An LRP5 Receptor with Internal Deletion in Hyperparathyroid Tumors with Implications for Deregulated WNT/β-Catenin Signaling

Westin G (2007) An LRP5 receptor with internal deletion in hyperparathyroid tumors with implications for deregulated WNT/b- catenin signaling. PLoS Med 4(11): e328. doi:10.1371/journal.pmed. 0040328 An LRP5 Receptor with Internal Deletion in Hyperparathyroid Tumors with Implications for Deregulated WNT/b-Catenin Signaling Peyman Bjo rklund 0 1 G oran A kerstro m 0 1 Gunnar Westin 0 1 0 Academic Editor: Hans Clevers, Utrecht University , The Netherlands 1 Department of Surgical Sciences, Uppsala University, Endocrine Unit, Uppsala University Hospital , Uppsala , Sweden 2 www.plosmedicine.org - The internally truncated LRP5 receptor is strongly implicated in deregulated activation of the WNT/b-catenin signaling pathway in hyperparathyroid tumors, and presents a potential target for therapeutic intervention. The Editors Summary of this article follows the references. Primary hyperparathyroidism (pHPT) is characterized by hypersecretion of parathyroid hormone and generally also hypercalcemia, due to one or several parathyroid tumors (adenoma). Secondary hyperparathyroidism (sHPT) develops in patients with uremia because of phosphate retention, hypocalcemia, and reduced 1,25-dihydroxyvitamin D3 levels, causing parathyroid hyperplasia and eventually development of parathyroid tumors and hypercalcemia [14]. Parathyroidectomy is the only considered therapy for most patients. We recently reported aberrant b-catenin (CTNNB1) accumulation in all analyzed parathyroid tumors from patients with pHPT and in hyperplastic parathyroid glands from patients with uremia secondary to HPT [5]. MYC, a direct target of the Wnt/b-catenin signaling pathway in colorectal cancer cells and established as the critical mediator of the early stages of intestinal neoplasia [6,7], was found to be overexpressed at the protein level in 79% of parathyroid tumors [5]. Maintained activity of endogenous b-catenin was found to be necessary for the expression of MYC and cyclin D1 (CCND1), as well as growth and survival of a unique human parathyroid tumor cell line [8]. Overexpression of cyclin D1 has been reported in 20%40% of pHPT tumors [2], and overexpression of cyclin D1 in the parathyroid glands of transgenic mice caused development of pHPT [9]. In a small fraction of parathyroid adenomas, overexpression is due to activation of the CCND1 gene by pericentromeric inversions of Chromosome 11, involving the parathyroid hormone (PTH) promoter [10]. Augmented cyclin D1 expression in some parathyroid adenomas could also be a consequence of aberrant b-catenin accumulation [5], although it remains to be determined whether CCND1 constitutes a b-catenin target [11] in parathyroid cells. We also reported CTNNB1 stabilizing mutations in a few cases (3 out of 20) of pHPT tumors, while no mutation was found in uremic secondary HPT tumors, and inactivating truncations of adenomatosis polyposis coli (APC) were not seen [5]. Mutation or deregulated expression of other Wnt-signaling components leading to bcatenin accumulation was therefore anticipated. Dysregulated Wnt signaling with accumulation of b-catenin in the cytoplasm/nucleus plays an important role in a variety of human cancers. The stability of b-catenin is regulated by Wnt ligands through a destruction complex consisting of APC/Axin/GSK-3b/Ck1/Dvl and other factors. In the absence of Wnt ligand, free cytoplasmic b-catenin is rapidly degraded by the proteasome after phosphorylation of its amino terminus at residues serine 33, serine 37, threonine 41, and serine 45 [1215]. Wnt ligands bind to cell-surface Frizzled receptors and LRP5/6 coreceptors and result in changes in phosphorylation of several intracellular signaling components with the subsequent accumulation of nonphosphorylated b-catenin [1619]. According to a current model, the destruction complex is inactivated through recruitment of Axin to the intracellular domain of LRP5 [20]. b-catenin binds the LEF/TCF family of transcription factors to positively or negatively regulate transcription of target genes. Many mutant proteins of the Wnt signaling pathway, such as b-catenin, APC, Axin, and beta-transducin repeat-containing protein (b-Trcp), are associated with specific forms of cancer. For instance, aberrant accumulation of b-catenin through stabilizing mutations in CTNNB1 or inactivating mutations in APC is strongly implicated in the cause of approximately 10% and 80% of colorectal cancers, respectively [13,14]. A mutant of LRP5 lacking the extracellular domain was demonstrated to be constitutively active in vitro [20]. In this study, we aimed at investigating the potential role of LRP5 in parathyroid tumorigenesis. Tissue Specimens Parathyroid adenomas (n 37) and hyperplastic glands (n 20) from patients with pHPT and sHPT, respectively, were acquired from patients diagnosed and operated on in the clinical routine. Each patient contributed with one tumor. All 57 tumors displayed aberrant accumulation of b-catenin (unpublished data), of which 14 parathyroid adenomas and all 20 hyperplastic parathyroid glands were described previously [5]. Normal parathyroid tissue (n 6) was obtained from glands inadvertently removed in conjunction with thyroid surgery where autotransplantation was not required or as normal parathyroid gland biopsies in patients subjected to parathyroidectomy. All tissues were intraoperatively snapfrozen, and cryosections were used in the analyses. Written informed consent and approval of local ethics committee was obtained. Detection of Normal and Internally Truncated LRP5 Transcripts by PCR and DNA Sequencing Total RNA was extracted with TriZol Reagent (Gibco BRL, Life Technologies) according to the manufacturers instructions and the RNA was subsequently treated with RQ1 DNase I (Promega) and proteinase K. Alternatively, DNA-free RNA was prepared using the Nucleospin RNA II kit (MachereyNagel). Successful DNase treatments were established by PCR analysis of all RNA preparations. Reverse transcription of total DNA-free RNA was performed with random hexamer primers using the First-Strand cDNA Synthesis kit (Amersham Pharmacia Biotech) according to the manufacturers instructions. cDNA was amplified by primary or nested PCR using mRNA-specific primers spanning positions 19922932 of LRP5 (GenBank accession number AF064548; http://www. ncbi.nlm.nih.gov/Genbank). A total of 1%2% of the primary PCR product was used for nested PCR. Primers used were the following: forward primer, 59-CTTCACCAGCAGAGCCGCCATCCACAG-39; nested forward, 59-GGATCTCCCTCGAGACCAATAACAACG-39; and reverse, 59-CCGGGATCATCC GACTGATG-39. The PCR amplifications were performed with cDNA, 25 pmol of each primer, 0.2 mM dNTPs, 13 PCR buffer, 1.5 mM MgCl2, and 0.25 U Platinum Taq DNA polymerase (Invitrogen). The PCR conditions were: denaturation at 95 8C for 60 s, followed by 40 cycles of denaturation for 20 s, annealing at 58 8C for 20 s and extension at 72 8C for 90 s, and a final extension at 72 8C for 7 min. An anne (...truncated)