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Multicenter Study
. 2008 Jan 15;111(2):806-15.
doi: 10.1182/blood-2007-07-101139. Epub 2007 Oct 12.

Overexpression and involvement in migration by the metastasis-associated phosphatase PRL-3 in human myeloma cells

Unn-Merete Fagerli  1 , Randi U Holt, Toril Holien, Thea K Vaatsveen, Fenghuang Zhan, Kjartan W Egeberg, Bart Barlogie, Anders Waage, Harald Aarset, Hong Yan Dai, John D Shaughnessy Jr, Anders Sundan, Magne Børset
Affiliations
Multicenter Study

Overexpression and involvement in migration by the metastasis-associated phosphatase PRL-3 in human myeloma cells

Unn-Merete Fagerli et al. Blood. .

Abstract

Multiple myeloma (MM) is characterized by accumulation and dissemination of malignant plasma cells (PCs) in the bone marrow (BM). Gene expression profiling of 2 MM cell lines (OH-2 and IH-1) indicated that expression of PRL-3, a metastasis-associated tyrosine phosphatase, was induced by several mitogenic cytokines. Cytokine-driven PRL-3 expression could be shown in several myeloma cell lines at both the mRNA and protein levels. There was significantly higher expression of the PRL-3 gene in PCs from patients with monoclonal gammopathy of undetermined significance (MGUS), smoldering myeloma (SMM), and myeloma than in PCs from healthy persons. Among 7 MM subgroups identified by unsupervised hierarchical cluster analysis, PRL-3 gene expression was significantly higher in the 3 groups denoted as "proliferation," "low bone disease," and "MMSET/FGFR3." PRL-3 protein was detected in 18 of 20 BM biopsies from patients with MM. Silencing of the PRL-3 gene by siRNA reduced cell migration in the MM cell line INA-6, but had no detectable effect on proliferation and cell-cycle phase distribution of the cells. In conclusion, PRL-3 is a gene product specifically expressed in malignant plasma cells and may have a role in migration of these cells.

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Figures

Figure 1
Figure 1
Expression of PRL-3 mRNA and protein after cytokine stimulation. (A) Quantification of PRL-3 mRNA by real-time RT-PCR was performed after stimulation of IH-1 and OH-2 cells either with or without IL-6 and IL-21 for 24 hours. The ΔCt value between PRL-3 and β-actin from the unstimulated cells reflects the expression of PRL-3 relative to its internal control β-actin. The ΔCt value for the unstimulated IH-1 cells was 2.68 with a standard variation of 0.17 and for the unstimulated OH-2 cells it was 22.49 with a standard variation of 0.31. Thus, standard variation for all ΔCt values from RT-PCR reactions was between 1% and 2% of its ΔCt value. The relative expression level of PRL-3 to β-actin in the unstimulated cells was represented by 2−ΔCt and was arbitrarily set to 1. The relative expression of PRL-3 to β-actin in the stimulated cell culture, 2−ΔCt, was then normalized to that of the unstimulated cell culture, 2−ΔCt(unstim) / 2−ΔCt(stim), and is illustrated on the y-axis. (B) PRL-3 expression was determined by Western analysis after an 18-hour incubation with various cytokines. Cytokine concentrations were as follows: IL-6, 5 ng/mL; IL-21, 20 ng/mL; IL-15, 20 ng/mL; TNF, 10 ng/mL; HGF, 150 ng/mL; IGF-1, 100 ng/mL, and SDF-1, 75 ng/mL. The loading control was GAPDH (bottom panels).
Figure 2
Figure 2
Expression of PRL-3 mRNA in myeloma and nonmyeloma cell lines. The ΔCt between PRL-3 and β-actin for CAG was 8.1 (± 0.58). The relative expression level of PRL-3 to β-actin in the CAG cells, 2−ΔCt, was arbitrarily set to 1. The relative expression level of other cell lines was normalized to that of CAG cells and was represented by 2−ΔΔCt, as is illustrated in this figure.
Figure 3
Figure 3
PRL-3 gene expression in normal PCs, MM cell lines, and PCs from patients with MGUS, SMM, and MM. (A) PRL-3 gene expression in PCs from 22 control subjects with normal BM, 44 patients with MGUS, 12 patients with SMM, 256 patients with MM, and in 45 myeloma cell lines. The Affymetrix signal, a quantitative measure of gene expression, is indicated on the y-axis. The level of expression of PRL-3 in each sample is indicated by the height of the bar. Samples are ordered from the lowest to highest level of expression of the PRL-3 gene, from left to the right on the x-axis. The table shows the results of the chi-square test by comparison of the Affymetrix Detection signal among the groups. The numbers in boxes above panels A and B indicate the Affymetrix annotation number of the PRL-3 gene. (B) PRL-3 gene expression in MM subgroups. The correlation of the Affymetrix signal (expression level: vertical axis) of PRL-3 with 7 myeloma subgroups from the 256 cases is shown on the top panel. The expression levels for PRL-3 are proportional to the height of each bar (representing a single patient sample). PRL-3 was significantly overexpressed in the proliferation (PR), MMSET/FGFR3, and low-bone disease (LB) groups. The table in the bottom panel shows the chi-square test for Affymetrix detection of PRL-3 among the 7 subgroups.
Figure 4
Figure 4
Fluorescent in situ hybridization with centromer and PRL-3 probe on MM cell lines and CRO-AP5. The results of FISH analysis with PRL-3 probe (SpectrumOrange) and centromer probe (SpectrumAqua). The red signals mark PRL-3 (8q24.3) and the aqua marks the centromer on chromosome 8. The cell nucleus is stained with DAPI. Original magnification is ×1000. (A) The OH-2 cell line has a normal chromosome 8 with 2 centromer signals and 2 PRL-3 signals. (B) The RPMI-8226 cell line has 2 centromer signals and 3 PRL-3 signals. (C) The CRO-AP5 cell line has 2 centromer signals and 4 to 5 PRL-3 signals, indicating that the area where the PRL-3 gene is located is amplified. (D) The IH-1 cell line has 5 PRL-3 signals and 5 centromer signals, demonstrating polyploidy of chromosome 8.
Figure 5
Figure 5
Expression of PRL-3 in malignant PCs from myeloma patients evaluated by immunohistochemistry. We stained BM biopsies from 20 patients, and found that 18 patients were PRL-3–positive to different degrees. In 11 of the biopsies more than 50% of the PCs were stained. A representative biopsy is illustrated. Original magnification ×400.
Figure 6
Figure 6
Staining of OH-2 cells in interphase and patient cells in interphase and metaphase with PRL-3. Goat polyclonal antibody against PRL-3 is shown in green. Nuclei visualized with Draq 5 are shown in red. (A) OH-2 cells in interphase. (B) Patient cells in interphase and metaphase. Examination by confocal microscopy.
Figure 7
Figure 7
The effect of down-regulation of PRL-3 expression on migration, proliferation, and cell cycle in INA-6 cells. (A) Transfection of PRL-3 siRNA reduced SDF-1–induced migration in INA-6 cells as compared with transfection with control siRNAs. Wild-type INA-6 cells are cells stimulated with SDF-1 but not transfected. (B) PRL-3 siRNA as well as control siRNAs did not influence the proliferation of INA-6 cells. (C) PRL-3 siRNA did not induce cell-cycle arrest. The percentages of cells in G0/G1, S, and G2M in wild-type INA-6 were 35.2%, 44.9%, and 19.9%, respectively. In transfected cells, the percentages were 32.3%, 44.4%, and 23.4% in siRNA-negative control; 29.7%, 46.2%, and 24.1% in positive control siRNA cyclophilin B; and 32.2%, 46.6%, and 21.3% in siRNA PRL-3, respectively. (D) The down-regulation of the PRL-3 protein was verified on Western blot. Similar results were shown in 3 independent experiments. Error bars represent +1 standard deviation of 5 (migration) and 4 (thymidine incorporation) measurements.
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