ErbB4 splice variants Cyt1 and Cyt2 differ by 16 amino acids and exert opposing effects on the mammary epithelium in vivo.

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 s80(Cyt1) 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 s80(Cyt1) decreased growth and increased the rate of three-dimensional lumen formation, that of s80(Cyt2) increased proliferation without promoting lumen formation. These results were recapitulated in vivo, using doxycycline-inducible, 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 s80(Cyt1) occurred simultaneously with lobuloalveolar growth that was unimpeded by s80(Cyt1), 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 beta-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.

HER4 D-box sequences regulate mitotic progression and degradation of the nuclear HER4 cleavage product s80HER4.

Heregulin-mediated activation of HER4 initiates receptor cleavage (releasing an 80-kDa HER4 intracellular domain, s80(HER4), containing nuclear localization sequences) and results in G(2)-M delay by unknown signaling mechanisms. We report herein that s80(HER4) contains a functional cyclin B-like sequence known as a D-box, which targets proteins for degradation by anaphase-promoting complex (APC)/cyclosome, a multisubunit ubiquitin ligase. s80(HER4) ubiquitination and proteasomal degradation occurred during mitosis but not during S phase. Inhibition of an APC subunit (APC2) using short interfering RNA knockdown impaired s80(HER4) degradation. Mutation of the s80(HER4) D-box sequence stabilized s80(HER4) during mitosis, and s80(HER4)-dependent growth inhibition via G(2)-M delay was significantly greater with the D-box mutant. Polyomavirus middle T antigen-transformed HC11 cells expressing s80(HER4) resulted in smaller, less proliferative, more differentiated tumors in vivo than those expressing kinase-dead s80(HER4) or the empty vector. Cells expressing s80(HER4) with a disrupted D-box did not form tumors, instead forming differentiated ductal structures. These results suggest that cell cycle-dependent degradation of s80(HER4) limits its growth-inhibitory action, and stabilization of s80(HER4) enhances tumor suppression, thus providing a link between HER4-mediated growth inhibition and cell cycle control.