Overexpression of Lrp5 enhanced the anti-breast cancer effects of osteocytes in bone.

Osteocytes are the most abundant cells in bone, which is a frequent site of breast cancer metastasis. Here, we focused on Wnt signaling and evaluated tumor-osteocyte interactions. In animal experiments, mammary tumor cells were inoculated into the mammary fat pad and tibia. The role of Lrp5-mediated Wnt signaling was examined by overexpressing and silencing Lrp5 in osteocytes and establishing a conditional knockout mouse model. The results revealed that administration of osteocytes or their conditioned medium (CM) inhibited tumor progression and osteolysis. Osteocytes overexpressing Lrp5 or ß-catenin displayed strikingly elevated tumor-suppressive activity, accompanied by downregulation of tumor-promoting chemokines and upregulation of apoptosis-inducing and tumor-suppressing proteins such as p53. The antitumor effect was also observed with osteocyte-derived CM that was pretreated with a Wnt-activating compound. Notably, silencing Lrp5 in tumors inhibited tumor progression, while silencing Lrp5 in osteocytes in conditional knockout mice promoted tumor progression. Osteocytes exhibited elevated Lrp5 expression in response to tumor cells, implying that osteocytes protect bone through canonical Wnt signaling. Thus, our results suggest that the Lrp5/ß-catenin axis activates tumor-promoting signaling in tumor cells but tumor-suppressive signaling in osteocytes. We envision that osteocytes with Wnt activation potentially offer a novel cell-based therapy for breast cancer and osteolytic bone metastasis.

Mechanical stimulations can inhibit local and remote tumor progression by downregulating WISP1.

Mechanical stimulations can prevent bone loss, but their effects on the tumor-invaded bone or solid tumors are elusive. Here, we evaluated the effect of knee loading, dynamic loads applied to the knee, on metastasized bone and mammary tumors. In a mouse model, tumor cells were inoculated to the mammary fat pad or the proximal tibia. Daily knee loading was then applied and metabolic changes were monitored mainly through urine. Urine samples were also collected from human subjects before and after step aerobics. The result showed that knee loading inhibited tumor progression in the loaded tibia. Notably, it also reduced remotely the growth of mammary tumors. In the urine, an altered level of cholesterol was observed with an increase in calcitriol, which is synthesized from a cholesterol derivative. In urinary proteins, knee loading in mice and step aerobics in humans markedly reduced WNT1-inducible signaling pathway protein 1, WISP1, which leads to poor survival among patients with breast cancer. In the ex vivo breast cancer tissue assay, WISP1 promoted the growth of cancer fragments and upregulated tumor-promoting genes, such as Runx2, MMP9, and Snail. Collectively, the present preclinical and human study demonstrated that mechanical stimulations, such as knee loading and step aerobics, altered urinary metabolism and downregulated WISP1. The study supports the benefit of mechanical stimulations for locally and remotely suppressing tumor progression. It also indicated the role of WISP1 downregulation as a potential mechanism of loading-driven tumor suppression.

Skeletal loading regulates breast cancer-associated osteolysis in a loading intensity-dependent fashion.

Osteocytes are mechanosensitive bone cells, but little is known about their effects on tumor cells in response to mechanical stimulation. We treated breast cancer cells with osteocyte-derived conditioned medium (CM) and fluid flow-treated conditioned medium (FFCM) with 0.25 Pa and 1 Pa shear stress. Notably, CM and FFCM at 0.25 Pa induced the mesenchymal-to-epithelial transition (MET), but FFCM at 1 Pa induced the epithelial-to-mesenchymal transition (EMT). This suggested that the effects of fluid flow on conditioned media depend on flow intensity. Fluorescence resonance energy transfer (FRET)-based evaluation of Src activity and vinculin molecular force showed that osteopontin was involved in EMT and MET switching. A mouse model of tumor-induced osteolysis was tested using dynamic tibia loadings of 1, 2, and 5 N. The low 1 N loading suppressed tumor-induced osteolysis, but this beneficial effect was lost and reversed with loads at 2 and 5 N, respectively. Changing the loading intensities in vivo also led to changes in serum TGFß levels and the composition of tumor-associated volatile organic compounds in the urine. Collectively, this study demonstrated the critical role of intensity-dependent mechanotransduction and osteopontin in tumor-osteocyte communication, indicating that a biophysical factor can tangibly alter the behaviors of tumor cells in the bone microenvironment.

Pitavastatin slows tumor progression and alters urine-derived volatile organic compounds through the mevalonate pathway.

Bone is a frequent site of metastasis from breast cancer, and a desirable drug could suppress tumor growth as well as metastasis-linked bone loss. Currently, no drug is able to cure breast cancer-associated bone metastasis. In this study, we focused on statins that are known to inhibit cholesterol production and act as antitumor agents. After an initial potency screening of 7 U.S. Food and Drug Administration-approved statins, we examined pitavastatin as a drug candidate for inhibiting tumor and tumor-induced bone loss. In vitro analysis revealed that pitavastatin acted as an inhibitor of tumor progression by altering stress to the endoplasmic reticulum, down-regulating peroxisome proliferator-activated receptor γ, and reducing Snail and matrix metalloproteinase 9. In bone homeostasis, it blocked osteoclast development by suppressing transcription factors c-Fos and JunB, but stimulated osteoblast mineralization by regulating bone morphogenetic protein 2 and p53. In a mouse model, pitavastatin presented a dual role in tumor inhibition in the mammary fat pad, as well as in bone protection in the osteolytic tibia. In mass spectrometry-based analysis, volatile organic compounds (VOCs) that were linked to lipid metabolism and cholesterol synthesis were elevated in mice from the tumor-grown placebo group. Notably, pitavastatin-treated mice reduced specific VOCs that are linked to lipid metabolites in the mevalonate pathway. Collectively, the results lay a foundation for further investigation of pitavastatin's therapeutic efficacy in tumor-induced bone loss, as well as VOC-based diagnosis of tumor progression and treatment efficacy.-Wang, L., Wang, Y., Chen, A., Teli, M., Kondo, R., Jalali, A., Fan, Y., Liu, S., Zhao, X., Siegel, A., Minami, K., Agarwal, M., Li, B.-Y., Yokota, H. Pitavastatin slows tumor progression and alters urine-derived volatile organic compounds through the mevalonate pathway.

Finite-element analysis of the mouse proximal ulna in response to elbow loading.

Bone is a mechano-sensitive tissue that alters its structure and properties in response to mechanical loading. We have previously shown that application of lateral dynamic loads to a synovial joint, such as the knee and elbow, suppresses degradation of cartilage and prevents bone loss in arthritis and postmenopausal mouse models, respectively. While loading effects on pathophysiology have been reported, mechanical effects on the loaded joint are not fully understood. Because the direction of joint loading is non-axial, not commonly observed in daily activities, strain distributions in the laterally loaded joint are of great interest. Using elbow loading, we herein characterized mechanical responses in the loaded ulna focusing on the distribution of compressive strain. In response to 1-N peak-to-peak loads, which elevate bone mineral density and bone volume in the proximal ulna in vivo, we conducted finite-element analysis and evaluated strain magnitude in three loading conditions. The results revealed that strain of ~ 1000 µstrain (equivalent to 0.1% compression) or above was observed in the limited region near the loading site, indicating that the minimum effective strain for bone formation is smaller with elbow loading than axial loading. Calcein staining indicated that elbow loading increased bone formation in the regions predicted to undergo higher strain.

Effects of a checkpoint kinase inhibitor, AZD7762, on tumor suppression and bone remodeling.

Chemotherapy for suppressing tumor growth and metastasis tends to induce various effects on other organs. Using AZD7762, an inhibitor of checkpoint kinase (Chk) 1 and 2, the present study examined its effect on mammary tumor cells in addition to bone cells (osteoclasts, osteoblasts and osteocytes), using monolayer cell cultures and three-dimensional (3D) cell spheroids. The results revealed that AZD7762 blocked the proliferation of 4T1.2 mammary tumor cells and suppressed the development of RAW264.7 pre-osteoclast cells by downregulating nuclear factor of activated T cells cytoplasmic 1. AZD7762 also promoted the mineralization of MC3T3 osteoblast-like cells and 3D bio-printed bone constructs of MLO-A5 osteocyte spheroids. While a Chk1 inhibitor, PD407824, suppressed the proliferation of tumor cells and the differentiation of pre-osteoclasts, its effect on gene expression in osteoblasts was markedly different compared with AZD7762. Western blotting indicated that the stimulating effect of AZD7762 on osteoblast development was associated with the inhibition of Chk2 and the downregulation of cellular tumor antigen p53. The results of the present study indicated that in addition to acting as a tumor suppressor, AZD7762 may prevent bone loss by inhibiting osteoclastogenesis and stimulating osteoblast mineralization.

Osteocyte-Driven Downregulation of Snail Restrains Effects of Drd2 Inhibitors on Mammary Tumor Cells.

While bone is a frequent target of breast cancer-associated metastasis, little is known about the effects of tumor-bone interactions on the efficacy of tumor-suppressing agents. Here we examined the effect of two FDA-approved dopamine modulators, fluphenazine and trifluoperazine, on mammary tumor cells, osteoclasts, osteoblasts, and osteocytes. These agents suppressed proliferation and migration of mammary tumor cells chiefly by antagonizing dopamine receptor D2 and reduced bone resorption by downregulating nuclear factor of activated T cells, cytoplasmic 1 (Nfatc1). Three-dimensional spheroid formation assays revealed that tumor cells have high affinity to osteocytes and type I collagen, and interactions with osteocytes as well as administration of fluphenazine and trifluoperazine downregulated Snail and suppressed migratory behaviors. Unlike the inhibitory action of fluphenazine and trifluoperazine on tumor growth, tumor-osteocyte interactions stimulated tumor proliferation by upregulating NFκB and Akt. In the bone microenvironment, osteocytes downregulated Snail and acted as an attractant as well as a stimulant to mammary tumor cells. These results demonstrate that tumor-osteocyte interactions strengthen dopamine receptor-mediated suppression of tumor migration but weaken its inhibition of tumor proliferation in the osteocyte-rich bone microenvironment.Significance: These findings provide novel insight into the cellular cross-talk in the bone microevironment and the effects of dopamine modulators on mammary tumor cells and osteocytes. Cancer Res; 78(14); 3865-76. ©2018 AACR.