Mitigating aging and doxorubicin induced bone loss in mature mice via mechanobiology based treatments.

Aging leads to a reduced anabolic response to mechanical stimuli and a loss of bone mass and structural integrity. Chemotherapy agents such as doxorubicin exacerbate the degeneration of aging skeleton and further subject older cancer patients to a higher fracture risk. To alleviate this clinical problem, we proposed and tested a novel mechanobiology-based therapy. Building upon prior findings that i) Yoda1, the Piezo1 agonist, promoted bone growth in young adult mice and suppressed bone resorption markers in aged mice, and ii) moderate tibial loading protected bone from breast cancer-induced osteolysis, we hypothesized that combined Yoda1 and moderate loading would improve the structural integrity of adult and aged skeletons in vivo and protect bones from deterioration after chemotherapy. We first examined the effects of 4-week Yoda1 (dose 5 mg/kg, 5 times/week) and moderate tibial loading (4.5 N peak load, 4 Hz, 300 cycles for 5 days/week), individually and combined, on mature mice (∼50 weeks of age). Combined Yoda1 and loading was found to mitigate age-associated cortical and trabecular bone loss better than individual interventions. As expected, the non-treated controls experienced an average drop of cortical polar moment of inertia (Ct.pMOI) by -4.3 % over four weeks and the bone deterioration occurred in the majority (64 %) of the samples. Relative to no treatment, loading alone, Yoda1 alone, and combined Yoda1 and loading increased Ct.pMOI by +7.3 %, +9.5 %, +12.0 % and increased the % of samples with positive Ct.pMOI changes by +32 %, +26 %, and +43 %, respectively, suggesting an additive protection of aging-related bone loss for the combined therapy. We further tested if the treatment efficacy was preserved in mature mice following two weeks (six injections) of doxorubicin at the dose of 2.5 or 5 mg/kg. As expected, doxorubicin increased osteocyte apoptosis, altered bone remodeling, and impaired bone structure. However, the effects induced by DOX were too severe to be rescued by Yoda1 and loading, alone or combined, although loading and Yoda1 individually, or combined, increased the number of mice showing positive responsiveness by 0 %, +15 %, and +29 % relative to no intervention after doxorubicin exposure. Overall, this study supported the potentials and challenges of the Yoda1-based strategy in mitigating the detrimental skeletal effects caused by aging and doxorubicin.

BIGH3 mediates apoptosis and gap junction failure in osteocytes during renal cell carcinoma bone metastasis progression.

Renal cell carcinoma (RCC) bone metastatis progression is driven by crosstalk between tumor cells and the bone microenvironment, which includes osteoblasts, osteoclasts, and osteocytes. RCC bone metastases (RCCBM) are predominantly osteolytic and resistant to antiresorptive therapy. The molecular mechanisms underlying pathologic osteolysis and disruption of bone homeostasis remain incompletely understood. We previously reported that BIGH3/TGFBI (transforming growth factor-beta-induced protein ig-h3, shortened to BIGH3 henceforth) secreted by colonizing RCC cells drives osteolysis by inhibiting osteoblast differentiation, impairing healing of osteolytic lesions, which is reversible with osteoanabolic agents. Here, we report that BIGH3 induces osteocyte apoptosis in both human RCCBM tissue specimens and in a preclinical mouse model. We also demonstrate that BIGH3 reduces Cx43 expression, blocking gap junction (GJ) function and osteocyte network communication. BIGH3-mediated GJ inhibition is blocked by the lysosomal inhibitor hydroxychloroquine (HCQ), but not osteoanabolic agents. Our results broaden the understanding of pathologic osteolysis in RCCBM and indicate that targeting the BIGH3 mechanism could be a combinational strategy for the treatment of RCCBM-induced bone disease that overcomes the limited efficacy of antiresorptives that target osteoclasts.

Yoda1 Enhanced Low-Magnitude High-Frequency Vibration on Osteocytes in Regulation of MDA-MB-231 Breast Cancer Cell Migration.

Low-magnitude (≤1 g) high-frequency (≥30 Hz) (LMHF) vibration has been shown to enhance bone mineral density. However, its regulation in breast cancer bone metastasis remains controversial for breast cancer patients and elder populations. Yoda1, an activator of the mechanosensitive Piezo1 channel, could potentially intensify the effect of LMHF vibration by enhancing the mechanoresponse of osteocytes, the major mechanosensory bone cells with high expression of Piezo1. In this study, we treated osteocytes with mono- (Yoda1 only or vibration only) or combined treatment (Yoda1 and LMHF vibration) and examined the further regulation of osteoclasts and breast cancer cells through the conditioned medium. Moreover, we studied the effects of combined treatment on breast cancer cells in regulation of osteocytes. Combined treatment on osteocytes showed beneficial effects, including increasing the nuclear translocation of Yes-associated protein (YAP) in osteocytes (488.0%, p < 0.0001), suppressing osteoclastogenesis (34.3%, p = 0.004), and further reducing migration of MDA-MB-231 (15.1%, p = 0.02) but not Py8119 breast cancer cells (4.2%, p = 0.66). Finally, MDA-MB-231 breast cancer cells subjected to the combined treatment decreased the percentage of apoptotic osteocytes (34.5%, p = 0.04) but did not affect the intracellular calcium influx. This study showed the potential of stimulating Piezo1 in enhancing the mechanoresponse of osteocytes to LMHF vibration and further suppressing breast cancer migration via osteoclasts.

Moderate tibial loading and treadmill running, but not overloading, protect adult murine bone from destruction by metastasized breast cancer.

Osteolytic bone lesions, which develop in many metastatic breast cancer patients, impair bone integrity and lead to adverse skeletal related events that are difficult to treat and sometimes fatal. Moderate mechanical loading has been shown to suppress osteolysis in young mice with breast cancer. In this study, we aimed to investigate the dose-dependent effects of mechanical loading on protecting the integrity of adult skeletons with breast cancer. Localized tibial loading and aerobic treadmill running with three levels of varying intensity were tested in a syngeneic mammary tumor bone metastasis model. Adult C57BL/6J female mice (14-week-old, N = 88 mice) received intra-tibial injections of Py8119 triple-negative murine breast cancer cells or PBS and underwent 4 to 5 weeks of exercise or acted as sedentary/non-loaded controls. The bone structure was monitored longitudinally with weekly in vivo micro-computed tomography imaging, while the cellular responses in bone and marrow were examined using immunohistochemistry. Moderate treadmill running (16 m/min, 50 min/day, 5 days/week, and 5 weeks) and tibial loading (4.5 N, 630 µÎµ, 4 Hz, 300 cycles/day, 5 days/week, and 4 weeks) suppressed tumor-induced bone destruction, as evaluated by full-thickness perforation of tibial cortex and the volume of osteolytic lesions in the cortex. In contrast, tibial loading at higher magnitude (8 N, 1100 µÎµ) induced woven bone and accelerated bone destruction, compared with the non-loaded controls. The three exercise regimens differentially affected osteocyte apoptosis, osteocyte hypoxia, osteoclast activity, bone marrow vasculature, and tumor proliferation. In conclusion, the relationship between exercise intensity and the risk of breast cancer-induced osteolysis was found to follow a J-shaped curve in a preclinical model, suggesting the need to optimize exercise parameters in order to harness the skeletal benefits of exercise in metastatic breast cancers.