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. 2013 Mar 18;15(2):R25.
doi: 10.1186/bcr3403.

Embryonic mammary signature subsets are activated in Brca1-/- and basal-like breast cancers

Marketa Zvelebil, Erik Oliemuller, Qiong Gao, Olivia Wansbury, Alan Mackay, Howard Kendrick, Matthew J Smalley, Jorge S Reis-Filho, Beatrice A Howard

Embryonic mammary signature subsets are activated in Brca1-/- and basal-like breast cancers

Marketa Zvelebil et al. Breast Cancer Res. .

Abstract

Introduction: Cancer is often suggested to result from development gone awry. Links between normal embryonic development and cancer biology have been postulated, but no defined genetic basis has been established. We recently published the first transcriptomic analysis of embryonic mammary cell populations. Embryonic mammary epithelial cells are an immature progenitor cell population, lacking differentiation markers, which is reflected in their very distinct genetic profiles when compared with those of their postnatal descendents.

Methods: We defined an embryonic mammary epithelial signature that incorporates the most highly expressed genes from embryonic mammary epithelium when compared with the postnatal mammary epithelial cells. We looked for activation of the embryonic mammary epithelial signature in mouse mammary tumors that formed in mice in which Brca1 had been conditionally deleted from the mammary epithelium and in human breast cancers to determine whether any genetic links exist between embryonic mammary cells and breast cancers.

Results: Small subsets of the embryonic mammary epithelial signature were consistently activated in mouse Brca1-/- tumors and human basal-like breast cancers, which encoded predominantly transcriptional regulators, cell-cycle, and actin cytoskeleton components. Other embryonic gene subsets were found activated in non-basal-like tumor subtypes and repressed in basal-like tumors, including regulators of neuronal differentiation, transcription, and cell biosynthesis. Several embryonic genes showed significant upregulation in estrogen receptor (ER)-negative, progesterone receptor (PR)-negative, and/or grade 3 breast cancers. Among them, the transcription factor, SOX11, a progenitor cell and lineage regulator of nonmammary cell types, is found highly expressed in some Brca1-/- mammary tumors. By using RNA interference to silence SOX11 expression in breast cancer cells, we found evidence that SOX11 regulates breast cancer cell proliferation and cell survival.

Conclusions: Specific subsets of embryonic mammary genes, rather than the entire embryonic development transcriptomic program, are activated in tumorigenesis. Genes involved in embryonic mammary development are consistently upregulated in some breast cancers and warrant further investigation, potentially in drug-discovery research endeavors.

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Figures

Figure 1
Figure 1
Embryonic mammary bud epithelial cells share key marker profiles with Brca1-/- and basal-like breast cancers. Many E12.5-stage embryonic mammary bud epithelial cells display a triple-negative profile. Immunofluorescence (IF) with ERα shows stain throughout the mammary mesenchymal tissue and no epithelial stain. IF with PR shows no staining of either mammary epithelial or mesenchymal cells. Control tissue shows staining in some luminal mammary epithelial cells in postnatal tissue. Erbb2 is expressed at low to moderate levels by some embryonic mammary epithelial cells, whereas other cells do not stain. Krt5 and Krt14 are highly expressed by many, but not all, embryonic mammary epithelial cells. All embryonic mammary epithelial cells express p63; many express low to moderate levels of Egfr. Scale bar, 50 μm.
Figure 2
Figure 2
Subsets of the embryonic mammary epithelial signature are activated in Brca1-/- mouse tumors. (A) Unsupervised hierarchic clustering of embryonic epithelial signature expression in Brca1-/- mouse mammary tumor dataset. (B) Five clusters of Brca1-/- tumor-associated embryonic genes and functional annotation. EMB, embryonic mammary cells; POST, postnatal mammary epithelial cells; F, mammary fibroblasts [10]. TUMOURS are Brca1-/- mammary tumors from [12].
Figure 3
Figure 3
Subsets of the embryonic epithelial mammary signature are activated in microdissected human breast cancers. (A) Unsupervised hierarchic clustering of embryonic epithelial signature expressed in Natrajan tumor dataset. Breast cancer subtypes were defined by the research version of PAM50 classification [18]. (B) Two subsets of embryonic genes from embryonic signature activated in basal-like breast cancers and three subsets found repressed in basal-like breast cancers and activated in luminal tumors and their functional annotation. (C) Proliferating cells are observed in the embryonic mammary bud epithelium with Ki67 stain. (D) Network of embryonic genes found activated and repressed in basal-like breast cancers in Natrajan data set. (E) Box plots showing the average expression levels of the 37 embryonic genes in the breast cancer subtypes classified by using PAM50 SSP on the NKI295 dataset. (F) Kaplan-Meier analysis shows significantly reduced distant metastasis-free survival in patients with tumors with activation of embryonic mammary signature in the van de Vijver dataset [15] (χ2 P value = 0.0028; log-rank P value = 0.0044).
Figure 4
Figure 4
Core tumor-associated embryonic mammary genes associate significantly with key clinical parameters in breast cancers. (A) Expression levels of core network activated across independent tumor datasets in ER+ versus ER- breast cancers. Red indicates expression levels upregulated in ER- versus ER+ tumors; green indicates expression levels up in ER+ versus ER- tumors. (B) Five genes (ASPM, BCL11A, SOX11, TPX2, and UCHL1) from the core network in Figure 5A show at least a twofold increase in expression levels in ER- versus ER+ breast cancers in seven datasets [16,38-43]. (C) Five genes (ASPM, BCL11A, SOX11, TPX2, and UCHL1) from the core network shown in Figure 5A show at least a twofold increase in expression levels in PR- versus PR+ breast cancers in eight datasets [16,39,41-46]. (D) Expression levels of core network activated across six independent tumor datasets [16,40,42,47-49] in HER2- versus HER+ breast cancers. Red, expression levels upregulated in HER2- versus HER2+ tumors; green, expression levels upregulated in HER2+ versus HER2- tumors. (E) Significance analysis of microarray (SAM) analysis of ASPM, BCL11A, SOX11, UCHL1, and TPX2 expression according to tumor subtype, as defined by PAM50 [17], in breast cancers in the Lu dataset [40]. (F) SAM analysis of ASPM, SOX11, and TPX2 expression according to grade in breast cancers in Miller dataset [41]. (G) Kaplan-Meier analysis shows significantly reduced overall survival in the high SOX11 as compared with the low-SOX11 subgroup in the van de Vijver dataset [15] (χ2 P value = 0.004; log-rank P value = 0.002133).
Figure 5
Figure 5
Embryonic mammary transcription factors are highly expressed in some Brca1-/- mammary tumor cells. (A) qRT-PCR data confirming embryonic-enriched expression of several basal-like tumor-associated transcription factors when compared with postnatal MEC subpopulations (described in [10] and [13]). MP, mammary primordium; MBE, E12.5-stage mammary bud epithelium; MM, E12.5-stage mammary mesenchyme; FB, fibroblast; ER-, luminal estrogen receptor negative; ER+, luminal estrogen-receptor positive; MYO, myoepithelial. (B) IHC showing cell types expressing embryonic mammary marker, Sox11 (guinea-pig antiserum) within embryonic mammary primordium. (C) IHC showing low level of Sox11 expression (guinea-pig antiserum) within 10-week-old postnatal mammary gland. (D-G) IHC showing Sox11 expression (guinea-pig antiserum) in some, but not all, Brca1-/- tumors. Scale bar, 50 μm.
Figure 6
Figure 6
Effects of SOX11 knockdown on cell proliferation and viability of breast cancer cells. (A) SOX11 expression levels in BT474 cells transfected with either SOX11 or control siRNAs. SOX11 was detected with immunoblotting. (B) BT474 cell number, represented as measured by absorbance by using PrestoBlue cell-viability reagent, after transfection with SOX11 or nontargeting siRNAs at daily intervals. Values represent mean ± SEM for three different experiments. (C) Change in percentage of viable cells was assessed by using PrestoBlue cell-viability assay of BT474 cells 72 hours after transfection with SOX11 siRNAs compared with control siRNA. Values represent mean ± SD for three different experiments; *P < 0.001, compared with the control. (D) Caspase-3 and cleaved caspase-3 levels detected by immunoblotting of lysates of adherent and floating BT474 cells transfected with either SOX11 or control siRNAs. (E) After transfection with either SOX11 or control siRNA, adherent BT474 cells were collected and stained with 7AAD. Cell-cycle phase was determined with FACS analysis. Values represent mean ± SD for four independent experiments. *P < 0.05; and **P < 0.01 compared with the control.
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