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HER4 intracellular domain (4ICD) activity in the developing mammary gland and breast cancer.

ABSTRACT: The HER4 receptor tyrosine kinase was the final member of the EGFR-family to be discovered. In contrast to the other three members of this receptor family which function primarily as mitogenic effectors in the breast, HER4 appears to have multiple divergent functions in the normal and malignant breast. Interestingly, the majority of HER4 activities in the breast including pregnancy induced differentiation and lactation initiation, transcriptional activation, tumor cell proliferation, growth suppression, and induction of apoptosis appear to be mediated by an independently signaling soluble HER4 intracellular domain (4ICD). The 4ICD can accumulate within the nucleus or mitochondria and subcellular localization of 4ICD in part determines the physiological response of breast cells to 4ICD action. Here I will discuss the evidence supporting the role of 4ICD as the critical effector of HER4 signaling in the breast. In addition a developmental and temporal model of 4ICD action in the normal breast and during the progression of breast cancer will be presented to explain the paradox of divergent HER4 and 4ICD activities.

SUBMITTER: Jones FE

PROVIDER: S-EPMC2442669 | biostudies-literature | 2008 Jun

REPOSITORIES: biostudies-literature

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HER4 intracellular domain (4ICD) activity in the developing mammary gland and breast cancer.

Jones Frank E FE

Journal of mammary gland biology and neoplasia 20080513 2

The HER4 receptor tyrosine kinase was the final member of the EGFR-family to be discovered. In contrast to the other three members of this receptor family which function primarily as mitogenic effectors in the breast, HER4 appears to have multiple divergent functions in the normal and malignant breast. Interestingly, the majority of HER4 activities in the breast including pregnancy induced differentiation and lactation initiation, transcriptional activation, tumor cell proliferation, growth supp ...[more]

PMID: 18473151

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Project description:The HER4 receptor tyrosine kinase and STAT5A cooperate to promote mammary luminal progenitor cell maturation and mammary epithelial cell differentiation. Coupled HER4 and STAT5A signaling is mediated, in part, through association of the HER4 intracellular domain (4ICD) with STAT5A at STAT5A target gene promoters where 4ICD functions as a STAT5A transcriptional coactivator. Despite an essential role for coupled 4ICD and STAT5A signaling in mammary gland development, the mechanistic basis of 4ICD and STAT5A cooperative signaling remains unexplored. Here we show for the first time that 4ICD and STAT5A directly interact through STAT5A recruitment and binding to HER4/4ICD residue Y984. Accordingly, altering the 4ICD Y984 to phenylalanine results in a dramatic reduction of STAT5A and 4ICD-Y984F interacting complexes coimmunoprecipitated with HER4 or STAT5A specific antibodies. We further show that disrupting the 4ICD and STAT5A interaction has an important physiological impact on mammary epithelial cell differentiation. HC11 mammary epithelial cells with stable expression of 4ICD undergo differentiation with significantly increased expression of the STAT5A target genes and differentiation markers ?-casein and WAP. In contrast, HC11 cells stably expressing 4ICD-Y984F failed to undergo differentiation with basal expression levels of ?-casein and WAP. Differentiation in this cell system was induced in the absence of exogenous prolactin indicating that 4ICD activity is sufficient to induce mammary epithelial cell differentiation. Finally, we show that suppression of STAT5A expression abolishes the ability of 4ICD to induce HC11 differentiation and activate ?-casein or WAP expression. Taken together our results demonstrate for the first time that direct coupling of 4ICD and STAT5A is both necessary and sufficient to drive mammary epithelial differentiation. In conclusion, our findings that 4ICD and STAT5A directly interact to form a physiologically important transcriptional activation complex, provide a mechanistic basis for the in vivo observations that HER4/4ICD and STAT5A cooperate to promote mammary gland progenitor cell maturation and initiate lactation at parturition.

Project description:The HER4 intracellular domain (4ICD) is a potent estrogen receptor (ERalpha) coactivator with activities in breast cancer and the developing mammary gland that appear to overlap with progesterone receptor (PgR). In fact, 4ICD has recently emerged as an important regulator and predictor of tamoxifen response, a role previously thought to be fulfilled by PgR. Here we investigated the possibility that the 4ICD coactivator regulates PgR expression thereby providing a mechanistic explanation for their partially overlapping activities in breast cancer. We show that 4ICD is both sufficient and necessary to potentiate estrogen stimulation of gene expression. Suppression of HER4/4ICD expression in the MCF-7 breast tumor cell line completely eliminated estrogen stimulated expression of PgR. In addition, the HER4/4ICD negative MCF-7 variant, TamR, failed to express PgR in response to estrogen. Reintroduction of wild-type HER4 but not the gamma-secretase processing mutant HER4V673I into the TamR cell line restored PgR expression indicating that 4ICD is an essential PgR coactivator in breast tumor cells. These results were substantiated in vivo using two different physiologically relevant experimental systems. In the mouse mammary gland estrogen regulates expression of PgR-A whereas expression of PgR-B is estrogen independent. Consistent with a role for 4ICD in estrogen regulated PgR expression in vivo, PgR-A, but not PgR-B, expression was abolished in HER4-null mouse mammary glands during pregnancy. Coexpression of PgR and 4ICD is also commonly observed in ERalpha positive breast carcinomas. Using quantitative AQUA IHC technology we found that 4ICD potentiated PgR expression in primary breast tumors and the highest levels of PgR expression required coexpression of ERalpha and the 4ICD coactivator. In summary, our results provide compelling evidence that 4ICD is a physiologically important ERalpha coactivator and 4ICD cooperates with ERalpha to potentiate PgR expression in the normal and malignant breast. We propose that direct coupling of these signaling pathways may have important implications for mammary development, breast carcinogenesis, and patient response to endocrine therapy.

Project description:Unlike the proliferative action of other epidermal growth factor (EGF) receptor family members, HER4/ErbB4 is often associated with growth-inhibitory and differentiation signaling. These actions may involve HER4 two-step proteolytic processing by intramembraneous gamma-secretase, releasing the soluble, intracellular 80-kDa HER4 cytoplasmic domain, s80HER4. We demonstrate that pharmacological inhibition of either gamma-secretase activity or HER4 tyrosine kinase activity blocked heregulin-dependent growth inhibition of SUM44 breast cancer cells. We next generated breast cell lines stably expressing GFP-s80HER4 [green fluorescent protein (GFP) fused to the N terminus of the HER4 cytoplasmic domain, residues 676-1308], GFP-CT(HER4) (GFP fused to N terminus of the HER4 C-terminus distal to the tyrosine kinase domain, residues 989-1308), or GFP alone. Both GFP-s80HER4 and GFP-CTHER4 were found in the nucleus, but GFP-s80HER4 accumulated to a greater extent and sustained its nuclear localization. s80HER4 was constitutively tyrosine phosphorylated, and treatment of cells with a specific HER family tyrosine kinase inhibitor 1) blocked tyrosine phosphorylation; 2) markedly diminished GFP-s80HER4 nuclear localization; and 3) reduced signal transducer and activator of transcription (STAT)5A tyrosine phosphorylation and nuclear localization as well as GFP-s80HER4:STAT5A interaction. Multiple normal mammary and breast cancer cell lines, stably expressing GFP-s80HER4 (SUM44, MDA-MB-453, MCF10A, SUM102, and HC11) were growth inhibited compared with the same cell line expressing GFP-CTHER4 or GFP alone. The s80HER4-induced cell number reduction was due to slower growth because rates of apoptosis were equivalent in GFP-, GFP-CTHER4-, and GFP-s80HER4-expressing cells. Lastly, GFP-s80HER4 enhanced differentiation signaling as indicated by increased basal and prolactin-dependent beta-casein expression. These results indicate that surface HER4 tyrosine phosphorylation and ligand-dependent release of s80HER4 are necessary, and s80HER4 signaling is sufficient for HER4-dependent growth inhibition.

Project description:INTRODUCTION: We have previously linked HER4 expression with increased survival in breast cancer. However, other reports have associated HER4 with adverse prognostic significance. One possible explanation for the conflicting reports may be that these results are antibody dependent. The HER4 protein is enzymatically cleaved, which may alter the function of its intracellular domain (ICD). We have therefore compared the staining patterns of antibodies against its intracellular and extracellular domains using tissue microarray technology. METHODS: Immunohistochemistry was performed and evaluated on tumours from 402 tamoxifen treated oestrogen receptor positive patients. The HFR1 antibody recognises the ICD of HER4 and thus recognises both the intact receptor and the cleaved ICD. The H4.77.16 clone recognises an extracellular domain of HER4 and thus detects the full length receptor only. RESULTS: Both antibodies demonstrated nuclear, cytoplasmic and membranous staining. Concordance between the membrane staining patterns was high (88.44%, kappa 0.426). The HFR1 antibody, however, demonstrated generally higher levels of cytoplasmic staining (concordance 74.77%, kappa 0.351). The antibodies demonstrated very different patterns of nuclear staining. Over 60% of patients stained with the H4.77.16 had no nuclear staining whereas the vast majority showed staining with the HFR1 antibody (concordance 40.12%, kappa 0.051). Neither antibody demonstrated relationships between membranous or cytoplasmic HER4 staining and survival, although associations were seen with known poor prognostic markers. Cases with H4.77.16-determined nuclear staining had significantly poorer survival outcomes. CONCLUSION: The difference in antigen site may explain the different staining patterns we have seen with respect to location; with each antibody appearing to select for distinct compartments. Thus, HFR1 may select for cytoplasmic and nuclear HER4 ICD, whilst H4.77.16 selects for membranous HER4 and/or HER4 being recycled in cytoplasm or nucleus. This ability to distinguish between site and function of HER4 and its fragments is particularly important, with recent evidence highlighting the different functions of nuclear and mitochondrial HER4.

Dataset Information

HER4 intracellular domain (4ICD) activity in the developing mammary gland and breast cancer.

Publications

HER4 intracellular domain (4ICD) activity in the developing mammary gland and breast cancer.

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