Page 188 - 2022_03-Haematologica-web

Letters to the Editor
Mechanical unloading aggravates bone destruc- tion and tumor expansion in myeloma
The importance of retaining physical functions has been increasingly emphasized to maintain the quality of life in patients with a variety of cancers, especially those with bone metastasis. Moreover, physical functions may impact prognosis of patients with cancers. Multiple myeloma (MM) has a unique propensity to develop and expand almost exclusively in the bone marrow and to generate destructive bone disease. Patients with MM in advanced stages often suffer from immobilization or are in a bed-ridden state with vertebral fracture and/or lower limb paralysis due to spinal cord compression by tumors expanding outside of bone.
The skeleton and skeletal muscles are sensitive to their mechanical environment such as mechanical loading with exercise. Patients in a bed-ridden state or those with lower limb paralysis are exposed to mechanical unloading to decrease bone volume and strength along with muscle atrophy. However, the effect of mechanical unloading on the progression of MM tumor has not been studied. We hypothesized that immobilization or a par- alytic state not only negatively affect bone health but also may aggravate tumor growth in patients with MM. In the present study, we therefore aimed to clarify the deleterious impact of paralytic immobilization and mechanical unloading on tumor expansion and bone destruction in MM.
Unilateral hind legs of mice were immobilized to expose to mechanical unloading by sciatic denervation1 or casting with an adhesive bandage.2 These procedures reduced hind leg muscle volume as shown by the weight as well as the outer appearance of the anterior tibial and gastrocnemius muscles at 2 weeks (Online Supplementary Figure S1A and B). Atrophy was more marked in the muscles in the hind legs paralyzed with sciatic denerva- tion than those immobilized by casting with adhesive bandage. Micro–computed tomography (mCT) revealed substantial reduction of the bone volume in the trabecu- lar bone in the tibiae in the immobilized hind legs (Figure 1A). Bone morphometric analysis also showed the reduction of bone volume in the immobilized hind legs, as indicated by an increase of bone volume over total volume and trabecular numbers with reduced trabecular separation (Figure 1B). These mCT findings were consis- tent with the previous results in mice upon mechanical unloading with hind legs paralyzed by surgical denerva- tion1 and tail suspension.3 Tartrate-resistant acid phos- phatase (TRAP)-positive multinucleated osteoclasts increased in number on the surface of the trabecular bone in the immobilized hind legs (Figure 1C and D). TRAP-5b, a bone resorption marker, was increased in sera from the mice with hind legs paralyzed by sciatic denervation, while the levels of bone formation markers, bone alkaline phosphatase and osteocalcin, were not sig- nificantly changed in their sera (Figure 1E). These results demonstrate acute activation of bone resorption by osteoclasts and thereby trabecular bone reduction alone with muscle atrophy in immobilized hind legs.
Osteocytes are embedded in the bone matrix, and major sensors of mechanical stress to regulate bone remodeling through interaction with bone marrow cells by their dendritic processes.4 Osteocytes produce critical molecules for bone metabolism, including receptor acti- vator of nuclear factor-κB ligand (RANKL) and its inhibitor osteoprotegerin (OPG), and sclerostin (SOST). After flushing out bone cavities to remove bone marrow
cells, femurs were used for gene analysis in osteocytes embedded in bone. Consistent with a previous report, 1 the gene expression of Rankl but neither Opg nor Sost was upregulated in the femurs from the hind legs immo- bilized with the sciatic denervation or casting (Figure 1F). Serum levels of Rankl were significantly increased in the mechanical unloading with sciatic denervation (Figure 1F). These results suggest that the role of RANKL upregulated in osteocytes in osteoclastogenesis is enhanced in immobilized hind legs.
We next looked at the effects of the immobilization or mechanical unloading of hind legs on MM tumor growth in bone. We inoculated luciferase-transfected mouse 5TGM1 MM cells into tibiae 2 weeks after sciatic dener- vation or sham operation, and compared tumor growth in the hind legs with or without mechanical loading. Tumor growth was more robust in the immobilized hind legs with the denervation than in sham-operated hind legs as shown in IVIS images (Figure 1G). Previous reports demonstrated that osteoclasts directly enhance MM cell proliferation5 and that RANKL-stimulated osteoclastogenesis triggers the proliferation of MM cells in vivo.6 Indeed, osteoclasts generated from mouse bone marrow cells directly enhanced the growth of 5TGM1 cells (Online Supplementary Figure S2A). As immobiliza- tion of legs acutely enhanced osteoclastogenesis (Figure 1C and D), osteoclasts induced in bone are suggested to play a causative role in MM cell expansion accelerated in mechanical unloading. Therefore, we next looked at the effects of the anti-bone resorbing agent zoledronic acid on MM tumor growth under hind leg immobilization. Treatment with zoledronic acid twice weekly after sciat- ic denervation resulted in the maintenance of bone vol- ume (Figure 2A) along with the reduction of osteoclast numbers (Figure 2B and C). The treatment with zole- dronic acid retarded MM tumor growth in the immobi- lized hind legs (Figure 2D), suggesting the role of osteo- clasts in the acceleration of MM tumor growth in immo- bilized hind legs.
In order to further confirm the acceleration of tumor growth in immobilized hind legs, we next simultaneous- ly inoculated 5TGM1 MM cells into bilateral tibiae in immobilized (right) and intact (left) hind legs in the same mice, and compared tumor growth between the immo- bilized or intact hind legs. MM tumor growth was assessed with IVIS images, and was more accelerated in the immobilized legs with sciatic denervation (Figure 3A, top) or in those in a cast (Online Supplementary Figure S2B). We reported that proviral integrations of Moloney virus 2 kinase (PIM2) is constitutively overexpressed and further upregulated in MM cells through the interaction with cellular components in MM bone marrow microen- vironment, including osteaolclasts (OC).7 We subse- quently reported that TGF-β-activated kinase-1 (TAK1) is also overexpressed and phosphorylated to transcrip- tionally induce PIM2 expression in MM cells and OC.8 Consistently, CD138-positive MM cells and cathepsin K- positive OC expressed both PIM2 and phosphorylated TAK1 in the tibiae with 5TGM1 MM cell inoculation (Online Supplementary Figure S3A). The TAK1 inhibitor LL-Z1640-2 as well as PIM inhibitor SMI16a are able to efficaciously reduce MM growth and osteoclastic bone destruction in in vivo MM models with 5TGM1 MM cells,8,9 suggesting the pivotal role of the TAK1-PIM2 pathway in MM cells in MM tumor growth and bone destruction. Interestingly, TAK1 phosphorylation and PIM2 protein levels are further upregulated in MM tumor lesions in immobilized hind legs by sciatic dener- vation compared to those in intact hind legs (Figure 3B).
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haematologica | 2022; 107(3)