Mammary Serine Protease Inhibitor (Maspin) Binds Directly to Interferon Regulatory Factor 6

Abstract

Since its reported discovery in 1994, maspin (mammary serine protease inhibitor) has been characterized as a class II tumor suppressor by its ability to promote apoptosis and inhibit cell invasion. Maspin is highly expressed in normal mammary epithelial cells but reduced or absent in aggressive breast carcinomas. However, despite efforts to characterize the mechanism(s) by which maspin functions as a tumor suppressor, its molecular characterization has remained somewhat elusive. Therefore, in an attempt to identify maspin-interacting proteins and thereby gain insight into the functional pathways of maspin, we employed a maspin-baited yeast two-hybrid system and subsequently identified Interferon Regulatory Factor 6 (IRF6) as a maspin-binding protein. IRF6 belongs to the IRF family of transcription factors, which is best known for its regulation of interferon and interferon-inducible genes following a pathogenic stimulus. Although many of the IRF family members have been well characterized, IRF6 remains poorly understood. We report that IRF6 is expressed in normal mammary epithelial cells and that it directly associates with maspin in a yeast two-hybrid system and in vitro. The interaction occurs via the conserved IRF protein association domain and is regulated by phosphorylation of IRF6. We have shown that, similar to maspin, IRF6 expression is inversely correlated with breast cancer invasiveness. We further demonstrated that the transient re-expression of IRF6 in breast cancer cells results in an increase of N-cadherin and a redistribution of vimentin commensurate with changes in cell morphology, suggestive of an epithelial-to-mesenchymal transition event. Concomitantly, we showed that maspin acts as a negative regulator of this process. These findings help to elucidate the molecular mechanisms of maspin and suggest an interactive role between maspin and IRF6 in regulating cellular phenotype, the loss of which can lead to neoplastic transformation. Since its reported discovery in 1994, maspin (mammary serine protease inhibitor) has been characterized as a class II tumor suppressor by its ability to promote apoptosis and inhibit cell invasion. Maspin is highly expressed in normal mammary epithelial cells but reduced or absent in aggressive breast carcinomas. However, despite efforts to characterize the mechanism(s) by which maspin functions as a tumor suppressor, its molecular characterization has remained somewhat elusive. Therefore, in an attempt to identify maspin-interacting proteins and thereby gain insight into the functional pathways of maspin, we employed a maspin-baited yeast two-hybrid system and subsequently identified Interferon Regulatory Factor 6 (IRF6) as a maspin-binding protein. IRF6 belongs to the IRF family of transcription factors, which is best known for its regulation of interferon and interferon-inducible genes following a pathogenic stimulus. Although many of the IRF family members have been well characterized, IRF6 remains poorly understood. We report that IRF6 is expressed in normal mammary epithelial cells and that it directly associates with maspin in a yeast two-hybrid system and in vitro. The interaction occurs via the conserved IRF protein association domain and is regulated by phosphorylation of IRF6. We have shown that, similar to maspin, IRF6 expression is inversely correlated with breast cancer invasiveness. We further demonstrated that the transient re-expression of IRF6 in breast cancer cells results in an increase of N-cadherin and a redistribution of vimentin commensurate with changes in cell morphology, suggestive of an epithelial-to-mesenchymal transition event. Concomitantly, we showed that maspin acts as a negative regulator of this process. These findings help to elucidate the molecular mechanisms of maspin and suggest an interactive role between maspin and IRF6 in regulating cellular phenotype, the loss of which can lead to neoplastic transformation. Maspin 2The abbreviations used are: maspin, mammary serine protease inhibitor; IRF6, interferon regulatory factor 6; IAD, IRF association domain; m.o.i., multiplicity of infection; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PBS, phosphate-buffered saline; HMEpC, human mammary epithelial cell. (mammary serine protease inhibitor, SerpinB5) was first isolated by subtractive hybridization and differential display as a protein that is expressed in normal mammary epithelial cells but reduced or absent in breast carcinomas (1Zou Z. Anisowicz A. Hendrix M.J.C. Thor A. Neveu M. Sheng S. Rafidi K. Seftor E. Sager R. Science. 1994; 263: 526-529Crossref PubMed Scopus (829) Google Scholar). Since its initial discovery, maspin has been shown to inhibit tumor invasion and metastasis in breast cancer cells (2Sheng S. Carey J. Seftor E. Dias L. Hendrix M.J.C. Sager R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11669-11674Crossref PubMed Scopus (328) Google Scholar). Further studies implicate maspin as an angiogenesis inhibitor by its ability to block neovascularization and reduce tumor-associated microvessels and also demonstrate a role for maspin in the induction of apoptosis of tumor cells (3Zhang M. Volpert O. Shi Y.H. Bouck N. Nat. Med. 2000; 6: 196-199Crossref PubMed Scopus (410) Google Scholar, 4Jiang N. Meng Y. Zhang S. Mensah-Osman E. Sheng S. Oncogene. 2002; 21: 4089-4098Crossref PubMed Scopus (115) Google Scholar). In addition, the overexpression of maspin in transgenic mice disrupts normal mammary gland development by increasing apoptosis and disrupting cell differentiation (5Zhang M. Magit D. Botteri F. Shi H.Y. He K. Li M. Furth P. Sager R. Dev. Biol. 1999; 215: 278-287Crossref PubMed Scopus (63) Google Scholar). Despite the characterization of maspin as a tumor suppressor, the molecular mechanisms underlying maspin function are complex and remain predominantly unknown. Therefore, in an effort to decipher the molecular mechanisms of maspin, we employed a yeast two-hybrid system, in which we expressed full-length maspin as bait, to identify maspin-binding proteins. This approach led to the identification of Interferon Regulatory Factor 6 (IRF6) as a possible maspin-binding protein. IRF6 belongs to the nine-member Interferon Regulatory Factor (IRF) family of transcription factors. IRFs are characterized by two conserved functional domains, namely a distinctive N-terminal DNA binding domain and a protein interaction domain. The protein interaction domain, termed the IRF association domain (IAD), is located near the C-terminal end of the protein. It is thought that protein-protein interactions involving the IAD, including homo- and heterodimerization among other IRF family members, are mediated through phosphorylation of one or multiple serine residues present within the serine-rich region near the C terminus, thus allowing interaction with other proteins (6Mori M. Yoneyama M. Ito T. Takahashi K. Inagaki F. Fujita T. J. Biol. Chem. 2004; 279: 9698-9702Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 7Lin R. Mamane Y. Hiscott J. J. Biol. Chem. 2000; 275: 34320-34327Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar, 8Meraro D. Hashmueli S. Koren B. Azriel A. Oumard A. Kirchhoff S. Hauser H. Nagulapalli S. Atchison M.L. Levi B. J. Immunol. 1999; 163: 6468-6478PubMed Google Scholar, 9Qin B.Y. Liu C. Lam S.S. Srinath H. Delston R. Correia J.J. Derynck R. Lin K. Nat. Struct. Biol. 2003; 10: 913-921Crossref PubMed Scopus (174) Google Scholar). For several IRFs, this activation step is initiated by various pathogenic stimuli that induce IRF phosphorylation, leading to dimerization and subsequent translocation to the nucleus, where IRFs function to modulate the activation of type I interferons (10Barnes B.J. Kellum M.J. Field A.E. Pitha P.M. Mol. Cell. Biol. 2002; 22: 5721-5740Crossref PubMed Scopus (210) Google Scholar, 11Lin R. Heylbroeck C. Pith P.M. Hiscott J. Mol. Cell. Biol. 1998; 18: 2986-2996Crossref PubMed Scopus (756) Google Scholar). Additionally, apart from their role in host defense, IRFs have been linked to the cellular processes of apoptosis, tumor suppression, and embryonic development (12Barnes B.J. Kellum M.J. Pinder K.E. Frisancho J.A. Pitha P.M. Cancer Res. 2003; 63: 6424-6431PubMed Google Scholar, 13Tanaka N. Ishihara M. Kitagawa M. Harada H. Kimura T. Matsuyama T. Lamphier M.S. Aizawa S. Mak T.W. Taniguchi T. Cell. 1994; 77: 829-839Abstract Full Text PDF PubMed Scopus (466) Google Scholar, 14Duguay D. Mercier F. Stagg J. Martineau D. Branson J. Servant M. Lin R. Galipeau J. Hiscott J. Cancer Res. 2002; 62: 5148-5152PubMed Google Scholar, 15Moriyama Y. Nishiguchi S. Tamori A. Koh N. Yano Y. Kubo S. Hirohashi K. Otani S. Clin. Cancer Res. 2001; 7: 1293-1298PubMed Google Scholar, 16Hatada S. Kinoshita M. Takahashi S. Nishihara R. Sakumoto H. Fukui A. Noda M. Asashima M. Gene (Amst.). 1997; 203: 183-188Crossref PubMed Scopus (22) Google Scholar, 17De Creus A. Van Beneden K. Stevenaert F. Debacker V. Plum J. Leclercq G. J. Immunol. 2002; 168: 6486-6493Crossref PubMed Scopus (68) Google Scholar). Although sequence alignment suggests that IRF6 possesses the conserved functional domains of the IRF family, there is no evidence to suggest that IRF6 functions in a similar manner to that of other IRFs nor has the initiating signal for IRF6 activation yet been determined. Here, we have demonstrated that IRF6 is expressed in normal mammary epithelial cells and that it interacts with maspin. IRF6 expression, similar to that of maspin, is reduced or lost as cells acquire a neoplastic state and progress toward a metastatic phenotype. We have also provided evidence implicating IRF6 in the regulation of cell differentiation through alterations in N-cadherin and vimentin, important phenotype markers associated with epithelial-to-mesenchymal transition. Importantly, changes induced by the re-expression of IRF6 are mitigated by the presence of maspin, all of which furthers our understanding of the molecular mechanisms of maspin. Cell Culture and Adenoviral Transfection—Normal, primary human mammary epithelial cells (HMEC 1331) were purchased from BioWhittaker, Inc. (Walkersville, MD) and maintained in defined mammary epithelial cell basal medium provided by the company. The 1436N1 cell line, an HPV16E6 immortalized normal mammary epithelial cell line, was a gift from Dr. Shijie Sheng, Wayne State University, Detroit, MI and was maintained in D-complete medium (1:1 α-minimal essential medium and HAM F-12 supplemented with 1% fetal calf serum, 1 ng/ml cholera toxin, 10 mm HEPES, pH 7.7, 50 μm ascorbic acid, and Mito+ with gentamicin. The human breast cancer cell lines MCF-7, T47-D, MDA-MB-231, and HS578T were maintained in RPMI supplemented with 10% fetal calf serum and gentamicin. IRF6 transfection of MDA-MB-231 and MDA-MB-231 green fluorescent protein-maspin was performed using the pacAd5CMV construct developed at the University of Iowa DNA vector core (supplied at 1011 particles/ml) at varying multiplicities of infection (m.o.i.) ranging from 50 to 250. Cells were treated in RPMI containing 1% serum with no for following which RPMI medium was to the was by of were performed at and and were performed using Seftor P. K. Hendrix M.J.C. Cancer Res. 2002; 62: Google for IRF6, and and for maspin, and for were purchased from was performed for with a of For maspin and GAPDH, were reported For was using the to the was performed a using expression human for the genes IRF6 and N-cadherin of of Gene and of in a of were with the following 1 at 50 for 1 at for 10 and at for for 1 were with The expression of was to an and of cell was performed similar to that Z. M. Seftor Hendrix M.J. Clin. Cancer Res. 2004; 10: PubMed Scopus Google Scholar). cells were in and in mm mm 10 mm mm with mm 1 mm mm protease Cells were and the was for Cells were at for 10 at the the was and the was in A. The was with mm mm mm mm protease in for and at for to the of cellular protein were to and using to maspin or IRF6 IRF6 was from using the and The was and its was using the two mammary epithelial cells were a and to Cells were and as Z. M. Seftor Hendrix M.J. Clin. Cancer Res. 2004; 10: PubMed Scopus Google Scholar). cells were in and in for cells were in and with 10% fetal calf serum in for 1 was and for 1 cells were in and with the for 1 Cells were in and with for IRF6 was used at and for maspin at was used at and was used at for N-cadherin and vimentin was performed and cells with to N-cadherin was used at and vimentin with was used at of cell from cells was treated with of with the and for 1 at The was using and with IRF6. of protein from of 1436N1 cell was with of protein The was with IRF6 for at complex was by the of at in PBS, and using for at were using 10% For cell was treated with of for at to the of the primary two-hybrid were performed using the from We the full-length maspin sequence into to the in which maspin is with the DNA binding domain. The human mammary gland was expressed in the vector as were performed to the was performed medium containing as a were isolated from yeast using a with of the was performed at the University of Iowa DNA For of IRF6 yeast was using a In yeast was in mm pH 1 mm pH and were to cells in the presence of 10 mm and to for at DNA was used as a was with and interactions were medium were from and from with no was from the of at The University of Iowa and the of at University, H. Cancer and were used in with of the for the of were at and to a using Maspin and IRF6 expression were the using the to maspin was used at a as M.J.C. Seftor Seftor J. 1997; Google Scholar). to IRF6 was used at a was by of the cells and the by the were by a R. was to the of the been the of the is in the and in of the tumor functions of maspin were first identified in mammary epithelial we a from normal human mammary epithelial cells in a maspin-baited yeast two-hybrid of the IRF6 as a maspin-binding with the sequence from residues to the IRF6 region and of the domain identified as the interaction in the the identification of IRF6 as a possible for maspin we the presence of IRF6 in human mammary epithelial cells by and and the interaction between maspin and IRF6 in through from 1436N1 cells and MDA-MB-231 cells were by and of the with to demonstrate the of the IRF6 and protein of IRF6 are demonstrated in and the immortalized mammary epithelial cell Importantly, cell used in the the where IRF6 and maspin the no IRF6 was in the This is by which the cellular of proteins in the of mammary epithelial cells by IRF6 as a with molecular of and it is known that phosphorylation of IRFs is important for IRF we the IRF6 phosphorylation of IRF6 in of cell with the in the of the the of the that the a of IRF6 further the phosphorylation of IRF6 and identify the acid, we IRF6 with to and We were to the phosphorylation using this and no to IRF6 is yet further is to this important the interaction in cell we performed a the from 1436N1 shown in with IRF6 and maspin, of an interaction between two proteins in mammary epithelial It is important to that the of maspin with IRF6 a of the maspin in the and further to the of this protein the role of IRF6 phosphorylation in regulating the we performed with cell with This led to a in the of maspin with IRF6, a role for phosphorylation in the regulation of the These were further by IRF6 protein The IRF family at two domains, namely an N-terminal DNA binding domain and a protein association domain termed the IRF association domain the is the primary of protein interaction among the IRFs, we that the for an interaction with maspin. we a of IRF6 and with the vector in a yeast two-hybrid system to interaction The results demonstrate that the is and for the IRF6 as the of interaction with maspin. Importantly, the of in the IRF6 vector was to this an interaction is Maspin and IRF6 in we to the of the we our studies breast where the expression of maspin is inversely correlated with the of tumor In maspin is present in but reduced in mammary and absent in a of highly metastatic breast carcinomas. of in breast cancer development and we IRF6 expression as it to maspin expression in a of neoplastic cell of the poorly breast cancer cell lines and a in IRF6 with and 1436N1 IRF6 was in the highly metastatic cell lines MDA-MB-231 and which the expression of maspin in This IRF6 expression was at the protein by where the loss of IRF6 in the poorly and highly cell lines is These were by in of of maspin and IRF6 in from normal mammary in and an expression in similar to that in the cell lines where neoplastic cells demonstrated a of IRF6 and the loss of maspin and IRF6 with the of cancer cells and of IRF6 and maspin in normal and neoplastic of maspin and IRF6 in from normal mammary and breast cancer with in and was used as the of in of maspin and IRF6 in in a In an attempt to decipher a possible role for IRF6 in mammary epithelial cells and the from the loss of IRF6 and maspin, we MDA-MB-231 and MDA-MB-231 green fluorescent cells with an containing IRF6 at m.o.i., ranging from 50 to 250. an phenotype was in However, no changes were in cells following IRF6 infection The presence of maspin and IRF6 was by of the cells and and changes in N-cadherin expression following IRF6 infection of MDA-MB-231 of IRF6 expression in and cells following infection with various of IRF6 changes in N-cadherin expression in the cells with IRF6 We to the phenotype induced by IRF6 re-expression by the expression of the known markers vimentin and of and cells an increase in the expression of N-cadherin This was further at the protein by of the cells and For vimentin, there was no at the but at the protein the presence of IRF6 a in the of vimentin and the alterations in N-cadherin and vimentin were in the cells with IRF6 C and This findings from a yeast two-hybrid system in which we identified a interaction between the tumor suppressor maspin and IRF6, a of the IRF transcription factor studies that IRF6 is expressed in normal human mammary epithelial and this interaction with maspin has been by IRF6 is the of the nine-member IRF for this family of transcription have been associated with the induction and regulation of type I interferons and interferon-inducible genes following pathogenic but have also been linked to the cellular processes of apoptosis, tumor suppression, and embryonic development (12Barnes B.J. 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IRF6 we have demonstrated a similar by the IRF6 C-terminal serine-rich We have shown that the IRF6 is and for an interaction with maspin and that the of the C-terminal serine-rich region this our that IRF6 phosphorylation a role in the of cell with to in a in the of maspin with IRF6. we that phosphorylation of IRF6 its interaction with maspin by the of the C in a manner similar to other studies also suggest that in normal mammary epithelial cells a of maspin is with IRF6. it that the interaction between maspin and IRF6 occurs in the it is possible that the interaction with maspin to IRF6 by its translocation to the This type of negative regulation has been reported with other IRFs, as the of by its binding J. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google Scholar). this is by our transfection studies that demonstrated the ability of maspin to the of IRF6 of the role of maspin in tumor suppression, we the expression of IRF6 in poorly and highly breast cancer cell of maspin and IRF6 expression by demonstrated a in IRF6 from a normal to a poorly phenotype, IRF6 expression was absent in the highly and metastatic This from maspin expression, which is highly reduced or absent in the poorly have shown that of the maspin is in maspin and is an in breast cancer development Hendrix M.J. J. 2000; PubMed Scopus Google Scholar). In the of IRF6 in poorly cells suggests an regulatory The of IRF6 is at the of protein expression, where of IRF6 in the poorly cell Importantly, the re-expression of maspin in MDA-MB-231 cells in changes in IRF6 expression nor the re-expression of IRF6 lead to changes in maspin expression, that of genes directly the expression of the We that the of IRF6 of a role for IRF6 in regulating cellular the poorly cells epithelial the metastatic cells have a phenotype. that of the maspin is one of the initial in tumor Hendrix M.J. J. 2000; PubMed Scopus Google Scholar). It is to that this in IRF6, leading to activation of genes in epithelial-to-mesenchymal transition. This is by our IRF6 transfection which that in the of maspin IRF6 to expression of epithelial-to-mesenchymal genes as It is also possible that IRF6 is for regulation is the in S. Y. E. S. A. L. M. C. C. M. D. A. M.J. Nat. 2002; PubMed Scopus Google and the presence of maspin this process. In the of maspin, activation of IRF6 lead to its which the loss of IRF6 in highly breast cancer The increase in N-cadherin in in cancer cells with epithelial has been reported to the of cancer cells into the host studies have identified the of N-cadherin as of a and breast cancer cell phenotype M.J. J. Cell. PubMed Scopus Google Scholar, L. C. N. M. C. B. M. Y. M. J. Cell. 2004; PubMed Scopus Google Scholar, L. J. Cell Biol. 2000; PubMed Scopus Google Scholar). in findings are further by of breast These studies that maspin and IRF6 are expressed in the and of normal and reduced or absent in neoplastic in normal the cell which the a of maspin but is of IRF6. This of IRF6 expression in cells the of an differentiation the and epithelial cells are from a mammary further a role for IRF6 and maspin in regulating the mammary epithelial cell phenotype H. Cell 2003; PubMed Scopus Google Scholar). In we have shown that the transcription factor IRF6 is expressed in normal mammary and that it interacts with the tumor suppressor maspin. We further demonstrated that IRF6 expression is lost in breast cancer cells and that the interaction is important in a normal mammary epithelial cell phenotype. We further that the interaction an important role in by regulating cell an important of mammary gland This elucidate the tumor mechanisms of maspin and a in which maspin its function in through the regulation of IRF6. the molecular mechanisms of maspin and its interaction with IRF6 to the of maspin as a possible and in breast We Dr. Dr. and Dr. and and Seftor for and with