Activating Canonical Wnt/β-catenin Pathway Enhances DNA Repair And Promotes Cell Survival In Osteoblasts:a Novel Anabolic Treatment For Radiotherapy Associated Bone Damage

Introduction:Radiotherapy is a common cancer treatment for eliminating tumor cells, but it has inevitable adverse effects on bones within irradiated region and causes problems, such as osteoporosis, osteoradionecrosis, and fractures. To date, there is no preventive or curative treatment for radiation-induced bone damage.Methods:Animal protocols-Three-month-old female Sprague Dawley rats (n=5/group) were radiated by SARRP at their right tibialmetaphyseal region at a dose of8Gy twice (day1and3). This radiation protocol mimics the dose to the hip during hypofractionated radiotherapy for prostate cancer. The contralateral tibiae that did not receive radiation served as paired controls. Rats were then separated into two groups receiving either vehicle or rhPTH (1-34,60μg/kg/day) treatment for4weeks. Micro-computed tomography (μCT) scanning-Bilateral tbiae were harvested on d28for μCT measurement of trabecular structural parameters and stiffness based on finite-element modeling. Radiation on osteoblast lineage cells and Wnt3a treatment-Xrad320i was used to deliver a dose of8Gy for osteoblastic (UMR106-01) and osteocytic (Ocy454) cells and24Gy for primary calvarial osteoprogenitors. Wnt3a conditioned medium was collected from Wnt3a overexpressing L cells and applied to osteoblast lineage cells at30min after radiation. Cell death detection-Ethidium Bromide (EB)/Acridine Orange (AO) staining was used for detection of apoptotic cells. Immunofluorescence-After radiation, cells were fixed with4%paraformaldehyde and incubated with antibodies against γ-H2AX, caspase3and Ku70followed by Alexa-conjugated fluorescent secondary antibodies and DAPI staining. Single cell gel electrophoresis-To measure the extent of DNA damage at a single cell level, comet assay was performed using the alkaline conditions of the Trevigen Comet Assay(?) kit.Results:In the current report, we established a clinically relevant radiation model using skeletally mature rats and a newly available small animal radiation research platform (SARRP) that replicates focal clinical radiotherapy in rodents.Within the model, we reported that focal radiation on rat bone causes a loss of small trabecular elements with decreases in bone mass and strength based on standard image analysis and finite element analysis (FEA). Further histological analysis indicated that diminished bone formation, but not enhanced bone resorption is a major contributor to such bone loss. Interestingly, daily injections of parathyroid hormone (PTH1-34, teriparatide), a FDA-approved anabolic treatment for severe osteoporosis, blocks post-radiation bone damage via protecting osteoblasts and osteocytes from apoptosis.Inhibitor assays revealed that the survival action of PTH is mediated by PKA and Wnt/β-catenin. Indeed, PTH enhanced β-catenin amount and its nuclear translocation in osteoblasts after radiation via PKA pathway. Radiation induces highly lethal DNA damage, among which double-strand breaks (DSBs) is the major factor responsible for cell death. A sensitive method to detect DSBs is the immunofluorescence staining of γ-H2AX. Detecting DNA double strand breaks (DSBs) by y-H2AX foci staining and comet assay revealed that PTH efficiently promotes the repair of DSBs in irradiated osteoblasts via activating β-catenin pathway. Interestingly, Wnt3a alone also blocked cell death and accelerated DNA repair in primary osteoprogenitors, osteoblastic and osteocytic cells after radiation through the canonical signaling. Further investigations revealed that both Wnt3a and PTH increase the amount of Ku70, a core protein for initiating the assembly of DSB repair machinery, in osteoblasts after radiation. Mechanistic analyses using siRNA demonstrated that Ku70is important for mediating DNA repair and cell survival by PTH and Wnt3a in osteoblasts.Conlusion:The improved survivorship rate and the increased age of cancer patients receiving radiotherapy emphasize the importance of understanding the mechanism of radiation-induced osteoporosis and identifying atreatment for this disease. Our results identify a novel role of PTH and canonical Wnt signaling in regulating DSB repair machinery and apoptosis in osteoblasts and shed light on using PTH1-34or Wnt agonist as possible therapy for radiation-induced osteoporosis.