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01.12.2017 | Research article | Ausgabe 1/2017 Open Access

Breast Cancer Research 1/2017

ErbB3 drives mammary epithelial survival and differentiation during pregnancy and lactation

Zeitschrift:
Breast Cancer Research > Ausgabe 1/2017
Autoren:
Michelle M. Williams, David B. Vaught, Meghan Morrison Joly, Donna J. Hicks, Violeta Sanchez, Philip Owens, Bushra Rahman, David L. Elion, Justin M. Balko, Rebecca S. Cook
Wichtige Hinweise

Electronic supplementary material

The online version of this article (doi:10.​1186/​s13058-017-0893-7) contains supplementary material, which is available to authorized users.

Abstract

Background

During pregnancy, as the mammary gland prepares for synthesis and delivery of milk to newborns, a luminal mammary epithelial cell (MEC) subpopulation proliferates rapidly in response to systemic hormonal cues that activate STAT5A. While the receptor tyrosine kinase ErbB4 is required for STAT5A activation in MECs during pregnancy, it is unclear how ErbB3, a heterodimeric partner of ErbB4 and activator of phosphatidyl inositol-3 kinase (PI3K) signaling, contributes to lactogenic expansion of the mammary gland.

Methods

We assessed mRNA expression levels by expression microarray of mouse mammary glands harvested throughout pregnancy and lactation. To study the role of ErbB3 in mammary gland lactogenesis, we used transgenic mice expressing WAP-driven Cre recombinase to generate a mouse model in which conditional ErbB3 ablation occurred specifically in alveolar mammary epithelial cells (aMECs).

Results

Profiling of RNA from mouse MECs isolated throughout pregnancy revealed robust Erbb3 induction during mid-to-late pregnancy, a time point when aMECs proliferate rapidly and undergo differentiation to support milk production. Litters nursed by ErbB3 KO dams weighed significantly less when compared to litters nursed by ErbB3 WT dams. Further analysis revealed substantially reduced epithelial content, decreased aMEC proliferation, and increased aMEC cell death during late pregnancy. Consistent with the potent ability of ErbB3 to activate cell survival through the PI3K/Akt pathway, we found impaired Akt phosphorylation in ErbB3 KO samples, as well as impaired expression of STAT5A, a master regulator of lactogenesis. Constitutively active Akt rescued cell survival in ErbB3-depleted aMECs, but failed to restore STAT5A expression or activity. Interestingly, defects in growth and survival of ErbB3 KO aMECs as well as Akt phosphorylation, STAT5A activity, and expression of milk-encoding genes observed in ErbB3 KO MECs progressively improved between late pregnancy and lactation day 5. We found a compensatory upregulation of ErbB4 activity in ErbB3 KO mammary glands. Enforced ErbB4 expression alleviated the consequences of ErbB3 ablation in aMECs, while combined ablation of both ErbB3 and ErbB4 exaggerated the phenotype.

Conclusions

These studies demonstrate that ErbB3, like ErbB4, enhances lactogenic expansion and differentiation of the mammary gland during pregnancy, through activation of Akt and STAT5A, two targets crucial for lactation.
Zusatzmaterial
Additional file 1: is Figure S1 showing ErbB3 IHC of mammary glands harvested at 16.5 d.p.c. and L1, Figure S2 showing immunofluorescence for ErbB3 and Cytokeratin-8 (K8) in mammary glands harvested at 16.5 d.p.c., Figure S3 showing mammary glands harvested from 12–15-week-old mice at 16.5 d.p.c., L1 and L5, and stained using IHC for Ki67 (representative images, original magnification 400×), Figure S4 showing mammary glands harvested from 12–15-week-old mice at 16.5 d.p.c. and L1, assessed by TUNEL analysis in situ (representative images, original magnification 400×), Figure S5 showing P-Akt S473 IHC of mammary glands harvested from 12–15-week-old mice at 16.5 d.p.c., L1, and L5, and stained using IHC for P-Akt Serine 473 (representative images, original magnification 400×), Figure S6 showing HC11 cells treated with BKM120 (1 μM) or AZD6244 (1 μM) for 4 h: A western blot analysis using antibodies listed at the left (representative images, repeated three times) and B cell cultures stained with Annexin V-FITC to detect apoptotic cells, and Annexin V+ cells quantitated in digital images (experiments assessed in duplicate and repeated three times; data points are the average of the experimental duplicates; midlines are the average (± SD) of the biological replicates; Student’s unpaired t test), Figure S7 showing immunohistochemical detection of P-STAT5 A/B in mammary glands, Figure S8 showing HC11 cells expressing shErbB3 or shScr serum and EGF-starved for 24 h in the presence or absence of NRG1β (2 ng/ml), then stained with Annexin V-FITC to detect apoptotic cells, and Annexin V+ cells quantitated in digital images (experiments assessed in duplicate and repeated three times; data points are the average of the experimental duplicates; midlines are the average (± SD) of the biological replicates; Student’s unpaired t test), Figure S9 showing A HC11 cells expressing shErbB3 or shScr serum and EGF-starved for 24 h in the presence or absence of PRL, then whole RNA assessed by RT-qPCR for transcripts encoding ErbB2 and EGFR (experiments assessed in duplicate, repeated three times; data points are the average of the experimental duplicates; midlines are the average (± SD) of the biological replicates; Student’s unpaired t test) and B, C IHC used to visualize ErbB2 expression in mammary glands harvested at 16.5 d.p.c., L1, and L5 from uniparous ErbB3 WT and ErbB3 KO mice (panels in (C) identical to panels in (B), but include a wider field of view), and Figure S10 showing A HC11 cells expressing shErbB3 or shScr serum and EGF-starved for 30 min in the presence or absence of NRG1β or PRL (whole cell lysates assessed by western blot analysis using the antibodies indicated at the left; Mw molecular weight marker) and B HC11 cells expressing shErbB3 or shScr serum and EGF-starved for 48 h in the presence of PRL, BKM120 (1 μM) or BSK805 (1 μM), with Annexin V-FITC added to cultures for the final 3 h, and the number of Annexin V+ cells counted (each data point shows the average of samples assessed in duplicate, relative to the average value of the number of Annexin V+ cells seen in untreated HC11-shScr cells; midlines are the average (± SD); N = 3) (PPTX 49017 kb)
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