RANK is a poor prognosis marker and a therapeutic target in ER-negative postmenopausal breast cancer.

Despite strong preclinical data, the therapeutic benefit of the RANKL inhibitor, denosumab, in breast cancer patients, beyond the bone, is unclear. Aiming to select patients who may benefit from denosumab, we hereby analyzed RANK and RANKL protein expression in more than 2,000 breast tumors (777 estrogen receptor-negative, ER- ) from four independent cohorts. RANK protein expression was more frequent in ER- tumors, where it associated with poor outcome and poor response to chemotherapy. In ER- breast cancer patient-derived orthoxenografts (PDXs), RANKL inhibition reduced tumor cell proliferation and stemness, regulated tumor immunity and metabolism, and improved response to chemotherapy. Intriguingly, tumor RANK protein expression associated with poor prognosis in postmenopausal breast cancer patients, activation of NFKB signaling, and modulation of immune and metabolic pathways, suggesting that RANK signaling increases after menopause. Our results demonstrate that RANK protein expression is an independent biomarker of poor prognosis in postmenopausal and ER- breast cancer patients and support the therapeutic benefit of RANK pathway inhibitors, such as denosumab, in breast cancer patients with RANK+ ER- tumors after menopause.

[Inhibitory effect of recombinant receptor activator of nuclear factor kappaB protein on bone loss in ovariectomized mice].

OBJECTIVE: To compare inhibitory effects of recombinant receptor activator of nuclear factor kappaB protein with bisphosphonate treatment (ALN) on osteoclasts activity and bone loss in ovariectomized mice. METHODS: Twenty-four female KM mice were ovariectomized bilaterally and treated with recombinant receptor activator of nuclear factor kappaB protein, alendronate, or PBS. Twelve weeks later, body weight, biochemical markers of bone metabolism, Micro CT scan and bone morphology were examined. RESULTS: After 12 weeks administration, the Micro CT scan and bone morphology values of each group were as follow. The control group: BMD (92.600 +/- 14.319) mg/cc, Tb.Th (0.094 +/- 0.011) mm, Tb.Sp (0.455 +/- 0.124) mm, BVF 0.192 +/- 0.023, SMI 1.388 +/- 0.328; the recombinant receptor activator of nuclear factor kappaB protein group: BMD (133.050 +/- 13.022) mg/cc, Tb.Th (0.098 +/- 0.009) mm, Tb.Sp (0.365 +/- 0.105) mm,BVF (0.291 +/- 0.025)%, SMI 0.661 +/- 0.384; the ALN group: BMD(128.013 +/- 16.040) mg/cc, Tb.Th (0.097 +/- 0.011) mm, Tb.Sp (0.376 +/- 0.104) mm, BVF 0.281 +/- 0.024, SMI 0.753 +/- 0.307. In the ovariectomized mice experiments, both recombinant receptor activator of nuclear factor kappaB protein and ALN significantly inhibited ovariectomy-induced bone loss. Compared to the control group (PBS), the recombinant receptor activator of nuclear factor kappaB protein group showed increased distal femur BMD and decreased trabecular spacing (Tb.Sp), whereas the control group had significantly decreased distal femur BMD, significantly decreased Tb.Th, and increased Tb.Sp. There was a significant difference in bone volume fraction among the groups. The TRAP-positive osteoclasts in distal femur bone slices were nearly complete inhibited for Recombinant receptor activator of nuclear factor kappaB protein group and alendronate group. CONCLUSION: In vivo, recombinant receptor activator of nuclear factor kappaB protein effectively inhibits the activity of osteoclasts and the resulting bone loss, which has a similar effect as alendronate.

Receptor activator of nuclear factor kappa B ligand/osteoprotegerin pathway is a promising target to reduce atherosclerotic plaque calcification.

Atherosclerotic plaque calcification represents a common pathophysiologic process in the advanced phases of the disease. Both inflammatory and vascular cells (such as osteoblast-like cells, osteoclast-like cells, dendritic cells, macrophages, smooth muscle cells, and endothelial cells) are active players in the balance between intraplaque bone deposition and resorption. Inflammatory processes underlying plaque calcification are regulated by soluble mediators that also contribute to plaque destabilization and increased vulnerability. Among different mediators, the receptor activator of nuclear factor-kappa B (NF-kappa B) ligand (RANKL)/osteoprogerin (OPG) system is a major determinant in inflammatory cell differentiation toward osteoclast-like cells. Thus, this system might be a promising parameter to be investigated as a marker of calcification-related cardiovascular risk and a therapeutic target. Despite some promising results, several limitations have been shown in the potential clinical use of serum RANKL/OPG to better assess the cardiovascular risk. At present, the potential relationship between RANKL/OPG and the content of calcium within the intima of the coronary arteries (CAC score, assessed by computed tomography) needs to be explored in large clinical studies. On the other hand, the antiatherosclerotic relevance of treatments antagonizing RANKL is still under investigation. Despite that clinical evidence is needed, this therapeutic approach might be of particular benefit in selective populations (such as rheumatoid arthritis patients) with an increased cardiovascular risk.

Inhibition of the osteoclast activity with the application of recombinant murine RANK protein.

OBJECTIVE: To investigate the effects of recombinant Murine RANK on the osteoclast activity. METHODS: Osteoclast was observed with soluble RANK. Female Wistar rats were bilaterally ovariectomized, and intra-abdominally injected with 5 mg/Kg soluble RANK. The bone metabolism, bone density, and bone histomorphology were observed. RESULTS: Compared with the control group, the quantity of TRAP-positive osteoclasts and bone resorption pit counting in the rest groups were significantly reduced. The bone density of the dosed groups was significantly increased and TRAP-stained osteoclasts in bone tissue sections were almost inhibited. CONCLUSION: rh-Murine RANK was able to inhibit osteoclast differentiation and prevent ovariectomy-induced bone loss.

Therapeutic efficacy of soluble receptor activator of nuclear factor-kappa B-Fc delivered by nonviral gene transfer in a mouse model of osteolytic osteosarcoma.

Osteosarcoma is the most frequent primary bone tumor that develops mainly during youth, the median age of diagnosis being 18 years. Despite improvement in osteosarcoma treatment, survival rate is only 30% after 5 years for patients with pulmonary metastases at diagnosis. This warrants exploration of new therapeutic options. The anti-bone resorption molecule receptor activator of NF-kappaB (RANK) is very promising, as it may block the vicious cycle between bone resorption and tumor proliferation that takes place during tumor development in bone site. The cDNA encoding murine RANK-Fc (mRANK-Fc) was administered by gene transfer using an amphiphilic polymer in a mouse model of osteolytic osteosarcoma. Clinical and bone microarchitecture variables were assessed by radiography and micro-CT analyses. In vitro experiments were designed to determine the mechanism of action of RANK-Fc on tumor cell proliferation (XTT assays), apoptosis (caspase activation), cell cycle distribution (fluorescence-activated cell sorting analysis), or gene expression (reverse transcription-PCR). RANK-Fc was effective in preventing the formation of osteolytic lesions associated with osteosarcoma development and in reducing the tumor incidence, the local tumor growth, and the lung metastases dissemination leading to a 3.9-fold augmentation of mice survival 28 days after implantation. On the contrary, mRANK-Fc did not prevent the development of nonosseous tumor nodules, suggesting that bone environment is necessary for mRANK-Fc therapeutic efficacy. Furthermore, mRANK-Fc has no direct activity on osteosarcoma cells in vitro. mRANK-Fc exerts an indirect inhibitory effect on osteosarcoma progression through inhibition of bone resorption.