Critical Role of AKT Protein in Myeloma-induced Osteoclast Formation and Osteolysis*

Background: Myeloma cells cause abnormal osteoclast formation and osteolysis. Results: Myeloma cells up-regulate AKT in osteoclast precursors and promote osteoclast formation. Systemic AKT inhibition blocks the myeloma-induced osteolysis and tumor growth in bone. Conclusion: AKT is critical for the myeloma promotion of osteoclast formation and osteolysis. Significance: AKT could be a useful target for treating patients with myeloma bone disease. Abnormal osteoclast formation and osteolysis are the hallmarks of multiple myeloma (MM) bone disease, yet the underlying molecular mechanisms are incompletely understood. Here, we show that the AKT pathway was up-regulated in primary bone marrow monocytes (BMM) from patients with MM, which resulted in sustained high expression of the receptor activator of NF-κB (RANK) in osteoclast precursors. The up-regulation of RANK expression and osteoclast formation in the MM BMM cultures was blocked by AKT inhibition. Conditioned media from MM cell cultures activated AKT and increased RANK expression and osteoclast formation in BMM cultures. Inhibiting AKT in cultured MM cells decreased their growth and ability to promote osteoclast formation. Of clinical significance, systemic administration of the AKT inhibitor LY294002 blocked the formation of tumor tissues in the bone marrow cavity and essentially abolished the MM-induced osteoclast formation and osteolysis in SCID mice. The level of activating transcription factor 4 (ATF4) protein was up-regulated in the BMM cultures from multiple myeloma patients. Adenoviral overexpression of ATF4 activated RANK expression in osteoclast precursors. These results demonstrate a new role of AKT in the MM promotion of osteoclast formation and bone osteolysis through, at least in part, the ATF4-dependent up-regulation of RANK expression in osteoclast precursors.

(MIP)-1␣ promotes the development of osteolytic lesions in patients with MM (19,20). Enhanced RANKL expression plays an important role in tumor-induced osteoclast formation and bone destruction (13,21). However, molecular mechanisms whereby the MM cells promote osteoclast formation and activation and osteolysis are not completely understood.
AKT, also known as protein kinase B (PKB), is a serine/threonine-specific protein kinase that plays a critical role in multiple cellular processes such as cell proliferation, survival, and migration, glucose metabolism, and gene transcription (22,23). Accumulative evidence shows that the PI3K/AKT pathway plays a critical role in activating osteoclast differentiation (24,25). However, if and how AKT mediates the MM-induced osteoclast formation and osteolysis have not been addressed.

EXPERIMENTAL PROCEDURES
Reagents-Tissue culture media, fetal bovine serum, and horse serum were obtained from Thermo Scientific HyClone (Logan, UT). LY294002 was purchased from LC Laboratories (Woburn, MA); ascorbic acid and DMSO were from Sigma; human recombinant M-CSF, RANKL, and TNF-␣ were from R&D Systems Inc. (Minneapolis, MN). All other chemicals were of analytical grade.
Human Bone Marrow Samples-Bone marrow aspirates were collected in heparin from normal donors (ND) and patients with MM. Protocols were approved by the respective Institutional Review Board committees of the University of Pittsburgh and the Institute of Hematology and Blood Diseases, Chinese Academy of Medical Sciences.
DNA Constructs, transfection, and Adenoviral Infection-pCMV/␤-gal, pCMV/ATF4, pCMV/AKT-CA, and wild-type and mutant p4OSE1-luc plasmids (an ATF4 reporter plasmid) were previously described (26,27). Rank-luc reporter plasmid was constructed by PCR subcloning of a Ϫ1073/ϩ79 mouse Rank gene promoter into the pGL3-luc vector (Promega, Madison, WI) in the project laboratory. For transfection experiments, the amounts of plasmid DNAs were balanced as necessary with ␤-galactosidase expression plasmid such that the total DNA was constant in each group. Adenoviruses expressing ATF4 or EGFP were described previously (28,29). The amount of adenovirus was balanced as necessary with a control adenovirus expressing EGFP such that the total amount was constant in each group.
Gene Expression Studies-RNA isolation and reverse transcription (RT) were previously described (30). Quantitative real time RT-PCR analysis was performed to measure the relative mRNA levels using the SYBR Green kit (Bio-Rad). Melting curve analysis was used to confirm the specificity of the PCR products. Four to six samples were run for each primer set. The levels of mRNA were calculated by the ⌬⌬Ct method. Samples were normalized to Gapdh expression. The DNA sequences of human and mouse primers used for qPCR were summarized in Tables 1 and 2. Western blot analysis was performed as described previously (26,30). Antibodies used were from the following sources: antibodies against NFATc1, c-FOS, RANK, ATF4, and anti-rabbit or anti-mouse antibodies conjugated with horseradish peroxidase were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA); antibodies recognizing phosphorylated and total AKT and PU.1 were from Cell Signaling Technology Inc. (Beverly, MA); and mouse monoclonal antibody against ␤-actin was from Sigma.
In Vitro Osteoclast Assays-For osteoclast studies using bone marrow cells from humans, nonadherent BMMs were isolated from patients with MM and NDs. For undifferentiated cultures, cells were cultured in the proliferation media (␣-MEM containing 20% horse serum and 10 ng/ml human recombinant M-CSF) for 10 days. For differentiation studies, cells were cultured in the differentiation media (proliferation media containing 50 ng/ml human recombinant RANKL) for 21 days, followed by TRAP or 23C6 staining or gene expression studies. The anti-23C6 antibody, which recognizes the ␣v␤3 integrin, a receptor for vitronectin on osteoclasts, was described previously (31)(32)(33). The TRAP-or 23C6-positive multinucleated cells (MNCs) (Ն3 nuclei) were scored using an inverted microscope. For osteoclast studies using primary mouse bone marrow cells (BMMs), nonadherent BMMs were isolated from total bone marrow cells and cultured on tissue culture dishes for 48 h. For differentiation, cells were first cultured in the proliferation media (␣-MEM containing 10% FBS and 10 ng/ml M-CSF) for 3 days and switched to differentiation media (proliferation media plus 50 ng/ml RANKL) for 5-7 days, followed by TRAP staining or gene expression studies. The TRAP-positive MNCs (Ն3 nuclei) were scored using an inverted microscope.
Mouse Model and Histological Analysis and Bone Histomorphometry-For intratibial injection, both the left and right tibiae of 4-week-old male SCID mice (10 mice/group or 20 tibiae/group) were injected with 1 ϫ 10 6 5TGM1 cells in 20 l of PBS or 20 l of PBS alone (control group without MM injec- TTG TGC TAG TGC CCT CGA GAA CTG GAG GAA AAA CTG GGG TGA tion). One week later, mice were subcutaneously injected with LY294002 (40 mg/kg body weight) or vehicle (DMSO) twice a week for 3 weeks. Protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of the Nankai University. All specimens were fixed in 10% formalin at 4°C for 24 h, decalcified in 10% EDTA (pH 7.4) for 10 -14 days, and embedded in paraffin. Then serial sections were prepared and stained with hematoxylin and eosin. Sections of tibiae from each group were used for TRAP staining as described previously (34). Bone histomorphometry such as osteoclast surface/ bone surface (Oc.S/BS) and osteoclast number/bone perimeter (Oc.Nb/BPm) of tibiae was measured using an Image Pro Plus 7.0 software as described previously (25,35). Statistical Analysis-Data were analyzed with a GraphPad Prism software (4.0) (San Diego). A one-way analysis of variance was used followed by the Tukey test. Results were expressed as means Ϯ S.D. Differences with a p Ͻ 0.05 was considered as statistically significant. All experiments were repeated at least two times, and similar results were obtained.

MM Activates Osteoclast Formation in Primary BMM Cultures in the Presence and Absence of Exogenously Added RANKL-
To study the mechanisms whereby MM cells activate osteoclast formation, we measured osteoclast differentiation in primary BMM cultures from patients with MM in vitro compared with BMM cultures from ND with or without the addition of exogenous RANKL. We measured the numbers of TRAP-positive multinucleated osteoclasts (MNCs) generated by each. Results showed that the number of TRAP ϩ MNCs (Ն3 nuclei) in the RANKL-differentiated MM BMM cultures was dramatically increased compared with that in the ND BMM cultures (Fig. 1,  A and B). The MNCs formed in the RANKL-differentiated MM BMM cultures were much larger than those from the ND BMM cultures (Fig. 1B). Surprisingly, we found a number of TRAP ϩ MNCs in the MM BMM cultures in the absence of exogenously added RANKL, which were absent from the ND BMM control cultures (Fig. 1, A and C). This could result from potential contamination of osteoblasts and stromal cells and bone marrow lymphocytes, which produce RANKL and OPG (36). For this reason, we measured the expression levels of both factors by qPCR analysis. Results showed that, although both mRNAs were detected in both cultures, the RANKL/OPG ratio was actually reduced in the MM versus ND cultures (Fig. 1D). Therefore, the enhanced osteoclast formation in the MM cultures is not due to increased RANKL and/or reduced OPG expression produced by cells in the cultures. qPCR analysis showed that the mRNA levels of osteoclast differentiation marker genes, including those encoding cathepsin K, integrin ␤3 (␤3), NFATc1 (a master regulator of osteoclast differentiation), and matrix metalloproteinase-9 (MMP-9) were all signif-icantly increased in the differentiated MM relative to ND BMM cultures (Fig. 1E). The mRNA level of c-FMS, the gene that encodes the receptor for the macrophage colony-stimulating factor (CSF-1R), was slightly but significantly increased in the RANKL-differentiated MM versus ND BMM cultures (Fig. 1E). In contrast, the mRNA level of PU.1 (an Ets family transcription factor that regulates osteoclast differentiation) was not different in the RANKL-differentiated MM and ND BMM cultures (Fig. 1E). Western blot analysis revealed that the levels of NFATc1 protein were increased in the RANKL-differentiated BMM cultures from five patients with MM compared with those in five NDs (Fig. 1F). Collectively, these results suggest that osteoclast differentiation capacity of the MM BMMs is increased in both RANKL-dependent and RANKL-independent manners.
Sustained High Expression of RANK Is a Major Feature of the BMMs from MM Patients-To study the early molecular events in the MM BMM cultures, we measured the expression levels of genes that are known to regulate early osteoclast differentiation in undifferentiated BMM cultures from patients with MM and NDs. Results showed that the level of RANK mRNA was increased by more than 10-fold in undifferentiated BMM cultures from patients with MM compared with that from NDs ( Fig. 2A). In contrast, the levels of c-FMS, PU.1, c-FOS, TRAF6, and DAP-12 mRNAs were not significantly increased in the undifferentiated MM versus ND BMM cultures ( Fig. 2A). Western blot analysis confirmed that the level of RANK protein was increased in the undifferentiated MM relative to ND BMM cultures (Fig. 2B). Importantly, the levels of RANK mRNA were significantly increased in the uncultured BMMs from 11 patients with MM compared with those from the uncultured BMMs from six NDs (Fig. 2C). Because RANK expression is a critical step for generating osteoclast precursors, these results suggest that MM cells greatly promote early osteoclast differentiation. Surprisingly, although RANK is a marker of osteoclast precursors, we found that its expression continued to increase even in the terminally differentiated osteoclast cultures from patients with MM (i.e. in the presence of RANKL for 21 days) (Fig. 2D). Western blot analysis confirmed that the level of RANK protein was increased in the RANKL-differentiated MM relative to ND BMM cultures (Fig. 2E).
Critical Role of AKT in RANK Expression and Osteoclast formation in BMM Cultures from MM Patients-Although the above results clearly showed that RANK expression was upregulated in osteoclast precursors from patients with MM, the underlying mechanisms remain unknown. Recent studies showed that AKT plays a role in promotion of early osteoclast differentiation (24,25). We investigated whether AKT is upregulated in the MM BMM cultures. As shown in Fig. 3A, in the RANKL-differentiated MM BMM cultures, the levels of both  3B). In contrast, the inhibitor did not reduce the expression of PU.1 mRNA in the MM BMMs (Fig. 3B). LY294002 similarly decreased RANK protein expression in the MM BMM cultures in a dose-dependent manner (Fig. 3C). Importantly, the AKT inhibitor blocked formation of the MNCs in the MM BMM cultures (Fig. 3, D-F). Furthermore, overexpression of a constitutively active form of AKT (AKT-CA) increased the mouse Rank promoter activity in COS-7 cells (Fig. 3F). Taken together, these results suggest that MM may activate RANK expression and osteoclast formation through, at least in part, up-regulation of AKT in osteoclast precursors. Soluble Factors Produced by MM Cells Increase AKT Phosphorylation, RANK Expression, and Osteoclast Formation-To determine whether MM cells produce soluble factors responsi-ble for the increased AKT and RANK expression and osteoclast formation, primary mouse BMMs were treated with the conditioned media (CM) from human MM1.S or mouse 5TGM1 MM cell lines. Western blot analysis showed that AKT phosphorylation was increased by CM from both MM cell lines (Fig.  4A). The MM1.S-CM increased RANK expression at both the mRNA and protein levels in dose-and time-dependent manners (Fig. 4, B and C). The MM1.S-CM dose-dependently promoted formation of both mononuclear and multinucleated osteoclasts (Fig. 4, D and E). However, the MM1.S-CM did not alter the formation of colony-forming units-granulocyte-macrophage (CFU-GM) (Fig. 4, F and G), which contains the earliest osteoclast precursors (37).
ATF4 Protein Is Up-regulated in MM Osteoclast Cultures and by 5TGM1-CM and TNF-␣-Our recent study demonstrates that loss of ATF4 impaired M-CSF induction of RANK expression in osteoclast precursors (25). Interestingly, we found that the level of ATF4 protein was increased in the RANKL-differentiated BMM cultures from four patients with MM compared with that of four NDs (Fig. 5A). The level of ATF4 mRNA was not increased in MM versus ND cultures (Fig. 5B), suggesting a post-transcriptional regulation. The 5TGM1-CM and TNF-␣, a well known osteoclastogenic factor produced by many cancer cells, including MM cells, increased the ATF4 protein levels in the BMM cultures (Fig. 5, C and E). In contrast, 5TGM1-CM and TNF-␣ did not increase the levels of Atf4 mRNA (Fig. 5, D  and F). Interestingly, slower migrating bands indicated by an arrow were observed in the MM BMM cultures (Fig. 5A) and increased by treatments with 5TGM1-CM (Fig. 5C) or TNF-␣ (Fig. 5E). These bands were probably phosphorylated forms of ATF4 because similar bands in primary mouse BMMs disappeared with calf intestinal phosphatase treatment (25).
ATF4 Is Phosphorylated and Up-regulated by AKT-To determine whether ATF4 is downstream of AKT, we performed several experiments. First, we found that AKT inhibitor LY294002 decreased the ATF4 protein levels in primary BMM cultures from patients with MM in a dose-dependent manner (Fig. 6A). Second, overexpression of a constitutively active form of AKT (AKT-CA) increased the level of ATF4 protein in COS-7 cells (Fig. 6B). There was a slower migrating ATF4 band on Western blots that was observed in samples from the AKT-CA-transfected cells (Fig. 6B). Third, transfection assays revealed that AKT-CA increased ATF4-dependent transcriptional activity (Fig. 6C), which was abolished by introduction of a 3-bp point mutation in the ATF4-binding core sequence (Fig.   6D). Fourth, we tested whether ATF4 can be directly phosphorylated by AKT enzyme by performing in vitro kinase assays using purified GST-ATF4 protein and AKT1 enzyme in the presence of [␥-32 P]ATP. As shown in Fig. 6E, purified GST-ATF4 but not GST protein was directly and strongly phosphorylated by the AKT1 enzyme in vitro. Finally, adenoviral overexpression of ATF4 increased the level of Rank mRNA by 10-fold in osteoclast precursors (Fig. 6F). In contrast, Dap-12 (DNAXactivating protein 12), a membrane protein expressed in both macrophages and osteoclasts, was not increased by ATF4 in BMMs (Fig. 6E).

Blocking AKT Reduces Tumor Burden in Bone Marrow Cavity and Inhibits the MM-induced Osteoclast Formation and
Osteolysis in SCID Mice-To determine whether AKT plays a role in the MM-induced osteoclast formation and osteolysis in vivo, we injected 5TGM1 MM cells into the tibial marrow cavity of SCID mice. One week after the injection, animals were subcutaneously injected with LY294002 (40 mg/kg body weight) or vehicle (DMSO) twice per week for 3 weeks. This LY294002 dose is in the range used by other investigators to efficiently block the PI3K/AKT pathway in mice (38,39). We examined whether the MM cells stimulated osteoclast formation by staining histological sections for the osteoclast enzyme TRAP. Results showed that 5TGM1 cells increased osteoclast surface/

. RANK expression levels are dramatically up-regulated in uncultured, undifferentiated, and RANKL-differentiated BMMs from patients with MM.
A and B, MM and ND BMMs were seeded at the density of 1 ϫ 10 6 /dish into a 35-mm dish in ␣-MEM containing 20% horse serum and proliferated by 10 ng/ml M-CSF for 10 days, followed by qPCR (A) and Western blot analysis (B). Experiments were repeated at least three times using samples from different patients with MM and NDs. Similar results were obtained. *, p Ͻ 0.01 (versus ND). C, RNAs from uncultured BMMs from 11 patients with MM and six NDs were used for qPCR analysis for RANK mRNA. *, p Ͻ 0.01 (versus ND). D, MM and ND BMMs were seeded at the density of 1 ϫ 10 6 /dish on 35-mm dish in ␣-MEM containing 20% horse serum and 10 ng/ml M-CSF with or without 50 ng/ml RANKL for 21 days, followed by qPCR analysis for RANK mRNA. Experiments were repeated at least three times using samples from different patients with MM and NDs. Similar results were obtained. *, p Ͻ 0.01 (versus ND). E, BMMs from three patients with MM and three NDs were differentiated for 21 days as in Fig. 1F, followed by Western blot analysis for RANK protein.
bone surface (Oc.S/BS) and osteoclast number/bone perimeter (Oc.Nb/BPm) of tibiae by 2.3-and 1.8-fold, respectively (p Ͻ 0.05 for both PBS/DMSO versus 5TGM1/DMSO) (Fig. 7, A-C). Strikingly, the MM-induced increases in Oc.S/BS and Oc.Nb/ BPm were completely inhibited by LY294002 (Fig. 7, A-C). We further determined whether blocking AKT affects the MM-induced osteolysis by examining histological sections. Results showed that, in DMSO-injected mice, the majority of the trabecular bone of the proximal tibial metaphysis was destroyed and that the marrow cavity was replaced by tumor tissues. In contrast, in those LY294002-injected mice, the bone destruction was largely prevented (Fig. 7, D and E). The formation of tumor tissues in bone marrow cavity was suppressed by AKT inhibition (Fig. 7D). It should be noted that although AKT was reported to regulate osteoclast and osteoblast function in mice (24), the LY294002 treatment under our experimental conditions only slightly decreased the basal level of the Oc.S/BS (p ϭ 0.087, PBS/DMSO versus 5TGM1/LY). In addition, LY294002 did not cause any reductions in the Oc.Nb/BPm and trabecular bone area (p Ͼ 0.12 for both parameters, PBS/DMSO versus 5TGM1/LY).

AKT Is Critical for the MM Cell Growth and Promotion of
Osteoclast Formation in Vitro-We next performed in vitro experiments to examine whether the AKT pathway in the MM cells is required for the tumor cell growth and promotion of osteoclast formation. Results showed that the LY294002 treatment significantly decreased the growth of MM1.S cells in vitro (Fig. 8A). Western blot analysis confirmed that the level of phospho-AKT but not total AKT protein was decreased by the AKT inhibitor (Fig. 8B). Finally, osteoclast formation induced by the CM from MM1.S cells was significantly reduced by treatment of the tumor cells with LY294002 (Fig. 8, C and D).

DISCUSSION
Abnormal osteoclast differentiation is a major contributor to osteolysis caused by major cancers such as MM, breast, lung, and prostate cancers. Bone-residing cancer cells promote osteoclast formation and activation (16, 40 -45). Increased osteoclast formation and activity and bone resorption increase releases of growth factors from the bone matrix that stimulate cancer cell growth (40 -46). Therefore, there is a vicious cycle between osteoclast-mediated osteolysis and tumor growth and  Fig. 1F, followed by Western blot analysis for total and phospho-AKT using specific antibodies. B and C, BMMs from patients with MM were differentiated for 10 days. On day 11, cells were treated by the indicated concentrations of LY294002 (LY) for 24 h, followed by qPCR (B) or Western blot analysis (C). D, BMMs from patients with MM were differentiated for 10 days. On day 11, cells were treated by LY294002 for 24 h and switched to differentiation media for another 10 days, followed by staining for the 23C6 ϩ (top) and TRAP ϩ (bottom) MNCs. E and F, Statistical analysis of D. *, p Ͻ 0.01 (versus 0 M LY294002). G, COS-7 cells were co-transfected with RANK-luc, in which a Ϫ1073/ϩ79 mouse Rank gene promoter driving firefly luciferase gene expression, pRL-SV40 (for normalization), and the indicated amounts of pCMV/AKT-CA or pCMV/␤-gal, followed by dual-luciferase assays. Experiments for B-F were repeated 2-4 times, and similar results were obtained. progression in bone marrow. Breaking the vicious cycle is of major clinical significance because osteolytic lesions and related complications such as severe bone pain, pathological fractures, spinal cord compression, and hypercalcemia of malignancy are common causes of morbidity and sometimes mortality (42,47).
In this study, we found that AKT in osteoclasts and their precursors plays an important role in the MM promotion of osteoclast formation and activation and osteolytic lesions. Both AKT expression and phosphorylation are up-regulated in pri-mary bone marrow osteoclast cultures from patients with MM. Blocking AKT inhibits the MM-induced osteoclast formation in vitro and abolishes the MM-induced increase in osteoclast differentiation and osteolysis in bone. We demonstrate that AKT promotes osteoclast formation through, at least in part, up-regulation of RANK in osteoclast precursors. The expression of AKT and RANK are both increased in primary bone marrow osteoclast cultures from patients with MM. AKT inhibition reduces the level of RANK mRNA and osteoclast formation in the MM BMM cultures. Overexpression of the consti- tutively active form of AKT increases the RANK gene promoter activity. We further demonstrate that ATF4, which is up-regulated by AKT, TNF-␣, and soluble factors produced by the MM cells, is a new upstream transcriptional activator of RANK gene expression in osteoclast precursors.
Increased RANK expression in osteoclast precursors in the MM bone marrow could greatly increase the sensitivity for RANKL to induce osteoclast formation and maturation, suggesting that, even under low concentrations of RANKL, osteoclast differentiation can be enhanced in the bone marrow of patients with MM. This notion is supported by the following evidence. (i) Osteoclast formation is greatly enhanced in the MM BMM cultures in the absence of exogenously added RANKL, in which the RANKL/ OPG ratio is reduced (Fig. 1D). (ii) The levels of RANKL from many tumor cells that can activate osteoclast formation are very low (17). Furthermore, sustained high expression of RANK even in  Fig. 1F, followed by Western blot analysis. ␤-Actin was used for a loading control. B, BMMs from one patient with MM and one ND were differentiated by M-CSF and RANKL for 21 days as in Fig. 1F, followed by qPCR analysis. ATF4 mRNA levels were normalized to GAPDH mRNA. C-F, primary mouse BMMs were cultured in M-CSF-containing media for 3 days and treated with 5TGM1-CM for the indicated times (C and D) or the indicated concentrations of TNF-␣ for 48 h (E and F), followed by Western blot (C and E) or qPCR analysis (D and F). The mRNAs were normalized to Gapdh mRNA. Experiments for B-F were repeated 2-3 times, and similar results were obtained. FIGURE 6. Effects of AKT activation or inhibition in the ATF4 expression and activity and phosphorylation. A, nonadherent BMMs from patients with MM were differentiated for 10 days. On day 11, cells were treated by the indicated concentrations of LY294002 for 24 h, followed by Western blot analysis for ATF4 expression. B, COS-7 cells were co-transfected with pCMV/ATF4 and increasing amounts of expression vectors for a AKT-CA, followed by Western blot analysis for ATF4 and AKT. C and D, COS-7 cells were co-transfected wild-type (wt) (C) and mutant (mt) (D) ATF4 reporter plasmid (pOSE1-luc) (49), pRL-SV40 (for normalization), and pCMV/ATF4 with or without the indicated amounts of AKT-CA expression vector, followed by dual-luciferase assays. *, p Ͻ 0.01 versus 0 g of AKT-CA. E, purified GST-ATF4 and GST proteins were incubated with AKT1 (Cell Signaling Technology, Inc.) in the presence of [␥-32 P]ATP. F, primary mouse BMMs were cultured in M-CSF-containing media for 3 days and infected with equal amounts of ATF4 or EGFP adenovirus. Twenty hours later, cells were harvested for qPCR for Rank and Dap-12 mRNAs. The mRNAs were normalized to Gapdh mRNA. *, p Ͻ 0.01 versus Ad/EGFP. Experiments for A-F were repeated 2-3 times, and similar results were obtained.
highly differentiated osteoclast cultures (i.e. 21 days in the presence of RANKL) from patients with MM could also suggest that part of the BMM cells in the MM bone marrow are maintained at the osteoclast precursor state because RANK expression is a marker of osteoclast precursors. These precursor cells can actively proliferate and increase the osteoclast numbers in bone marrow, which could further contribute to the increased osteoclast formation and osteolysis in patients with MM. A-C, 1 ϫ 10 6 5TGM1 cells in 20 l of PBS or an equal volume PBS alone were injected into both left and right tibiae of 4-week-old male SCID mice (10 mice/group or 20 tibiae/group). One week later, mice were subcutaneously injected with LY294002 (40 mg/kg body weight) or an equal volume of vehicle (DMSO) twice a week for 3 weeks. Oc.S/BS and Oc.Nb/BPm of tibiae were measured using an Image Pro Plus 7.0 software as described previously (25). D and E, tibiae from each group were sectioned from the midtibial metaphysis for at least 10 sections from each tibia (six tibiae/group) and stained with H&E. Trabecular bone area versus total bone marrow area was measured using an Image Pro Plus 7.0 software. *, p Ͻ 0.01 (versus PBS/DMSO); #, p Ͻ 0.01 (versus 5TGM1/DMSO).
Although results from this study demonstrate that AKT is critical for the MM-induced osteoclast formation, how MM cells activate AKT remains unclear. Cancer cells can produce and induce factors that can promote osteoclast formation and activity, including granulocyte-macrophage colony-stimulating factor (GM-CSF) (16), MCP-1 (17), MIP-1␣ and MIP-1␤ FIGURE 8. Blocking AKT inhibits the growth of MM cells and reduces their ability to activate osteoclast differentiation in vitro. A, MM1. S cells were seeded at 5 ϫ 10 3 /well on a 96-well plate and cultured for 2 days in 10% FBS/DMEM. Cells were then treated with LY294004 (10 M) or equal volume of vehicle (DMSO) for the indicated times followed by the methanethiosulfonate assay as described previously (35). *, p Ͻ 0.05, versus DMSO. B, MM1.S cells were seeded at 5 ϫ 10 5 /dish on 6-well plate and cultured for 2 days in 10% FBS/DMEM and then treated with LY294004 (10 M) or DMSO for 2 days, followed by Western blotting. C and D, MM1.S cells were seeded in 10% FBS/DMEM and treated with or without LY294004 (10 M) or equal volume vehicle (DMSO) for 24 h. Cells were then switched to 2% FBS/DMEM for another 24 h. CM from each group were harvested. 2% FBS/DMEM was used as control. For in vitro osteoclast differentiation, primary mouse BMMs were differentiated by M-CSF (10 ng/ml) and RANKL (30 ng/ml) in the presence of an equal volume of the indicated CMs or control media (1:200, v/v) for 7 days, followed by TRAP staining (C). TRAP-positive MNCs (3Ն nuclei/cell) were counted (D). Experiments for A-D were repeated two times, and similar results were obtained. *, p Ͻ 0.05 (versus Ctrl Media); #, p Ͻ 0.05 (versus MM1CM/DMSO). (19,20), TNF-␣ (48), and parathyroid hormone-related protein (14,15). Future studies will determine whether any of those factors are responsible for MM cell activation of AKT in osteoclast precursors. It will be interesting to determine whether AKT also mediates the osteoclast formation and osteolysis caused by other cancers such as breast, lung, and prostate in the future.
It should be noted that blocking AKT in MM cells decreases their growth in vitro and that systemic inhibition of AKT blocks the formation of tumor tissues in the bone marrow cavity in SCID mice. Interestingly, our in vitro studies show that AKT inhibition in the MM cells also reduces the ability of tumor cells to activate osteoclast formation, which should contribute to the reduced osteoclast formation induced by the tumor cells. Finally, although the AKT inhibitor essentially abolishes the MM-induced osteolysis and bone loss, it does not significantly affect the basal level osteoclast formation and bone mass under our experimental conditions. Collectively, these results suggest that AKT could be a potential target for inhibiting the MM-induced osteoclast osteolytic lesions as well as tumor growth and progression, thus breaking the vicious cycle in the patients with MM.