Members of the ErbB/HER family of receptor tyrosine kinases are known to stimulate the proliferation of breast epithelial cells and to play causative roles in the formation and progression of breast cancer. However, recent evidence suggests that HER4 activation prevents the growth of breast epithelial cells while promoting cellular differentiation. Most studies agree that HER4 expression correlates with better outcomes for human breast cancer patients. However, the mechanism by which HER4 promotes a better prognosis or decreases the growth of breast cancer cells is currently unknown. We describe results herein suggesting that HER4 impairs the growth of breast cancer cells through a BRCA1-dependent delay in mitotic progression.
It is known that BRCA1 mRNA expression is regulated in a cell cycle-specific manner, with the lowest levels of BRCA1 expression detected during G1
and with increasing levels detected as cells transition through S phase and G2
). This is consistent with the proposed functions of BRCA1 in mediating DNA repair and checkpoint controls in the later phases of the cell cycle. Because BRCA1 expression is influenced by cell cycle dynamics, it is possible that the regulation of BRCA1 expression by HER4 activity might be an indirect reflection of the demonstrated HER4-induced cell cycle delay in late G2
or early M phase. However, our data with SUM102-HER4 cells demonstrated that HRG increased BRCA1 expression >4-fold (Fig. ), while cells accumulated in G1
(Fig. ). Furthermore, HRG treatment of synchronized BT-474 cells increased BRCA1 expression regardless of the cell cycle phase in which cells were synchronized. Therefore, it seems as if HER4 increases BRCA1 expression by mechanisms that are independent, at least in part, of cell cycle dynamics.
The mechanism by which HER4 triggers BRCA1 expression is currently unknown. Two likely possibilities are that HER4 stabilizes BRCA1 transcripts and that HER4 induces the transcription of BRCA1. Given that the intracellular domain of HER4 becomes liberated upon ligand binding to form a constitutively active tyrosine kinase that can translocate to the nucleus (25
), bind transcription factors such as p53, YAP, and STAT5A (4
), and perhaps exhibit transcriptional activity on its own (25
), it is interesting to speculate that HER4 may contribute directly to the transcriptional activation of the BRCA1 gene. Alternatively, HER4 could activate gene transcription of BRCA1 through a conventional RTK signaling cascade. The observations that JNK signaling was triggered by HER4 activity and that JNK was required for HRG-induced BRCA1 induction (Fig. ) suggest that classical RTK signaling pathways, or perhaps tyrosine kinase signaling by the liberated intracellular domain of HER4, may be involved in the regulation of BRCA1, as opposed to direct transcriptional activation by the HER4 intracellular domain. It is clearly possible that both signaling pathways (classical signaling and a novel action of the intracellular fragment of HER4) may simultaneously be involved in BRCA1 induction. Similarly, while Fig. demonstrates that overexpression of BRCA1 produces the same G2
/M delay as does HRG-induced HER4 activity, it is possible that maximal growth inhibition via HER4 involves both BRCA1 induction and other HER4-dependent signaling steps.
The observation that molecular signals initiated at or emanating from the cell surface could enhance expression of the BRCA1 gene may hold therapeutic potential, given the importance of BRCA1 as a tumor suppressor. In addition to the numerous inactivating mutations or deletions in the BRCA1 gene detected in hereditary breast cancer families (9
), decreased BRCA1 expression is often detected in sporadic cases of breast cancer (42
). Our studies showed that human breast cancers expressing higher levels of HER4 mRNA generally displayed greater BRCA1 mRNA levels (Fig. ), supporting the idea that HER4 expression may confer a better breast cancer prognosis, in part by virtue of elevated BRCA1 expression. Although our translational analysis examined only a small number of tumors (n
= 19), we found repeated evidence that HER4-expressing breast cancer-derived cell lines had increased BRCA1 expression at both the protein and mRNA levels when treated with HRG (Fig. ), while HER4−
cell lines did not. HRG-dependent induction of BRCA1 mRNA required HER4 tyrosine kinase activation (Fig. ) but was independent of HER2 and, in fact, might be enhanced in the absence of HER2 (Fig. ). The introduction of HER4 into a HER4-negative breast cancer cell line conferred HRG-mediated BRCA1 induction. Because BRCA1 is a protein with numerous and diverse functions, including control of genomic stability, regulation of cell cycle checkpoints, DNA repair, and regulation of gene transcription, BRCA1 has many pathways by which it can carry out its critically important tumor suppressor activity (58
). The interest in this bona fide human tumor suppressor has produced much data and many hypotheses to explain how it works; our findings add to this list the possibility that increased expression of BRCA1 in response to HER4 activation may confer greater control over cellular decisions regarding growth, particularly in the G2
/M region of the cell cycle (see below).
Although the accumulation of cells with 4N DNA in response to HRG may have suggested a delay in G2
or at the G2
/M transition, our data suggest that HER4-positive breast cancer cells are delayed in early mitosis. Mitotic delay was demonstrated by increased phosphorylation of histone H3, sustained cyclin B expression and cyclin B-cdc2 interaction, and sustained cdc2 activity, each of which initially becomes evident in early mitosis. As cells progress through mitosis and chromosomes align in anaphase, cyclin B dissociates from cdc2, cdc2 becomes inactivated, and cyclin B is targeted for degradation by the anaphase promoting complex. Because we observed sustained cyclin B-cdc2 association and cdc2 activity in response to HRG, it is likely that HRG resulted in a delay at a checkpoint in mitosis known as the spindle assembly checkpoint (SAC). Most of what is known with regard to the SAC has been studied in response to cellular damage rather than in response to a physiologic ligand such as HRG. We examined several SAC proteins in HRG-treated BT-474 cells but did not find any difference in the subcellular localization of BRCA1, cdc20, MAD2, or BubR1 (data not shown), nor did we observe any overall change in the phosphorylation status of these four proteins, as assessed by phosphoserine and phosphothreonine Western analysis of immunoprecipitates from cells treated with or without HRG. This does not rule out HRG-dependent posttranslational modifications of BRCA1. In fact, HRG-dependent BRCA1 phosphorylation has been reported previously (3
). The data herein showing that BRCA1S1473A
overexpression did not result in a G2
/M delay may suggest that HRG-dependent modification by a serine-threonine kinase is involved.
The role of BRCA1 as a necessary intermediary in HRG- and HER4-dependent growth delay was demonstrated by independent means in assays with human breast cancer cell lines, using RNA interference (Fig. ), and in genetic experiments using isogenic mouse mammary cell lines that either express BRCA1 or do not. HRG-dependent mitotic delay occurred in BRCA1+
cells and was absent in BRCA1−
cells (Fig. ). Although the precise role of BRCA1 in regulating mitotic progression in response to HER4 activation is still under investigation, it may relate to the ability of BRCA1 to activate the SAC, a checkpoint that ensures the accurate alignment and segregation of chromosomes and prevents cells with misaligned chromosomes from exiting mitosis. The requirement for BRCA1 in the SAC is highlighted by the remarkable number of aneuploid cells derived from mice carrying a homozygous targeted deletion of BRCA1 exon 11, the inability of these cells to inhibit the anaphase promoting complex in the presence of mitotic poisons, and their inability to arrest in mitosis (6
Evidence from animal models demonstrates that HER4 activity is required for lactational differentiation of the mammary epithelium. Mammary glands from mice that lack HER4 activity as a result of multiple genetic strategies each have lactational defects due to an impaired program of differentiation (16
), as measured by a decrease in the expression of milk proteins (β-casein and whey acidic protein) and a decrease in activity of the transcription factor STAT5a, which is required for lactation. Interestingly, the role of BRCA1 in the differentiation of mammary epithelial cells has recently become apparent. BRCA1 expression is spatially and temporally regulated at each distinct stage of mammary gland development (21
). Also, mammary glands from mice with a conditional BRCA1 knockout displayed an impaired program of growth and differentiation (55
). In cell culture models, the role of BRCA1 in morphological differentiation has been studied in three-dimensional cultures of MCF10A cells. Depletion of BRCA1 using RNA interference revealed that acinus formation, the formation of a single-layered, polarized epithelial structure surrounding a lumen, relied on BRCA1 expression and inversely correlated with increased growth of the MCF10A cells in the absence of BRCA1 expression (11
). These studies support a hypothesis currently under investigation that the mechanism by which HER4 regulates mammary differentiation may rely on BRCA1 activity, similar to the BRCA1-dependent mechanism by which HER4 decreases the growth of breast cancer cells, as demonstrated herein.
In summary, our data suggest that HER4-mediated induction of BRCA1 expression may inhibit the growth of breast cancer cells by two mechanisms, by decreasing progression through mitosis and by enhancing cellular differentiation. These data warrant further investigations examining the potential synergy between HER4 and BRCA1 in mitotic delay with regard to tumor formation and progression.