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The anaplastic lymphoma kinase (ALK), tyrosine kinase oncogene is implicated in a wide variety of cancers. In this study we used conditional onco-ALK (NPM-ALK and TPM3-ALK) mouse MEF cell lines (ALK+ fibroblasts) and transgenic models (ALK+ B-lymphoma) to investigate the involvement and regulation of angiogenesis in ALK tumor development. First, we observed that ALK expression leads to downregulation of miR-16 and increased Vascular Endothelial Growth Factor (VEGF) levels. Second, we found that modification of miR-16 levels in TPM3-ALK MEF cells greatly affected VEGF levels. Third, we demonstrated that miR-16 directly interacts with VEGF mRNA at the 3'-untranslated region and that the regulation of VEGF by miR-16 occurs at the translational level. Fourth, we showed that expression of both the ALK oncogene and hypoxia-induced factor 1α (HIF1α) is a prerequisite for miR-16 downregulation. Fifth, in vivo, miR-16 gain resulted in reduced angiogenesis and tumor growth. Finally, we highlighted an inverse correlation between the levels of miR-16 and VEGF in human NPM-ALK+ Anaplastic Large Cell Lymphomas (ALCL). Altogether, our results demonstrate, for the first time, the involvement of angiogenesis in ALK+ ALCL and strongly suggest an important role for hypoxia-miR-16 in regulating VEGF translation.

Although deregulated expression of specific microRNAs (miRNAs) has been described in solid cancers and leukemias, little evidence of miRNA deregulation has been reported in ALK-positive (ALK(+)) anaplastic large cell lymphomas (ALCL). These tumors overexpress the major antiapoptotic protein myeloid cell leukemia 1 (MCL-1), a situation that could compensate for the lack of BCL-2. We report that ALK(+) ALCL cell lines and biopsy specimens (n = 20) express a low level of miR-29a and that this down-modulation requires an active NPM-ALK kinase. Murine models (transgenic mice and mouse embryonic fibroblast [MEF] cells), which allow conditional NPM-ALK fusion protein expression, showed an increase of miR-29a expression in the absence of NPM-ALK. Concordant results were observed after the abolition of NPM-ALK kinase activity (siALK or PF-2341066) in NPM-ALK(+) ALCL cell lines. In addition, we showed that low expression of miR-29a, probably through methylation repression, plays an important regulatory role in MCL-1 overexpression that could promote tumor cell survival by inhibiting apoptosis. Enforced miR-29a expression was found to modulate apoptosis through inhibition of MCL-1 expression in ALCL cell lines and in a xenografted model, with a concomitant tumor growth reduction. Thus, synthetic miR-29a represents a potential new tool to affect tumorigenesis in these lymphomas.

The CCAAT/enhancer-binding protein β (C/EBPβ) plays a major role in the pathogenesis of anaplastic large cell lymphomas (ALCL) that express the nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) tyrosine kinase (ALK(+)). Although ALK-mediated C/EBPβ transcriptional activation has been reported, C/EBPβ mRNA possesses U- and AU-rich domains in its 3'-untranslated region (3'-UTR) that might be privileged targets for posttranscriptional control in ALK(+) ALCLs. The purpose of this study was to explore this possibility. By using human ALCL-derived cells and a murine model of ALK-transformed cells, we show that the AU-binding protein HuR binds to the 3'-UTR of C/EBPβ mRNA, as previously reported in adipocytes, and that NPM-ALK enhances this interaction. Interaction between HuR and C/EBPβ mRNA impacts on C/EBPβ gene expression at both the mRNA and protein levels. Indeed, C/EBPβ mRNA stability following HuR silencing is reduced and reaches the value observed in ALK-inactivated cells. Remarkably, HuR expression is not modified by NPM-ALK, but its association with actively translating polysomes is dramatically increased in ALK(+) cells. HuR/polysomes association diminishes when NPM-ALK activity is inhibited and is accompanied by a concomitant decrease of C/EBPβ mRNA translation. Finally, we show that HuR and NPM-ALK colocalized in cytoplasmic granules and HuR is phosphroylated on tyrosine residues in ALK(+) ALCL cells. Our study thus demonstrates that C/EBPβ is indeed regulated at the posttranscriptional level by HuR in ALK(+) cells, leading us to propose that part of NPM-ALK oncogenic properties relies on its ability to modify HuR properties in the cytoplasm and hence to alter expression of key actors of transformation., (©2011 AACR.)