Activating transcription factor 4, an ER stress mediator, is required for, but excessive ER stress suppresses osteoblastogenesis by bortezomib.

Endoplasmic reticulum (ER) stress is induced in matrix-producing osteoblasts and plays an essential role in osteoblastogenesis. Although the bone anabolic activity of proteasome inhibitors has been demonstrated, the roles of ER stress induced by proteasome inhibition in osteoblastogenesis remain largely unknown. Here we show that bortezomib translationally increases protein levels of activating transcription factor 4 (ATF4), a downstream mediator of ER stress, in bone marrow stromal cells and MC3T3-E1 preosteoblastic cells. The suppression of ATF4 expression by siRNA abrogated osteocalcin expression and mineralized nodule formation by MC3T3-E1 cells induced by bortezomib, indicating a critical role for ATF4 in bortezomib-mediated osteoblastogenesis. However, bortezomib at 20 nM or higher abolished the mineralized nodule formation along with reductions in the expression of osteoblastogenesis mediators ß-catenin and Osterix. Furthermore, at 50 nM, bortezomib induced the expression of C/EBP homologous protein (CHOP), suggesting activation of the ATF4-CHOP pro-apoptotic pathway. These results suggest that a low dose of bortezomib induces osteogenic activity, but that, in contrast, excessive ER stress caused by bortezomib at higher doses hampers osteoblastogenesis. Therefore, dosing schedules for proteasome inhibitors warrant further study to maximize anabolic actions without compromising anti-MM activity in patients with multiple myeloma (MM).

Inhibition of TACE activity enhances the susceptibility of myeloma cells to TRAIL.

BACKGROUND: TNF-related apoptosis-inducing ligand/Apo2 ligand (TRAIL/Apo2L) selectively induces apoptosis in various cancer cells including myeloma (MM) cells. However, the susceptibility of MM cells to TRAIL is largely low in most of MM cells by yet largely unknown mechanisms. Because TNF-α converting enzyme (TACE) can cleave some TNF receptor family members, in the present study we explored the roles of proteolytic modulation by TACE in TRAIL receptor expression and TRAIL-mediated cytotoxicity in MM cells. METHODOLOGY/PRINCIPAL FINDINGS: MM cells preferentially expressed death receptor 4 (DR4) but not DR5 on their surface along with TACE. Conditioned media from RPMI8226 and U266 cells contained a soluble form of DR4. The DR4 levels in these conditioned media were reduced by TACE inhibition by the TACE inhibitor TAPI-0 as well as TACE siRNA. Conversely, the TACE inhibition restored surface levels of DR4 but not DR5 in these cells without affecting DR4 mRNA levels. The TACE inhibition was able to restore cell surface DR4 expression in MM cells even in the presence of bone marrow stromal cells or osteoclasts, and enhanced the cytotoxic effects of recombinant TRAIL and an agonistic antibody against DR4 on MM cells. CONCLUSIONS/SIGNIFICANCE: These results demonstrate that MM cells post-translationally down-modulate the cell surface expression of DR4 through ectodomain shedding by endogenous TACE, and that TACE inhibition is able to restore cell surface DR4 levels and the susceptibility of MM cells to TRAIL or an agonistic antibody against DR4. Thus, TACE may protect MM cells from TRAIL-mediated death through down-modulation of cell-surface DR4. It can be envisaged that TACE inhibition augments clinical efficacy of TRAIL-based immunotherapy against MM, which eventually becomes resistant to the present therapeutic modalities.

Up-regulation of hexokinaseII in myeloma cells: targeting myeloma cells with 3-bromopyruvate.

Hexokinase II (HKII), a key enzyme of glycolysis, is widely over-expressed in cancer cells. However, HKII levels and its roles in ATP production and ATP-dependent cellular process have not been well studied in hematopoietic malignant cells including multiple myeloma (MM) cells.We demonstrate herein that HKII is constitutively over-expressed in MM cells. 3-bromopyruvate (3BrPA), an inhibitor of HKII, promptly and substantially suppresses ATP production and induces cell death in MM cells. Interestingly, cocultures with osteoclasts (OCs) but not bone marrow stromal cells (BMSCs) enhanced the phosphorylation of Akt along with an increase in HKII levels and lactate production in MM cells. The enhancement of HKII levels and lactate production in MM cells by OCs were mostly abrogated by the PI3K inhibitor LY294002, suggesting activation of glycolysis in MM cells by OCs via the PI3K-Akt-HKII pathway. Although BMSCs and OCs stimulate MM cell growth and survival, 3BrPA induces cell death in MM cells even in cocultures with OCs as well as BMSCs. Furthermore, 3BrPA was able to diminish ATP-dependent ABC transporter activity to restore drug retention in MM cells in the presence of OCs. These results may underpin possible clinical application of 3BrPA in patients with MM.

Glycolysis inhibition inactivates ABC transporters to restore drug sensitivity in malignant cells.

Cancer cells eventually acquire drug resistance largely via the aberrant expression of ATP-binding cassette (ABC) transporters, ATP-dependent efflux pumps. Because cancer cells produce ATP mostly through glycolysis, in the present study we explored the effects of inhibiting glycolysis on the ABC transporter function and drug sensitivity of malignant cells. Inhibition of glycolysis by 3-bromopyruvate (3BrPA) suppressed ATP production in malignant cells, and restored the retention of daunorubicin or mitoxantrone in ABC transporter-expressing, RPMI8226 (ABCG2), KG-1 (ABCB1) and HepG2 cells (ABCB1 and ABCG2). Interestingly, although side population (SP) cells isolated from RPMI8226 cells exhibited higher levels of glycolysis with an increased expression of genes involved in the glycolytic pathway, 3BrPA abolished Hoechst 33342 exclusion in SP cells. 3BrPA also disrupted clonogenic capacity in malignant cell lines including RPMI8226, KG-1, and HepG2. Furthermore, 3BrPA restored cytotoxic effects of daunorubicin and doxorubicin on KG-1 and RPMI8226 cells, and markedly suppressed subcutaneous tumor growth in combination with doxorubicin in RPMI8226-implanted mice. These results collectively suggest that the inhibition of glycolysis is able to overcome drug resistance in ABC transporter-expressing malignant cells through the inactivation of ABC transporters and impairment of SP cells with enhanced glycolysis as well as clonogenic cells.

KRN5500, a spicamycin derivative, exerts anti-myeloma effects through impairing both myeloma cells and osteoclasts.

The spicamycin analogue KRN5500 alters glycoprotein processing and induces damage in the endoplasmic reticulum (ER)-Golgi apparatus in cancer cells. In the present study, we explored the cytotoxic effects of KRN5500 on multiple myeloma (MM) cells and the bone marrow microenvironment with special reference to ER stress. Cell proliferation assay showed that KRN5500 induced G1 arrest and apoptosis in MM cells in a time- and dose-dependent manner. KRN5500 enhanced ER stress independently of caspase activation in MM cells. This cell death was observed even in the presence of bone marrow stroma cells or osteoclasts. Notably, KRN5500 induced cell death also in osteoclasts. In vivo effects of KRN5500 were evaluated using two xenograft models established in severe combined immunodeficient (SCID) mice by either subcutaneous injection of RPMI 8226 cells or intra-bone injection of INA-6 cells to subcutaneously implanted rabbit bones (SCID-rab model). KRN5500 significantly inhibited tumour growth in both animal models, and decreased the number of osteoclasts, which resulted in prevention of bone destruction in the SCID-rab model. These results suggest that KRN5500 exerts anti-MM effects through impairing both MM cells and osteoclasts. Therefore, this unique mechanism of KRN5500 might be a useful therapeutic option in patients with MM.

Targeting myeloma-osteoclast interaction with Vγ9Vδ2 T cells.

Multiple myeloma (MM) cells stimulate osteoclastogenesis, and osteoclasts (OCs) in turn enhance MM growth and drug resistance, resulting in a vicious cycle. Vγ9Vδ2 T cells exert potent anti-tumor effects, making T cell-based immunotherapies using these cells attractive candidates for currently incurable malignancies, such as MM. However, the impact of such treatments on the MM-OC interaction is largely unknown. We demonstrate here that Vγ9Vδ2 T cells expanded by zoledronic acid and IL-2 exerted potent cytotoxic effects on both MM cells and OCs, even in coculture settings, but showed no such effect on bone marrow stromal cells. Vγ9Vδ2 T cells marginally affected colony formation from normal hematopoietic progenitors, and furthermore migrated toward osteopontin and MIP-1α, factors produced by the MM-OC interaction. These results suggest that Vγ9Vδ2 T cells expanded by zoledronic acid and IL-2 are able to migrate to MM bone lesions and preferentially target OCs as well as MM cells, thereby inhibiting both tumor expansion and bone destruction.

Delayed treatment with vitamin C and N-acetyl-L-cysteine protects Schwann cells without compromising the anti-myeloma activity of bortezomib.

Bortezomib-induced peripheral neuropathy (BIPN) emerges as a disabling adverse effect. As rat models for BIPN have demonstrated damage in nerve Schwann cells, we screened for cytoprotective agents to devise a method of rescuing Schwann cells from the cytotoxic effects of bortezomib without compromising its anti-myeloma effects. Schwann cells underwent macroautophagy along with cytoplasmic inclusion body and vacuole formation, and appeared much less susceptible to bortezomib-induced cytotoxicity than did myeloma cells. Vitamin C or N-acetyl-L-cysteine (NAC) achieved near-complete rescue of Schwann cells treated with bortezomib at 30 nM or less, and these agents in combination are able to cooperatively inhibit the morphological changes and the cytotoxicity in Schwann cells with higher doses of bortezomib. The delayed addition of vitamin C and/or NAC after the exposure to bortezomib alleviated the cytotoxicity in Schwann cells but not myeloma cells. These results suggest that delayed treatment with these agents may be instrumental in prophylaxis of BIPN.

Tgf-Beta inhibition restores terminal osteoblast differentiation to suppress myeloma growth.

BACKGROUND: Multiple myeloma (MM) expands almost exclusively in the bone marrow and generates devastating bone lesions, in which bone formation is impaired and osteoclastic bone resorption is enhanced. TGF-beta, a potent inhibitor of terminal osteoblast (OB) differentiation, is abundantly deposited in the bone matrix, and released and activated by the enhanced bone resorption in MM. The present study was therefore undertaken to clarify the role of TGF-beta and its inhibition in bone formation and tumor growth in MM. METHODOLOGY/PRINCIPAL FINDINGS: TGF-beta suppressed OB differentiation from bone marrow stromal cells and MC3T3-E1 preosteoblastic cells, and also inhibited adipogenesis from C3H10T1/2 immature mesenchymal cells, suggesting differentiation arrest by TGF-beta. Inhibitors for a TGF-beta type I receptor kinase, SB431542 and Ki26894, potently enhanced OB differentiation from bone marrow stromal cells as well as MC3T3-E1 cells. The TGF-beta inhibition was able to restore OB differentiation suppressed by MM cell conditioned medium as well as bone marrow plasma from MM patients. Interestingly, TGF-beta inhibition expedited OB differentiation in parallel with suppression of MM cell growth. The anti-MM activity was elaborated exclusively by terminally differentiated OBs, which potentiated the cytotoxic effects of melphalan and dexamethasone on MM cells. Furthermore, TGF-beta inhibition was able to suppress MM cell growth within the bone marrow while preventing bone destruction in MM-bearing animal models. CONCLUSIONS/SIGNIFICANCE: The present study demonstrates that TGF-beta inhibition releases stromal cells from their differentiation arrest by MM and facilitates the formation of terminally differentiated OBs, and that terminally differentiated OBs inhibit MM cell growth and survival and enhance the susceptibility of MM cells to anti-MM agents to overcome the drug resistance mediated by stromal cells. Therefore, TGF-beta appears to be an important therapeutic target in MM bone lesions.

GM-CSF and IL-4 induce dendritic cell differentiation and disrupt osteoclastogenesis through M-CSF receptor shedding by up-regulation of TNF-alpha converting enzyme (TACE).

Monocytes give rise to macrophages, osteoclasts (OCs), and dendritic cells (DCs). Macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-kappaB (RANK) ligand induce OC differentiation from monocytes, whereas granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) trigger monocytic differentiation into DCs. However, regulatory mechanisms for the polarization of monocytic differentiation are still unclear. The present study was undertaken to clarify the mechanism of triggering the deflection of OC and DC differentiation from monocytes. GM-CSF and IL-4 abolished monocytic differentiation into OCs while inducing DC differentiation even in the presence of M-CSF and RANK ligand. GM-CSF and IL-4 in combination potently up-regulate tumor necrosis factor-alpha (TNF-alpha) converting enzyme (TACE) and activity in monocytes, causing ectodomain shedding of M-CSF receptor, resulting in the disruption of its phosphorylation by M-CSF as well as the induction of osteoclastogenesis from monocytes by M-CSF and RANK ligand. Interestingly, TACE inhibition robustly causes the resumption of the surface expression of M-CSF receptor on monocytes, facilitating M-CSF-mediated phosphorylation of M-CSF receptor and macrophage/OC differentiation while impairing GM-CSF- and IL-4-mediated DC differentiation from monocytes. These results reveal a novel proteolytic regulation of M-CSF receptor expression in monocytes to control M-CSF signaling and monocytic differentiation into macrophage/OC-lineage cells or DCs.

Valproic acid exerts anti-tumor as well as anti-angiogenic effects on myeloma.

Multiple myeloma is still an incurable disease, most commonly occurring in the elderly. The myeloma-induced bone marrow microenvironment protects myeloma cells from drug-induced apoptosis. Therefore, the development of novel and tolerable therapeutic alternatives to overcome the drug resistance is an important clinical issue. Valproic acid (VPA), a safe and widely used anti-epileptic agent, is revisited as a class I- and IIa-specific histone deacetylase inhibitor. In the present study, we evaluated the effect as well as a mechanism of actions of VPA on myeloma cell growth and survival, with special reference to the myeloma-induced bone marrow microenvironment. VPA at therapeutic concentrations for epilepsy induced cell death in primary CD138-positive myeloma cells as well as myeloma cell lines, but not in CD138-negative bone marrow cells. VPA suppressed osteoclastogenesis as well as osteoclast-mediated myeloma cell growth. VPA also inhibited vascular tubule formation enhanced by co-cultures of myeloma cells and osteoclasts in concert with thalidomide. In addition, VPA induced both caspase-dependent and -independent cell death in myeloma cells, and potentiated the anti-myeloma effects of melphalan and dexamethasone. Collectively, VPA is suggested to exert multi-factorial anti-myeloma actions, and may serve as a safe adjuvant to be included in conventional chemotherapies against myeloma.

Myeloma cell-osteoclast interaction enhances angiogenesis together with bone resorption: a role for vascular endothelial cell growth factor and osteopontin.

PURPOSE: Similar to osteoclastogenesis, angiogenesis is enhanced in the bone marrow in myeloma in parallel with tumor progression. We showed previously that myeloma cells and osteoclasts are mutually stimulated to form a vicious cycle to lead to enhance both osteoclastogenesis and tumor growth. The present study was undertaken to clarify whether myeloma cell-osteoclast interaction enhances angiogenesis and whether there is any mutual stimulation between osteoclastogenesis and angiogenesis. EXPERIMENTAL DESIGN: Myeloma cells and monocyte-derived osteoclasts were cocultured, and angiogenic activity produced by the cocultures was assessed with in vitro vascular tubule formation assays and human umbilical vascular endothelial cell (HUVEC) migration and survival. Osteoclastogenic activity was determined with rabbit bone cell cultures on dentine slices. RESULTS: Myeloma cells and osteoclasts constitutively secrete proangiogenic factors, vascular endothelial growth factor (VEGF) and osteopontin, respectively. A cell-to-cell interaction between myeloma cells and osteoclasts potently enhanced vascular tubule formation. Blockade of both VEGF and osteopontin actions almost completely abrogated such vascular tubule formation as well as migration and survival of HUVECs enhanced by conditioned medium from cocultures of myeloma cells and osteoclasts. Furthermore, these factors in combination triggered the production of osteoclastogenic activity by HUVEC. CONCLUSIONS: Osteoclast-derived osteopontin and VEGF from myeloma cells cooperatively enhance angiogenesis and also induce osteoclastogenic activity by vascular endothelial cells. These observations suggest the presence of a close link between myeloma cells, osteoclasts, and vascular endothelial cells to form a vicious cycle between bone destruction, angiogenesis, and myeloma expansion.