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Comparative Study
. 2010 May;95(5):785-93.
doi: 10.3324/haematol.2009.014464. Epub 2009 Dec 16.

Cancer-testis antigens MAGE-C1/CT7 and MAGE-A3 promote the survival of multiple myeloma cells

Djordje Atanackovic  1 , York Hildebrandt, Adam Jadczak, Yanran Cao, Tim Luetkens, Sabrina Meyer, Sebastian Kobold, Katrin Bartels, Caroline Pabst, Nesrine Lajmi, Maja Gordic, Tanja Stahl, Axel R Zander, Carsten Bokemeyer, Nicolaus Kröger
Affiliations
Comparative Study

Cancer-testis antigens MAGE-C1/CT7 and MAGE-A3 promote the survival of multiple myeloma cells

Djordje Atanackovic et al. Haematologica. 2010 May.

Abstract

Background: Multiple myeloma is a life-threatening disease and despite the introduction of stem cell transplantation and novel agents such as thalidomide, lenalidomide, and bortezomib most patients will relapse and develop chemoresistant disease. Therefore, alternative therapeutic modes for myeloma are needed and cancer-testis antigens such as MAGE-C1/CT7 and MAGE-A3 have been suggested to represent a class of tumor-specific proteins particularly suited for targeted immunotherapies. Surprisingly, the biological role of cancer-testis genes in myeloma remains poorly understood.

Design and methods: We performed the first investigation of the function of two cancer-testis antigens most commonly expressed in myeloma, MAGE-C1/CT7 and MAGE-A3, using an RNA interference-based gene silencing model in myeloma cell lines. Functional assays were used to determine changes in proliferation, cell adhesion, chemosensitivity, colony formation, and apoptosis resulting from gene-specific silencing.

Results: We show that the investigated genes are not involved in regulating cell proliferation or adhesion; however, they play an important role in promoting the survival of myeloma cells. Accordingly, knock-down of MAGE-C1/CT7 and MAGE-A3 led to the induction of apoptosis in the malignant plasma cells and, importantly, both genes were also essential for the survival of clonogenic myeloma precursors. Finally, silencing of cancer-testis genes further improved the response of myeloma cells to conventional therapies.

Conclusions: Cancer-testis antigens such as MAGE-C1/CT7 and MAGE-A3 play an important role in promoting the survival of myeloma cells and clonogenic precursors by reducing the rate of spontaneous and chemotherapy-induced apoptosis and might, therefore, represent attractive targets for novel myeloma-specific therapies.

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Figures

Figure 1.
Figure 1.
Cancer-testis genes MAGE-A3 and MAGE-C1/CT7 are constitutively expressed in myeloma cells and transfection with inhibitory RNAi results in gene-specific down-regulation of both proteins. (A) ten myeloma cell lines were investigated for the expression of MAGE-A3 and MAGE-C1/CT7 protein by western blot. β-actin (ACTB) served as an internal control for protein quality and protein lysate of normal human testis was used as a positive control. Notably, all ten myeloma cell lines showed constitutive expression of both CT antigens. (B) Myeloma cell lines Molp-8 (left column) and RPMI-8226 (right column) were transfected using two RNAi constructs specific for CT genes MAGE-A3 and MAGE-C1/CT7, respectively, or with scrambled control RNAi. Treatment resulted in knockdown of the expression of MAGE-A3 and MAGE-C1/CT7 protein in both cell lines starting 48–72 h after transfection as indicated by immunoblotting. Gene-silencing lasted at least until day 7 after transfection. (C) Importantly, treatment with gene-specific RNAi only affected the expression of the target genes and not the expression of other CT antigens tested (MAGE-A4, Ropporin-1, NY-ESO-1). The only exception was CT gene MAGE-C2/CT10, the expression of which was suppressed following transfection with RNAi targeting MAGE-C1/CT7. Comparable findings were observed following gene-silencing in cell line RPMI-8226 and using a second gene-specific RNAi construct (data not shown).
Figure 2.
Figure 2.
Silencing of CT genes MAGE-A3 or MAGE-C1/CT7 exerts a strong pro-apoptotic effect on myeloma cells. (A) Myeloma cell lines Molp-8 (left column) and RPMI-8226 (right column) were transfected with two RNAi constructs specific for CT genes MAGE-A3 or MAGE-C1/CT7. Numbers of viable myeloma cells transfected with RNAi specific for the target genes or of cells transfected with scrambled control RNAi were normalized to the number of viable untreated cells. Silencing of MAGE-A3 or MAGE-C1/CT7 resulted in significantly decreased cell viability at 72 h after transfection as indicated by an MTT assay. Results show mean values [+ standard error of means (SEM)] of three separate experiments and asterisks indicate statistically significant differences between untreated cells and cells transfected with scrambled control or one gene-specific RNAi (*P<0.05). (B) Percentages of apoptotic cells in Molp-8 cultures were analyzed in a flow cytometry-based TUNEL assay at 72 h after transfection with RNAi specific for CT genes MAGE-A3 or MAGE-C1/CT7. Bars indicate mean values (+SEM) of three separate experiments. Percentages of apoptotic myeloma cells transfected with specific or scrambled control RNAi were compared to those of untreated cells and asterisks indicate statistically significant differences (*P<0.05). Silencing of MAGE-A3-or MAGE-C1/CT7, resulted in significantly and specifically increased rates of apoptosis. Histograms indicate findings of a representative experiment analyzing TUNEL expression in cells transfected with control RNAi (gray area) or gene-specific RNAi (black line). Cell sorting according to annexin V-staining and subsequent immunoblot indicated that, in contrast to the apoptotic population, viable cells showed significant protein expression of either target gene following specific siRNA transfection. Comparable findings were observed following gene-silencing in cell line RPMI-8226 (data not shown). (C) Microscopic analysis of one representative experiment shows strong expression of green fluorescence in TUNEL-positive myeloma cells following silencing of MAGE-A3 or MAGE-C1/CT7. (D) Increased apoptosis in Molp-8 myeloma cells was accompanied by an increased expression of caspases 12 and 9, but not caspase-8 or caspase-3, in a western blot assay at 72 h after MAGE-A3 or MAGE-C1/CT7 knockdown.
Figure 3.
Figure 3.
Silencing of CT genes MAGE-A3 or MAGE-C1/CT7 results in a decreased outgrowth of myeloma precursors in a colony formation assay. Previous studies have shown that myeloma precursors are also present in the bulk population of myeloma cell lines and that these cells can be identified by their lack of CD138 expression. FACS-sorting CD138+ and CD138- subpopulations we show here by western blot analysis that myeloma precursors derived from cell lines Molp-8 and KMS-12-BM as well as conventional myeloma plasma cells express MAGE-A3 and MAGE-C1/CT7 (A). In a clonogenic growth assay, colonies of myeloma precursors were counted 7–10 days after culture initiation using myeloma cell lines Molp-8 (left column) and RPMI-8226 (right column) separately transfected with two RNAi constructs specific for CT genes MAGE-A3 or MAGE-C1/CT7, respectively (B). Bars indicate mean values (+SEM) of three separate experiments. Numbers of colonies produced by myeloma cells transfected with RNAi specific for the target genes or of cells transfected with scrambled control RNAi were compared with colony numbers in cultures with untreated cells and asterisks indicate statistically significant differences (*P<0.05). Photos show results of representative analyses. Silencing of MAGE-A3- or MAGE-C1/CT7 resulted in significantly and specifically decreased outgrowth of myeloma precursors.
Figure 4
Figure 4
Silencing of CT genes MAGE-A3 or MAGE-C1/CT7 only exerts a minor influence on the proliferation of myeloma cells. Proliferation of myeloma cell line Molp-8 following silencing of MAGE-A3- or MAGE-C1/CT7 was assessed by a BrdU incorporation assay. Proliferation was measured 72 h after transfection with RNAi specific for the target gene or with scrambled control RNAi and results were compared to untreated cells. Bars show mean values (+SEM) of three separate experiments and asterisks indicate statistically significant differences between untreated cells and scrambled control or gene specific RNAi (*P<0.05).
Figure 5.
Figure 5.
Silencing of MAGE-A3- or MAGE-C1/CT7 has an additive effect on cell death when combined with conventional myeloma-specifif therapies. The myeloma cell line Molp-8 was transfected with two RNAi constructs specific for CT genes MAGE-A3 (left column) or MAGE-C1/CT7 (right column) or with scrambled control RNAi. Starting 72 h after transfection, myeloma cells were treated with conventional myeloma-specific therapies melphalan (A) or bortezeomib (B). Twelve hours later, myeloma cell viability was assessed by an MTT assay and was normalized using untreated cells. Results show mean values (+SEM) of three separate experiments and asterisks indicate statistically significant differences between untreated cells and scrambled control or gene-specific RNAi (*P<0.05, **P<0.01). Comparable findings were observed following gene-silencing in the RPMI-8226 cell line and using a second gene-specific RNAi construct (data not shown).
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