ErbB4 Splice Variants Cyt1 and Cyt2 Differ by 16 Amino Acids and Exert Opposing Effects on the Mammary Epithelium In Vivo

Abstract

Data concerning the prognostic value of ErbB4 in breast cancer and effects on cell growth have varied in published reports, perhaps due to the unknown signaling consequences of expression of the intracellular proteolytic ErbB4 s80 HER4 fragment or due to differing signaling capabilities of alternatively spliced ErbB4 isoforms. One isoform (Cyt1) contains a 16-residue intracellular sequence that is absent from the other (Cyt2). We expressed s80Cyt1 and s80 Cyt2 in HC11 mammary epithelial cells, finding diametrically opposed effects on the growth and organization of colonies in three-dimensional matrices. Whereas expression of s80Cyt1 decreased growth and increased the rate of three-dimensional lumen formation, that of s80Cyt2 increased proliferation without promoting lumen formation. These results were recapitulated in vivo, using doxycyclineinducible, mouse breast-transgenic expression of s80 Cyt1 amd s80 Cyt2 . Expression of s80 Cyt1 decreased growth of the mammary ductal epithelium, caused precocious STAT5a activation and lactogenic differentiation, and increased cell surface E-cadherin levels. Remarkably, ductal growth inhibition by s80Cyt1 occurred simultaneously with lobuloalveolar growth that was unimpeded by s80Cyt1 , suggesting that the response to ErbB4 may be influenced by the epithelial subtype. In contrast, expression of s80 Cyt2 caused epithelial hyperplasia, increased Wnt and nuclear ␤-catenin expression, and elevated expression of c-myc and cyclin D1 in the mammary epithelium. These results demonstrate that the Cyt1 and Cyt2 ErbB4 isoforms, differing by only 16 amino acids, exhibit markedly opposing effects on mammary epithelium growth and differentiation.Activation of the ErbB/HER/epidermal growth factor receptor (ErbB/HER/EGFR) family of receptor tyrosine kinases (RTKs) is often equated with proliferation and oncogenesis. The family consists of four members, HER1/ErbB1/EGFR, HER2/ErbB2/Neu, HER3/ErbB3, and HER4/ErbB4. Each protein has a large NH 2 -terminal extracellular domain, a transmembrane domain, and a large intracellular domain with a tyrosine-rich carboxy-terminal region and a tyrosine kinaselike sequence (12). HER1/ErbB1, HER2/ErbB2, and HER4/ ErbB4 exhibit ligand-inducible tyrosine kinase activity, whereas HER3/ErbB3 does not.ErbB receptor activation at the cell surface targets multiple cytoplasmic signaling cascades that transmit signals to the nucleus. In addition to this classic RTK signaling model, ErbB RTKs have exhibited nuclear localization (4,17,19,27,28,37,47,48). While ErbB1, -2, and -3 are transported to the nucleus as full-length receptors, one isoform of ErbB4 is cleaved upon ligand binding, liberating the entire soluble intracellular domain. This intracellular, kinase-active 80-kDa cleavage product (s80 HER4 ) exhibits nuclear-cytoplasmic shuttling (8,20,25,27 (14,21,44). Inhibition of ErbB4 cleavage or its nuclear localization impairs ErbB4-mediated STAT5a activation (25,46,48).Evidence suggests that, whereas ErbB1, -2, and -3 all contribute to proliferation and/or survi...

Protein Inhibitor of Activated STAT3 (PIAS3) Protein Promotes SUMOylation and Nuclear Sequestration of the Intracellular Domain of ErbB4 Protein

“…When ErbB4 cleavage was inhibited by GSI IX, cells expressing ErbB4 showed a shift toward a more differentiated phenotype (Fig. 6B), indicating that the soluble ErbB4 ICD2 had an inhibitory effect on differentiation, consistent with previous findings (13). To address the effect of PIAS3 and PML on ErbB4 ICD function in this system, MDA-MB-468 cells expressing ErbB4 JM-a CYT-2 and the vector control cells were transfected with either non-targeting siRNA or two independent siRNAs targeting PIAS3 or PML.…”

“…We and others have previously utilized three-dimensional cultures of MDA-MB-468 human breast cancer (31) and HC11 mouse mammary epithelial cells (13,34) as models to address the role of ErbB4 isoforms in mammary differentiation. In a reconstituted basement membrane matrix (Matrigel), mammary epithelial cells are able to differentiate morphologically and functionally, forming small, organized colonies that resemble mammary acini, whereas transformed cells grow in large, disorganized colonies (36).…”

“…The released ErbB4 ICD can translocate into the nucleus, where it associates with transcriptional regulators including yes-associated protein (YAP) (6), STAT5 (7), estrogen receptor (8), Eto2 (9), TAB2-N-CoR complex (10), AP-2 (11), and HIF-1␣ (12), and modifies their activity. In vivo, the soluble ErbB4 ICD has been shown to regulate astrogenesis in the developing brain (10) as well as mammary epithelial cell differentiation and proliferation (13). In addition to nuclear localization and regulation of transcriptional events, the ErbB4 ICD has been reported to localize in the cytosol in mitochondria, where it regulates apoptosis (14).…”

Developmental pruning of excitatory synaptic inputs to parvalbumin interneurons in monkey prefrontal cortex

“…Moreover, ErbB4 transcripts are alternatively spliced at two loci, generating four different splice variants with distinct downstream signaling pathways (21). Although their functional consequences are not known in PV interneurons, ErbB4 splice variants display different levels of tyrosine kinase activity and exert different physiological effects in nonneuronal cells (22)(23)(24), suggesting that the effect of ErbB4 signaling could be modulated by alternative splicing. Thus, shifts in the expression level and/or splicing of ErbB4 in PV interneurons during adolescence might function as a developmental switch regulating the pruning of excitatory synapses on these neurons.…”

Expression of the Epidermal Growth Factor Receptors and Ligands in Paired Samples of Normal Breast Tissue, Primary Breast Carcinomas and Lymph Node Metastases

“…The functional significance of this is controversial [39]. The HER4 receptor appears to possess divergent functions demonstrated in vitro to depend on the isoform of the receptor [40] [41]. The HER4 response can also depend on the activating ligand [42], or the localization in the cell compartments (membrane bound, cytoplasmatic or in the nucleus) [43].…”