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The Embryonic Transcription Cofactor LBH Is a Direct Target of the Wnt Signaling Pathway in Epithelial Development and in Aggressive Basal Subtype Breast Cancers

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The Embryonic Transcription Cofactor LBH Is a Direct Target of the Wnt Signaling Pathway in Epithelial Development and in Aggressive Basal Subtype Breast Cancers Edinburgh Research Explorer The embryonic transcription cofactor LBH is a direct target of the Wnt signaling pathway in epithelial development and in aggressive basal subtype breast cancers Citation for published version: Rieger, ME, Sims, AH, Coats, ER, Clarke, RB & Briegel, KJ 2010, 'The embryonic transcription cofactor LBH is a direct target of the Wnt signaling pathway in epithelial development and in aggressive basal subtype breast cancers', Molecular and Cellular Biology, vol. 30, no. 17, pp. 4267-79. https://doi.org/10.1128/MCB.01418-09 Digital Object Identifier (DOI): 10.1128/MCB.01418-09 Link: Link to publication record in Edinburgh Research Explorer Document Version: Publisher's PDF, also known as Version of record Published In: Molecular and Cellular Biology General rights Copyright for the publications made accessible via the Edinburgh Research Explorer is retained by the author(s) and / or other copyright owners and it is a condition of accessing these publications that users recognise and abide by the legal requirements associated with these rights. Take down policy The University of Edinburgh has made every reasonable effort to ensure that Edinburgh Research Explorer content complies with UK legislation. If you believe that the public display of this file breaches copyright please contact [email protected] providing details, and we will remove access to the work immediately and investigate your claim. Download date: 07. Oct. 2021 MOLECULAR AND CELLULAR BIOLOGY, Sept. 2010, p. 4267–4279 Vol. 30, No. 17 0270-7306/10/$12.00 doi:10.1128/MCB.01418-09 Copyright © 2010, American Society for Microbiology. All Rights Reserved. The Embryonic Transcription Cofactor LBH Is a Direct Target of the Wnt Signaling Pathway in Epithelial Development and in Aggressive Basal Subtype Breast Cancersᰔ† Megan E. Rieger,1 Andrew H. Sims,2‡ Ebony R. Coats,1§ Robert B. Clarke,2 and Karoline J. Briegel1* Department of Biochemistry and Molecular Biology, Braman Family Breast Cancer Institute at the Sylvester Comprehensive Cancer Center, University of Miami Miller School of Medicine, Miami, Florida 33136,1 and Breast Biology Group, Paterson Institute for Cancer Research, Manchester Academic Health Sciences Centre, University of Manchester, Manchester M20 4BX, United Kingdom2 Received 28 October 2009/Returned for modification 29 December 2009/Accepted 22 June 2010 Limb-bud and heart (LBH) is a novel key transcriptional regulator of vertebrate development. However, the molecular mechanisms upstream of LBH and its role in adult development are unknown. Here we show that in epithelial development, LBH expression is tightly controlled by Wnt signaling. LBH is transcriptionally induced by the canonical Wnt pathway, as evident by the presence of conserved functional T-cell factor (TCF)/lymphoid enhancer-binding factor (LEF) binding sites in the LBH locus and rapid ␤-catenin-dependent upregulation of endogenous LBH by Wnt3a. In contrast, LBH induction by Wnt/␤-catenin signaling is inhibited by Wnt7a, which in limb development signals through a noncanonical pathway involving Lmx1b. Furthermore, we show that LBH is aberrantly overexpressed in mammary tumors of mouse mammary tumor virus (MMTV)- Wnt1-transgenic mice and in aggressive basal subtype human breast cancers that display Wnt/␤-catenin hyperactivation. Deregulation of LBH in human basal breast cancer appears to be Wnt/␤-catenin dependent, as DKK1 and Wnt7a inhibit LBH expression in breast tumor cells. Overexpression studies indicate that LBH suppresses mammary epithelial cell differentiation, an effect that could contribute to Wnt-induced tumorigen- esis. Taken together, our findings link LBH for the first time to the Wnt signaling pathway in both development and cancer and highlight LBH as a potential new marker for therapeutically challenging basal-like breast cancers. Increasing evidence suggests that embryonic development and corepressor functions (7, 8). Recent biophysical analysis and tumorigenesis share some of the same molecular mecha- has revealed a high degree of structural disorder in LBH, nisms. In particular, aberrant reactivation of latent develop- suggesting that conformational plasticity may play a signif- mental signaling pathways and transcription factors in tumor icant role in modulating LBH-dependent transcriptional cells has been associated with and shown to play causal roles in processes (2). advanced-stage, invasive cancers (5, 6, 80). Aberrant gain of LBH function is associated with partial Limb-bud and heart (LBH) is a highly conserved, novel trisomy 2p syndrome (7), a rare human autosomal disorder tissue-specific transcription cofactor in vertebrates with impor- that is characterized by multiple congenital anomalies, includ- tant roles in embryonic development (7, 8, 19). We have pre- ing cardiovascular, skeletal, and postaxial limb defects (46). viously identified Lbh as a novel mouse gene with unique Partial trisomy 2p syndrome patients harbor a triplication of spatiotemporal expression during early embryogenesis that re- chromosomal region 2p23, where LBH maps, indicating that flects pattern formation in the developing limb buds and heart increased LBH gene dosage is pathological in humans (7). (8). Lbh encodes a small acidic protein (molecular mass, 12.3 Transgenic misexpression of Lbh during embryonic heart de- kDa) that contains a conserved putative nuclear localization velopment in mice phenocopies congenital heart disease ob- signal and a glutamate-rich putative transcriptional activation served in these patients (7) and indicates that Lbh functions to domain but lacks a DNA binding domain (DBD) (8). In mam- attenuate cardiac chamber differentiation through corepres- malian reporter assays, LBH has both transcriptional activator sion of key cardiac transcription factors NKX2.5 and TBX5 (7). Interestingly, gain of function of LBH during mouse heart * Corresponding author. Mailing address: Department of Biochem- development also causes various growth defects, such as ven- istry and Molecular Biology, University of Miami Miller School of tricular hyperplasia and increased cardiac valve formation, as Medicine, 1011 15th NW Street, 33136 Miami, FL. Phone: (305) 243- well as the abnormally sustained self-renewal of cardiomyo- 4770. Fax: (305) 243-9249. E-mail: [email protected]. cytes after birth, suggesting that LBH has promitogenic activity † Supplemental material for this article may be found at http://mcb .asm.org/. (7). Consistent with these findings, retroviral overexpression of ‡ Present address: Applied Bioinformatics of Cancer Research Lbh in chicken embryonic limbs leads to increased cell prolif- Group, Breakthrough Research Unit, Edinburgh Cancer Research eration of immature chondrocytes and markedly delays bone Centre, Crewe Road South, Edinburgh EH4 2XR, United Kingdom. differentiation (19). However, the regulatory pathways acting § Present address: Department of Microbiology, Immunology and In- fectious Diseases, University of Virginia, Charlottesville, VA 22908. upstream of LBH and its role in adult development have re- ᰔ Published ahead of print on 6 July 2010. mained obscure. 4267 4268 RIEGER ET AL. MOL.CELL.BIOL. Wnt signaling plays a fundamental role in embryonic devel- induction is efficiently blocked by Wnt7a. Given the parallels opment by regulating pattern formation, cell proliferation, dif- between Wnt signaling in development and cancer, we hypoth- ferentiation, and migration (44). Wnt ligands are secreted esized that LBH, as a canonical Wnt target gene, might be lipid-modified glycoproteins that act as morphogens and elicit deregulated in breast cancer. Indeed, we found that LBH is different cell behaviors depending on whether receptor inter- aberrantly overexpressed in mammary tumors of MMTV-Wnt1 action activates a canonical ␤-catenin-dependent transduction transgenic mice as well as in highly aggressive basal subtype pathway or other ␤-catenin-independent noncanonical path- human breast cancers. Overexpression of Lbh in HC11 mam- ways (39, 73). In adults, canonical Wnt signaling promotes the mary epithelial cells (MECs) further demonstrates that LBH self-renewal and maintenance of stem cells required for nor- suppresses terminal cell differentiation, an effect that could mal tissue homeostasis (52), a function that becomes onco- contribute to Wnt-induced tumorigenesis. Collectively, our genic when this pathway is deregulated (18). Activation of data suggest that LBH is a direct Wnt target gene that is canonical Wnt signaling leads to the stabilization of cyto- reactivated in a particularly lethal form of human breast plasmic ␤-catenin and its subsequent translocation into the cancer. nucleus, where it forms a heteromeric complex with DNA- binding proteins of the T-cell factor (TCF)/lymphoid en- MATERIALS AND METHODS hancer-binding factor (LEF) family to activate target gene transcription (18). In the absence of Wnt, TCF/LEF factors Genomic DNA isolation and plasmids. A Lambda gt11-129EV mouse genomic DNA library (Stratagene) was screened with Lbh-specific cDNA probes (8). are bound to target gene promoters but act as transcrip- Several overlapping genomic clones comprising approximately 30 kb of the tional repressors by forming a complex with Groucho/Grg/ murine Lbh gene locus were isolated and mapped by restriction analysis. A TLE corepressors (18). The amplitude of canonical Wnt SexAI-NotI genomic fragment containing approximately 1.5 kb of Lbh promoter signaling is autoregulated via multiple positive and negative region and 283 bp
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