FRZB and WNT Biology in Osteoarthritis

Osteoarthritis (OA) is the most prevalent chronic joint disorder and a major cause of pain and disability affecting millions of people worldwide. The impact of osteoarthritis on society is enormous from the individual perspective on quality of life as well as from the economical perspective with high medical costs and work disability. Osteoarthritis incidence and severity increase with age and loading of joints. The burden of disease is expected to grow due to the obesity epidemic and overall ageing of the population. Treatment strategies for osteoarthritis are currently limited to the management of risk factors including loss of weight and changes in lifestyle, and to exercise in order to improve muscle strength and joint control, often combined with the use of painkillers and anti-inflammatory drugs that offer symptom relief. Effective interventions that slow down disease progression are however still unavailable. 10 to 15% of patients ultimately require joint-replacement surgery, in particular when large joints such as knees and hip are affected. A large unmet need for more effective therapies that would delay or arrest progression of disease therefore exists. Such therapies can only be developed by an in-depth understanding of joint biology and the disease processes. Increasing evidence supports a role for signaling pathways, such as the wingless-type (WNT) pathway, in joint and skeletal development and in the maintenance of homeostasis in the postnatal joint. WNTs are secreted growth factors with roles in proliferation, differentiation and behavior of cells during embryonic axis formation and in the development of many organs, including the bones and joints. Also postnatally, they play a role in the homeostasis of these tissues. Changes in the balance of WNT signaling are associated with different diseases including cancer, bone and joint disorders. The main goal of this PhD research was to further investigate the role of WNT signaling in the biology of the postnatal joint and in the pathogenesis of osteoarthritis. We studied the effects of modulation of 2 WNT pathway components in mouse models of arthritis. FRZB is a secreted WNT antagonist that acts as a decoy receptor for WNT ligands. LRP5 is an essential co-receptor in the canonical WNT signaling cascade. We have used gain and loss of function strategies to understand their role in homeostasis and disease. In the first part, we showed by microarray gene expression analysis that WNT signaling is tightly regulated in the joint, as deficiency of Frzb increases the expression of other WNT antagonists in the articular cartilage and subchondral bone, likely representing a compensatory effort to maintain WNT signaling balance. Our data further demonstrated that Frzb is involved in chondrocyte proliferation and extracellular matrix synthesis, with loss of the gene resulting in molecular changes that are detrimental to cartilage integrity upon challenge. In the second part, we demonstrated that adenoviral overexpression of FRZB in the arthritic mouse knee joint does not protect the cartilage against degradation. Restricted adenoviral transgene expression within the articular cartilage and limited diffusion of the translated proteins from the synovium into the cartilage may be limiting factors, although some modulation of WNT target genes Mmp3 and Wisp1 expression revealed effects at the molecular level. Surprisingly, we found that FRZB overexpression increased inflammation and pro-inflammatory cytokine expression in the synovium of the mBSA-induced arthritis knee joint, and contributed to increased Adamts5 expression. Furthermore, the experiments provided evidence for a role of Frzb in adaptive immune responses. These data highlight the complexity of the signaling pathway and the multifaceted function of FRZB which interactions may not be limited to WNT antagonism. In the third part, we demonstrated that loss of Lrp5, a WNT co-receptor, not only decreases the bone mineral density in mice and man, but also increases cartilage degradation after induction of instability in the knee joint. These data agree with the concept that homeostasis in the joint is dependent on a molecular balance in the bone-cartilage biomechanical unit, which is defined by pathways, such as WNT signaling. This molecular balance is likely necessary to provide an optimal bone-cartilage architecture and interface. The observations in the Lrp5-/- mice also demonstrate that mice with low bone mass and abnormal bone architecture can be prone to osteoarthritis development. In summary, this work provides further insights into the complex regulation of WNT signaling in joint biology and disease. From a therapeutic perspective, the WNT signaling pathway remains an interesting therapeutic target for bone and joint diseases. Further work should focus on the specificity and fine regulation of this signaling pathway within cartilage and bone to define precise targets and avoid undesired secondary effects.nrpages: 153status: publishe

Genetic deletion of low-density lipoprotein receptor-related protein 5 increases cartilage degradation in instability-induced osteoarthritis

Objective. The wingless-type MMTV integration site family (WNT) signalling pathway plays an important role in embryonic joint and bone development and has been associated with osteoporosis and osteoarthritis (OA). Loss-of-function mutations in low-density lipoprotein receptor-related protein 5 (LRP5), a WNT co-receptor, result in low bone mass. Lrp5(-)(/)(-) mice also have low bone mass phenotypes. Recently an OA-susceptibility locus containing the LRP5 gene was suggested. We investigated the effects of loss of Lrp5 in joint biology in three different mouse models of OA.Methods. Total body bone mineral parameters were measured by dual-energy X-ray absorptiometry. Trabecular and cortical bone parameters of tibia and femur were assessed ex vivo by peripheral quantitative CT. Osteoarthritic changes were induced in Lrp5(-)(/)(-) and wild-type C57Bl/6J mice using the surgically induced destabilization of the medial meniscus model and the chemically induced papain and collagenase model. The severity of joint disease was investigated by histological analysis of the knee joints.Results. Bone mineral density and weight were significantly decreased in Lrp5(-)(/)(-) C57Bl/6J mice compared with their wild-type littermates. Surgically induced destabilization of the knee joint resulted in significantly increased cartilage degradation in the medial tibia of Lrp5(-)(/)(-) mice compared with wild-type control mice. In the medial femur, a similar trend was found but did not reach statistical significance. In the papain- and collagenase-induced models, these differences were not observed. Inflammation scores were comparable between wild-type and Lrp5(-)(/)(-) mice.Conclusion. These data show that loss of function of Lrp5 increases cartilage degradation in mild instability-induced OA models in mice. Low bone mass density could have contributed to this effect.status: publishe

Tight regulation of wingless-type signaling in the articular cartilage - subchondral bone biomechanical unit: transcriptomics in Frzb-knockout mice

ABSTRACT: INTRODUCTION: The aim was to study molecular changes in the articular cartilage and subchondral bone of the tibial plateau from mice deficient in frizzled-related protein (Frzb) compared to wild-type mice by transcriptome analysis. METHODS: Gene-expression analysis of the articular cartilage and subchondral bone of three wild-type and three Frzb-/- mice was performed by microarray. Data from three wild-type and two Frzb-/- samples could be used for pathway analysis of differentially expressed genes and were explored with PANTHER, DAVID and GSEA bioinformatics tools. Activation of the wingless-type (WNT) pathway was analyzed using western blot. The effects of Frzb gain and loss of function on chondrogenesis and cell proliferation was examined using ATDC5 micromasses and mouse ribcage chondrocytes. RESULTS: Extracellular matrix-associated integrin and cadherin pathways, as well as WNT pathway genes were upregulated in Frzb-/- samples. Several WNT receptors, target genes, and other antagonists were upregulated, but no difference in active beta-catenin was found. Analysis of ATDC5 cell micromasses overexpressing FRZB indicated an upregulation of aggrecan and Col2a1, and downregulation of molecules related to damage and repair in cartilage, Col3a1 and Col5a1. Silencing of Frzb resulted in downregulation of aggrecan and Col2a1. Pathways associated with cell cycle were downregulated in this transcriptome analysis. Ribcage chondrocytes derived from Frzb-/- mice showed decreased proliferation compared to wild-type cells. CONCLUSIONS: Our analysis provides evidence for tight regulation of WNT signaling, shifts in extracellular matrix components and effects on cell proliferation and differentiation in the articular cartilage - subchondral bone unit in Frzb-/- mice. These data further support an important role for FRZB in joint homeostasis and highlight the complex biology of WNT signaling in the joint.status: publishe

Real-World Treatment Patterns, Epidermal Growth Factor Receptor (EGFR) Testing and Outcomes in EGFR-Mutated Advanced Non-small Cell Lung Cancer Patients in Belgium: Results from the REVEAL Study.

Treatment of patients with epidermal growth factor receptor-mutated (EGFRm) non-small cell lung cancer (NSCLC) continues to evolve expeditiously. This retrospective study investigated real-world treatment patterns and EGFR mutation testing in patients with EGFRm advanced NSCLC in Belgium. Data were extracted from medical records of adults diagnosed with EGFRm locally advanced/metastatic NSCLC between 1 September 2015 and 31 December 2017. Patients were followed retrospectively from diagnosis until 1 September 2018, end of clinical activity or death. Data on demographics, patient outcomes and disease characteristics, treatment patterns and EGFR mutation testing at diagnosis and progression were analyzed descriptively. A total of 141 patients were enrolled. At diagnosis, median age was 69 years, 63.1% were female, 88.7% had metastatic disease, 94.3% had adenocarcinoma histology, 76.6% had ECOG 0/1, 70.9% had common EGFR mutations and 29.1% had only rare mutations. In first line, 73.8% of patients received first/second-generation EGFR-tyrosine kinase inhibitors (1G/2G EGFR-TKIs), while 21.9% received other systemic treatments. Among 61 patients progressing on and discontinuing a first 1G/2G EGFR-TKI, 45 (73.8%) received subsequent systemic treatment while 16 (26.2%) did not; 20 (32.8%) received osimertinib. Among 65 patients progressing on a first 1G/2G EGFR-TKI, 47 (72.3%) were tested for T790M, of whom 25 (53.2%) were positive. These real-world data from Belgium show that a substantial fraction of patients with EGFRm NSCLC do not receive 1G/2G EGFR-TKIs in first line and do not receive subsequent systemic treatment after progression on 1G/2G EGFR-TKIs. Only a third receive osimertinib upon progression on 1G/2G EGFR-TKIs. These observations should be considered in first-line treatment decisions. ClinicalTrials.gov: NCT03761901-December 3, 2018