Oncogene (2003) 22, 3875–3887 & 2003 Nature Publishing Group All rights reserved 0950-9232/03 $25.00 www.nature.com/onc Activation of b-catenin in prostate epithelium induces hyperplasias and squamous transdifferentiation Brian Bierie1,7, Masahiro Nozawa1,7, Jean-Pierre Renou1,2, Jonathan M Shillingford1, Fanta Morgan1, Takami Oka1, Makoto M Taketo3, Robert D Cardiff4, Keiko Miyoshi1,5, Kay-Uwe Wagner6, Gertraud W Robinson1 and Lothar Hennighausen*,1 1Laboratory of Genetics and Physiology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA; 2Institut National de la Recherche Agronomique, Jouy-en-Josas Cedex, France; 3Department of Pharmacology, Kyoto University Graduate School of Medicine, Sakyo, Kyoto 606-8501, Japan; 4Center for Comparative Medicine, University of California, Davis, CA 95616, USA; 5Department of Biochemistry, School of Dentistry, The University of Tokushima, Tokushima 770-8504, Japan; 6Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Nebraska Medical Center, Omaha, Nebraska The Wnt/b-catenin signaling pathway is critical for intra-acinous hyperplasias or neoplastic foci is a later normal mammalian development, the specification of event. epidermal cells and neoplastic transformation of intestinal Oncogene (2003) 22, 3875–3887. doi:10.1038/sj.onc.1206426 epithelium. However, precise molecular information regarding cell-specific responses to b-catenin signaling Keywords: b-catenin; cell identity; transdifferentiation; has been limited. This question was addressed using a neoplasia; prostate mouse model in which exon 3 of the b-catenin gene was deleted in several cell types with loxP-mediated recombi- nation utilizing a Cre transgene under control of the mouse mammary tumor virus-long terminal repeat Introduction (MMTV-LTR). The stabilization of b-catenin in prostate epithelium resulted in hyperplasias and extensive trans- b-Catenin is an integral part of the Wnt signaling differentiation into epidermal-like structures, which ex- pathway and has been linked to developmental pro- pressed keratins 1 and 6, filaggrin, loricrin and involucrin. cesses including cell fate specification, polarity and The cell-specific loss of NKCC1 protein and reduced migration (McCrea et al., 1991; Gat et al., 1998; Brault nuclear Stat5a is further suggestive of a loss of prostate et al., 2001; Merrill et al., 2001; Niemann et al., 2002). epithelial characteristics. In addition to the prostate, Wnt proteins bind to Frizzled receptors and induce a hyperplasias and squamous metaplasias were detected in signaling cascade that leads to the stabilization of b- epithelia of the epididymis, vas deferens, coagulating catenin. The stabilized b-catenin is able to interact with gland, preputial gland and salivary gland. However, and in the lymphoid enhancer transcription factors (LEFs) and contrast to a recent study, no lesions reminiscent of high- T-cell factors (TCFs), which subsequently activate grade prostate intraepithelial neoplasia were detected. genetic programs (Roose et al., 1999; de Lau and Since b-catenin was activated in several cell types and Clevers, 2001; Hovanes et al., 2001). Genetic loss and impinged upon the viability of these mice, it was not gain of function experiments (Haegel et al., 1995; Gat possible to evaluate the cumulative effect over more than 3 et al., 1998; Huelsken et al., 2000, 2001; Widelitz et al., months. To assess long-term consequences of b-catenin 2000) have revealed the importance of b-catenin in the activation, mutant and control prostate tissues were determination of the anterior–posterior axis and meso- transplanted into the mammary fat pads of wild-type dermstructures during fetal development,and its role in males. Notably, squamous metaplasias, intra-acinous the specification of cell fate. Furthermore, b-catenin hyperplasia and possible neoplastic transformation were signaling is essential for the follicular differentiation of observed after a total of 18 weeks of b-catenin stimula- epidermal cells and the expression of a stabilized b- tion. This suggests that the transdifferentiation into catenin in these cells results in the formation of squamous metaplasias is an early response of endoderm- unscheduled hair follicles (Gat et al., 1998). In support derived cells to b-catenin, and that the development of of this observation, an epidermal fate is adopted by stem cells in the skin in the absence of b-catenin (Huelsken and Birchmeier, 2001; Huelsken et al., 2001). Such *Correspondence: L Hennighausen, Laboratory of Genetics and lineage decisions appear to be regulated through specific Physiology, NIDDK, NIH Building 8, Room101, Bethesda, MD genetic programs initiated by TCF3/LEF complexes 20892-0822, USA; E-mail [email protected] 7Both contributed equally to this work (Merrill et al., 2001). Recently, we demonstrated that the Received 29 July 2002; revised 28 January 2003; accepted 28 January stabilization of b-catenin in differentiated mammary 2003 epitheliumresults in a loss of differentiation and the Stabilized b-catenin induces squamous transdifferentiation B Bierie et al 3876 transdifferentiation into epidermal-like structures (Miyoshi et al., 2002a), further demonstrating the ability of this signaling pathway to determine cell fate. Aberrant signaling through b-catenin has been found in many tumors, in particular those of the colon (e.g. Polakis, 2000). Mutations in the b-catenin pathway have been observed in approximately 5% of human prostate cancers (Voeller et al., 1998; Chesire et al., 2000) and Brain Lung Heart Liver Kidney Small Intestine Skin Gland Salivary Testis Epididymis Deferens Vas Gland Coagulating Seminal Vesicle Prostate Ventral there is evidence to suggest that aberrant b-catenin a signaling contributes to prostate growth and tumorigen- esis (Chesire et al., 2002; Gerstein et al., 2002). To establish whether tumorigenesis in the prostate can be induced by activated b-catenin alone, mice (Catnb+/ lox(ex3)) were generated (Harada et al., 1999) that express ( ) stabilized b-catenin in prostate epitheliumupon Cre- Catnbb+/+/ loxPP :MMTVVcrec mediated excision of exon 3 (amino acids 5–80). b Deletion of exon 3 in several cell types, including prostate epithelium, vas deferens, coagulating gland, preputial gland, basal cells in skin and salivary epithelium, was achieved through the use of transgenic mice that carry the Cre gene under the control of the MMTV-LTR (Wagner et al., 1997, 2001). To determine the cellular changes induced by b-catenin immunohis- tochemical approaches and microarray analyses were Figure 1 Expression of stabilized b-catenin in Catnb+/Dex3: used to identify proteins and genes, respectively, whose MMTVcre mice. Proteins were extracted from different tissues types expressions were altered. of 8-week-old Catnb+/Dex3:MMTVcre mice (a) and age-matched littermate controls (b). Stabilized b-catenin was detected in skin, salivary gland, epididymis, vas deferens, coagulating gland, seminal vesicle and the ventral prostate (arrow) Results Stabilization of b-catenin in different tissues catenin was evident in skin, salivary gland, epididymis, vas deferens, coagulating gland, seminal vesicle, ventral Exon 3 of the b-catenin gene (amino acids 5–80) encodes prostate (Figure 1a), spleen and mammary tissue (data critical serine and threonine residues that, upon not shown) of Catnb+/Dex3:MMTVcre mice. The appear- phosphorylation, target the b-catenin protein for ance of N-terminal truncated b-catenin in these tissues is ubiquitin-mediated degradation. Mice have been gener- consistent with the cell-specificity of Cre activity in ated that carry exon 3 of the b-catenin gene flanked by MMTV-Cre transgenic mice (Wagner et al., 2001). loxP sites. Using these mice, it has been demonstrated that Cre-mediated loss of exon 3 specifically in the Stabilization of b-catenin causes hyperproliferation and intestine results in the generation of a truncated squamous transdifferentiation of prostate epithelium stabilized b-catenin and the formation of intestinal polyposis (Harada et al., 1999). To further assess the Histological analysis of the ventral prostate from consequence of b-catenin stabilization in other cell Catnb+/Dex3:MMTVcre mice at 8 weeks of age revealed types, the conditionally targeted b-catenin mice were papillary epithelial hyperplasias and evidence of epithe- crossed with a mouse line expressing the Cre transgene lial cells growing into the ductal lumina (Figure 2a). under the control of the MMTV-LTR (Wagner et al., Some ducts were filled with epithelial cells (Figure 2a) 1997, 2001), which is referred to as Catnb+/Dex3:MMTVcre and also contained extensive squamous metaplasias mice (Figure 2b), which were similar to those observed in Catnb+/Dex3:MMTVcre mice could be identified visually the mammary tissue expressing stabilized b-catenin at 10 days of age because they exhibited sparse and (Miyoshi et al., 2002a). Furthermore, comparable to irregular hair growth (data not shown). In addition, mammary tissue, the early lesions were characterized by these mice developed thickness of the eyelids, nose and an abundance of ghost cells (Figure 2b). Prostate tissue ears within 6 weeks after birth (data not shown). An fromlittermates that did not carry the Cre transgene increase in the number of hair follicles and the presence exhibited normal histology consisting of regular acini of epithelioid cysts was observed (data not shown) and ducts and normal nuclei (Figure