Bortezomib Reduces the Tumorigenicity of Multiple Myeloma via Downregulation of Upregulated Targets in Clonogenic Side Population Cells

<div><p>Side population (SP) cells in cancers, including multiple myeloma, exhibit tumor-initiating characteristics. In the present study, we isolated SP cells from human myeloma cell lines and primary tumors to detect potential therapeutic targets specifically expressed in SP cells. We found that SP cells from myeloma cell lines (RPMI 8226, AMO1, KMS-12-BM, KMS-11) express CD138 and that non-SP cells include a CD138-negative population. Serial transplantation of SP and non-SP cells into NOD/Shi-scid IL-2γnul mice revealed that clonogenic myeloma SP cells are highly tumorigenic and possess a capacity for self-renewal. Gene expression analysis showed that SP cells from five MM cell lines (RPMI 8226, AMO1, KMS-12-BM, KMS-11, JJN3) express genes involved in the cell cycle and mitosis (e.g., <i>CCNB1, CDC25C, CDC2</i>, <i>BIRC5, CENPE, SKA1</i>, <i>AURKB, KIFs</i>, <i>TOP2A, ASPM</i>), polycomb (e.g., <i>EZH2, EPC1</i>) and ubiquitin-proteasome (e.g., <i>UBE2D3, UBE3C, PSMA5</i>) more strongly than do non-SP cells. Moreover, <i>CCNB1, AURKB</i>, <i>EZH2</i> and <i>PSMA5</i> were also upregulated in the SPs from eight primary myeloma samples. On that basis, we used an aurora kinase inhibitor (VX-680) and a proteasome inhibitor (bortezomib) with RPMI 8226 and AMO1 cells to determine whether these agents could be used to selectively target the myeloma SP. We found that both these drugs reduced the SP fraction, though bortezomib did so more effectively than VX-680 due to its ability to reduce levels of both phospho-histone H3 (p-hist. H3) and EZH2; VX-680 reduced only p-hist. H3. This is the first report to show that certain oncogenes are specifically expressed in the myeloma SP, and that bortezomib effectively downregulates expression of their products. Our approach may be useful for screening new agents with which to target a cell population possessing strong tumor initiating potential in multiple myeloma.</p> </div

Cell cycle analysis of SP and MP of MM cell lines.

<p>(A). Cell cycle pattern of RPMI 8226 cell with and without Hoechst 33342 stain. (B). Cell cycle analysis of RPMI 8226 SP cells and CD138⁺ and CD138⁻ MP cells. % of each cell cycle phase is as follows; SP: G₀/G₁ 34.5%, S 35.1%, G₂/M 30.4%, CD138⁺MP: G₀/G₁ 44.0%, S 50.5%, G₂/M 0.90%, CD138⁻MP: G₀/G₁ 84.1%, S 15.3%, G₂/M 0.61%. (C). G₀/G₁, S, and G₂/M fractions (%) among SP, CD138⁺ MP and CD138⁻ MP cells in RPMI 8226. Bars are means ± SD of three independent experiments. (D–F). Cell cycle analysis of SP and MP cells from the AMO1 (D), KMS-12-BM (E) and KMS-11 (F) cell lines. Cell cycle patterns in SP and MP cells from the indicated lines are shown beside bar graphs of %SP and %MP. Bars are means ± SD of three independent experiments.</p

Serial transplantation of myeloma SP into NOG mice.

<p>(A). Serial transplantation of SP and MP cells into NOG mice. Sorted SP and MP cells collected 31 days (RPMI 8226, upper panel) or 57 days (KMS-12-BM, under panel) after subcutaneous inoculation into NOG mice were restained with Hoechst 33342 dye (shown as 1^st generation). Sorted SP cells from 1^st generation collected 49 days (RPMI 8226) or 68 days (KMS-12-BM) are also shown (shown as 2^nd generation). Shown is the % SP. (B). CD138 staining of SP and MP cells of 1^st generation from NOG mice. Data of RPMI 8226 (upper panel) and KMS-12-BM (under panel) are shown.</p

Information of primary MM samples.

#1<p>PCM: plasma cell myeloma.</p>#2<p>ID: Initial Diagnosis.</p>#3<p>R: Recurrence.</p>#4<p>FISH: fluorescence in situ hybridization (100 count).</p>#5<p>gain: DNA copy number gain.</p>#6<p>ND: Not Done.</p>#7<p>del (deletion): DNA copy number loss.</p>#8<p>MP: melphalan+prednisolone.</p>#9<p>61,X,-X,-X,t(1;18)(p13;q21),+5,−8, −10, −12, −13, −14, +15, −16, add(17)(p11.2), −18,+19, −20, −22[5%].</p>#10<p>45,X,der(Y)t(Y;1)(q11.2;q12),add(1)(p13),add(5)(p15), −13, −14,+der(?)t(?;7)(?;q11.2);2/20; 46,XY [85%]. ^#1158<2n>,XY,+3,+5,+del(5)(q?),+6,+7,+8,+9,+10,+18,+19,+mar1[10%]/46,XY[90%]%].</p>#12<p>43,XX,t(6;14)(p21;q32), −13,?t(14;16)(q32;q23), −16, −22 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056954#pone.0056954-Greipp1" target="_blank">[4]</a>; 46,XX[80%].</p

SP cells of myeloma cell lines are showing CD138.

<p>(A). Fluorescence immunophenotyping assay of indicated cells for CD38 and CD138. X-axis: FITC; Y-axis: PE. (B). Flow cytometric dot plots for SP and MP of RPMI 8226 cell line, representative and CD138-PE staining in SP and MP. (C). Flow cytometric dot plots for myeloma SP (AMO1, KMS-12-BM, KMS-11) and representative CD138-PE staining in SP are shown. All immunophenotyping assays were performed by three individual experiments and obtained similar results. %CD138+ cells of AMO1 SP were ranged from 96.6 to 98.0%, KMS-12-BM SP were from from 99.1 to 100%, KMS-11 SP were from 98.5 to 100%.</p

Reduction of the SP in MM cells treated with bortezomib.

<p>(A). Frequency of apoptosis of RPMI 8226 and AMO1. Left panels: dot plots showing the frequency of apoptosis at the indicated bortezomib (Bor.) concentrations (48 hr exposure). X-axis: cells stained with AnnexinV-PE. Y-axis: cells stained with 7-AAD. Right panels: bar graphs showing the % apoptotic cells (R1+R2) among examined cells treated with indicated concentration of bortezomib at 24 hr and 48 hr as indicated. Asterisks (*) indicate statistical significance: *0.01≤<i>P</i><0.05, **0.001≤<i>P</i><0.01, ***<i>P</i><0.001. NS: not significant. (B). Cell cycle analysis RPMI 8226 and AMO1 treated with 10 nM bortezomib (24 hr). DMSO served as the control. RPMI 8226 control (+DMSO): subG₁ 3.9%, G₀/G₁ 48.3%, S 19.2%, G₂/M 28.6%; RPMI 8226+ bortezomib (10 nM): subG₁ 5.0%, G₀/G₁ 20.1%, S 20.4%, G₂/M 54.4%. AMO1 control (+DMSO): subG₁ 1.3%, G₀/G₁ 58.4%, S 18.5%, G₂/M 21.7%; AMO1+ bortezomib (10 nM): subG₁ 14.8%, G₀/G₁ 31.6%, S 23.6%, G₂/M 29.6% (C). Detection of M phase cells among bortezomib-treated (48 hr) myeloma cells. Bar graphs showing the numbers of M phase cells after treatment with DMSO, 1 nM, 10 nM and 100 nM bortezomib (24 hr exposure.) (D).Western blot analysis of p-Hist.H3 and EZH2 in RPMI 8226 (left panel) and AMO1 (right panel) cells after treatment with the indicated concentration of bortezomib and dexamethasone (48 hr). (E). Flow cytometric analysis of RPMI 8226 SP cells treated with bortezomib. Upper left panels: Dot plots of cells stained with Hoechst 33342 alone, Hoechst 33342 in the presence of 1 nM bortezomib for 48 h, or Hoechst 33342 in the presence of 10 nM bortezomib for 48 h. Lower left panels: cells treated as in the upper panels with 50 µM reserpine (shown as “res”). SP cell fractions (%) after treating RPMI 8226 and AMO1 cells are also shown besides the flow cytometric analysis. (F). CFC assay. Colonies of SP and MP by dexamethasone (left panel, Control (1 µl of 100% ethanol), 0.1 µM, 1 µM) and bortezomib (right pane, DMSO, 1 nM or 10 nM) for indicated cell lines. Asterisks (*) indicate statistical significance: *0.01≤<i>P</i><0.05, **0.001≤<i>P</i><0.01, ***<i>P</i><0.001. Bars are means ± SD of three independent experiments.</p

SP analysis in primary MM samples and gene expression analysis.

<p>(A). SP of primary samples (M4, M7 and M8). Left panel: cells obtained through bone marrow aspiration gated for CD138⁺ with and without 50 µM reserpine. SP fractions (%) are shown beside the SP gates surrounded by black lines. (B). Real time PCR analysis of eight samples of MM primary tumor cells. Shown bar graphs are <i>CCNB1, EZH2, AURKB</i> and <i>PSMA5</i> in SP and MP cells of indicated five myeloma cell lines. Y-axis: gray and white bars depict 2^−ΔΔCt values for gene expression. Asterisks (*) indicate statistical significance: *0.01≤<i>P</i><0.05, **0.001≤<i>P</i><0.01, ***<i>P</i><0.001. Bars are means ± SD of triplicate samples.</p

Real time quantitative PCR and western blot analyses of candidate markers of the SP in MM cells.

<p>(A). Real time quantitative PCR of cell cycle and mitosis related genes (<i>CCNB1,CDC2, CDC20, CDC25C, AURKB, BIRC5, TOP2A, ASPM),</i> Polycomb related genes (<i>EPC1, EZH2),</i> and ubiquitin-proteasome related gene (<i>UBE2D3</i> and <i>PSMA5)</i> against RPMI 8226, AMO1, KMS-12-BM, JJN3 and KMS-11 cells. Y-axis: gray and white bars depict 2^−ΔΔCt values for gene expression. Asterisks (*) indicate statistical significance: *0.01≤<i>P</i><0.05, **0.001≤<i>P</i><0.01, ***<i>P</i><0.001. Bars are means ± SD of three independent experiments. (B). Western blot analysis of Cyclin B1, CDC2, p-WEE1, p-CDC2, Aurora B, p-Aurora B, p-Hist.H3, EZH2, PSMA5 and GAPDH in SP and MP against RPMI 8226 and AMO1 cell lines.</p

Effect of Aurora kinase inhibitor (VX-680) on myeloma SP cells.

<p>(A). Cell cycle analysis of RPMI 8226 and AMO1 cells exposed toVX-680 (1 µM). X-axis, PI; Y-axis, cell count. RPMI 8226+DMSO: subG1 1.1%, G₀/G₁ 47.3%, S 18.5%, G₂/M 33.2%; RPMI 8226+VX-680 (1 uM): subG₁ 1.7%, G₀/G₁ 3.4%, S 16.4%, G₂/M 78.5%. AMO1+DMSO: subG₁ 1.9%, G₀/G₁ 82.5%, S 22.4%, G₂/M 12.9%; AMO1+VX-680 (1 uM): subG₁ 8.4%, G₀/G₁ 54.8%, S 10.5%, G₂/M 30.7%. (B). Detection of M phase cells among VX-680-treated MM cells. Upper panels: DAPI and p-Hist.H3 staining (green) of cells treated with DMSO, 1 µM, and 10 µM VX-680 (24 hr exposure). Under panels: bar graphs showing the numbers of M phase cells after treatment with the indicated concentration of VX-680 (24 hr exposure). (C). Western blot analysis of p-Hist.H3, EZH2 in RPMI 8226 (left panel) and AMO1 (right panel) cells; Tubulin is the control. (D). Flow cytometric analysis of RPMI 8226 SP cells. Dot plots of cells stained with Hoechst 33342 alone, Hoechst 33342 in the presence of 1 µM VX-680 or Hoechst 33342 in the presence of 10 µM VX-680. Left upper panels: 24 h exposure to VX-680; left lower panels: 48 h exposure to VX-680. Bar graphs of SP cell fractions (%) of indicated cells treated with VX-680 (DMSO, 1 µM, 10 µM) for 24 hr or 48 hr are also shown besides the flow cytometric analysis. DMSO is the control. Asterisks (*) indicate statistical significance: *0.01≤<i>P</i><0.05, **0.001≤<i>P</i><0.01, ***<i>P</i><0.001. Bars are means ± SD of triplicate samples. (E). CFC assay. Colonies of SP by VX-680 (DMSO, 1 µM, 10 µM) for RPMI 8226 and AMO1 cell lines. Colony count was examined after 10 days from SP or MP distribution.</p