CCN3 impairs osteoblast and stimulates osteoclast differentiation to favor breast cancer metastasis to bone.

Bone is a preferred site for breast cancer metastasis, causing pain, fractures, spinal cord compressions, and hypercalcemia, all of which can significantly diminish the patient's quality of life. We identified CCN3 as a novel factor that is highly expressed in bone metastatic breast cancer cells from a xenograft mouse model and in bone metastatic lesions from patients with breast cancer. We demonstrate that CCN3 overexpression enhances the ability of weakly bone metastatic breast cancer cells to colonize and grow in the bone without altering their growth in the mammary fat pad. We further demonstrated that human recombinant CCN3 inhibits osteoblast differentiation from primary bone marrow cultures, leading to a higher receptor activator of NF-κB ligand (RANKL)/osteoprotegerin (OPG) ratio. In conjunction with its ability to impair osteoblast differentiation, we uncovered a novel role for CCN3 in promoting osteoclast differentiation from RANKL-primed monocyte precursors. CCN3 exerts its pro-osteoclastogenic effects by promoting calcium oscillations and nuclear factor of activated T cells c1 (NFATc1) nuclear translocation. Together, these results demonstrate that CCN3 regulates the differentiation of bone resident cells to create a resorptive environment that promotes the formation of osteolytic breast cancer metastases.

Tumor-supportive and osteoclastogenic changes induced by breast cancer-derived factors are reversed by inhibition of {gamma}-secretase.

During breast cancer metastasis to bone, tumor cells home to bone marrow, likely targeting the stem cell niche, and stimulate osteoclasts, which mediate osteolysis required for tumor expansion. Although osteoblasts contribute to the regulation of the hematopoietic stem cell niche and control osteoclastogenesis through production of proresorptive cytokine RANKL (receptor activator of NF-κB ligand), their role in cancer metastases to bone is not fully understood. C57BL/6J mouse bone marrow cells were treated for 3-12 days with ascorbic acid (50 µg/ml) in the presence or absence of 10% medium conditioned by breast carcinoma cells MDA-MB-231, 4T1, or MCF7. Treatment with cancer-derived factors resulted in a sustained 40-60% decrease in osteoblast differentiation markers, compared with treatment with ascorbic acid alone, and induced an osteoclastogenic change in the RANKL/osteoprotegerin ratio. Importantly, exposure of bone cells to breast cancer-derived factors stimulated the subsequent attachment of cancer cells to immature osteoblasts. Inhibition of γ-secretase using pharmacological inhibitors DAPT and Compound E completely reversed cancer-induced osteoclastogenesis as well as cancer-induced enhancement of cancer cell attachment, identifying γ-secretase activity as a key mediator of these effects. Thus, we have uncovered osteoblasts as critical intermediary of premetastatic signaling by breast cancer cells and pinpointed γ-secretase as a robust target for developing therapeutics potentially capable of reducing both homing and progression of cancer metastases to bone.

The Movember Prostate Cancer Landscape Analysis: an assessment of unmet research needs.

Prostate cancer is a heterogeneous cancer with widely varying levels of morbidity and mortality. Approaches to prostate cancer screening, diagnosis, surveillance, treatment and management differ around the world. To identify the highest priority research needs across the prostate cancer biomedical research domain, Movember conducted a landscape analysis with the aim of maximizing the effect of future research investment through global collaborative efforts and partnerships. A global Landscape Analysis Committee (LAC) was established to act as an independent group of experts across urology, medical oncology, radiation oncology, radiology, pathology, translational research, health economics and patient advocacy. Men with prostate cancer and thought leaders from a variety of disciplines provided a range of key insights through a range of interviews. Insights were prioritized against predetermined criteria to understand the areas of greatest unmet need. From these efforts, 17 research needs in prostate cancer were agreed on and prioritized, and 3 received the maximum prioritization score by the LAC: first, to establish more sensitive and specific tests to improve disease screening and diagnosis; second, to develop indicators to better stratify low-risk prostate cancer for determining which men should go on active surveillance; and third, to integrate companion diagnostics into randomized clinical trials to enable prediction of treatment response. On the basis of the findings from the landscape analysis, Movember will now have an increased focus on addressing the specific research needs that have been identified, with particular investment in research efforts that reduce disease progression and lead to improved therapies for advanced prostate cancer.

Regulation of Osteoclast Growth and Fusion by mTOR/raptor and mTOR/rictor/Akt.

Osteoclasts are giant bone cells formed by fusion from monocytes and uniquely capable of a complete destruction of mineralized tissues. Previously, we have demonstrated that in energy-rich environment not only osteoclast fusion index (the number of nuclei each osteoclast contains), but also cytoplasm volume per single nucleus was increased. The goal of this study was to investigate the regulation of metabolic sensor mTOR during osteoclast differentiation in energy-rich environment simulated by addition of pyruvate. We have found that in the presence of pyruvate, the proportion of mTOR associated with raptor increased, while mTOR-rictor-mediated Akt phosphorylation decreased. Inhibition of mTOR with rapamycin (10 nM) significantly interfered with all aspects of osteoclastogenesis. However, rapamycin at 1 nM, which preferentially targets mTOR-raptor complex, was only effective in control cultures, while in the presence of pyruvate osteoclast fusion index was successfully increased. Inhibition of Akt drastically reduced osteoclast fusion, however in energy-rich environment, osteoclasts of comparable size were formed through increased cytoplasm growth. These data suggest that mTOR-rictor mediated Akt signaling regulates osteoclast fusion, while mTOR-raptor regulation of protein translation contributes to fusion-independent cytoplasm growth. We demonstrate that depending on the bioenergetics microenvironment osteoclastogenesis can adjust to occur through preferential multinucleation or through cell growth, implying that attaining large cell size is part of the osteoclast differentiation program.

Moderate excess of pyruvate augments osteoclastogenesis.

Cell differentiation leads to adaptive changes in energy metabolism. Conversely, hyperglycemia induces malfunction of many body systems, including bone, suggesting that energy metabolism reciprocally affects cell differentiation. We investigated how the differentiation of bone-resorbing osteoclasts, large polykaryons formed through fusion and growth of cells of monocytic origin, is affected by excess of energy substrate pyruvate and how energy metabolism changes during osteoclast differentiation. Surprisingly, small increases in pyruvate (1-2 mM above basal levels) augmented osteoclastogenesis in vitro and in vivo, while larger increases were not effective in vitro. Osteoclast differentiation increased cell mitochondrial activity and ATP levels, which were further augmented in energy-rich conditions. Conversely, the inhibition of respiration significantly reduced osteoclast number and size. AMP-activated protein kinase (AMPK) acts as a metabolic sensor, which is inhibited in energy-rich conditions. We found that osteoclast differentiation was associated with an increase in AMPK levels and a change in AMPK isoform composition. Increased osteoclast size induced by pyruvate (1 mM above basal levels) was prevented in the presence of AMPK activator 5-amino-4-imidazole carboxamide ribonucleotide (AICAR). In keeping, inhibition of AMPK using dorsomorphin or siRNA to AMPKγ increased osteoclast size in control cultures to the level observed in the presence of pyruvate. Thus, we have found that a moderate excess of pyruvate enhances osteoclastogenesis, and that AMPK acts to tailor osteoclastogenesis to a cell's bioenergetics capacity.

Ascorbic acid accelerates osteoclast formation and death.

Ascorbic acid (AA) plays a key role in bone formation. However controversy remains about the effect of AA on cells responsible for bone destruction, osteoclasts. We investigated the effect of AA on osteoclastogenesis using primary mouse bone marrow cultures and monocytic RAW 264.7 cells treated with osteoclastogenic factors RANKL and MCSF. Treatment with AA resulted in significant increase in osteoclast number, size and nucleation. To assess osteoclast oxidative stress level, a ratio of reduced (GSH) to oxidized (GSSG) glutathione and the total glutathione content (GSH(t)) were evaluated. Osteoclast differentiation was associated with a decrease in GSH/GSSG and GSH(t). AA induced further decrease in both parameters, and resulted in significant production of H(2)O(2), indicating its pro-oxidant action. At low concentration, H(2)O(2) induced similar effects to AA, although less potently, and catalase partially inhibited AA-induced osteoclastogenesis. To assess the modification in osteoclast metabolism, the mitochondrial activity was evaluated using JC-1 and the ATP levels were assessed. Osteoclast formation was associated with the increase in mitochondrial activity and ATP concentration, which were further increased in the presence of AA. Importantly, the stimulatory effect of AA was only evident at early phase of osteoclastogenesis, whereas at the late stage AA significantly accelerated osteoclast death. Thus, during osteoclastogenesis AA acts as an oxidant, first stimulating osteoclast formation, but later limiting osteoclast lifespan. This duality of AA action allows reconciling the stimulatory action of AA on osteoclastogenesis observed in vitro with an overall attenuation of bone resorption in the presence of AA observed in vivo.