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 ErbB3^MMTV-KO TEBs. B, body layer; C, cap layer. (Lower) Whole-mount hematoxylin-stained mammary glands from 8-wk-old ErbB3^MMTV-KO mice display reduced rate of ductal lengthening during puberty upon ErbB3 loss. (B) Histological analysis of ErbB3^MMTV-KO and ErbB3^Flox/+ 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 ErbB3^MMTV-KO and ErbB3^Flox/+ × MMTV-Cre mice. (D) Twelve-week-old ErbB3^DOX-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 ErbB3^MMTV-KO and ErbB3^Flox/+ × MMTV-Cre mice. (G) Twelve-week old ErbB3^DOX-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.

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) ErbB3^MMTV-KO and ErbB3^FL/+ × MMTV-Cre. (B) ErbB3^CK14-KO and ErbB3^FL/+ × CK14-Cre. (C) ErbB3^MMTV-KO × MMTV-PyVmT and ErbB3^FL/+ × MMTV-Cre × MMTV-PyVmT. n = 7/group. Arrows in C indicate keratinizing squamous metaplasia. (D) Representative image of squamous metaplasia observed in ErbB3^MMTV-KO mammary glands, but not in ErbB3^FL/+ × MMTV-Cre glands. (E and F) Representative images of PCNA IHC in ErbB3^MMTV-KO, ErbB3^FL/+ × MMTV-Cre, ErbB3^CK14-KO, and ErbB3^FL/+ × CK14-Cre mammary glands. Basal epithelium (BE) is outlined. Quantification is provided in Fig. S3D.

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 ErbB3^DOX-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) ErbB3^flox/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.

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 ErbB3^DOX-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 ErbB3^DOX-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) ErbB3^FL/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.