The acid-sensing nociceptor TRPV1 controls breast cancer progression in bone via regulating HGF secretion from sensory neurons.

Cancers showing excessive innervation of sensory neurons (SN) in their microenvironments are associated with poor outcomes due to promoted growth, increased tumor recurrence, metastasis, and cancer pain, suggesting SNs play a regulatory role in cancer aggressiveness. Using a preclinical model in which mouse 4T1 breast cancer (BC) cells were injected into the bone marrow of tibiae, we found 4T1 BC cells aggressively colonized bone with bone destruction and subsequently spread to the lung. Of note, 4T1 BC colonization induced the acidic tumor microenvironment in bone in which SNs showed increased innervation and excitation with elevated expression of the acid-sensing nociceptor transient receptor potential vanilloid-1 (TRPV1), eliciting bone pain (BP) assessed by mechanical hypersensitivity. Further, these excited SNs produced increased hepatocyte growth factor (HGF). Importantly, the administration of synthetic and natural TRPV1 antagonists and genetic deletion of TRPV1 decreased HGF production in SNs and inhibited 4T1 BC colonization in bone, pulmonary metastasis from bone, and BP induction. Our results suggest the TRPV1 of SNs promotes BC colonization in bone and lung metastasis via up-regulating HGF production in SNs. The SN TRPV1 may be a novel therapeutic target for BC growing in the acidic bone microenvironment and for BP.

Lactate secreted via MCT4 from bone­colonizing breast cancer excites sensory neurons via GPR81.

Breast cancer (BC) bone metastasis causes bone pain (BP), which detrimentally damages the quality of life and outcome of patients with BC. However, the mechanism of BC­BP is poorly understood, and effective treatments are limited. The present study demonstrated a novel mechanism of BC­BP using a mouse model of bone pain, in which mouse (EO771) and human (MDA­MB­231) BC cells were injected in the bone marrow cavity of tibiae. Western blot analysis using sensory nerves, in vivo assessment of cancer pain and in vitro calcium flux analysis were performed. These mice developed progressive BC­BP in tibiae in conjunction with an upregulation of phosphorylated pERK1/2 and cAMP­response element­binding protein (pCREB), which are molecular indicators of neuron excitation, in the dorsal root ganglia (DRG) of sensory nerves. Importantly, mice injected with BC cells, in which the expression of the lactic acid transporter monocarboxylate transporter 4 (MCT4) was silenced, exhibited decreased BC­BP with downregulated expression of pERK1/2 and pCREB in the DRG and reduced circulating levels of lactate compared with mice injected with parental BC cells. Further, silencing of the cell­surface orphan receptor for lactate, G protein­coupled receptor 81 (GPR81), in the F11 sensory neuron cells decreased lactate­promoted upregulation of pERK1/2 and Ca2+ influx, suggesting that the sensory neuron excitation was inhibited. These results suggested that lactate released from BC cells via MCT4 induced BC­BP through the activation of GPR81 of sensory neurons. In conclusion, the activation of GPR81 of sensory neurons by lactate released via MCT4 from BC was demonstrated to contribute to the induction of BC­BP, and disruption of the interactions among lactate, MCT4 and GPR81 may be a novel approach to control BC­BP.

Cancer-nerve interplay in cancer progression and cancer-induced bone pain.

INTRODUCTION: Cancer-induced bone pain (CIBP) is one of the most common and debilitating complications associated with bone metastasis. Although our understanding of the precise mechanism is limited, it has been known that bone is densely innervated, and that CIBP is elicited as a consequence of increased neurogenesis, reprogramming, and axonogenesis in conjunction with sensitization and excitation of sensory nerves (SNs) in response to the noxious stimuli that are derived from the tumor microenvironment developed in bone. Recent studies have shown that the sensitized and excited nerves innervating the tumor establish intimate communications with cancer cells by releasing various tumor-stimulating factors for tumor progression. APPROACHES: In this review, the role of the interactions of cancer cells and SNs in bone in the pathophysiology of CIBP will be discussed with a special focus on the role of the noxious acidic tumor microenvironment, considering that bone is in nature hypoxic, which facilitates the generation of acidic conditions by cancer. Subsequently, the role of SNs in the regulation of cancer progression in the bone will be discussed together with our recent experimental findings. CONCLUSION: It is suggested that SNs may be a newly-recognized important component of the bone microenvironment that contribute to not only in the pathophysiology of CIBP but also cancer progression in bone and dissemination from bone. Suppression of the activity of bone-innervating SNs, thus, may provide unique opportunities in the treatment of cancer progression and dissemination, as well as CIBP.

The lactate sensor GPR81 regulates glycolysis and tumor growth of breast cancer.

Metabolic reprogramming is a malignant phenotype of cancer. Cancer cells utilize glycolysis to fuel rapid proliferation even in the presence of oxygen, and elevated glycolysis is coupled to lactate fermentation in the cancer microenvironment. Although lactate has been recognized as a metabolic waste product, it has become evident that lactate functions as not only an energy source but a signaling molecule through the lactate receptor G-protein-coupled receptor 81 (GPR81) under physiological conditions. However, the pathological role of GPR81 in cancer remains unclear. Here, we show that GPR81 regulates the malignant phenotype of breast cancer cell by reprogramming energy metabolism. We found that GPR81 is highly expressed in breast cancer cell lines but not in normal breast epithelial cells. Knockdown of GPR81 decreased breast cancer cell proliferation, and tumor growth. Mechanistically, glycolysis and lactate-dependent ATP production were impaired in GPR81-silenced breast cancer cells. RNA sequencing accompanied by Gene Ontology enrichment analysis further demonstrated a significant decrease in genes associated with cell motility and silencing of GPR81 suppressed cell migration and invasion. Notably, histological examination showed strong expression of GPR81 in clinical samples of human breast cancer. Collectively, our findings suggest that GPR81 is critical for malignancy of breast cancer and may be a potential novel therapeutic target for breast carcinoma.

Sensory nerves: A driver of the vicious cycle in bone metastasis?

Bone is one of the preferential target organs of cancer metastasis. Bone metastasis is associated with various complications, of which bone pain is most common and debilitating. The cancer-associated bone pain (CABP) is induced as a consequence of increased neurogenesis, reprogramming and axonogenesis of sensory nerves (SNs) in harmony with sensitization and excitation of SNs in response to the tumor microenvironment created in bone. Importantly, CABP is associated with increased mortality, of which precise cellular and molecular mechanism remains poorly understood. Bone is densely innervated by autonomic nerves (ANs) (sympathetic and parasympathetic nerves) and SNs. Recent studies have shown that the nerves innervating the tumor microenvironment establish intimate communications with tumors, producing various stimuli for tumors to progress and disseminate. In this review, our current understanding of the role of SNs innervating bone in the pathophysiology of CABP will be overviewed. Then the hypothesis that SNs facilitate cancer progression in bone will be discussed in conjunction with our recent findings that SNs play an important role not only in the induction of CABP but also the progression of bone metastasis using a preclinical model of CABP. It is suggested that SNs are a critical component of the bone microenvironment that drives the vicious cycle between bone and cancer to progress bone metastasis. Suppression of the activity of bone-innervating SNs may have potential therapeutic effects on the progression of bone metastasis and induction of CABP.

Effects of Denosumab in Japanese Patients With Rheumatoid Arthritis Treated With Conventional Antirheumatic Drugs: 36-month Extension of a Phase III Study.

OBJECTIVE: To evaluate the safety and efficacy of long-term denosumab 60 mg every 6 months (Q6M) or every 3 months (Q3M) in patients with rheumatoid arthritis (RA). METHODS: This 12-month, randomized, double-blind, placebo-controlled, multicenter, phase III trial with an open-label extension period from 12 to 36 months (DESIRABLE) enrolled Japanese patients with RA treated with placebo (P) for 12 months followed by either denosumab Q6M (P/Q6M) or denosumab Q3M (P/Q3M) for 24 months; denosumab Q6M for 36 months (Q6M/Q6M); or denosumab Q3M for 36 months (Q3M/Q3M). Efficacy was assessed by van der Heijde modified total Sharp score (mTSS), bone erosion score (BES), and joint space narrowing (JSN) score. RESULTS: Long-term treatment better maintained mTSS and BES suppression in the P/Q3M and Q3M/Q3M vs P/Q6M and Q6M/Q6M groups; changes from baseline in total mTSS (standard error) at 36 months were 2.8 (0.4) and 1.7 (0.3) vs 3.0 (0.4) and 2.4 (0.3), respectively, and corresponding changes in BES were 1.3 (0.2) and 0.4 (0.2) vs 1.4 (0.2) and 1.1 (0.2), respectively. No JSN effect was observed. Bone mineral density consistently increased in all groups after denosumab initiation, regardless of concomitant glucocorticoid administration. Serum C-terminal telopeptide of type I collagen decreased rapidly at 1 month postdenosumab administration (in both the initial 12-month [Q3M and Q6M groups] and long-term treatment [P/Q3M and P/Q6M groups] phases). Adverse event incidence leading to study drug discontinuation was similar across treatment groups. CONCLUSION: Denosumab treatment maintained inhibition of progression of joint destruction up to 36 months. Based on effects on BES progression, higher dosing frequency at an earlier treatment stage may be needed to optimize treatment. Denosumab was generally well tolerated. (ClinicalTrials.gov: NCT01973569).

Dmrt2 promotes transition of endochondral bone formation by linking Sox9 and Runx2.

Endochondral bone formation is fundamental for skeletal development. During this process, chondrocytes undergo multiple steps of differentiation and coordinated transition from a proliferating to a hypertrophic stage, which is critical to advance skeletal development. Here, we identified the transcription factor Dmrt2 (double-sex and mab-3 related transcription factor 2) as a Sox9-inducible gene that promotes chondrocyte hypertrophy in pre-hypertrophic chondrocytes. Epigenetic analysis further demonstrated that Sox9 regulates Dmrt2 expression through an active enhancer located 18 kb upstream of the Dmrt2 gene and that this enhancer's chromatin status is progressively activated through chondrocyte differentiation. Dmrt2-knockout mice exhibited a dwarf phenotype with delayed initiation of chondrocyte hypertrophy. Dmrt2 augmented hypertrophic chondrocyte gene expression including Ihh through physical and functional interaction with Runx2. Furthermore, Dmrt2 deficiency reduced Runx2-dependent Ihh expression. Our findings suggest that Dmrt2 is critical for sequential chondrocyte differentiation during endochondral bone formation and coordinates the transcriptional network between Sox9 and Runx2.

The HMGB1/RAGE axis induces bone pain associated with colonization of 4T1 mouse breast cancer in bone.

Bone pain is a common complication of breast cancer (BC) bone metastasis and is a major cause of increased morbidity and mortality. Although the mechanism of BC-associated bone pain (BCABP) remains poorly understood, involvement of BC products in the pathophysiology of BCABP has been proposed. Aggressive cancers secrete damage-associated molecular patterns (DAMPs) that bind to specific DAMP receptors and modulate cancer microenvironment. A prototypic DAMP, high mobility group box 1 (HMGB1), which acts as a ligand for the receptor for advanced glycation end products (RAGE) and toll-like receptors (TLRs), is increased in its expression in BC patients with poor outcomes. Here we show that 4T1 mouse BC cells colonizing bone up-regulate the expression of molecular pain markers, phosphorylated ERK1/2 (pERK) and pCREB, in the dorsal root ganglia (DRGs) innervating bone and induced BCABP as evaluated by hind-paw mechanical hypersensitivity. Importantly, silencing HMGB1 in 4T1 BC cells by shRNA reduced pERK and pCREB and BCABP with decreased HMGB1 levels in bone. Further, administration of a neutralizing antibody to HMGB1 or an antagonist for RAGE, FPS-ZM1, ameliorated pERK, pCREB and BCABP, while a TLR4 antagonist, TAK242, showed no effects. Consistent with these in vivo results, co-cultures of F11 sensory neuron-like cells with 4T1 BC cells in microfluidic culture platforms increased neurite outgrowth of F11 cells, which was blocked by HMGB1 antibody. Our results show that HMGB1 secreted by BC cells induces BCABP via binding to RAGE of sensory neurons and suggest that the HMGB1/RAGE axis may be a potential novel therapeutic target for BCABP.

High-mobility group box 1 induces bone destruction associated with advanced oral squamous cancer via RAGE and TLR4.

Bone destruction of maxillary and mandibular bone by invasive oral squamous cell cancer (OSCC) raises various problems in the management of patients, resulting in poor outcomes and survival. However, the mechanism behind bone destruction by OSCC remains unclear. High-mobility group box 1 (HMGB1), a highly conserved ubiquitous nuclear non-histone DNA-binding protein, has been demonstrated to be secreted by aggressive cancers and regulate osteoclastogenesis, a central player during bone destruction. We therefore reasoned that HMGB1 secreted by OSCCs contributes to bone destruction. Our results showed that HMGB1 is produced by human cell lines of OSCC and promotes osteoclastogenesis via up-regulation of the expression of receptor activator of nuclear factor kappa-Β ligand in osteoblasts and osteocytes, and consequently osteoclastic bone destruction in mice. Further, we found that these actions of HMGB1 are mediated via the receptor for advanced glycation end products and toll-like receptors. These findings suggest that HMGB1 of OSCC and its down-stream signal pathways are potential targets for the treatment of bone destruction associated with advanced OSCC.

Identifying the preferable rheumatoid arthritis subgroups for intervention with the anti-RANKL antibody denosumab to reduce progression of joint destruction.

OBJECTIVES: To clarify which rheumatoid arthritis (RA) patients benefit most from the anti-receptor activator of nuclear factor-κB ligand antibody denosumab to reduce the progression of joint destruction. METHODS: We pooled patient data from the 12-month, double-blind, placebo-controlled DRIVE (phase II) and DESIRABLE (phase III) studies. In DRIVE, concomitant treatment was limited to methotrexate, salazosulfapyridine and bucillamine. In DESIRABLE, patients could receive any disease-modifying antirheumatic drug. RA patients were randomised to denosumab 60 mg every 6 months (Q6M), every 3 months (Q3M) or placebo. Efficacy was assessed by van der Heijde-modified total Sharp score (mTSS), bone erosion score (ES) and joint space narrowing score (JSNS). Change in mTSS was assessed in subgroups stratified by risk factors for radiographic damage if the interaction factor was significant. RESULTS: The pooled analysis included 909 patients. Denosumab reduced worsening of mTSS (mean (SD)) at 12 months in the Q6M (0.88 (3.30), p=0.0024) and Q3M (0.66 (2.16), p=0.0002) groups versus placebo (1.50 (3.73)). This reduction in mTSS progression was due to the change in ES (Q6M, 0.44 (1.89), p=0.0006; Q3M, 0.20 (0.86), p<0.0001) versus placebo (0.98 (2.54)); no effect was observed on JSNS. Anti-cyclic citrullinated peptide (CCP) antibodies, glucocorticoid use and baseline ES showed a significant interaction. Denosumab was particularly effective in patients who were anti-CCP antibody positive (p<0.05). Changes in mTSS versus placebo were observed in all denosumab dose groups, regardless of glucocorticoid use and baseline ES. CONCLUSIONS: Denosumab broadly reduced the progression of joint destruction in RA patients with risk factors for radiographic damage such as especially anti-CCP antibody positivity.

Management manual for cancer treatment-induced bone loss (CTIBL): position statement of the JSBMR.

Androgen deprivation therapy and aromatase inhibitors are known to cause a decrease in bone mineral density and an increase in fractures. Patients receiving these treatments have been shown to have a fracture risk equal to or greater than that of patients with osteoporosis with prevalent fractures. This manual was created to prevent fractures in patients with cancer treatment-induced bone loss with high fracture risks who cannot be treated under the current Japanese guideline for the prevention and treatment of osteoporosis. This manual recommends drug treatment for patients with BMD - 2.0 ≤ T score < - 1.5 with the family history of hip fracture or 15% or more 10-year probability of major osteoporotic fractures by FRAX®; or in patients with BMD T score < - 2.0. It is important to verify whether the use of this manual can reduce fractures and improve the quality of life of patients with cancer treatment-induced bone loss by prospective studies.

Effects of the anti-RANKL antibody denosumab on joint structural damage in patients with rheumatoid arthritis treated with conventional synthetic disease-modifying antirheumatic drugs (DESIRABLE study): a randomised, double-blind, placebo-controlled phase 3 trial.

OBJECTIVE: To evaluate the efficacy of denosumab in suppressing joint destruction when added to conventional synthetic disease-modifying antirheumatic drug (csDMARD) therapy in patients with rheumatoid arthritis (RA). METHODS: This was a multi-centre, randomised, double-blind, parallel-group, placebo-controlled phase 3 study in Japan. Patients with RA aged ≥20 years receiving csDMARDs were randomly assigned (1:1:1) to denosumab 60 mg every 3 months (Q3M), denosumab 60 mg every 6 months (Q6M) or placebo. The change in the modified total Sharp score (mTSS) and effect on bone mineral density (BMD) at 12 months was evaluated. RESULTS: In total, 654 patients received the trial drugs. Denosumab groups showed significantly less progression of joint destruction. The mean changes in the mTSS at 12 months were 1.49 (95% CI 0.99 to 1.99) in the placebo group, 0.99 (95% CI 0.49 to 1.49) in the Q6M group (p=0.0235) and 0.72 (95% CI 0.41 to 1.03) in the Q3M group (p=0.0055). The mean changes in bone erosion score were 0.98 (95% CI 0.65 to 1.31) in the placebo group, 0.51 (95% CI 0.22 to 0.80) in the Q6M group (p=0.0104) and 0.22 (95% CI 0.09 to 0.34) in the Q3M group (p=0.0001). No significant between-group difference was observed in the joint space narrowing score. The per cent change in lumbar spine (L1-L4) BMD in the placebo, Q6M and Q3M groups were -1.03%, 3.99% (p<0.0001) and 4.88% (p<0.0001). No major differences were observed among safety profiles. CONCLUSIONS: Denosumab inhibits the progression of joint destruction, increases BMD and is well tolerated in patients with RA taking csDMARD.

Efficacy of denosumab with regard to bone destruction in prognostic subgroups of Japanese rheumatoid arthritis patients from the phase II DRIVE study.

OBJECTIVES: To evaluate the efficacy of denosumab for progressive bone erosion in risk factor subgroups of Japanese RA patients. METHODS: This study included 340 RA patients on MTX from the dose-response study of Denosumab in patients with RheumatoId arthritis on methotrexate to Validate inhibitory effect on bone Erosion (DRIVE study-a 12-month, multicentre, randomized, double-blind, placebo-controlled, phase II study). The patients were randomized to receive placebo or denosumab 60 mg every 6 months, 3 months or 2 months. Subgroup analyses involved baseline RF, ACPA, swollen joint count, CRP level, RA duration, ESR and glucocorticoid use. RESULTS: Patients with risk factor positivity generally showed consistent results for the primary endpoint of the change in the modified Sharp erosion score at 12 months from baseline. In the placebo, every 6 months, every 3 months and every 2 months groups, the mean changes in the erosion score, according to the RF status (RF-positive vs -negative subgroups), were 1.18 vs 0.59, 0.25 (P = 0.0601 vs placebo) vs 0.31 (P = 0.0827), 0.21 (P = 0.0422) vs -0.02 (P = 0.0631) and 0.15 (P = 0.0010) vs -0.05 (P = 0.0332), respectively, while the mean changes in the erosion score, according to the ACPA status (ACPA-positive vs -negative subgroups), were 1.30 vs 0.07, 0.26 (P = 0.0142) vs 0.33 (P = 0.2748), 0.16 (P = 0.0058) vs 0.08 (P = 0.7166) and 0.09 (P < 0.0001) vs 0.08 (P = 0.8939), respectively. CONCLUSION: Denosumab is a potentially useful treatment option for RA patients who are positive for RF, ACPA and other possible risk factors. TRIAL REGISTRATION: JAPIC Clinical Trials Information, http://www.clinicaltrials.jp/user/cteSearch_e.jsp, JapicCTI-101263.

Crosstalk Between Sensory Nerves and Cancer in Bone.

PURPOSE OF REVIEW: Sensory nerves (SNs) richly innervate bone and are a component of bone microenvironment. Cancer metastasis in bone, which is under the control of the crosstalk with bone microenvironment, induces bone pain via excitation of SNs innervating bone. However, little is known whether excited SNs in turn affect bone metastasis. RECENT FINDINGS: Cancer cells colonizing bone promote neo-neurogenesis of SNs and excite SNs via activation of the acid-sensing nociceptors by creating pathological acidosis in bone, evoking bone pain. Denervation of SNs or inhibition of SN excitation decreases bone pain and cancer progression and increases survival in preclinical models. Importantly, patients with cancers with increased SN innervation complain of cancer pain and show poor outcome. SNs establish the crosstalk with cancer cells to contribute to bone pain and cancer progression in bone. Blockade of SN excitation may have not only analgesic effects on bone pain but also anti-cancer actions on bone metastases.

Decreased sensory nerve excitation and bone pain associated with mouse Lewis lung cancer in TRPV1-deficient mice.

Bone pain is one of the most common and life-limiting complications of cancer metastasis to bone. Although the mechanism of bone pain still remains poorly understood, bone pain is evoked as a consequence of sensitization and excitation of sensory nerves (SNs) innervating bone by noxious stimuli produced in the microenvironment of bone metastases. We showed that bone is innervated by calcitonin gene-related protein (CGRP)+ SNs extending from dorsal root ganglia (DRG), the cell body of SNs, in mice. Mice intratibially injected with Lewis lung cancer (LLC) cells showed progressive bone pain evaluated by mechanical allodynia and flinching with increased CGRP+ SNs in bone and augmented SN excitation in DRG as indicated by elevated numbers of pERK- and pCREB-immunoreactive neurons. Immunohistochemical examination of LLC-injected bone revealed that the tumor microenvironment is acidic. Bafilomycin A1, a selective inhibitor of H+ secretion from vacuolar proton pump, significantly alleviated bone pain, indicating that the acidic microenvironment contributes to bone pain. We then determined whether the transient receptor potential vanilloid 1 (TRPV1), a major acid-sensing nociceptor predominantly expressed on SNs, plays a role in bone pain by intratibially injecting LLC cells in TRPV1-deficient mice. Bone pain and SN excitation in the DRG and spinal dorsal horn were significantly decreased in TRPV1 -/- mice compared with wild-type mice. Our results suggest that TRPV1 activation on SNs innervating bone by the acidic cancer microenvironment in bone contributes to SN activation and bone pain. Targeting acid-activated TRPV1 is a potential therapeutic approach to cancer-induced bone pain.

Japanese Expert Panel Meeting on the Management of Prostate Cancer with Bone Metastases.

INTRODUCTION: The incidence of prostate cancer in Japan continues to increase, necessitating the continued development of effective therapies and strategies. Recent advances in treatments have improved the prognosis of metastatic disease and highlighted the importance of treating bone metastases to reduce the incidence of skeletal complications and improve patients' quality of life. With the increasing number of treatment options that have become available, including bone-targeted therapy with the alpha emitter radium-223 dichloride (Ra-223), Japanese clinicians are faced with making difficult decisions on the choice of optimal treatment strategy. In such situations, guidance based on expert opinions can be beneficial. METHODS: A panel meeting of 27 Japanese experts in the management of prostate cancer was held to share opinions and to establish consensus recommendations on key clinical questions. Panelists were asked to vote on more than 40 questions pertinent to prostate cancer, and the answers helped guide a comprehensive discussion. RESULTS: The panel reached a consensus on key topics related to the optimal treatment strategy for Ra-223 therapy, namely, that patients with symptomatic, metastatic castration-resistant prostate cancer (CRPC) would benefit most from the use of this agent and that this treatment therapy should be provided before chemotherapy. Other topics that achieved consensus included: monitoring for osteoporosis and providing treatment if necessary during androgen deprivation therapy; performing magnetic resonance imaging in the presence of discrepancies in bone scintigram and computed tomography scans; monitoring alkaline phosphatase during CRPC treatment; using osteoclast-targeting in patients with CRPC with bone metastases; and using osteoclast-targeted agents combined with Ra-223. CONCLUSION: These consensus recommendations and the updated information which became available subsequent to the panel meeting included here provide useful information for clinicians to aid in designing optimal treatment strategies for their patients. FUNDING: Bayer Yakuhin Ltd.

Disruption of the Cx43/miR21 pathway leads to osteocyte apoptosis and increased osteoclastogenesis with aging.

Skeletal aging results in apoptosis of osteocytes, cells embedded in bone that control the generation/function of bone forming and resorbing cells. Aging also decreases connexin43 (Cx43) expression in bone; and osteocytic Cx43 deletion partially mimics the skeletal phenotype of old mice. Particularly, aging and Cx43 deletion increase osteocyte apoptosis, and osteoclast number and bone resorption on endocortical bone surfaces. We examined herein the molecular signaling events responsible for osteocyte apoptosis and osteoclast recruitment triggered by aging and Cx43 deficiency. Cx43-silenced MLO-Y4 osteocytic (Cx43def ) cells undergo spontaneous cell death in culture through caspase-3 activation and exhibit increased levels of apoptosis-related genes, and only transfection of Cx43 constructs able to form gap junction channels reverses Cx43def cell death. Cx43def cells and bones from old mice exhibit reduced levels of the pro-survival microRNA miR21 and, consistently, increased levels of the miR21 target phosphatase and tensin homolog (PTEN) and reduced phosphorylated Akt, whereas PTEN inhibition reduces Cx43def cell apoptosis. miR21 reduction is sufficient to induce apoptosis of Cx43-expressing cells and miR21 deletion in miR21fl/fl bones increases apoptosis-related gene expression, whereas a miR21 mimic prevents Cx43def cell apoptosis, demonstrating that miR21 lies downstream of Cx43. Cx43def cells release more osteoclastogenic cytokines [receptor activator of NFκB ligand (RANKL)/high-mobility group box-1 (HMGB1)], and caspase-3 inhibition prevents RANKL/HMGB1 release and the increased osteoclastogenesis induced by conditioned media from Cx43def cells, which is blocked by antagonizing HMGB1-RAGE interaction. These findings identify a novel Cx43/miR21/HMGB1/RANKL pathway involved in preventing osteocyte apoptosis that also controls osteoclast formation/recruitment and is impaired with aging.

Bone Pain Induced by Multiple Myeloma Is Reduced by Targeting V-ATPase and ASIC3.

Multiple myeloma patients experience severe bone pain (MMBP) that is undertreated and poorly understood. In this study, we studied MMBP in an intratibial mouse xenograft model that employs JJN3 human multiple myeloma cells. In this model, mice develop MMBP associated in bone with increased sprouting of calcitonin gene-related peptide-positive (CGRP+) sensory nerves and in dorsal root ganglia (DRG) with upregulation of phosphorylated ERK1/2 (pERK1/2) and pCREB, two molecular indicators of neuron excitation. We found that JJN3 cells expressed a vacuolar proton pump (V-ATPase) that induced an acidic bone microenvironment. Inhibition of JJN3-colonized bone acidification by a single injection of the selective V-ATPase inhibitor, bafilomycin A1, decreased MMBP, CGRP+ sensory neuron sprouting, and pERK1/2 and pCREB expression in DRG. CGRP+ sensory nerves also expressed increased levels of the acid-sensing nociceptor ASIC3. Notably, a single injection of the selective ASIC3 antagonist APETx2 dramatically reduced MMBP in the model. Mechanistic investigations in primary DRG neurons cocultured with JJN3 cells showed increased neurite outgrowth and excitation inhibited by bafilomycin A1 or APETx2. Furthermore, combining APETx2 with bafilomycin A1 reduced MMBP to a greater extent than either agent alone. Finally, combining bafilomycin A1 with the osteoclast inhibitor zoledronic acid was sufficient to ameliorate MMBP, which was refractory to zoledronic acid. Overall, our results show that osteoclasts and multiple myeloma cooperate to induce an acidic bone microenvironment that evokes MMBP as a result of the excitation of ASIC3-activated sensory neurons. Furthermore, they present a mechanistic rationale for targeting ASIC3 on neurons along with the multiple myeloma-induced acidic bone microenvironment as a strategy to relieve MMBP in patients. Cancer Res; 77(6); 1283-95. ©2017 AACR.