The receptor tyrosine kinase ErbB3 maintains the balance between luminal and basal breast epithelium - PubMed (original) (raw)

. 2012 Jan 3;109(1):221-6.

doi: 10.1073/pnas.1115802109. Epub 2011 Dec 16.

Todd W Miller, Meghan M Morrison, Katherine Hutchinson, Christian Young, Cammie Rinehart, Violeta Sánchez, David Jee, Kornelia Polyak, Aleix Prat, Charles M Perou, Carlos L Arteaga, Rebecca S Cook

Affiliations

The receptor tyrosine kinase ErbB3 maintains the balance between luminal and basal breast epithelium

Justin M Balko et al. Proc Natl Acad Sci U S A. 2012.

Abstract

ErbB3 harbors weak kinase activity, but strongly activates downstream phosphatidylinositol 3-kinase/Akt signaling through heterodimerization with and activation by other ErbB receptor tyrosine kinases. We report here that ErbB3 loss in the luminal mammary epithelium of mice impaired Akt and MAPK signaling and reduced luminal cell proliferation and survival. ERBB3 mRNA expression levels were highest in luminal mammary populations and lowest in basal cell/stem cell populations. ErbB3 loss in mammary epithelial cells shifted gene expression patterns toward a mammary basal cell/stem cell signature. ErbB3 depletion-induced gene expression changes were rescued upon activation of Akt and MAPK signaling. Interestingly, proliferation and expansion of the mammary basal epithelium (BE) occurred upon ErbB3 targeting in the luminal epithelium, but not upon its targeting in the BE. Multiple cytokines, including interleukin 6, were induced upon ErbB3 depletion in luminal epithelium cells, which increased growth of BE cells. Taken together, these results suggest that ErbB3 regulates the balance of differentiated breast epithelial cell types by regulating their growth and survival through autocrine- and paracrine-signaling mechanisms.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

ErbB3 phosphorylation induces PI3K and MAPK signaling to promote cell growth, survival, and organization of mammary LE. (A) (Upper) IHC detection of ErbB3 in TEBs from 6-wk-old mice demonstrates expression in body cell layers with the lowest expression in the cap layer. ErbB3 expression is lost in ErbB3MMTV-KO TEBs. B, body layer; C, cap layer. (Lower) Whole-mount hematoxylin-stained mammary glands from 8-wk-old ErbB3MMTV-KO mice display reduced rate of ductal lengthening during puberty upon ErbB3 loss. (B) Histological analysis of ErbB3MMTV-KO and ErbB3Flox/+ control mammary glands from 6-wk-old mice by hematoxylin and eosin (H&E) staining and IHC detection of Ki67, SMA, and E-cadherin shows alterations in TEB body cell proliferation and structural organization. B, body layer; C, cap layer. (C_–_E) Western analysis of whole mammary lysates from virgin female mice was performed for the indicated proteins and phosphoproteins. (C) Six-week-old ErbB3MMTV-KO and ErbB3Flox/+ × MMTV-Cre mice. (D) Twelve-week-old ErbB3DOX-KO mice treated 7 d with or without DOX. (E) Six-week-old WT mice treated 3 wk with or without lapatinib. (F_–_H) TUNEL analysis of mammary glands. (F) Six-week-old ErbB3MMTV-KO and ErbB3Flox/+ × MMTV-Cre mice. (G) Twelve-week old ErbB3DOX-KO mice treated 7 d with or without DOX. (H) Six-week-old WT mice treated 3 wk with or without lapatinib. (Scale bars, 50 μm.) Average percentage of total epithelial nuclei that were TUNEL+ (±SD) was calculated from five random 400× fields/sample; n = 7 per condition, compared by t test.

Fig. 2.

Fig. 2.

ErbB3 expression in mammary LE is required to maintain its differentiated molecular phenotype. (A) Examination of ERBB3 relative mRNA across normal mouse (Left) and human (Right) mammary cell fractions demonstrated that ErbB3 expression is highest in LE cell populations. Str, stromal; BE/MaSC, basal epithelium/mammary stem cell; pL, luminal progenitor; mL, mature luminal cell. Differences in ErbB3 expression were tested by ANOVA. (B) ERBB3 mRNA expression is maximal during late puberty in mice. Data and plot were extracted and generated from the National Center for Biotechnology Information Gene Expression Omnibus dataset GDS2721 using the probeset 1434606_at. Details of the study were previously published (34). (C) Positive correlation between ERBB3 gene expression in normal breast specimens and a molecular score of the degree of luminal differentiation (20). (D) Western analysis of ErbB3DOX-KO organoids treated with or without DOX × 7 d confirmed results of cDNA microarrays showing altered expression of the genes encoding Elf5, cyclin B1, E2F1, β-casein, p27, and Twist. (E) Rank correlation of expression patterns comparing mammary subpopulation signatures to gene expression data derived from ErbB3-deficient PMECs and control WT cells. The Spearman's correlation is plotted for each treated sample (n = 3) and control (n = 3) across all four mammary subpopulation signatures. Bars represent the mean correlation of the three experiments. (F and G) ErbB3flox/flox PMECs were infected ex vivo with Ad.Cre or Ad.LacZ and with adenovirally encoded myrAkt, dnAkt, caMek1, or dnMek1. Ad.GFP was used as a secondary negative control for dual infections. Seven days later, cell lysates and RNA were harvested. (F) Western analysis of cell lysates for the indicated proteins. (G) qRT-PCR to measure ELF5, KIT, CDKN1B, and CCN1B. *P < 0.05, ***P < 0.01, ***P < 0.001.

Fig. 3.

Fig. 3.

Expansion of the basal epithelium in untransformed mammary glands and mammary tumors lacking ErbB3. (A_–_C) Representative images of CK5 IHC in 12- to 20-wk-old virgin females. (A) ErbB3MMTV-KO and ErbB3FL/+ × MMTV-Cre. (B) ErbB3CK14-KO and ErbB3FL/+ × CK14-Cre. (C) ErbB3MMTV-KO × MMTV-PyVmT and ErbB3FL/+ × MMTV-Cre × MMTV-PyVmT. n = 7/group. Arrows in C indicate keratinizing squamous metaplasia. (D) Representative image of squamous metaplasia observed in ErbB3MMTV-KO mammary glands, but not in ErbB3FL/+ × MMTV-Cre glands. (E and F) Representative images of PCNA IHC in ErbB3MMTV-KO, ErbB3FL/+ × MMTV-Cre, ErbB3CK14-KO, and ErbB3FL/+ × CK14-Cre mammary glands. Basal epithelium (BE) is outlined. Quantification is provided in

Fig. S3_D_

.

Fig. 4.

Fig. 4.

IL-6 produced by ErbB3-deficient luminal epithelium increases growth of basal epithelium. (A) RNA from T47D cells transfected with control or ErbB3-specific siRNA was used for reverse transcription and quantitative PCR for 84 individual transcripts. Values shown are the mean fold-change (log2) relative to control-siRNA–transfected cells, normalized to GAPDH. Only transcripts demonstrating greater than twofold changes are shown. Samples were analyzed in triplicate, and experiments were repeated three times. Bars represent mean of three experiments ± SD. (B) Primary ErbB3DOX-KO MECs were cultured in serum-free medium (SFM) for 7 d with or without DOX. Cultured media was assessed by cytokine array to detect secreted factors. Cytokines outlined in solid black indicate DOX-induced cytokines, and dashed lines indicate cytokines down-regulated by DOX. (C) Primary ErbB3DOX-KO PMECs were cultured in SFM ± lapatinib (1 μM), BKM120 (0.5 μM), or AZD6244 (1 μM). Cultured media were collected and assayed by ELISA to measure IL-6 (Left), RANTES (Center), and VEGF-A (Right). Results indicate mean ± SD (n = 3). *P < 0.05, **P < 0.01, and ***P < 0.001. (D) Human mammary BE cells were cultured in SFM with neutralizing antibody (Ab) against IL-6 (10 μg/mL) or control IgG and then treated +/− IL-6 (10 ng/mL) or EGF (10 ng/mL). Cell viability was measured after 96 h using WST-1 assay. Bars represent mean ± SD. ***P < 0.001. (E) ErbB3FL/FL × MMTV-PyVmT mammary tumor cells were infected with Ad.Cre or Ad.GFP and then cultured for 7 d in SFM. Conditioned media were collected, filtered, and used to culture human mammary BE cells for 96 h and BE cell viability was measured by WST-1 assay. Where indicated, BE cells were pretreated for 1 h with neutralizing IL-6 Ab (10 μg/mL) or control IgG. Bars represent mean ± SD. ***P < 0.001.

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