This study presents new findings from a yeast two-hybrid system in which we identified a novel interaction between the tumor suppressor maspin and IRF6, a member of the IRF transcription factor family. Our studies indicate that IRF6 is expressed in normal human mammary epithelial cells, and this interaction with maspin has been verified by co-immunoprecipitation. IRF6 is the least understood of the nine-member IRF family. Functions for this family of transcription factors have generally been associated with the induction and regulation of type I interferons and interferon-inducible genes following pathogenic insult but have also been linked to the cellular processes of growth control, apoptosis, tumor suppression, and embryonic development (12
). Notably, recent studies indicate a crucial role for IRF6 in palatal development (26
). Palatal fusion requires the dispersion and elimination of the medial edge epithelium lining opposing palatal shelves, which occurs through a combination of apoptosis, epithelial-to-mesenchymal transition, and cell migration (28
). IRF6 is highly expressed in the medial edge epithelium immediately prior to palatal fusion, indicating a possible role for IRF6 in one or more of these tissue-remodeling processes.
Our studies show for the first time that IRF6 is expressed in mammary epithelial cells where, under our experimental conditions, it resides primarily in the cytoplasm. SDS-PAGE of the cytosolic fraction demonstrated that IRF6 appears as two distinct bands with molecular masses of ~59 and ~63 kDa. Our data indicate that the upper ~63-kDa band represents a phosphorylated form of IRF6, as treatment with lambda-phosphatase abolished the upper band. Phosphorylation is a central step in the activation of many IRFs and acts to facilitate protein interactions involving the IAD. Phosphorylation normally occurs in the serine-rich region of the C terminus, which, in a non-phosphorylated state, exerts an auto-inhibitory effect by blocking protein-protein interactions (31
). Through detailed IRF6 truncation analysis, we have demonstrated a similar auto-inhibitory effect caused by the IRF6 C-terminal serine-rich region. We have shown that the IRF6 IAD is both necessary and sufficient for an interaction with maspin and that the inclusion of the C-terminal serine-rich region abrogates this interaction. Furthermore, our co-immunoprecipitation data indicate that IRF6 phosphorylation plays a significant role in the maspin-IRF6 interaction, because treatment of cell lysate with lambda-phosphatase prior to co-immunoprecipitation resulted in a significant decrease in the amount of maspin interacting with IRF6. Taken together, we postulate that phosphorylation of IRF6 facilitates its interaction with maspin by relieving the auto-inhibitory effect of the C terminus in a manner similar to other IRFs.
Co-immunoprecipitation studies also suggest that in normal mammary epithelial cells only a small portion of maspin is interacting with IRF6. Because it appears that the interaction between maspin and IRF6 occurs primarily in the cytoplasmic compartment, it is possible that the interaction with maspin serves to regulate IRF6 by controlling its translocation to the nucleus. This type of negative regulation has been reported with other IRFs, such as the cytoplasmic retention of IRF9 by its binding partner Stat2 (34
). Indeed, this is supported by our transfection studies that demonstrated the ability of maspin to protect against the effects of IRF6 re-expression.
Because of the role of maspin in tumor suppression, we evaluated the expression of IRF6 in both poorly invasive and highly invasive breast cancer cell lines. A comparison of maspin and IRF6 expression by semi-quantitative PCR demonstrated a moderate decrease in IRF6 mRNA levels during progression from a normal to a neoplastic, poorly invasive phenotype, whereas IRF6 expression was absent in the highly invasive and metastatic cells. This differs from maspin expression, which is highly reduced or completely absent in the poorly invasive cells. Studies have shown that methylation of the maspin promoter is involved in maspin silencing and is an early event in breast cancer development (35
). In contrast, the moderate down-regulation of IRF6 in poorly invasive cells suggests an altered regulatory pathway rather than epigenetic gene silencing. The reduction of IRF6 is more pronounced at the level of protein expression, where Western blot analysis revealed only minimal amounts of IRF6 in the poorly invasive cell lines. Importantly, the stable re-expression of maspin in MDA-MB-231 cells did not result in any changes in IRF6 expression nor did the re-expression of IRF6 lead to changes in maspin expression, suggesting that neither of these genes directly regulates the expression of the other. We postulate that the stepwise decline of IRF6 could be indicative of a role for IRF6 in regulating cellular differentiation, because the poorly invasive cells still maintain some epithelial characteristics, whereas the aggressive, metastatic cells have adopted a mesenchymal phenotype. Studies indicate that silencing of the maspin gene is one of the initial steps in tumor progression (35
). It is plausible to speculate that this could result in deregulated IRF6, leading to transcriptional activation of genes involved in epithelial-to-mesenchymal transition. This is supported by our IRF6 transfection approach, which indicates that in the absence of maspin IRF6 leads to increased expression of epithelial-to-mesenchymal transition-associated genes such as N-cadherin. It is also possible that IRF6 is temporally required for gene regulation (as is the case in palatal fusion (26
)) and the presence of maspin regulates this process. In the absence of maspin, continual activation of IRF6 might lead to its silencing, which might explain the loss of IRF6 in highly invasive breast cancer cells. The increase in N-cadherin (and decrease in E-cadherin) in cancer cells with epithelial origin has been reported to facilitate the infiltration of cancer cells into the host basement membrane. Indeed, studies have identified the up-regulation of N-cadherin as characteristic of a more invasive and less differentiated breast cancer cell phenotype (36
Our in vitro
findings are further supported by immunohistochemical analysis of breast tissue. These studies indicate that both maspin and IRF6 are moderately expressed in both the ductal and glandular epithelium of normal tissue and reduced or absent in neoplastic lesions. Interestingly, in normal tissue the myoepithelial cell layer, which surrounds the gland, expresses a high level of maspin but is devoid of IRF6. This lack of IRF6 expression in myoepithelial cells could be the result of an alternate differentiation pathway because both the myoepithelial and luminal epithelial cells are derived from a common mammary progenitor cell, further supporting a role for IRF6 and maspin in regulating the mammary epithelial cell phenotype (39
In conclusion, we have shown that the transcription factor IRF6 is expressed in normal mammary epithelium and that it interacts with the tumor suppressor maspin. We further demonstrated that IRF6 expression is lost in invasive breast cancer cells and hypothesize that the maspin-IRF6 interaction is important in maintaining a normal mammary epithelial cell phenotype. We further postulate that the maspin-IRF6 interaction plays an important role in tissue remodeling by regulating cell differentiation, an important aspect of mammary gland morphogenesis. This work helps elucidate the tumor suppressive mechanisms of maspin and supports a model in which maspin realizes its function in part through the regulation of IRF6. Understanding the molecular mechanisms of maspin and its interaction with IRF6 will allow us to translate the potential of maspin as a possible therapeutic agent and prognostic indicator in breast cancer.