Tumour metastasis is a complex process involving reciprocal interplay between cancer cells and host stroma at both primary and secondary sites, and is strongly influenced by microenvironmental factors such as hypoxia1. Tumour-secreted proteins play a crucial role in these interactions2-5 and present strategic therapeutic potential. Metastasis of breast cancer to the bone affects approximately 85% of patients with advanced disease and renders them largely untreatable6. Specifically, osteolytic bone lesions, where bone is destroyed, lead to debilitating skeletal complications and increased patient morbidity and mortality6,7. The molecular interactions governing the early events of osteolytic lesion formation are currently unclear. Here we show hypoxia to be specifically associated with bone relapse in ER-negative breast cancer patients. Global quantitative analysis of the hypoxic secretome identified Lysyl Oxidase (LOX) as significantly associated with bone-tropism and relapse. High expression of LOX in primary breast tumours or systemic delivery of LOX leads to osteolytic lesion formation whereas silencing or inhibition of LOX activity abrogates tumour-driven osteolytic lesion formation. We identify LOX as a novel regulator of NFATc1-driven osteoclastogenesis, independent of RANK Ligand, which disrupts normal bone homeostasis leading to the formation of focal pre-metastatic lesions. We show that these lesions subsequently provide a platform for circulating tumour cells to coloniseCorrespondence and requests for materials should be addressed to janine.erler@bric.ku.dk and a.gartland@shef.ac.uk.

More than 90% of cancer patient mortality is attributed to metastasis. In this study, we investigated a role for the lysyl oxidase-related enzyme lysyl oxidase-like 2 (LOXL2) in breast cancer metastasis, in both patient samples and in vivo models. Analysis of a published microarray data set revealed that LOXL2 expression is correlated with metastasis and decreased survival in patients with aggressive breast cancer. In immunocompetent or immunocompromised orthotopic and transgenic breast cancer models we showed that genetic, chemical or antibody-mediated inhibition of LOXL2 resulted in decreased metastasis. Mechanistic investigations revealed that LOXL2 promotes invasion by regulating the expression and activity of the extracellular proteins tissue inhibitor of metalloproteinase-1 (TIMP1) and matrix metalloproteinase-9 (MMP9). We found that LOXL2, TIMP1, and MMP9 are coexpressed during mammary gland involution, suggesting they function together in glandular remodeling after weaning. Finally, we found that LOXL2 is highly expressed in the basal/myoepithelial mammary cell lineage, like many other genes that are upregulated in basal-like breast cancers. Our findings highlight the importance of LOXL2 in breast cancer progression and support the development of anti-LOXL2 therapeutics for the treatment of metastatic breast cancer. Cancer Res; 71(5); 1561-72. Ó2011 AACR.

Multiple myeloma (MM) is associated with the development of osteolytic bone disease, mediated by increased osteoclastic bone resorption and impaired osteoblastic bone formation. Dickkopf-1 (Dkk1), a soluble inhibitor of wingless/int (Wnt) signaling and osteoblastogenesis, is elevated in patients with MM and correlates with osteolytic bone disease. In this study, we investigated the effect of inhibiting Dkk1 on the development of osteolytic lesions in the 5T2MM murine model of myeloma. We showed that Dkk1 is expressed by murine 5T2MM myeloma cells. Injection of 5T2MM cells into C57BL/KaLwRij mice resulted in the development of osteolytic bone lesions (p < 0.05), mediated by increased osteoclast numbers (p < 0.001) and a decrease in osteoblast numbers (p < 0.001) and mineralizing surface (p < 0.05). Mice bearing 5T2MM cells were treated with an anti-Dkk1 antibody (BHQ880, 10 mg/kg, IV, twice weekly for 4 wk) from time of paraprotein detection. Anti-Dkk1 treatment prevented 5T2MM-induced suppression of osteoblast numbers (p < 0.001) and surface (p < 0.001). Treatment increased mineralizing surface by 28% and bone formation rate by 25%; however, there was no change in mineral apposition rate. Inhibiting Dkk1 had no effect on osteoclast numbers. mCT analysis showed that anti-Dkk1 treatment significantly protected against 5T2MM-induced trabecular bone loss (p < 0.05) and reduced the development of osteolytic bone lesions (p < 0.05). Treatment had no significant effect on tumor burden. These data suggest that inhibiting Dkk1 prevents the suppression of bone formation and in doing so is effective in preventing the development of osteolytic bone disease in myeloma, offering an effective therapeutic approach to treating this clinically important aspect of myeloma.

Hypothyroidism and thyrotoxicosis are each associated with an increased risk of fracture. Although thyroxine (T4) is the predominant circulating thyroid hormone, target cell responses are determined by local intracellular availability of the active hormone 3,5,3′-L-triiodothyronine (T3), which is generated from T4 by the type 2 deiodinase enzyme (D2). To investigate the role of locally produced T3 in bone, we characterized mice deficient in D2 (D2KO) in which the serum T3 level is normal. Bones from adult D2KO mice have reduced toughness and are brittle, displaying an increased susceptibility to fracture. This phenotype is characterized by a 50% reduction in bone formation and a generalized increase in skeletal mineralization resulting from a local deficiency of T3 in osteoblasts. These data reveal an essential role for D2 in osteoblasts in the optimization of bone strength and mineralization.thyroid hormone metabolism | fracture | hypothyroidism | bone formation | skeleton T hyroid hormones are essential for linear growth and peak bone mass acquisition. In adults, thyrotoxicosis results in high bone turnover osteoporosis and increased susceptibility to fracture, whereas hypothyroidism reduces bone turnover (1-3). In previous studies, we identified thyroid hormone receptor α (TRα) as the critical mediator of 3,5,3′-L-triiodothyronine (T3) action in bone (4-7). The aim of this study is to investigate the role of the type 2 iodothyronine deiodinase (D2) as a local prereceptor modulator of T3 action in the skeleton.Thyroid hormone actions are determined by local availability of T3 to its nuclear receptor (8). D2 catalyzes removal of an outer ring 5′-iodine atom from the major circulating hormone thyroxine (T4) to generate the active metabolite T3. Conversely, the type 3 deiodinase (D3) inactivates both T4 and T3 by removal of an inner ring 5-iodine atom. Thus, D2 and D3, in conjunction with serum-derived T3, are important local modulators of thyroid hormone action in vivo. Expression of D2 and D3 is regulated in a temporo-spatial and tissue-specific manner, resulting in varying levels of T3 action in individual tissues despite relatively constant serum thyroid hormone levels (8).Mice deficient in D2 (D2KO) exhibit pituitary resistance to feedback regulation by T4 characterized by a 3-fold increase in serum thyroid-stimulating hormone (TSH), a 27% increase in the serum T4 level, but a normal T3 level. The increased TSH and T4 levels are evident as early as postnatal day (PD)10 (9). These changes are accompanied by cold intolerance, impaired hearing, and reduced brain T3 content (10). The type 1 deiodinase (D1) is widely believed to catalyze conversion of T4 to T3 in tissues such as liver and kidney, predominantly for export to plasma. Like the D2KO mice, D1/D2KO double-mutant mice exhibit increases in serum T4 and TSH and have a normal serum T3 level (10).The roles of D1 and D2 in regulating T3 action in the skeleton have not been studied, although limited information regarding growth is available. A minor and trans...

Osteoporosis is a common polygenic disease and global healthcare priority but its genetic basis remains largely unknown. We report a high-throughput multi-parameter phenotype screen to identify functionally significant skeletal phenotypes in mice generated by the Wellcome Trust Sanger Institute Mouse Genetics Project and discover novel genes that may be involved in the pathogenesis of osteoporosis. The integrated use of primary phenotype data with quantitative x-ray microradiography, micro-computed tomography, statistical approaches and biomechanical testing in 100 unselected knockout mouse strains identified nine new genetic determinants of bone mass and strength. These nine new genes include five whose deletion results in low bone mass and four whose deletion results in high bone mass. None of the nine genes have been implicated previously in skeletal disorders and detailed analysis of the biomechanical consequences of their deletion revealed a novel functional classification of bone structure and strength. The organ-specific and disease-focused strategy described in this study can be applied to any biological system or tractable polygenic disease, thus providing a general basis to define gene function in a system-specific manner. Application of the approach to diseases affecting other physiological systems will help to realize the full potential of the International Mouse Phenotyping Consortium.

SUMMARY Filarial worms cause highly morbid diseases such as elephantiasis and river blindness. Since the 1940s, researchers have conducted vaccine trials in 27 different animal models of filariasis. Although no vaccine trial in a permissive model of filariasis has provided sterilizing immunity, great strides have been made toward developing vaccines that could block transmission, decrease pathological sequelae, or decrease susceptibility to infection. In this review, we have organized, to the best of our ability, all published filaria vaccine trials and reviewed them in the context of the animal models used. Additionally, we provide information on the life cycle, disease phenotype, concomitant immunity, and natural immunity during primary and secondary infections for 24 different filaria models.

Cancers that grow in bone, such as myeloma and breast cancer metastases, cause devastating osteolytic bone destruction. These cancers hijack bone remodeling by stimulating osteoclastic bone resorption and suppressing bone formation. Currently, treatment is targeted primarily at blocking bone resorption, but this approach has achieved only limited success. Stimulating osteoblastic bone formation to promote repair is a novel alternative approach. We show that a soluble activin receptor type IIA fusion protein (ActRIIA.muFc) stimulates osteoblastogenesis ( p < .01), promotes bone formation ( p < .01) and increases bone mass in vivo ( p < .001). We show that the development of osteolytic bone lesions in mice bearing murine myeloma cells is caused by both increased resorption ( p < .05) and suppression of bone formation ( p < .01). ActRIIA.muFc treatment stimulates osteoblastogenesis ( p < .01), prevents myeloma-induced suppression of bone formation ( p < .05), blocks the development of osteolytic bone lesions ( p < .05), and increases survival ( p < .05). We also show, in a murine model of breast cancer bone metastasis, that ActRIIA.muFc again prevents bone destruction ( p < .001) and inhibits bone metastases ( p < .05). These findings show that stimulating osteoblastic bone formation with ActRIIA.muFc blocks the formation of osteolytic bone lesions and bone metastases in models of myeloma and breast cancer and paves the way for new approaches to treating this debilitating aspect of cancer. ß

Progeny of mice treated with the mutagen N-ethyl-N-nitrosourea (ENU) revealed a mouse, designated Longpockets (Lpk), with short humeri, abnormal vertebrae, and disorganized growth plates, features consistent with spondyloepiphyseal dysplasia congenita (SEDC). The Lpk phenotype was inherited as an autosomal dominant trait. Lpk/þ mice were viable and fertile and Lpk/Lpk mice died perinatally. Lpk was mapped to chromosome 15 and mutational analysis of likely candidates from the interval revealed a Col2a1 missense Ser1386Pro mutation. Transient transfection of wild-type and Ser1386Pro mutant Col2a1 c-Myc constructs in COS-7 cells and CH8 chondrocytes demonstrated abnormal processing and endoplasmic reticulum retention of the mutant protein. Histology revealed growth plate disorganization in 14-day-old Lpk/þ mice and embryonic cartilage from Lpk/þ and Lpk/Lpk mice had reduced safranin-O and type-II collagen staining in the extracellular matrix. The wild-type and Lpk/þ embryos had vertical columns of proliferating chondrocytes, whereas those in Lpk/Lpk mice were perpendicular to the direction of bone growth. Electron microscopy of cartilage from 18.5 dpc wild-type, Lpk/þ, and Lpk/Lpk embryos revealed fewer and less elaborate collagen fibrils in the mutants, with enlarged vacuoles in the endoplasmic reticulum that contained amorphous inclusions. Micro-computed tomography (CT) scans of 12-week-old Lpk/þ mice revealed them to have decreased bone mineral density, and total bone volume, with erosions and osteophytes at the joints. Thus, an ENU mouse model with a Ser1386Pro mutation of the Col2a1 C-propeptide domain that results in abnormal collagen processing and phenotypic features consistent with SEDC and secondary osteoarthritis has been established. ß