Page 298 - 2021_02-Haematologica-web

Letters to the Editor
ALCL tumors express different levels of miR-939 (Figure 1C and Online Supplementary Table S3).
To decipher the functional role of miR-939 in ALCL, we used Karpas299 and SUP-M2 cells, characterized by different basal levels of the miRNA (Figure 2A). We tran- siently overexpressed miR-939 or inhibited its expression in these cell lines (Online Supplementary Figure S1) that are more similar to ALK-high ALCL cells than to the ALK- low counterpart.6 The overexpression of miR-939 signifi- cantly reduced the migratory ability of both ALCL cell lines as compared to negative control cells, while its inhi- bition was shown to significantly increase ALCL cell migration (Figure 2B). Moreover, miR-939 overexpression reduced both Karpas299 and SUP-M2 clonogenic growth capacity (Figure 2C). In contrast, its inhibition significant- ly increased Karpas299 clonogenicity, while a less notice- able effect was observed on SUP-M2, which might be in line with the lower basal level of miR-939 in SUP-M2 compared to Karpas299 cells (Figure 2A). Of note, NPM- ALK expression was not affected by miR-939 overexpres- sion, thus suggesting that miR-939 does not contribute mechanistically to the ALK-high or ALK-low status (Online Supplementary Figure S2).
In silico prediction of miR-939 targets and Gene Ontology analysis were performed by using miRTarBase 7.0 (http://mirtarbase.mbc.nctu.edu.tw/php/index.php) and Panther 14.1 (http://pantherdb.org/data/) online tools. Among transcription factors and nucleic acid binding proteins, which were highly represented in the miR-939 putative target list, we focused on the transcriptional fac- tor JUNB as the most promising candidate for our analy-
ses, since its knockdown in NPM-ALK expressing cells was previously shown to impair cell proliferation.9 Indeed, transient overexpression of miR-939 decreased JUNB protein expression both in Karpas299 and SUP-M2 cells (Figure 2D). Moreover, direct modulation of JUNB by miR-939 was confirmed by 3’UTR reporter assay in both Karpas299 and SUP-M2 cell lines transfected with miR-939 or negative control and reporter vectors con- taining wild-type and mutated miR-939 binding site in JUNB 3’-UTR sequence (Figure 2E). The miR-939 recog- nition site was identified by using the online tools at http://www.microrna.org (Figure 2F).
JUNB is an activator protein-1 (AP-1) transcription factor, together with JUN, JUND and also members of the Fos/Fra, ATF and Maf subfamilies. AP-1 proteins exert their activity by forming homo- and hetero-com- plexes, and mainly regulate the expression of genes involved in cell proliferation.10 JUNB overexpression was shown in CD30+ lymphomas and its transcriptional activity was directly implicated in CD30 promoter induction in both Hodgkin lymphoma and ALCL.11 NPM-ALK itself contributes to JUNB activation and CD30 expression in ALK+ALCL via both ERK1/2-MAPK and mTOR pathways.9,12 JUNB and JUN activities were also shown to sustain PI3K-associated activation of AKT1, further suggesting the existence of a synergistic crosstalk between NPM-ALK signaling and AP-1 tran- scription factors.13 More recently, platelet-derived growth factor receptor B (PDGFRB) was confirmed as a JUNB transcriptional target in NPM-ALK-driven lym- phomas and the combination of ALK and PDGFR
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Figure 3. PDGFRB expression according to miR-939 levels in anaplastic lymphoma kinase-positive (ALK+) anaplastic large cell lymphoma (ALCL) cell lines and ALCL patients’ samples. (A) PDGFRB protein immunoprecipitation in Karpas299 and SUP-M2 cell lines transfected with miR-939 mimic or negative control at indicated post-transfection time points; western blotting analysis of γ-tubulin was used as immunoprecipitation input control. Blk: blank. (B) Box plot of PDGFRB mRNA expression in patients expressing high or low levels of miR-939 (n=25 miR-939_low [≤ median value]; n=24 miR-939_high [> median value]) detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR). *P=0.03. Data have been calculated according to the comparative dCt method and com- pared to reactive lymph node tissues (LN, n=11). ABL1 was used as endogenous control. (C) Dot plot representing PDGFRB protein expression assessed by RPPA in ten available ALCL tumor tissue samples (5 miR-939_low, 5 miR-939_high; *P=0.01). (D) Schematic representation of miR-939-mediated JUNB modulation in ALK-low/-high ALCL. In the presence of lower levels of NPM-ALK transcript, miR-939 is able to inhibit JUNB transcriptional activity, resulting in PDGFRB down- regulation (black arrows). Gray arrows are referred to previous data from the literature.12
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