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CDK4/CDK6 inhibitors in ALK-positive lymphomas
expressed by transfecting synthetic mimics into KARPAS- 299, SU-DHL1 and COST cell lines. We observed that upregulation of miR-195 by transfection with miR-195 mimic does not impair cell viability of NPM-ALK+ cells (Figure 3A). By contrast, upregulation of miR-497 signifi- cantly affects cell viability of the three cell lines (Figure 3B). The successful overexpression of miR-497 and of miR-195 were confirmed by qPCR (Online Supplementary Figure S3). Taken together, these data suggest that only miR-497 in the miR-497/miR-195 cluster plays a role in cell proliferation of NPM-ALK+ cells.
Since cell proliferation is closely associated with cell cycle control and progression, we performed flow cyto- metric analysis of cell cycle with propidium iodide DNA staining to examine the effects of miR-497 on cell cycle distribution in all three NPM-ALK+ cell lines. Accumulation in the G1 phase of the cell cycle was signif- icant in KARPAS-299 cells, 48 h post transfection with miR-497 compared with those transfected with miR-CTL (Figure 4A). In addition, in the same condition, ectopic expression of miR-497 in COST and SU-DHL1 cells led to a significant accumulation of cells in a sub-G1 phase of cell cycle suggesting cell death (Figure 4A). To test whether apoptosis is involved in the growth inhibition caused by miR-497 overexpression, we measured caspase 3 and 7
activity. As shown in Figure 4B, compared to that of miR- CTL, miR-497 overexpression induced activation of apop- tosis pathways in COST and SU-DHL1 cells (2.2- and 4- fold, respectively) but not in KARPAS-299 cells. These findings suggest that miR-497 overexpression induces growth-inhibitory effects on cancer cells by impeding cell cycle progression and leads to programmed cell death. We next investigated the effect of miR-497 transfection on tumor growth of NPM-ALK+ cell lines KARPAS-299, SU- DHL1 and COST in vivo. MiR-CTL or miR-497-transfected NPM-ALK+ cells were inoculated subcutaneously into the left or right flanks of each immunodeficient mouse, respectively (n = 6 for each condition). MiR-497-transfect- ed NPM-ALK+ cells resulted in significant reduction of tumor growth (Figure 4C) when compared to those trans- fected with miR-CTL. Morphological analyses showed that forced expression of miR-497 in NPM-ALK+ cells caused phenotypic hallmarks of cellular degeneration (uncommon chromatin condensation, nuclear piknosis, decreased cellular volume and disruption of the nuclear envelope) (Figure 4 D).These results show that miR-497 overexpression drastically reduces the growth of NPM- ALK+ cells in vivo. Taken together, these experiments sug- gest a tumor-suppressive function of miR-497 in NPM- ALK+ cells.
AB
C
Figure 5. MiR-497 targets G1/S regulators in anaplastic lymphoma kinase (ALK)-positive(+) lymphoma cells. (A) mRNA targets of miR-497 identification workflow. NPM-ALK+ KARPAS-299 cells transfected with biotinylated forms of human miR-497 (biotin-miR-497) or an irrelevant biotin-miRNA as negative control (biotin-miR- CTL) were used to co-purify associated mRNAs (enrichment ratio >1.5). Validated candidate targets of miR-497 were collected from miRTarBase (n=77) and experi- mentally validated miR-497 associated with the regulation of cell cycle from miRSystem database (n=16). CCNE1, CDC25A, CDK6, CCND3 and E2F3 were the only genes identified in the two computational analyses. (B) Microarray analysis of CCNE1, CDC25A, CDK6, CCND3 and E2F3 mRNA expression in human NPM-ALK+ (n=56; Daugrois et al., submitted publication). Data were normalized against equivalent miRNA levels from reactive lymph (RLN) node tissue samples (n=3). Data represent means±Standard Error of Mean (bars), *P<0.05; **P<0.001; ***P<0.0001; unpaired two-tailed Student’s t-test with Welch’s correction. (C) Quantitative real-time PCR (qRT-PCR) analysis of CCNE1, CDC25A, CDK6, CCND3 and E2F3 mRNA was performed after pulldown of the biotinylated miRNAs using streptavidin beads. The results are presented as the relative enrichment in miR-497 pulldown compared to miRNA control pulldown (enrichment ratio >2).
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