Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 7-Day Trial for You or Your Team.

Learn More →

Hypoxia reduces CD138 expression and induces an immature and stem cell-like transcriptional program in myeloma cells

Hypoxia reduces CD138 expression and induces an immature and stem cell-like transcriptional... INTERNATIONAL JOURNAL OF ONCOLOGY 43: 1809-1816, 2013 Hypoxia reduces CD138 expression and induces an immature and stem cell-like transcriptional program in myeloma cells 1 1 1 1 YAWARA KAWANO , YOSHITAKA KIKUKAWA , SHIHO FUJIWARA , NAOKO WADA , 1 1 2 YUTAKA OKUNO , HIROAKI MITSUYA and HIROYUKI HATA 1 2 Department of Hematology, Division of Informative Clinical Science, Kumamoto University School of Medicine, Kumamoto 860-8556, Japan Received August 8, 2013; Accepted September 16, 2013 DOI: 10.3892/ijo.2013.2134 Abstract. Although CD138 expression is a hallmark of plasma hypoxia. We propose that hypoxia induces immature and cells and myeloma cells, reduced CD138 expression is occa- stem cell-like transcription phenotypes in myeloma cells. sionally found. However, the mechanisms underlying CD138 Taken together with our previous observation that decreased downregulation in myeloma cells remain unclear. Previous CD138 expression is correlated with disease progression, the reports suggest that the bone marrow microenvironment may present data suggest that a hypoxic microenvironment affects contribute to CD138 downregulation. Among various factors the phenotype of MM cells, which may correlate with disease in the tumor microenvironment, hypoxia is associated with progression. tumor progression, poor clinical outcomes, dedifferentiation and the formation of cancer stem cell niches in solid tumors. Introduction Since recent findings showed that progression of multiple myeloma (MM) delivers hypoxia within the bone marrow, Multiple myeloma (MM) is characterized by clonal expansion we hypothesized that CD138 expression may be regulated of malignant plasma cells in the bone marrow. Although novel by hypoxia. In the present study, we examined whether the therapeutic agents and stem cell transplantation have improved expression of CD138 and transcription factors occurred in the survival of MM patients (1), MM remains an incurable myeloma cells under hypoxic conditions. MM cell lines disease. (KMS-12BM and RPMI 8226) were cultured under normoxic Cancer stem cells are often considered to contribute to or hypoxic conditions for up to 30 days. Changes in the pheno- relapse and drug resistance in various cancers (2). Matsui type and the expression of surface antigens and transcription et al (3) reported that myeloma stem cells are enriched in the factors were analyzed using flow cytometry, RT-PCR and CD138-negative population. During normal B-cell develop- western blotting. All-trans retinoic acid (ATRA) was used to ment, abundant CD138 (also known as syndecan-1: SDC1) examine the phenotypic changes under hypoxic conditions. expression is highly specific for terminally differentiated The expression levels of CD138, CS1 and plasma cell-specic fi plasma cells in the bone marrow (4). Since CD138 expression transcription factors decreased under hypoxic conditions, is also a hallmark of malignant plasma cells (myeloma cells), it while those of CD20, CXCR4 and B cell-specic fi transcription has been used for myeloma cell puric fi ation (5) and is consid- factors increased compared with those under normoxic condi- ered to be a target for treatment (6). While the majority of tions. Stem cell-specic fi transcription factors were upregulated myeloma cells express CD138, decreased expression of CD138 under hypoxic conditions, while no difference was observed in is occasionally found in clinical practice (7-9). Although the ALDH activity. The reduced CD138 expression under hypoxic association between CD138 expression and myeloma stem cells conditions recovered when cells were treated with ATRA, even remains a matter of debate (10), several reports have shown that under hypoxic conditions, along with decreases in the expres- CD138-low or -negative myeloma cells may contribute to drug sion of stem cell-specic fi transcription factor. Interestingly, resistance or relapse of the disease (9,11,12). Therefore, analysis ATRA treatment sensitized MM cells to bortezomib under of CD138 downregulation in myeloma cells is required for a better understanding of myeloma biology. Previous reports have indicated that the bone marrow microenvironment may contribute to CD138 downregulation (13-16). Among various factors in the tumor microenviron- Correspondence to: Dr Yawara Kawano, Department of Hemato- ment, hypoxia is one of the important factors associated with logy, Kumamoto University School of Medicine, Honjo 1-1-1, tumor progression, poor clinical outcomes, dedifferentiation, Chuo-ku, Kumamoto City, Kumamoto 860-8556, Japan E-mail: [email protected] and formation of cancer stem cell niches in solid tumors (17). Based on recent findings showing a correlation of MM at the Key words: myeloma, hypoxia, CD138, ATRA advanced stage with hypoxic conditions in the microenvi- ronment within the bone marrow (18), we hypothesized that CD138 expression may be inu fl enced by hypoxia. KAWANO et al: CD138 DECREASED MYELOMA UNDER HYPOXIA In the present study, we compared the changes in CD138 CTCATTTCACCAGGCC-3'; reverse, 5'-TGGGACTCCTCCG and various transcription factor expressions in myeloma cells GGTTTTG-3'); NANOG (forward, 5'-ACTGTCTCTCCTCT under hypoxic or normoxic conditions. We also attempted to TCCTTC-3'; reverse, 5'-CCTGTTTGTAGCTGAGGTTC-3'); revert CD138 expression in cells under hypoxia by treatment SOX2 (forward, 5'-ACAACTCGGAGATCAGCA-3'; reverse, with all-trans retinoic acid (ATRA). The inu fl ence of ATRA 5'- G CAG CGTGTACT TATCCT TC-3'); ACT B (forward, on the sensitivity to bortezomib under hypoxic conditions was 5'-GGACTTCGAGCAAGAGATGG-3'; reverse, 5'-AGCAC also examined. TGTGTTGGCGTACAG-3'). Quantitative real-time RT-PCR was performed using Materials and methods Assay-on-Demand primers and TaqMan Universal PCR Master Mix Reagent (Applied Biosystems, Foster City, NJ, Cell culture. Human myeloma cell lines, KMS-12BM (19) USA). Samples were analyzed using an ECO™ Real-Time and RPMI 8226 (20), were obtained from the Health Science PCR System (Illumina, San Diego, CA, USA). The ∆∆Ct Research Resources Bank (Osaka, Japan) and maintained in method was used to analyze the relative changes in gene RPMI-1640 medium supplemented with 10% heat-inactivated expression as previously described (21), using ACTB as a fetal bovine serum at 37˚C under 5% CO . The two myeloma normalization control. The following primers and probes were cell lines were cultured under normoxic (21% O ) and hypoxic used: SDC1 (Hs00896423_m1); IRF4 (Hs01056534_m1); (1% O ) conditions for up to 30 days, with fresh medium PRDM1 (Hs00153357_m1); XBP1 (Hs00964360_m1); and provided every 3 days. Experiments under hypoxic condi- ACTB (Hs99999903_m1). tions were performed in a Personal CO Multigas Incubator (ASTEC, Fukuoka, Japan). Intracellular staining of IRF4 followed by flow cytometric analysis. The MM cell lines cultured under normoxic or Flow cytometric analysis of surface antigens. MM cell lines hypoxic conditions were stained with an FITC-CD138 cultured under normoxic and hypoxic conditions were stained antibody (clone MI15; BD Biosciences), fixed and permea - with the following fluorescently-labeled antibodies: FITC- bilized using a FOXP3 Staining Buffer Set (eBioscience, CD138 (clone MI15), FITC-CD38 (clone HIT2), PE-CD44 San Diego, CA, USA), and then stained intracellularly (clone 515), PE-CD45 (clone HI30), FITC-CD49d (clone gf10) with an Alexa 647-IRF4 antibody (clone 3E4; eBioscience) (BD Biosciences, Fran k lin La kes, NJ, USA); PE- CD54 according to the manufacturer's protocol. Flow cytometric (clone HCD54), PE - CXCR4 (clone 12G5), PE -M DR-1 analysis was performed using the FACSCalibur (Becton- (clone UIC2), APC-ABCG2 (clone 5D3) (Biolegend, San Dickinson). Diego, CA, USA); FITC-CD19 (clone HD37), FITC-CD20 (clone B-Ly1) (Dako, Glostrup, Denmark); and Alexa 647-CS1 Western blot analysis. Cell lysates were prepared as reported (clone 162) (AbD Serotec, Oxford, UK). Density gradient previously (22). Quantic fi ation of total protein was performed centrifugation using Ficoll-Paque Plus (GE Healthcare, using a Pierce BCA Protein Assay Kit (Thermo Scientific, Uppsala, Sweden), the forward/side scatter profile and Waltham, MA, USA), and equal amounts of protein were 7-amino-actinomycin D (7-AAD) (BD Biosciences) labeling used for analysis. The cell lysates were separated in NuPAGE were used for exclusion of non-viable cells. Flow cytometric Bis-Tris precast gels (Invitrogen) and transferred to PVDF anal ysis was performed using a FACSCalibur or FACSVerse membranes using an iBlot Dry Blotting system (Invitrogen). o fl w cytometer (Becton-Dickinson, San Jose, CA, USA). The membranes were blocked with 5% non-fat dry milk for 1 h at room temperature, followed by incubation with primary Adhesion to type-1 collagen. MM cells were plated in antibodies at 4˚C for 18 h. The primary antibodies against quadruplicate at a concentration of 5x10 cells/ml on type-1 HIF-1α, HIF-2α, NANOG, and SOX2 were purchased from collagen-coated 96-well plates (Becton-Dickinson) and Cell Signaling Technology (Beverly, MA, USA), while those incubated for 1 h at 37˚C. After the incubation, the cells were against Oct-4, RARα, and actin were obtained from Santa washed twice with PBS and incubated with the WST-8 reagent Cruz Biotechnology (Santa Cruz, CA, USA). The membranes (Dojindo, Kumamoto, Japan). The ratios of adherent cells to were then incubated with horseradish peroxidase-conjugated total applied cells were quantie fi d by the light absorbance of secondary antibodies (GE Healthcare) for 1 h at room each well at 450 nm using a VMax absorbance microplate temperature. Antibody-bound proteins were visualized reader (Molecular Devices, Sunnyvale, CA, USA). using the ECL prime western blotting detection reagent (GE Healthcare) and an LAS-1000 bio-image analyzer (GE cDNA synthesis and reverse transcription-polymerase chain Healthcare). reaction (RT-PCR). RNA was extracted from the MM cell lines using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). cDNA Aldehyde dehydrogenase (ALDH) activity. The ALDH synthesis was performed using a SuperScript III First-Strand activities of the MM cell lines cultured under normoxic and Synthesis System for RT-PCR (Invitrogen) according to the hypoxic conditions were analyzed using Aldeu fl or (Stem Cell manufacturer's protocol. Technologies, Vancouver, Canada). After adding activated The expression levels of BCL6, PAX5, Oct-4, NANOG and Aldeu fl or reagent to the cell cultures, half of the cells were SOX2 were determined by RT-PCR. β-actin (ACTB) was used transferred to tubes containing an ALDH inhibitor, diethyl- as a normalization control. The primers for BCL6 and PAX5 aminobenzaldehyde (DEAB), to confirm specificity of the were described previously (9). The primers for Oct- 4, NANOG, reagent. Samples were incubated at 37˚C for 30 min and SOX2 and ACTB were as follows: Oct- 4 (forward, 5'-AGCC analyzed using the FACSCalibur (Becton-Dickinson). INTERNATIONAL JOURNAL OF ONCOLOGY 43: 1809-1816, 2013 Figure 1. Decrease in CD138 expression under hypoxia. (A) CD138 expression in the MM cell lines is decreased after 72 h of culture under hypoxic conditions. The left panel shows CD138 expression in KMS-12BM cells under normoxic and hypoxic conditions analyzed by o fl w cytometry (solid or dashed line, CD138; shadowed area, isotype control). Overlay plots of the CD138 expressions in KMS-12BM and RPMI 8226 cells under normoxic (solid line) and hypoxic (dashed line) conditions are shown in the right panel. (B) CD138 expression in KMS-12BM cells is decreased in a time-dependent manner. The increasing proportion of CD138-negative cells and decrease in CD138 u fl orescence intensity as judged by the mean u fl orescence intensity (MFI) ratio to the isotype control are shown in the left and right panels, respectively. White bars, normoxic conditions; black bars, hypoxic conditions. (C) Re-oxygenation recovers CD138 expression. KMS-12BM cells were cultured under hypoxic conditions for 72 h. The cells were then further cultured under normoxic (solid line) or hypoxic (dashed line) conditions for an additional 72 h. Analysis of apoptosis. The MM cell lines were incubated in the of culture under hypoxic conditions compared with normoxic presence of 1 µM ATRA (Sigma, St. Louis, MO, USA) or 5 nM conditions in both cell lines (Fig. 1A). As shown in Fig. 1B, bortezomib (Sigma) for 24 h. Apoptosis in the MM cell lines CD138 expression was reduced from 48 h, and subsequently was quantie fi d by staining with Annexin V (MBL, Nagoya, proceeded in a time-dependent manner. We then re-oxygenized Japan) and 7-AAD (BD Biosciences). The samples were the cells at 72 h after starting the hypoxic conditions and main- analyzed by flow cytometry (FACSVerse; Becton-Dickinson). tained the cells under normoxic conditions for an additional 72 h. Interestingly, we observed recovery of the CD138 expres- Statistical analysis. The data were analyzed statistically by sion, indicating that the reduction in CD138 expression under Student's t-test using GraphPad Prism version 5.0 (GraphPad hypoxia was a reversible phenomenon (Fig. 1C). Software, La Jolla, CA, USA). P-values of <0.05 were consid- ered statistically signic fi ant. Changes in CD138 and other surface antigens under long- term hypoxia. We further analyzed CD138 expression under Results long-term exposure (30 days) to hypoxic or normoxic condi- tions. After 30 days of incubation, CD138 expression was Hypoxia reduces CD138 expression in MM cell lines. To inves- markedly reduced in KMS-12BM and RPMI 8226 cells tigate whether CD138 expression in MM cells was inu fl enced cultured under hypoxia compared with those cultured under by oxygen levels, we cultured two MM cell lines (KMS-12BM normoxia (Fig. 2A). In KMS-12BM cells in particular, the and RPMI 8226) under normoxic and hypoxic conditions for positivity for CD138 expression under hypoxic conditions was up to 72 h and compared the surface CD138 expressions by flow reduced to only 10%. Real-time RT-PCR showed that CD138 cytometry. Since CD138 was reported to be downregulated in (SDC1) mRNA was downregulated after long-term hypoxia, non-viable cells (23), only viable cells were obtained by Ficoll indicating that CD138 expression was reduced at the gene density gradient centrifugation followed by gating of live cells in transcription level (Fig. 2B). combination with the forward/side scatter prol fi e and exclusion Since CD138 mediates the adhesion of MM cells to type-1 of 7-AAD-positive cells. CD138 expression was reduced by 72 h collagen (24), which is an important component of the bone KAWANO et al: CD138 DECREASED MYELOMA UNDER HYPOXIA Figure 2. Decrease in CD138 expression and changes in other surface antigens after long-term exposure to hypoxia. (A) KMS-12BM and RPMI 8226 cells were cultured under hypoxic or normoxic conditions for 30 days and the expression of CD138 was analyzed by flow cytometry. Overlay plots of CD138 expression are shown. Normoxic conditions, solid line; hypoxic conditions, dashed line. (B) A decrease in CD138 expression is found at the mRNA level. The expression of CD138 mRNA was quantie fi d by real-time RT-PCR. (C) Decrease in adhesion to collagen type-I under hypoxic conditions. Cells were treated as described in (A) and their adhesion capabilities to collagen type-I were analyzed. White bars, normoxic conditions; black bars, hypoxic conditions. (D) Increases in CD20 and CXCR4 expressions and decrease in CS1 expression under hypoxic conditions. KMS-12BM cells were treated as described in (A) and other surface antigens were analyzed by flow cytometry (solid or dashed line, CD138; shadowed area, isotype control). marrow microenvironment, the inu fl ence of oxygen concentra - and BCL6, was increased in the hypoxic state in KMS-12BM tions on the adhesion of MM cells was evaluated. The hypoxic and RPMI 8226 cells (Fig. 3A). The gene expressions of IRF4, MM cell lines adhered poorly to type-1 collagen, ree fl cting the PRDM1 and XBP1, which are plasma cell-specic fi transcrip - low expression of CD138 (Fig. 2C). tion factors, were decreased in the hypoxic cells compared The expression changes in other surface antigens were with the normoxic cells (Fig. 3B). The intracellular IRF4 compared between the hypoxic and normoxic MM cell lines. protein expression, which was analyzed by flow cytometry, The CD20 and CXCR4 expressions were increased, while the was decreased in the hypoxic cells (Fig. 3C), consistent with CS1 expression was decreased, under hypoxia compared with the results for the gene expressions. Analysis of subpopulations normoxia in KMS-12BM cells (Fig. 2D). Similar results were obtained as CD138-high and -low cells showed high and low observed in RPMI 8226 cells (normoxia vs. hypoxia: CD20, IRF4 expressions, respectively (Fig. 3D). These findings show 1.8 vs. 14.1%; CS1, 81.3 vs. 34.6%; CXCR4, 40.2 vs. 82.1%). that the hypoxic MM cell lines with low CD138 expression No changes in expression were observed for CD19, adhesion have an immature, so-called mature B cell-like rather than molecules other than CD138 (CD44, CD49d and CD54), and plasma cell, transcriptional status. ATP-binding cassette transporters (ABCG2 and MDR1) (data not shown). Hypoxia induces a stem cell-like transcriptional program in MM cells. Hypoxia-inducible factors (HIFs) are key molecules Hypoxic MM cell lines have an immature phenotype. Since for the cellular response to hypoxia. It was reported that a CD138 (4) and CS1 (25) expressions are highly specic fi for hypoxic environment and HIF activity are required not only terminally differentiated plasma cells, we hypothesized that for stem cells, but also for cancer stem cells (27). Since HIFs the hypoxic MM cell lines have a less mature phenotype than induce stem cell transcription factors in solid tumors (28), the normoxic cell lines. To prove this hypothesis, we assessed we analyzed the expression of HIFs and stem cell transcrip- the expressions of the BCL6 and PAX5 transcription factors, tion factors in the hypoxic MM cell lines. HIFs, especially which exist in the mature B-cell state and are decreased in HIF-2α, were increased in the hypoxic MM cell lines, as mature plasma cells (26), by RT-PCR in the MM cell lines evaluated by western blotting, indicating that the cells were after 30 days of culture under hypoxic or normoxic condi- actually responding to the hypoxic environment (Fig. 4A). The tions. Each of the mature B-cell transcription factors, PAX5 expression of stem cell transcription factors (Oct-4, SOX2, and INTERNATIONAL JOURNAL OF ONCOLOGY 43: 1809-1816, 2013 Figure 3. Exposure to hypoxia induces transcription factors specic fi for the B-cell phenotype. (A) Induction of BCL6 and PAX5 by exposure to hypoxia. KMS-12BM and RPMI 8226 cells were cultured under hypoxic conditions for 30 days. PAX5 expression is increased in hypoxic KMS-12BM cells, while BCL6 is increased in hypoxic RPMI 8226 cells. (B) Hypoxia reduces plasma cell-specic fi transcription factor expression. The gene expression levels of IRF4 (white bars), PRDM1 (striped bars), and XBP1 (black bars) were analyzed by real-time RT-PCR in KMS-12BM and RPMI 8226 cells. The expression of the three genes are decreased. (C) Reduction in intracellular IRF4 expression under hypoxic conditions detected by o fl w cytometry. The expression is decreased in hypoxic cells (dashed line) compared with normoxic cells (solid line). (D) The CD138-low subpopulation has lower IRF4 expression. RPMI 8226 cells were cultured under hypoxic conditions for 30 days. The expression of IRF4 was evaluated by gating the CD138-high (25% from the highest intensity; solid square in the left panel) and CD138-low (25% from the lowest intensity; dashed square in the left panel) subpopulations. The intracellular IRF4 expression in each compartment is shown in the right panel. The CD138-low population has lower intracellular IRF4 expression. Figure 4. Hypoxia induces a stem cell-like transcriptional program in MM cell lines. (A) KMS-12BM and RPMI 8226 cells were cultured under hypoxic or normoxic conditions for 30 days. Cell lysates were extracted and subjected to western blot analysis. HIF expression is increased in the hypoxic MM cell lines. (B and C) Expression of transcription factors related to the stem cell phenotype was analyzed by RT-PCR (B) or western blotting (C). Upregulation of transcription factors related to the stem cell phenotype is observed under hypoxia. (D) Analyses of ALDH activity in KMS-12BM cells. Cells were cultured under hypoxic or normoxic conditions and subjected to ALDH assays. DEAB was used as an ALDH inhibitor. No increase in ALDH activity is observed in hypoxic KMS-12BM cells. KAWANO et al: CD138 DECREASED MYELOMA UNDER HYPOXIA Figure 5. ATRA restores the phenotypic changes and sensitivity to bortezomib induced by hypoxic conditions. (A) Western blot analysis showing the expres- sion of RARα in both KMS-12BM and RPMI 8226 cells cultured under hypoxic conditions. (B) Recovery of CD138 expression after ATRA treatment in the MM cell lines under hypoxic conditions. Solid line, ATRA treatment for 48 h; dashed line, DMSO treatment. (C) Reductions in stem cell-related transcription factors after ATRA treatment in hypoxic MM cell lines evaluated by western blot analysis. (D) Analysis of cytotoxicity toward KMS-12BM cells cultured under hypoxic conditions with ATRA and bortezomib. Cells were treated with ATRA or bortezomib alone or in combination for 24 h. The cells were then subjected to Annexin V/7-AAD analysis. The upper panel shows the raw data of the scatter graph. The lower panel shows the proportion of early apoptotic + - cells (Annexin V /7-AAD ). ATRA augments early apoptosis in combination with bortezomib. NANOG) was increased in the hypoxic MM cell lines at both were then incubated with 1 µM ATRA or dimethyl sulfoxide the mRNA (Fig. 4B) and protein (Fig. 4C) levels. The only (DMSO) for 48 h under hypoxic conditions. Surprisingly, exception was the expression of SOX2 protein, which was not ATRA increased the CD138 expression compared with detected in hypoxic KMS-12BM cells, although its mRNA DMSO (Fig. 5B), especially in KMS-12BM cells, while the expression was upregulated. However, the ALDH activity, expression of all stem cell transcription factors was decreased which is characteristic of cancer stem cells including MM (Fig. 5C). These findings indicate that ATRA can induce the stem cells (29), was not increased in hypoxic KMS-12BM cells redifferentiation of immature MM cells, even under hypoxic (Fig. 4D) and RPMI 8226 cells (normoxia vs. hypoxia: 1.06 vs. conditions. 0.10%). The expression of ABCG2, which is highly expressed Next, we investigated whether the differentiation induced in stem cells and associated with side-population cells (30), by ATRA can sensitize the immature and hypoxic cells to was not increased in hypoxic cells, as described earlier in this bortezomib. Hypoxic KMS-12BM cells were treated with report. 1 µM ATRA and 5 nM bortezomib alone or in combination for 24 h. While ATRA alone slightly affected the viability ATRA induces differentiation of hypoxic MM cells and of hypoxic KMS-12BM cells, the combination of the two increase their sensitivity to bortezomib. ATRA induces cell reagents increased the cytotoxicity, especially the early apop- + - differentiation not only of stem cells (31), but also of MM cells tosis (Annexin V /7-AAD cells) (Fig. 5D). (32,33). ATRA is also known to repress the expression of stem cell transcription factors, such as Oct-4, NANOG and SOX2 Discussion (34,35). Based on these previous reports, we investigated whether ATRA could induce redifferentiation of the hypoxic Accumulating evidence indicates that CD138 is an important MM cell lines. We first confirmed the expression of RAR α, a molecule not only for the identic fi ation of plasma cells, but specic fi receptor for retinoic acid, in the MM cell lines cultured also for distinguishing myeloma stem cells. CD138-negative for 30 days under hypoxic conditions (Fig. 5A). The cell lines cells have recently been proposed as myeloma stem cells (3). INTERNATIONAL JOURNAL OF ONCOLOGY 43: 1809-1816, 2013 On the other hand, Jakubikova et al (10) showed that the cells expressions of stem cell transcription factors at both the in the side population analyzed by o fl w cytometry, which are mRNA and protein levels. Previous reports showing stem considered to be cancer stem cells, are CD138-positive cells cell marker expression in CD138-negative myeloma cells with clonogenic potential, while Hosen et al (36) reported that (29,43) support our findings. It was also reported that Oct4 both CD138-positive and -negative lesions have the capacity and SOX2 expression is essential for maintenance of the to propagate MM in vivo. Although it remains unclear side-population fraction in MM (44). These findings indicate whether CD138-negative myeloma cells are ‘myeloma stem that hypoxia induces a stem cell-like phenotype in MM cells. cells’, several reports have shown that CD138-low or -nega- However, we did not observe any increases in ALDH activity tive cells may contribute to drug resistance or relapse of the or ABCG2 expression, which are associated with cancer stem disease (9,11,12). Therefore, these previous findings allow us to cell function. Taken together, hypoxia induces stem cell-like consider that the expression of CD138 in myeloma cells is not features in MM cells, although further analyses are needed uniformly high. Analyses of the mechanisms underlying the to conclude that MM cells under hypoxic conditions are regulation of CD138 expression in myeloma cells may lead to cancer stem cells. a better understanding of myeloma biology. It is known that MM patients with SOX2 expression have a In the present study, we found that a reduced oxygen worse overall survival than patients without SOX2 expression level contributes to CD138 downregulation in MM cells. We (45), and this report is compatible with our previous finding observed a reduction in the CD138 level under hypoxia in a that MM patients with low CD138 expression showed a poor time-dependent manner that lasted for at least 30 days. The prognosis (9). MM cells under hypoxic conditions may become recovery of CD138 expression by re-oxygenation indicates that less sensitive to anticancer agents, thereby contributing to a the oxygen level plays a major role in MM cell regulation of poor prognosis through the acquisition of drug resistance. CD138 expression. Indeed, ATRA was able to sensitize MM cells to bortezomib When the expression of CD138 was decreased, the adhesion under hypoxic conditions by inducing a more differentiated of the MM cell lines to type-1 collagen was also decreased, phenotype. These findings allow us to propose a new thera - indicating a correlation between CD138 expression and adhe- peutic approach against CD138-low myeloma cells with drug sion of MM cells to the extracellular matrix. Indeed, several resistance by combining ATRA with other anti-myeloma reports have shown that CD138 downregulation may contribute reagents such as bortezomib. to a more metastatic potential not only in solid tumors (37,38), Taken together, the present data suggest that hypoxia but also in MM (12,39). It can be hypothesized that hypoxia reduces CD138 expression and provides stem cell-like features may promote the metastasis of myeloma cells partly through to myeloma cells. Further analyses of the mechanisms that CD138 downregulation. CXCR4, which is induced under regulate CD138 expression and related biological processes hypoxia and contributes to migration and homing of MM cells including cell adhesion or drug sensitivity should contribute (18), was upregulated in hypoxic MM cells. This finding may not only to a better understanding of the disease, but also to an also be associated with tumor progression under hypoxia. improvement of the prognosis of myeloma. Hypoxic MM cells had lower CD138 and CS1 expressions and higher CD20 expression than normoxic cells. Since the Acknowledgments expressions of CD138 (4) and CS1 (25) are highly specic fi for terminally differentiated plasma cells and previous reports This study was supported by a grant from the Amyloidosis showed an immature transcriptional profile in MM cells with Research Committee for Research on Intractable Diseases low expression of CD138 (9,11,16), we hypothesized that the from the Ministry of Health, Labour and Welfare, Japan. hypoxic MM cell lines may have a less mature phenotype than the normoxic cell lines. Expression analyses of transcription References factors associated with plasma cells (IRF4, PRDM1 and XBP1) 1. Kumar SK, Rajkumar SV, Dispenzieri A, et al: Improved survival and B cells (BCL6 and PAX5) revealed that the hypoxic MM in multiple myeloma and the impact of novel therapies. Blood cells had a relatively immature (B cell-like) transcriptional 111: 2516-2520, 2008. profile compared with normoxic cells. This indicates that the 2. Clarke MF, Dick JE, Dirks PB, et al: Cancer stem cells - perspec- tives on current status and future directions: AACR Workshop on oxygen levels inu fl ence not only the surface antigens, but also cancer stem cells. Cancer Res 66: 9339-9344, 2006. the transcription factor expressions in MM cells. Although 3. Matsui W, Huff CA, Wang Q, et al: Characterization of clono- hypoxic cells acquired a B cell-like phenotype, CD19 expres- genic multiple myeloma cells. Blood 103: 2332-2336, 2004. 4. Medina F, Segundo C, Campos-Caro A, Gonzalez-Garcia I and sion was negative. Since CD19 expression can distinguish MM Brieva JA: The heterogeneity shown by human plasma cells cells from B cells and normal plasma cells (40), this indicates from tonsil, blood, and bone marrow reveals graded stages of that hypoxia delivers an immature phenotype to MM cells, increasing maturity, but local profiles of adhesion molecule expression. Blood 99: 2154-2161, 2002. but they are not identical to normal mature B cells and can 5. Wijdenes J, Vooijs WC, Clement C, et al: A plasmocyte selective be distinguished as malignant plasma cells in terms of CD19 monoclonal antibody (B-B4) recognizes syndecan-1. Br J negativity. Haematol 94: 318-323, 1996. 6. Ikeda H, Hideshima T, Fulciniti M, et al: The monoclonal HIFs (HIF-1α and HIF-2α) mediate the cellular response antibody nBT062 conjugated to cytotoxic Maytansinoids has to hypoxia and activate transcription factors that control stem selective cytotoxicity against CD138-positive multiple myeloma cell self-renewal (27). It was reported that HIFs are positive cells in vitro and in vivo. Clin Cancer Res 15: 4028-4037, 2009. 7. Witzig TE, Kimlinger T, Stenson M and Therneau T: Syndecan-1 in myeloma cells in the bone marrow (41,42), suggesting that expression on malignant cells from the blood and marrow of myeloma cells are also hypoxic in vivo. Our analyses of stem patients with plasma cell proliferative disorders and B-cell chronic cell transcription factors revealed that hypoxia induces the lymphocytic leukemia. Leuk Lymphoma 31: 167-175, 1998. KAWANO et al: CD138 DECREASED MYELOMA UNDER HYPOXIA 8. Reid S, Yang S, Brown R, et al: Characterisation and relevance of 27. Keith B and Simon MC: Hypoxia-inducible factors, stem cells, CD138-negative plasma cells in plasma cell myeloma. Int J Lab and cancer. Cell 129: 465-472, 2007. Hematol 32: e190-e196, 2010. 28. Mathieu J, Zhang Z, Zhou W, et al: HIF induces human embryonic 9. Kawano Y, Fujiwara S, Wada N, et al: Multiple myeloma cells stem cell markers in cancer cells. Cancer Res 71: 4640-4652, expressing low levels of CD138 have an immature phenotype 2011. and reduced sensitivity to lenalidomide. Int J Oncol 41: 876-884, 29. Brennan SK, Wang Q, Tressler R, et al: Telomerase inhibition 2012. targets clonogenic multiple myeloma cells through telomere 10. Jakubikova J, Adamia S, Kost-Alimova M, et al: Lenalidomide length-dependent and independent mechanisms. PLoS One 5: targets clonogenic side population in multiple myeloma: patho- 2010. physiologic and clinical implications. Blood 117: 4409-4419, 30. Zhou S, Schuetz JD, Bunting KD, et al: The ABC transporter 2011. Bcrp1/ABCG2 is expressed in a wide variety of stem cells and is 11. Van Valckenborgh E, Matsui W, Agarwal P, et al: Tumor- a molecular determinant of the side-population phenotype. Nat - + initiating capacity of CD138 and CD138 tumor cells in the 5T33 Med 7: 1028-1034, 2001. multiple myeloma model. Leukemia 26: 1436-1439, 2012. 31. Gudas LJ and Wagner JA: Retinoids regulate stem cell differen- 12. Chaidos A, Barnes CP, Cowan G, et al: Clinical drug resistance tiation. J Cell Physiol 226: 322-330, 2011. linked to interconvertible phenotypic and functional states 32. Huang H, Wu D, Fu J, et al: All-trans retinoic acid can intensify of tumor-propagating cells in multiple myeloma. Blood 121: the growth inhibition and differentiation induction effect of 318-328, 2013. rosiglitazone on multiple myeloma cells. Eur J Haematol 83: 13. Yaccoby S: The phenotypic plasticity of myeloma plasma cells 191-202, 2009. as expressed by dedifferentiation into an immature, resilient, and 33. Gu JL, Li J, Zhou ZH, et al: Differentiation induction enhances apoptosis-resistant phenotype. Clin Cancer Res 11: 7599-7606, bortezomib efc fi acy and overcomes drug resistance in multiple 2005. myeloma. Biochem Biophys Res Commun 420: 644-650, 2012. 14. Zlei M, Egert S, Wider D, Ihorst G, Wasch R and Engelhardt M: 34. Akamatsu W, DeVeale B, Okano H, Cooney AJ and van der Characterization of in vitro growth of multiple myeloma cells. Kooy D: Suppression of Oct4 by germ cell nuclear factor restricts Exp Hematol 35: 1550-1561, 2007. pluripotency and promotes neural stem cell development in the 15. Dezorella N, Pevsner-Fischer M, Deutsch V, et al: Mesenchymal early neural lineage. J Neurosci 29: 2113-2124, 2009. stromal cells revert multiple myeloma cells to less differentiated 35. Gu P, LeMenuet D, Chung AC, Mancini M, Wheeler DA and phenotype by the combined activities of adhesive interactions Cooney AJ: Orphan nuclear receptor GCNF is required for the and interleukin-6. Exp Cell Res 315: 1904-1913, 2009. repression of pluripotency genes during retinoic acid-induced 16. Fuhler GM, Baanstra M, Chesik D, et al: Bone marrow stromal embryonic stem cell differentiation. Mol Cell Biol 25: 8507-8519, cell interaction reduces syndecan-1 expression and induces 2005. kinomic changes in myeloma cells. Exp Cell Res 316: 1816-1828, 36. Hosen N, Matsuoka Y, Kishida S, et al: CD138-negative clono- 2010. genic cells are plasma cells but not B cells in some multiple 17. Axelson H, Fredlund E, Ovenberger M, Landberg G and myeloma patients. Leukemia 26: 2135-2141, 2012. Pahlman S: Hypoxia-induced dedifferentiation of tumor cells - a 37. Matsumoto A, Ono M, Fujimoto Y, Gallo RL, Berne fi ld M and mechanism behind heterogeneity and aggressiveness of solid Kohgo Y: Reduced expression of syndecan-1 in human hepato- tumors. Semin Cell Dev Biol 16: 554-563, 2005. cellular carcinoma with high metastatic potential. Int J Cancer 18. Azab AK, Hu J, Quang P, et al: Hypoxia promotes dissemination 74: 482-491, 1997. of multiple myeloma through acquisition of epithelial to mesen- 38. Ishikawa T and Kramer RH: Sdc1 negatively modulates chymal transition-like features. Blood 119: 5782-5794, 2012 carcinoma cell motility and invasion. Exp Cell Res 316: 951-965, 19. Ohtsuki T, Yawata Y, Wada H, Sugihara T, Mori M and Namba M: 2010. Two human myeloma cell lines, amylase-producing KMS-12-PE 39. Purushothaman A, Uyama T, Kobayashi F, et al: Heparanase- and amylase-non-producing KMS-12-BM, were established from enhanced shedding of syndecan-1 by myeloma cells promotes a patient, having the same chromosome marker, t(11;14)(q13;q32). endothelial invasion and angiogenesis. Blood 115: 2449-2457, Br J Haematol 73: 199-204, 1989. 2010. 20. Matsuoka Y, Moore GE, Yagi Y and Pressman D: Production of 40. Harada H, Kawano MM, Huang N, et al: Phenotypic difference free light chains of immunoglobulin by a hematopoietic cell line of normal plasma cells from mature myeloma cells. Blood 81: derived from a patient with multiple myeloma. Proc Soc Exp Biol 2658-2663, 1993. Med 125: 1246-1250, 1967. 41. Giatromanolaki A, Bai M, Margaritis D, et al: Hypoxia and 21. Livak KJ and Schmittgen TD: Analysis of relative gene expres- activated VEGF/receptor pathway in multiple myeloma. sion data using real-time quantitative PCR and the 2(-Delta Delta Anticancer Res 30: 2831-2836, 2010. C(T)) method. Methods 25: 402-408, 2001. 42. Martin SK, Diamond P, Williams SA, et al: Hypoxia-inducible 22. Fujiwara S, Kawano Y, Yuki H, et al: PDK1 inhibition is a novel factor-2 is a novel regulator of aberrant CXCL12 expression in therapeutic target in multiple myeloma. Br J Cancer 108: 170-178, multiple myeloma plasma cells. Haematologica 95: 776-784, 2013. 2010. 23. Jourdan M, Ferlin M, Legouffe E, et al: The myeloma cell antigen 43. Spisek R, Kukreja A, Chen LC, et al: Frequent and specific syndecan-1 is lost by apoptotic myeloma cells. Br J Haematol immunity to the embryonal stem cell-associated antigen SOX2 in 100: 637-646, 1998. patients with monoclonal gammopathy. J Exp Med 204: 831-840, 24. Ridley RC, Xiao H, Hata H, Woodliff J, Epstein J and 2007. Sanderson RD: Expression of syndecan regulates human 44. Ikegame A, Ozaki S, Tsuji D, et al: Small molecule antibody myeloma plasma cell adhesion to type I collagen. Blood 81: targeting HLA class I inhibits myeloma cancer stem cells 767-774, 1993. by repressing pluripotency-associated transcription factors. 25. Shaffer AL, Emre NC, Lamy L, et al: IRF4 addiction in multiple Leukemia 26: 2124-2134, 2012. myeloma. Nature 454: 226-231, 2008. 45. Schoenhals M, Kassambara A, De Vos J, Hose D, Moreaux J and 26. Shapiro-Shelef M and Calame K: Regulation of plasma-cell Klein B: Embryonic stem cell markers expression in cancers. development. Nat Rev Immunol 5: 230-242, 2005. Biochem Biophys Res Commun 383: 157-162, 2009. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png International Journal of Oncology Pubmed Central

Hypoxia reduces CD138 expression and induces an immature and stem cell-like transcriptional program in myeloma cells

KAWANO, YAWARA; KIKUKAWA, YOSHITAKA; FUJIWARA, SHIHO; WADA, NAOKO; OKUNO, YUTAKA; MITSUYA, HIROAKI; HATA, HIROYUKI
International Journal of Oncology , Volume 43 (6) – Oct 10, 2013
Download PDF
8 pages

Loading next page...
 
/lp/pubmed-central/hypoxia-reduces-cd138-expression-and-induces-an-immature-and-stem-cell-NpUCDu1NH0

References (46)

Publisher
Pubmed Central
Copyright
Copyright © 2013, Spandidos Publications
ISSN
1019-6439
eISSN
1791-2423
DOI
10.3892/ijo.2013.2134
Publisher site
See Article on Publisher Site

Abstract

INTERNATIONAL JOURNAL OF ONCOLOGY 43: 1809-1816, 2013 Hypoxia reduces CD138 expression and induces an immature and stem cell-like transcriptional program in myeloma cells 1 1 1 1 YAWARA KAWANO , YOSHITAKA KIKUKAWA , SHIHO FUJIWARA , NAOKO WADA , 1 1 2 YUTAKA OKUNO , HIROAKI MITSUYA and HIROYUKI HATA 1 2 Department of Hematology, Division of Informative Clinical Science, Kumamoto University School of Medicine, Kumamoto 860-8556, Japan Received August 8, 2013; Accepted September 16, 2013 DOI: 10.3892/ijo.2013.2134 Abstract. Although CD138 expression is a hallmark of plasma hypoxia. We propose that hypoxia induces immature and cells and myeloma cells, reduced CD138 expression is occa- stem cell-like transcription phenotypes in myeloma cells. sionally found. However, the mechanisms underlying CD138 Taken together with our previous observation that decreased downregulation in myeloma cells remain unclear. Previous CD138 expression is correlated with disease progression, the reports suggest that the bone marrow microenvironment may present data suggest that a hypoxic microenvironment affects contribute to CD138 downregulation. Among various factors the phenotype of MM cells, which may correlate with disease in the tumor microenvironment, hypoxia is associated with progression. tumor progression, poor clinical outcomes, dedifferentiation and the formation of cancer stem cell niches in solid tumors. Introduction Since recent findings showed that progression of multiple myeloma (MM) delivers hypoxia within the bone marrow, Multiple myeloma (MM) is characterized by clonal expansion we hypothesized that CD138 expression may be regulated of malignant plasma cells in the bone marrow. Although novel by hypoxia. In the present study, we examined whether the therapeutic agents and stem cell transplantation have improved expression of CD138 and transcription factors occurred in the survival of MM patients (1), MM remains an incurable myeloma cells under hypoxic conditions. MM cell lines disease. (KMS-12BM and RPMI 8226) were cultured under normoxic Cancer stem cells are often considered to contribute to or hypoxic conditions for up to 30 days. Changes in the pheno- relapse and drug resistance in various cancers (2). Matsui type and the expression of surface antigens and transcription et al (3) reported that myeloma stem cells are enriched in the factors were analyzed using flow cytometry, RT-PCR and CD138-negative population. During normal B-cell develop- western blotting. All-trans retinoic acid (ATRA) was used to ment, abundant CD138 (also known as syndecan-1: SDC1) examine the phenotypic changes under hypoxic conditions. expression is highly specific for terminally differentiated The expression levels of CD138, CS1 and plasma cell-specic fi plasma cells in the bone marrow (4). Since CD138 expression transcription factors decreased under hypoxic conditions, is also a hallmark of malignant plasma cells (myeloma cells), it while those of CD20, CXCR4 and B cell-specic fi transcription has been used for myeloma cell puric fi ation (5) and is consid- factors increased compared with those under normoxic condi- ered to be a target for treatment (6). While the majority of tions. Stem cell-specic fi transcription factors were upregulated myeloma cells express CD138, decreased expression of CD138 under hypoxic conditions, while no difference was observed in is occasionally found in clinical practice (7-9). Although the ALDH activity. The reduced CD138 expression under hypoxic association between CD138 expression and myeloma stem cells conditions recovered when cells were treated with ATRA, even remains a matter of debate (10), several reports have shown that under hypoxic conditions, along with decreases in the expres- CD138-low or -negative myeloma cells may contribute to drug sion of stem cell-specic fi transcription factor. Interestingly, resistance or relapse of the disease (9,11,12). Therefore, analysis ATRA treatment sensitized MM cells to bortezomib under of CD138 downregulation in myeloma cells is required for a better understanding of myeloma biology. Previous reports have indicated that the bone marrow microenvironment may contribute to CD138 downregulation (13-16). Among various factors in the tumor microenviron- Correspondence to: Dr Yawara Kawano, Department of Hemato- ment, hypoxia is one of the important factors associated with logy, Kumamoto University School of Medicine, Honjo 1-1-1, tumor progression, poor clinical outcomes, dedifferentiation, Chuo-ku, Kumamoto City, Kumamoto 860-8556, Japan E-mail: [email protected] and formation of cancer stem cell niches in solid tumors (17). Based on recent findings showing a correlation of MM at the Key words: myeloma, hypoxia, CD138, ATRA advanced stage with hypoxic conditions in the microenvi- ronment within the bone marrow (18), we hypothesized that CD138 expression may be inu fl enced by hypoxia. KAWANO et al: CD138 DECREASED MYELOMA UNDER HYPOXIA In the present study, we compared the changes in CD138 CTCATTTCACCAGGCC-3'; reverse, 5'-TGGGACTCCTCCG and various transcription factor expressions in myeloma cells GGTTTTG-3'); NANOG (forward, 5'-ACTGTCTCTCCTCT under hypoxic or normoxic conditions. We also attempted to TCCTTC-3'; reverse, 5'-CCTGTTTGTAGCTGAGGTTC-3'); revert CD138 expression in cells under hypoxia by treatment SOX2 (forward, 5'-ACAACTCGGAGATCAGCA-3'; reverse, with all-trans retinoic acid (ATRA). The inu fl ence of ATRA 5'- G CAG CGTGTACT TATCCT TC-3'); ACT B (forward, on the sensitivity to bortezomib under hypoxic conditions was 5'-GGACTTCGAGCAAGAGATGG-3'; reverse, 5'-AGCAC also examined. TGTGTTGGCGTACAG-3'). Quantitative real-time RT-PCR was performed using Materials and methods Assay-on-Demand primers and TaqMan Universal PCR Master Mix Reagent (Applied Biosystems, Foster City, NJ, Cell culture. Human myeloma cell lines, KMS-12BM (19) USA). Samples were analyzed using an ECO™ Real-Time and RPMI 8226 (20), were obtained from the Health Science PCR System (Illumina, San Diego, CA, USA). The ∆∆Ct Research Resources Bank (Osaka, Japan) and maintained in method was used to analyze the relative changes in gene RPMI-1640 medium supplemented with 10% heat-inactivated expression as previously described (21), using ACTB as a fetal bovine serum at 37˚C under 5% CO . The two myeloma normalization control. The following primers and probes were cell lines were cultured under normoxic (21% O ) and hypoxic used: SDC1 (Hs00896423_m1); IRF4 (Hs01056534_m1); (1% O ) conditions for up to 30 days, with fresh medium PRDM1 (Hs00153357_m1); XBP1 (Hs00964360_m1); and provided every 3 days. Experiments under hypoxic condi- ACTB (Hs99999903_m1). tions were performed in a Personal CO Multigas Incubator (ASTEC, Fukuoka, Japan). Intracellular staining of IRF4 followed by flow cytometric analysis. The MM cell lines cultured under normoxic or Flow cytometric analysis of surface antigens. MM cell lines hypoxic conditions were stained with an FITC-CD138 cultured under normoxic and hypoxic conditions were stained antibody (clone MI15; BD Biosciences), fixed and permea - with the following fluorescently-labeled antibodies: FITC- bilized using a FOXP3 Staining Buffer Set (eBioscience, CD138 (clone MI15), FITC-CD38 (clone HIT2), PE-CD44 San Diego, CA, USA), and then stained intracellularly (clone 515), PE-CD45 (clone HI30), FITC-CD49d (clone gf10) with an Alexa 647-IRF4 antibody (clone 3E4; eBioscience) (BD Biosciences, Fran k lin La kes, NJ, USA); PE- CD54 according to the manufacturer's protocol. Flow cytometric (clone HCD54), PE - CXCR4 (clone 12G5), PE -M DR-1 analysis was performed using the FACSCalibur (Becton- (clone UIC2), APC-ABCG2 (clone 5D3) (Biolegend, San Dickinson). Diego, CA, USA); FITC-CD19 (clone HD37), FITC-CD20 (clone B-Ly1) (Dako, Glostrup, Denmark); and Alexa 647-CS1 Western blot analysis. Cell lysates were prepared as reported (clone 162) (AbD Serotec, Oxford, UK). Density gradient previously (22). Quantic fi ation of total protein was performed centrifugation using Ficoll-Paque Plus (GE Healthcare, using a Pierce BCA Protein Assay Kit (Thermo Scientific, Uppsala, Sweden), the forward/side scatter profile and Waltham, MA, USA), and equal amounts of protein were 7-amino-actinomycin D (7-AAD) (BD Biosciences) labeling used for analysis. The cell lysates were separated in NuPAGE were used for exclusion of non-viable cells. Flow cytometric Bis-Tris precast gels (Invitrogen) and transferred to PVDF anal ysis was performed using a FACSCalibur or FACSVerse membranes using an iBlot Dry Blotting system (Invitrogen). o fl w cytometer (Becton-Dickinson, San Jose, CA, USA). The membranes were blocked with 5% non-fat dry milk for 1 h at room temperature, followed by incubation with primary Adhesion to type-1 collagen. MM cells were plated in antibodies at 4˚C for 18 h. The primary antibodies against quadruplicate at a concentration of 5x10 cells/ml on type-1 HIF-1α, HIF-2α, NANOG, and SOX2 were purchased from collagen-coated 96-well plates (Becton-Dickinson) and Cell Signaling Technology (Beverly, MA, USA), while those incubated for 1 h at 37˚C. After the incubation, the cells were against Oct-4, RARα, and actin were obtained from Santa washed twice with PBS and incubated with the WST-8 reagent Cruz Biotechnology (Santa Cruz, CA, USA). The membranes (Dojindo, Kumamoto, Japan). The ratios of adherent cells to were then incubated with horseradish peroxidase-conjugated total applied cells were quantie fi d by the light absorbance of secondary antibodies (GE Healthcare) for 1 h at room each well at 450 nm using a VMax absorbance microplate temperature. Antibody-bound proteins were visualized reader (Molecular Devices, Sunnyvale, CA, USA). using the ECL prime western blotting detection reagent (GE Healthcare) and an LAS-1000 bio-image analyzer (GE cDNA synthesis and reverse transcription-polymerase chain Healthcare). reaction (RT-PCR). RNA was extracted from the MM cell lines using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). cDNA Aldehyde dehydrogenase (ALDH) activity. The ALDH synthesis was performed using a SuperScript III First-Strand activities of the MM cell lines cultured under normoxic and Synthesis System for RT-PCR (Invitrogen) according to the hypoxic conditions were analyzed using Aldeu fl or (Stem Cell manufacturer's protocol. Technologies, Vancouver, Canada). After adding activated The expression levels of BCL6, PAX5, Oct-4, NANOG and Aldeu fl or reagent to the cell cultures, half of the cells were SOX2 were determined by RT-PCR. β-actin (ACTB) was used transferred to tubes containing an ALDH inhibitor, diethyl- as a normalization control. The primers for BCL6 and PAX5 aminobenzaldehyde (DEAB), to confirm specificity of the were described previously (9). The primers for Oct- 4, NANOG, reagent. Samples were incubated at 37˚C for 30 min and SOX2 and ACTB were as follows: Oct- 4 (forward, 5'-AGCC analyzed using the FACSCalibur (Becton-Dickinson). INTERNATIONAL JOURNAL OF ONCOLOGY 43: 1809-1816, 2013 Figure 1. Decrease in CD138 expression under hypoxia. (A) CD138 expression in the MM cell lines is decreased after 72 h of culture under hypoxic conditions. The left panel shows CD138 expression in KMS-12BM cells under normoxic and hypoxic conditions analyzed by o fl w cytometry (solid or dashed line, CD138; shadowed area, isotype control). Overlay plots of the CD138 expressions in KMS-12BM and RPMI 8226 cells under normoxic (solid line) and hypoxic (dashed line) conditions are shown in the right panel. (B) CD138 expression in KMS-12BM cells is decreased in a time-dependent manner. The increasing proportion of CD138-negative cells and decrease in CD138 u fl orescence intensity as judged by the mean u fl orescence intensity (MFI) ratio to the isotype control are shown in the left and right panels, respectively. White bars, normoxic conditions; black bars, hypoxic conditions. (C) Re-oxygenation recovers CD138 expression. KMS-12BM cells were cultured under hypoxic conditions for 72 h. The cells were then further cultured under normoxic (solid line) or hypoxic (dashed line) conditions for an additional 72 h. Analysis of apoptosis. The MM cell lines were incubated in the of culture under hypoxic conditions compared with normoxic presence of 1 µM ATRA (Sigma, St. Louis, MO, USA) or 5 nM conditions in both cell lines (Fig. 1A). As shown in Fig. 1B, bortezomib (Sigma) for 24 h. Apoptosis in the MM cell lines CD138 expression was reduced from 48 h, and subsequently was quantie fi d by staining with Annexin V (MBL, Nagoya, proceeded in a time-dependent manner. We then re-oxygenized Japan) and 7-AAD (BD Biosciences). The samples were the cells at 72 h after starting the hypoxic conditions and main- analyzed by flow cytometry (FACSVerse; Becton-Dickinson). tained the cells under normoxic conditions for an additional 72 h. Interestingly, we observed recovery of the CD138 expres- Statistical analysis. The data were analyzed statistically by sion, indicating that the reduction in CD138 expression under Student's t-test using GraphPad Prism version 5.0 (GraphPad hypoxia was a reversible phenomenon (Fig. 1C). Software, La Jolla, CA, USA). P-values of <0.05 were consid- ered statistically signic fi ant. Changes in CD138 and other surface antigens under long- term hypoxia. We further analyzed CD138 expression under Results long-term exposure (30 days) to hypoxic or normoxic condi- tions. After 30 days of incubation, CD138 expression was Hypoxia reduces CD138 expression in MM cell lines. To inves- markedly reduced in KMS-12BM and RPMI 8226 cells tigate whether CD138 expression in MM cells was inu fl enced cultured under hypoxia compared with those cultured under by oxygen levels, we cultured two MM cell lines (KMS-12BM normoxia (Fig. 2A). In KMS-12BM cells in particular, the and RPMI 8226) under normoxic and hypoxic conditions for positivity for CD138 expression under hypoxic conditions was up to 72 h and compared the surface CD138 expressions by flow reduced to only 10%. Real-time RT-PCR showed that CD138 cytometry. Since CD138 was reported to be downregulated in (SDC1) mRNA was downregulated after long-term hypoxia, non-viable cells (23), only viable cells were obtained by Ficoll indicating that CD138 expression was reduced at the gene density gradient centrifugation followed by gating of live cells in transcription level (Fig. 2B). combination with the forward/side scatter prol fi e and exclusion Since CD138 mediates the adhesion of MM cells to type-1 of 7-AAD-positive cells. CD138 expression was reduced by 72 h collagen (24), which is an important component of the bone KAWANO et al: CD138 DECREASED MYELOMA UNDER HYPOXIA Figure 2. Decrease in CD138 expression and changes in other surface antigens after long-term exposure to hypoxia. (A) KMS-12BM and RPMI 8226 cells were cultured under hypoxic or normoxic conditions for 30 days and the expression of CD138 was analyzed by flow cytometry. Overlay plots of CD138 expression are shown. Normoxic conditions, solid line; hypoxic conditions, dashed line. (B) A decrease in CD138 expression is found at the mRNA level. The expression of CD138 mRNA was quantie fi d by real-time RT-PCR. (C) Decrease in adhesion to collagen type-I under hypoxic conditions. Cells were treated as described in (A) and their adhesion capabilities to collagen type-I were analyzed. White bars, normoxic conditions; black bars, hypoxic conditions. (D) Increases in CD20 and CXCR4 expressions and decrease in CS1 expression under hypoxic conditions. KMS-12BM cells were treated as described in (A) and other surface antigens were analyzed by flow cytometry (solid or dashed line, CD138; shadowed area, isotype control). marrow microenvironment, the inu fl ence of oxygen concentra - and BCL6, was increased in the hypoxic state in KMS-12BM tions on the adhesion of MM cells was evaluated. The hypoxic and RPMI 8226 cells (Fig. 3A). The gene expressions of IRF4, MM cell lines adhered poorly to type-1 collagen, ree fl cting the PRDM1 and XBP1, which are plasma cell-specic fi transcrip - low expression of CD138 (Fig. 2C). tion factors, were decreased in the hypoxic cells compared The expression changes in other surface antigens were with the normoxic cells (Fig. 3B). The intracellular IRF4 compared between the hypoxic and normoxic MM cell lines. protein expression, which was analyzed by flow cytometry, The CD20 and CXCR4 expressions were increased, while the was decreased in the hypoxic cells (Fig. 3C), consistent with CS1 expression was decreased, under hypoxia compared with the results for the gene expressions. Analysis of subpopulations normoxia in KMS-12BM cells (Fig. 2D). Similar results were obtained as CD138-high and -low cells showed high and low observed in RPMI 8226 cells (normoxia vs. hypoxia: CD20, IRF4 expressions, respectively (Fig. 3D). These findings show 1.8 vs. 14.1%; CS1, 81.3 vs. 34.6%; CXCR4, 40.2 vs. 82.1%). that the hypoxic MM cell lines with low CD138 expression No changes in expression were observed for CD19, adhesion have an immature, so-called mature B cell-like rather than molecules other than CD138 (CD44, CD49d and CD54), and plasma cell, transcriptional status. ATP-binding cassette transporters (ABCG2 and MDR1) (data not shown). Hypoxia induces a stem cell-like transcriptional program in MM cells. Hypoxia-inducible factors (HIFs) are key molecules Hypoxic MM cell lines have an immature phenotype. Since for the cellular response to hypoxia. It was reported that a CD138 (4) and CS1 (25) expressions are highly specic fi for hypoxic environment and HIF activity are required not only terminally differentiated plasma cells, we hypothesized that for stem cells, but also for cancer stem cells (27). Since HIFs the hypoxic MM cell lines have a less mature phenotype than induce stem cell transcription factors in solid tumors (28), the normoxic cell lines. To prove this hypothesis, we assessed we analyzed the expression of HIFs and stem cell transcrip- the expressions of the BCL6 and PAX5 transcription factors, tion factors in the hypoxic MM cell lines. HIFs, especially which exist in the mature B-cell state and are decreased in HIF-2α, were increased in the hypoxic MM cell lines, as mature plasma cells (26), by RT-PCR in the MM cell lines evaluated by western blotting, indicating that the cells were after 30 days of culture under hypoxic or normoxic condi- actually responding to the hypoxic environment (Fig. 4A). The tions. Each of the mature B-cell transcription factors, PAX5 expression of stem cell transcription factors (Oct-4, SOX2, and INTERNATIONAL JOURNAL OF ONCOLOGY 43: 1809-1816, 2013 Figure 3. Exposure to hypoxia induces transcription factors specic fi for the B-cell phenotype. (A) Induction of BCL6 and PAX5 by exposure to hypoxia. KMS-12BM and RPMI 8226 cells were cultured under hypoxic conditions for 30 days. PAX5 expression is increased in hypoxic KMS-12BM cells, while BCL6 is increased in hypoxic RPMI 8226 cells. (B) Hypoxia reduces plasma cell-specic fi transcription factor expression. The gene expression levels of IRF4 (white bars), PRDM1 (striped bars), and XBP1 (black bars) were analyzed by real-time RT-PCR in KMS-12BM and RPMI 8226 cells. The expression of the three genes are decreased. (C) Reduction in intracellular IRF4 expression under hypoxic conditions detected by o fl w cytometry. The expression is decreased in hypoxic cells (dashed line) compared with normoxic cells (solid line). (D) The CD138-low subpopulation has lower IRF4 expression. RPMI 8226 cells were cultured under hypoxic conditions for 30 days. The expression of IRF4 was evaluated by gating the CD138-high (25% from the highest intensity; solid square in the left panel) and CD138-low (25% from the lowest intensity; dashed square in the left panel) subpopulations. The intracellular IRF4 expression in each compartment is shown in the right panel. The CD138-low population has lower intracellular IRF4 expression. Figure 4. Hypoxia induces a stem cell-like transcriptional program in MM cell lines. (A) KMS-12BM and RPMI 8226 cells were cultured under hypoxic or normoxic conditions for 30 days. Cell lysates were extracted and subjected to western blot analysis. HIF expression is increased in the hypoxic MM cell lines. (B and C) Expression of transcription factors related to the stem cell phenotype was analyzed by RT-PCR (B) or western blotting (C). Upregulation of transcription factors related to the stem cell phenotype is observed under hypoxia. (D) Analyses of ALDH activity in KMS-12BM cells. Cells were cultured under hypoxic or normoxic conditions and subjected to ALDH assays. DEAB was used as an ALDH inhibitor. No increase in ALDH activity is observed in hypoxic KMS-12BM cells. KAWANO et al: CD138 DECREASED MYELOMA UNDER HYPOXIA Figure 5. ATRA restores the phenotypic changes and sensitivity to bortezomib induced by hypoxic conditions. (A) Western blot analysis showing the expres- sion of RARα in both KMS-12BM and RPMI 8226 cells cultured under hypoxic conditions. (B) Recovery of CD138 expression after ATRA treatment in the MM cell lines under hypoxic conditions. Solid line, ATRA treatment for 48 h; dashed line, DMSO treatment. (C) Reductions in stem cell-related transcription factors after ATRA treatment in hypoxic MM cell lines evaluated by western blot analysis. (D) Analysis of cytotoxicity toward KMS-12BM cells cultured under hypoxic conditions with ATRA and bortezomib. Cells were treated with ATRA or bortezomib alone or in combination for 24 h. The cells were then subjected to Annexin V/7-AAD analysis. The upper panel shows the raw data of the scatter graph. The lower panel shows the proportion of early apoptotic + - cells (Annexin V /7-AAD ). ATRA augments early apoptosis in combination with bortezomib. NANOG) was increased in the hypoxic MM cell lines at both were then incubated with 1 µM ATRA or dimethyl sulfoxide the mRNA (Fig. 4B) and protein (Fig. 4C) levels. The only (DMSO) for 48 h under hypoxic conditions. Surprisingly, exception was the expression of SOX2 protein, which was not ATRA increased the CD138 expression compared with detected in hypoxic KMS-12BM cells, although its mRNA DMSO (Fig. 5B), especially in KMS-12BM cells, while the expression was upregulated. However, the ALDH activity, expression of all stem cell transcription factors was decreased which is characteristic of cancer stem cells including MM (Fig. 5C). These findings indicate that ATRA can induce the stem cells (29), was not increased in hypoxic KMS-12BM cells redifferentiation of immature MM cells, even under hypoxic (Fig. 4D) and RPMI 8226 cells (normoxia vs. hypoxia: 1.06 vs. conditions. 0.10%). The expression of ABCG2, which is highly expressed Next, we investigated whether the differentiation induced in stem cells and associated with side-population cells (30), by ATRA can sensitize the immature and hypoxic cells to was not increased in hypoxic cells, as described earlier in this bortezomib. Hypoxic KMS-12BM cells were treated with report. 1 µM ATRA and 5 nM bortezomib alone or in combination for 24 h. While ATRA alone slightly affected the viability ATRA induces differentiation of hypoxic MM cells and of hypoxic KMS-12BM cells, the combination of the two increase their sensitivity to bortezomib. ATRA induces cell reagents increased the cytotoxicity, especially the early apop- + - differentiation not only of stem cells (31), but also of MM cells tosis (Annexin V /7-AAD cells) (Fig. 5D). (32,33). ATRA is also known to repress the expression of stem cell transcription factors, such as Oct-4, NANOG and SOX2 Discussion (34,35). Based on these previous reports, we investigated whether ATRA could induce redifferentiation of the hypoxic Accumulating evidence indicates that CD138 is an important MM cell lines. We first confirmed the expression of RAR α, a molecule not only for the identic fi ation of plasma cells, but specic fi receptor for retinoic acid, in the MM cell lines cultured also for distinguishing myeloma stem cells. CD138-negative for 30 days under hypoxic conditions (Fig. 5A). The cell lines cells have recently been proposed as myeloma stem cells (3). INTERNATIONAL JOURNAL OF ONCOLOGY 43: 1809-1816, 2013 On the other hand, Jakubikova et al (10) showed that the cells expressions of stem cell transcription factors at both the in the side population analyzed by o fl w cytometry, which are mRNA and protein levels. Previous reports showing stem considered to be cancer stem cells, are CD138-positive cells cell marker expression in CD138-negative myeloma cells with clonogenic potential, while Hosen et al (36) reported that (29,43) support our findings. It was also reported that Oct4 both CD138-positive and -negative lesions have the capacity and SOX2 expression is essential for maintenance of the to propagate MM in vivo. Although it remains unclear side-population fraction in MM (44). These findings indicate whether CD138-negative myeloma cells are ‘myeloma stem that hypoxia induces a stem cell-like phenotype in MM cells. cells’, several reports have shown that CD138-low or -nega- However, we did not observe any increases in ALDH activity tive cells may contribute to drug resistance or relapse of the or ABCG2 expression, which are associated with cancer stem disease (9,11,12). Therefore, these previous findings allow us to cell function. Taken together, hypoxia induces stem cell-like consider that the expression of CD138 in myeloma cells is not features in MM cells, although further analyses are needed uniformly high. Analyses of the mechanisms underlying the to conclude that MM cells under hypoxic conditions are regulation of CD138 expression in myeloma cells may lead to cancer stem cells. a better understanding of myeloma biology. It is known that MM patients with SOX2 expression have a In the present study, we found that a reduced oxygen worse overall survival than patients without SOX2 expression level contributes to CD138 downregulation in MM cells. We (45), and this report is compatible with our previous finding observed a reduction in the CD138 level under hypoxia in a that MM patients with low CD138 expression showed a poor time-dependent manner that lasted for at least 30 days. The prognosis (9). MM cells under hypoxic conditions may become recovery of CD138 expression by re-oxygenation indicates that less sensitive to anticancer agents, thereby contributing to a the oxygen level plays a major role in MM cell regulation of poor prognosis through the acquisition of drug resistance. CD138 expression. Indeed, ATRA was able to sensitize MM cells to bortezomib When the expression of CD138 was decreased, the adhesion under hypoxic conditions by inducing a more differentiated of the MM cell lines to type-1 collagen was also decreased, phenotype. These findings allow us to propose a new thera - indicating a correlation between CD138 expression and adhe- peutic approach against CD138-low myeloma cells with drug sion of MM cells to the extracellular matrix. Indeed, several resistance by combining ATRA with other anti-myeloma reports have shown that CD138 downregulation may contribute reagents such as bortezomib. to a more metastatic potential not only in solid tumors (37,38), Taken together, the present data suggest that hypoxia but also in MM (12,39). It can be hypothesized that hypoxia reduces CD138 expression and provides stem cell-like features may promote the metastasis of myeloma cells partly through to myeloma cells. Further analyses of the mechanisms that CD138 downregulation. CXCR4, which is induced under regulate CD138 expression and related biological processes hypoxia and contributes to migration and homing of MM cells including cell adhesion or drug sensitivity should contribute (18), was upregulated in hypoxic MM cells. This finding may not only to a better understanding of the disease, but also to an also be associated with tumor progression under hypoxia. improvement of the prognosis of myeloma. Hypoxic MM cells had lower CD138 and CS1 expressions and higher CD20 expression than normoxic cells. Since the Acknowledgments expressions of CD138 (4) and CS1 (25) are highly specic fi for terminally differentiated plasma cells and previous reports This study was supported by a grant from the Amyloidosis showed an immature transcriptional profile in MM cells with Research Committee for Research on Intractable Diseases low expression of CD138 (9,11,16), we hypothesized that the from the Ministry of Health, Labour and Welfare, Japan. hypoxic MM cell lines may have a less mature phenotype than the normoxic cell lines. Expression analyses of transcription References factors associated with plasma cells (IRF4, PRDM1 and XBP1) 1. Kumar SK, Rajkumar SV, Dispenzieri A, et al: Improved survival and B cells (BCL6 and PAX5) revealed that the hypoxic MM in multiple myeloma and the impact of novel therapies. Blood cells had a relatively immature (B cell-like) transcriptional 111: 2516-2520, 2008. profile compared with normoxic cells. This indicates that the 2. Clarke MF, Dick JE, Dirks PB, et al: Cancer stem cells - perspec- tives on current status and future directions: AACR Workshop on oxygen levels inu fl ence not only the surface antigens, but also cancer stem cells. Cancer Res 66: 9339-9344, 2006. the transcription factor expressions in MM cells. Although 3. Matsui W, Huff CA, Wang Q, et al: Characterization of clono- hypoxic cells acquired a B cell-like phenotype, CD19 expres- genic multiple myeloma cells. Blood 103: 2332-2336, 2004. 4. Medina F, Segundo C, Campos-Caro A, Gonzalez-Garcia I and sion was negative. Since CD19 expression can distinguish MM Brieva JA: The heterogeneity shown by human plasma cells cells from B cells and normal plasma cells (40), this indicates from tonsil, blood, and bone marrow reveals graded stages of that hypoxia delivers an immature phenotype to MM cells, increasing maturity, but local profiles of adhesion molecule expression. Blood 99: 2154-2161, 2002. but they are not identical to normal mature B cells and can 5. Wijdenes J, Vooijs WC, Clement C, et al: A plasmocyte selective be distinguished as malignant plasma cells in terms of CD19 monoclonal antibody (B-B4) recognizes syndecan-1. Br J negativity. Haematol 94: 318-323, 1996. 6. Ikeda H, Hideshima T, Fulciniti M, et al: The monoclonal HIFs (HIF-1α and HIF-2α) mediate the cellular response antibody nBT062 conjugated to cytotoxic Maytansinoids has to hypoxia and activate transcription factors that control stem selective cytotoxicity against CD138-positive multiple myeloma cell self-renewal (27). It was reported that HIFs are positive cells in vitro and in vivo. Clin Cancer Res 15: 4028-4037, 2009. 7. Witzig TE, Kimlinger T, Stenson M and Therneau T: Syndecan-1 in myeloma cells in the bone marrow (41,42), suggesting that expression on malignant cells from the blood and marrow of myeloma cells are also hypoxic in vivo. Our analyses of stem patients with plasma cell proliferative disorders and B-cell chronic cell transcription factors revealed that hypoxia induces the lymphocytic leukemia. Leuk Lymphoma 31: 167-175, 1998. KAWANO et al: CD138 DECREASED MYELOMA UNDER HYPOXIA 8. Reid S, Yang S, Brown R, et al: Characterisation and relevance of 27. Keith B and Simon MC: Hypoxia-inducible factors, stem cells, CD138-negative plasma cells in plasma cell myeloma. Int J Lab and cancer. Cell 129: 465-472, 2007. Hematol 32: e190-e196, 2010. 28. Mathieu J, Zhang Z, Zhou W, et al: HIF induces human embryonic 9. Kawano Y, Fujiwara S, Wada N, et al: Multiple myeloma cells stem cell markers in cancer cells. Cancer Res 71: 4640-4652, expressing low levels of CD138 have an immature phenotype 2011. and reduced sensitivity to lenalidomide. Int J Oncol 41: 876-884, 29. Brennan SK, Wang Q, Tressler R, et al: Telomerase inhibition 2012. targets clonogenic multiple myeloma cells through telomere 10. Jakubikova J, Adamia S, Kost-Alimova M, et al: Lenalidomide length-dependent and independent mechanisms. PLoS One 5: targets clonogenic side population in multiple myeloma: patho- 2010. physiologic and clinical implications. Blood 117: 4409-4419, 30. Zhou S, Schuetz JD, Bunting KD, et al: The ABC transporter 2011. Bcrp1/ABCG2 is expressed in a wide variety of stem cells and is 11. Van Valckenborgh E, Matsui W, Agarwal P, et al: Tumor- a molecular determinant of the side-population phenotype. Nat - + initiating capacity of CD138 and CD138 tumor cells in the 5T33 Med 7: 1028-1034, 2001. multiple myeloma model. Leukemia 26: 1436-1439, 2012. 31. Gudas LJ and Wagner JA: Retinoids regulate stem cell differen- 12. Chaidos A, Barnes CP, Cowan G, et al: Clinical drug resistance tiation. J Cell Physiol 226: 322-330, 2011. linked to interconvertible phenotypic and functional states 32. Huang H, Wu D, Fu J, et al: All-trans retinoic acid can intensify of tumor-propagating cells in multiple myeloma. Blood 121: the growth inhibition and differentiation induction effect of 318-328, 2013. rosiglitazone on multiple myeloma cells. Eur J Haematol 83: 13. Yaccoby S: The phenotypic plasticity of myeloma plasma cells 191-202, 2009. as expressed by dedifferentiation into an immature, resilient, and 33. Gu JL, Li J, Zhou ZH, et al: Differentiation induction enhances apoptosis-resistant phenotype. Clin Cancer Res 11: 7599-7606, bortezomib efc fi acy and overcomes drug resistance in multiple 2005. myeloma. Biochem Biophys Res Commun 420: 644-650, 2012. 14. Zlei M, Egert S, Wider D, Ihorst G, Wasch R and Engelhardt M: 34. Akamatsu W, DeVeale B, Okano H, Cooney AJ and van der Characterization of in vitro growth of multiple myeloma cells. Kooy D: Suppression of Oct4 by germ cell nuclear factor restricts Exp Hematol 35: 1550-1561, 2007. pluripotency and promotes neural stem cell development in the 15. Dezorella N, Pevsner-Fischer M, Deutsch V, et al: Mesenchymal early neural lineage. J Neurosci 29: 2113-2124, 2009. stromal cells revert multiple myeloma cells to less differentiated 35. Gu P, LeMenuet D, Chung AC, Mancini M, Wheeler DA and phenotype by the combined activities of adhesive interactions Cooney AJ: Orphan nuclear receptor GCNF is required for the and interleukin-6. Exp Cell Res 315: 1904-1913, 2009. repression of pluripotency genes during retinoic acid-induced 16. Fuhler GM, Baanstra M, Chesik D, et al: Bone marrow stromal embryonic stem cell differentiation. Mol Cell Biol 25: 8507-8519, cell interaction reduces syndecan-1 expression and induces 2005. kinomic changes in myeloma cells. Exp Cell Res 316: 1816-1828, 36. Hosen N, Matsuoka Y, Kishida S, et al: CD138-negative clono- 2010. genic cells are plasma cells but not B cells in some multiple 17. Axelson H, Fredlund E, Ovenberger M, Landberg G and myeloma patients. Leukemia 26: 2135-2141, 2012. Pahlman S: Hypoxia-induced dedifferentiation of tumor cells - a 37. Matsumoto A, Ono M, Fujimoto Y, Gallo RL, Berne fi ld M and mechanism behind heterogeneity and aggressiveness of solid Kohgo Y: Reduced expression of syndecan-1 in human hepato- tumors. Semin Cell Dev Biol 16: 554-563, 2005. cellular carcinoma with high metastatic potential. Int J Cancer 18. Azab AK, Hu J, Quang P, et al: Hypoxia promotes dissemination 74: 482-491, 1997. of multiple myeloma through acquisition of epithelial to mesen- 38. Ishikawa T and Kramer RH: Sdc1 negatively modulates chymal transition-like features. Blood 119: 5782-5794, 2012 carcinoma cell motility and invasion. Exp Cell Res 316: 951-965, 19. Ohtsuki T, Yawata Y, Wada H, Sugihara T, Mori M and Namba M: 2010. Two human myeloma cell lines, amylase-producing KMS-12-PE 39. Purushothaman A, Uyama T, Kobayashi F, et al: Heparanase- and amylase-non-producing KMS-12-BM, were established from enhanced shedding of syndecan-1 by myeloma cells promotes a patient, having the same chromosome marker, t(11;14)(q13;q32). endothelial invasion and angiogenesis. Blood 115: 2449-2457, Br J Haematol 73: 199-204, 1989. 2010. 20. Matsuoka Y, Moore GE, Yagi Y and Pressman D: Production of 40. Harada H, Kawano MM, Huang N, et al: Phenotypic difference free light chains of immunoglobulin by a hematopoietic cell line of normal plasma cells from mature myeloma cells. Blood 81: derived from a patient with multiple myeloma. Proc Soc Exp Biol 2658-2663, 1993. Med 125: 1246-1250, 1967. 41. Giatromanolaki A, Bai M, Margaritis D, et al: Hypoxia and 21. Livak KJ and Schmittgen TD: Analysis of relative gene expres- activated VEGF/receptor pathway in multiple myeloma. sion data using real-time quantitative PCR and the 2(-Delta Delta Anticancer Res 30: 2831-2836, 2010. C(T)) method. Methods 25: 402-408, 2001. 42. Martin SK, Diamond P, Williams SA, et al: Hypoxia-inducible 22. Fujiwara S, Kawano Y, Yuki H, et al: PDK1 inhibition is a novel factor-2 is a novel regulator of aberrant CXCL12 expression in therapeutic target in multiple myeloma. Br J Cancer 108: 170-178, multiple myeloma plasma cells. Haematologica 95: 776-784, 2013. 2010. 23. Jourdan M, Ferlin M, Legouffe E, et al: The myeloma cell antigen 43. Spisek R, Kukreja A, Chen LC, et al: Frequent and specific syndecan-1 is lost by apoptotic myeloma cells. Br J Haematol immunity to the embryonal stem cell-associated antigen SOX2 in 100: 637-646, 1998. patients with monoclonal gammopathy. J Exp Med 204: 831-840, 24. Ridley RC, Xiao H, Hata H, Woodliff J, Epstein J and 2007. Sanderson RD: Expression of syndecan regulates human 44. Ikegame A, Ozaki S, Tsuji D, et al: Small molecule antibody myeloma plasma cell adhesion to type I collagen. Blood 81: targeting HLA class I inhibits myeloma cancer stem cells 767-774, 1993. by repressing pluripotency-associated transcription factors. 25. Shaffer AL, Emre NC, Lamy L, et al: IRF4 addiction in multiple Leukemia 26: 2124-2134, 2012. myeloma. Nature 454: 226-231, 2008. 45. Schoenhals M, Kassambara A, De Vos J, Hose D, Moreaux J and 26. Shapiro-Shelef M and Calame K: Regulation of plasma-cell Klein B: Embryonic stem cell markers expression in cancers. development. Nat Rev Immunol 5: 230-242, 2005. Biochem Biophys Res Commun 383: 157-162, 2009.

Journal

International Journal of Oncology – Pubmed Central

Published: Oct 10, 2013

There are no references for this article.