described in Methods. Gated: upper left (FVB) and upper second from left (RARα1/KO) for CD24high/CD29low (luminal progenitors, upper left square) and for CD24low/CD29high (MaSC, lower right square). The third and fourth upper squares-negative controls, cells incubated with conjugated IgGs. The results shown are of one experiment, which was repeated once.
Figure 6
Figure 6
Wnt1-tumorigenesis and wt and RARα1/KO transplanted tumor growth. (a) Representative histograms of flow cytometry analysis of CD24low/ALDHhigh cells in FVB-wnt1 and FVB-wnt1-RARα1/KO- mammary glands. Primary mammary cells were isolated from glands of seven-week-old mice and processed for flow cytometry analysis as described in Figure 3. (b) Summary of flow cytometry experiments. The experiments were carried out as in A; each dot represents individually processed sample. Statistical analysis, two way ANOVA, P = 0.0025. (c) Tumor-free survival (Kaplan-Meier curve). Forty female mice transgenic for Wnt1 and wt-RAR and 42 RARα/KO were allowed to age and were examined at regular intervals for the appearance of a palpable tumor nodule. The difference in disease-free survival (percent of tumor-free mice), plotted as a function of post-natal age (Kaplan-Meier curve) was statistically significant (P = 0.0002). (d) Transplanted MMTV-RARα1/KO-wnt1 tumor fragments have slower growth rate than MMTV-wnt1 tumors. Fragments of randomly chosen pairs of MMTV-wnt1/wt and MMTV-wnt1/RARα1/KO tumors were transplanted into the opposite flanks of wt-FVB hosts. The tumors were measured every three days. The bars show mean and SE (n = 12 per group, P = 0.01. two-way ANOVA test). (e) Inoculation of MMTV-wnt1/RARα1/KO cell suspensions produces tumors after longer latency. Cell suspensions (105 and 104) prepared from a pair of similar size MMTV-wnt1/RARα1/KO and MMTV-wnt1 tumors were inoculated into eight to ten FVB-mice, (as in D) and appearance of palpable tumors was recorded and plotted as fraction of tumor-free mice vs. days post-inoculation (Kaplan-Meier).
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