1774 Wnt3/RhoA/ROCK signaling pathway is involved in adhesion-mediated drug resistance of multiple myeloma in an autocrine mechanism Masayoshi Kobune,1,2 Hiroki Chiba,1,2 Junji Kato,1 kinase pathway signal.This CAM-DR was also significantly Kazunori Kato,2 Kiminori Nakamura,2 reduced by Wnt3 small interfering RNA transfer to KMS-5. Yutaka Kawano,1 Kohichi Takada,1 These results indicate that Wnt3 contributes to VLA-6– Rishu Takimoto,1 Tetsuji Takayama,1 mediated CAM-DR via the Wnt/RhoA/ROCK pathway of Hirofumi Hamada,2 and Yoshiro Niitsu1 myeloma cells in an autocrine manner.Thus, the Wnt3 signaling pathway could be a promising molecular target to 1Fourth Department of Internal Medicine and 2Department of overcome CAM-DR of myeloma cells.[Mol Cancer Ther Molecular Medicine, Sapporo Medical University School of 2007;6(6):1774–84] Medicine, Sapporo, Japan Introduction Abstract Multiple myeloma is an end-stage B-cell neoplasia charac- Adhesion of myeloma cells to bone marrow stromal cells is terized by localization of malignant plasma cells in the bone now considered to play a critical role in chemoresistance. marrow and not in other locations. Despite tremendous However, little is known about the molecular mechanism efforts in developing effective treatment modalities such as governing cell adhesion–mediated drug resistance (CAM- autologous stem cell transplantation, exploring new mo- DR) of myeloma cells.In this study, we focused our interests lecular targeting drugs, etc., the disease still remains on the implication of the Wnt signal in CAM-DR.We first incurable, mainly due to the ultimate acquisition of drug screened the expression of Wnt family in myeloma cell lines resistance (1–7). and found that Wnt3 was overexpressed in all the myeloma One of the main mechanisms underlying the drug cells examined.KMS-5 and ARH77, which highly expressed resistance has been ascribed to the adhesive property of Wnt3 protein, tightly adhered to human bone marrow myeloma cells to the extracellular matrix or stromal cells in stromal cells, and accumulation of B-catenin and GTP- bone marrow (8–11). We and others have reported that in bounded RhoA was observed in these myeloma cell lines. various hematologic malignancies, including multiple Conversely, RPMI8226 and MM1S, which modestly myeloma, molecules responsible for the adhesive nature expressed Wnt3 protein, rather weakly adhered to human of tumor cells were integrins such as VLA-4 and VLA-5 bone marrow stromal.We then examined the relevance of (12–14). Through the interaction of integrins with their Wnt3 expression to adhesive property to stromal cells and ligands, up-regulation of antiapoptotic bcl-2 family mem- multidrug resistant gene 1 to CAM-DR of myeloma cells.KMS-5 and ARH-77 exhibited bers and/or overexpression of are induced, thereby causing tumor cells to become unrespon- apparent CAM-DR against doxorubicin.This CAM-DR was sive to anticancer drugs (12, 13, 15). This cell adhesion– significantly reduced by anti-integrin B antibody, anti- 1 mediated drug resistance (CAM-DR) has been overcome integrin A antibody and a Wnt-receptor competitor, 6 in vitro and in vivo by a combination of chemotherapy with secreted Frizzled-related protein-1, and Rho kinase inhibitor anti-integrin antibodies or inhibitors to drug resistance– Y27632, but not by the specific inhibitor of canonical related intracellular signal pathways (16). Furthermore, signaling (Dickkopf-1), indicating that Wnt-mediated CAM- recent studies have revealed that various humoral factors DR that is dependent on integrin A /B (VLA-6)–mediated 6 1 from tumor cells and/or stromal cells were implicated in attachment to stromal cells is induced by the Wnt/RhoA/Rho the adhesion and motility of myeloma plasma cells by their autocrine or paracrine action (17–21). Among the humoral factors, Wnt family members, which play a Received 11/7/06; revised 3/23/07; accepted 5/1/07. crucial role in differentiation and proliferation of normal Grant support: Ministry of Education, Culture, Sports, Science and Technology of Japan (H. Hamada) and Ministry of Health, Labour and and malignant cells in addition to the motility or adhesion Welfare of Japan (M. Kobune). mentioned above, are often activated in both bone marrow The costs of publication of this article were defrayed in part by the stromal cells and B-cell neoplasia including multiple payment of page charges. This article must therefore be hereby marked myeloma (4, 22–26). On the basis of these notions, in advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. the present study, we elucidated if any Wnt family Note: M. Kobune and H. Chiba contributed equally to this work. members are involved in CAM-DR of multiple myeloma. Requests for reprints: Yoshiro Niitsu, Fourth Department of Internal Medicine, Sapporo Medical University, Chuo-ku S1, W17, Sapporo, Materials and Methods 060-8556, Japan. Phone: 81-11-611-2111, ext. 3254; Fax: 81-11-612-7987. E-mail: [email protected] Cell Lines and Human Stromal Cells Copyright C 2007 American Association for Cancer Research. Human myeloma cell lines RPMI8226, EBV-transformed doi:10.1158/1535-7163.MCT-06-0684 ARH-77 (27, 28), MM1S, and KMS-5 (29, 30); lymphoma cell MolCancerTher2007;6(6).June2007 Downloaded from mct.aacrjournals.org on September 25, 2021. © 2007 American Association for Cancer Research. Molecular Cancer Therapeutics 1775 lines Ramos, PEER, and Molt-4; and leukemia cell lines were electrophoresed on an agarose gel and visualized by KG-1, HL-60, and U937 were cultured in RPMI 1640 ethidium bromide staining. containing 10% heat-inactivated FCS (Life Technologies), Transfection of Small Interfering RNA 2 mmol/L L-glutamine, 0.1% penicillin (100 units/mL), and Dicer substrate RNA duplexes for use in RNA interfer- streptomycin (100 mg/mL). hTERT gene–transduced ence against Wnt3 (NM_ 030753) were obtained from human stromal cells (hTERT-stromal cells) were generated iGENE Therapeutics. The sequences of dsRNA synthesized as previously described (31, 32) and were cultured in long- newly were as follows: sense, 5¶-ACGGAGAAGCGGAAG- term culture medium containing MEM-a, 12.5% horse GAAAAAUGCCAG-3¶; antisense, 5¶-UAUGCCUCUUCG- À serum (Life Technologies), 12.5% FCS, 1  10 6 mol/L CCUUCCUUUUUACGG-3¶. The non-silencing control À hydrocortisone (Sigma), 1  10 4 mol/L h-mercaptoetha- small interfering RNA (siRNA) for human was purchased nol (Sigma), 2 mmol/L L-glutamine, 0.1% penicillin from iGENE Therapeutics. (100 units/mL), and streptomycin (100 mg/mL). RNA duplexes specific for the human Wnt3 were Antibodies, Wnt Antagonists, and Chemical Reagents transfected into KMS-5 or ARH-77 cells using the DeliverX The specific antibody for h-catenin (clone 14) was transfection kit (Panomics) according to provided proto- purchased from BD. The specific antibodies for Wnt3 (sc- cols. Briefly, 5 Amol/L working stocks of siRNA were 5212), RhoA (sc-418), and Cdc42 (sc-8401) were from Santa diluted with Buffer-1, and siRNA Transfection Reagent was Cruz Biotechnology. The specific antibody for Rac (clone diluted with Buffer-2. Both solutions were mixed well and 23A8) was from Upstate, and the specific antibody for incubated at 37jC for 20 min to form working siRNA/ Wnt5a was purchased from R&D Systems. Function- siRNA transfection reagent complex. Cells were pelleted in h a  blocking anti-integrin 1 antibody 4B4, anti-integrin 4 a microtube (Falcon) by spinning for 5 min at 400 g. Cells a  antibody HP2/1, and anti-integrin 5 antibody SAM1were were washed thrice by PBS and pelleted again at 400 g obtained from Coulter, and anti-integrin ar AMF7, anti- for 5 min. After aspiration of the PBS, cells were a a a A integrin 2 Gi9, anti-integrin 3 C3, and anti-integrin 6 resuspended with 300 L of working siRNA/siRNA GoH3 (33) were obtained from Immunotech. Isotype transfection reagent complex and incubated at room control mouse IgG1antibody MOPC21(Sigma) and rat temperature for 5 min. After adding 300 AL of Opti-MEM IgG2a antibody DD13 (Chemicon) was used in the control (Invitrogen) to the microtubes, 600 AL of cells/transfection experiments. Recombinant secreted Frizzled-related pro- complex were transferred into one well of a six-well plate. tein-1 (sFRP-1) and Dickkopf-1 (DKK-1) were obtained Final concentration of Wnt3 siRNA in the sample well was from R&D Systems. Rho kinase (ROCK) inhibitor Y-27632 30 nmol/L. Cells/transfection complex was incubated for j and GRGDS (H-Gly-Arg-Gly-Asp-Ser-OH) peptide were 4 h at 37 C and 5% CO2. Subsequently, 1mL of complete purchased from Calbiochem. Doxorubicin was obtained growth media was added and incubated overnight. Next from Sigma and dissolved in sterile double-distilled water day, after cells were washed once, and cells were washed to before use. remove transfection complex and cultured in complete j Reverse Transcription-PCR growth media at 37 C and 5% CO2 before use. One microgram of total RNA was reverse transcribed by PlasmidVectors, GeneTransduction, andWnt3 Condi- SuperScript II (Invitrogen) and amplified using TaKaRa Ex tioned Medium Taq (TAKARA BIO, Inc.), with the primers described below. The CA promoter (chicken h-actin promoter with All PCR reactions were done using a PTC-200 Peltier cytomegalovirus enhancer)–based plasmid vector pCAcc Thermal Cycler (MJ Japan). PCR amplification using specific was employed in this study. Human Wnt3 cDNA contain- R primers for Wnt1 (5-GCTCCTGCTCAGAAGGTTCCAT-3, ing plasmid, pCR 2.1-hWnt3, was previously described R 5-GTCCGCTGTACGTGCAGAAGTT-3), Wnt2B (5- (34). To construct pCAcc-hWnt3, pCR 2.1-hWnt3 was GTAGCCAGGGTGAACTGAGTGT-3, 5-CTAGGGAAC- digested with EcoRI and ligated in pCAcc. The integrity CTGCAGCCTTGT-3), Wnt3 (5-ATCATAAGGGG- of the pCAcc-Wnt3 was confirmed by restriction endonu- CCGCCTGGCGAA-3, 5-CTTGCAGGTGTGCACGTCG- clease digestion and DNA sequencing.