miR-221/222: new insights in Burkitt Lymphoma

Burkitt Lymphoma (BL) is a highly aggressive B cell non Hodgkin lymphoma. It’s considered the fastest growing human tumor and it is commonly associated with EBV infection. It was the first type of cancer shown to have a chromosomal translocation that activates c-Myc to become an oncogene. This genetic rearrangement places myc that usually is on chromosome 8 under the immunoglobulin gene regulatory element on chromosome 14 resulting in tumor promoting effect. BL is a highly malignant B-cell neoplasm that occurs endemically in equatorial Africa and sporadically throughout the world. The endemic BL (eBL) is the pediatric form positive for EBV in most cases. The variant of BL affecting the rest of the world is the sporadic BL (sBL) which is found in older patients and it is considered EBV negative because only a minority is EBV infected. The World Health Organization recognizes also a third form, HIV-BL which develops in HIV positive patients. Although, until now the translocation 8-14 and its variants are considered the major mechanism for the pathogenesis of BL, other molecular mechanism such as microRNA expression profile have been used to characterize and classify different types of BL from other lymphoma malignancies. However, the differential expression of microRNAs between BL patients and healthy control has not been studied before. For this reason our goal is to investigate the functional role of microRNAs that are disregulated in BL patients compared to healthy (cancer free) individuals. MicroRNAs are noncoding RNA, 18-24 nucleotides long. They are transcribed in the nucleus as long primary transcripts, and then cut by Drosha and DGCR8 into 70 nucleotides long precursors (pre-miRNA). This Pre-Mir is exported to the cytoplasm by Exportin-5 and then cleaved into a mature dsRNA by Dicer. Only one strand of the duplex miRNA-miRNA* binds the target mRNA to modulate the gene expression through two principle mechanisms which are the degradation of mRNA or the inhibition of the protein translation. To gain further insight into the molecular pathology of BL, we performed miRNA expression profile using a set of 5 sporadic, and 2 endemic BL patients, compared to B cells from reactive lymph nodes of 9 healthy patients and 11 patients affected by mononucleosis. MiRNAs expression signature shows, among the group of downregulated miRNAs, miR-221 that usually is upregulated in solid tumors. This is the first microRNA profiling that has been done in BL using as negative control lymph nodes taken from reactive patients or patients affected by the EBV virus, whereas the literature shows microRNA profiles in BL using as negative control T cells or different type of B cell lymphomas like for example DLBL (diffuse large B cell lymphoma). To confirm the remarkable down-regulation of miR-221 a nanoString analysis in 2 different cohorts of BL cell lines was also performed. We observed a common trend of altered expression of microRNAs, highlighting once again the down-regulation of miR-221/222, suggesting a different role of these miRNA in liquid tumours compared to their well-known pro-tumorigenic function in epithelial tumors The down-modulation of miR-221/222 was also confirmed by the qRT-PCR method in a bigger cohort of BL cell lines compared to 4 normal B lymphoblast EBV transformed cell lines. The four cell lines representing the controls express high levels of miR-221 compared to the group that represents the BL cell lines where the miR-221 is lost. The same trend is shown for miR-222. We found that interesting considering the up-regulation of miR-221 and miR-222 previously confirmed in a lot of solid tumors by multiple studies, such as breast, liver and lung cancer. Here, we are investigating a different role of the cluster 221/222 in lymphomas that have a different process in carcinogenesis than solid tumors. To better understand the potential role of miR-221/222 in BL, we also analyzed their expression levels in EµMyc transgenic mouse model which has been considered for a decade a good in vivo model of BL. We investigated the expression of miR-221 and 222 in B cells extracted from both transgenic and wild type mice. The miRNA levels detected by qRT-PCR show a down-regulation in 80% of the transgenic samples when compared to normal B cells derived from the spleen of wild type mice littermates. Once we determined that miR-221 and 222 were down regulated in both human and mouse models, we wanted to understand what pathways both of the models had in common and how miR-221 and 222 play a role in these pathways. Therefore, in order to establish the effect of miR-221 and 222 in a human model, we transfected BL cell line Bjab, which lacks of miR-221 and 222 expression with mature miRNA. A gene expression analysis was then conducted on extracted RNA from treated Bjab cells collected 48 hours after transfection compared to its negative control collected at the same time point. We then performed a parallel gene expression profile on the mouse model using the RNA extracted from CD-19+ of the wild type spleen and the transgenic spleen of littermates. Then we picked the down-regulated gene of the human gene expression profiling and compared it to the gene expression profile of the mouse model. From this comparison, we found some genes that were up-regulated in the mouse model that were also down-regulated by miR-221 and 222 in the human model. One of these genes is DUSP6/MKP-3, a MAP kinase phosphatase that dephosphorylate phosphothreonine and phosphotyrosine within ERK pathway, playing a role in the induction of apoptosis. This dual specificity phosphatase has been found also acting as an oncogene but no further studies have been conducted, leaving its function in a contradictory background. The level of expression in BL cell lines of DUSP6 has been evaluated by qRT-PCR, compared to negative lymphoblastoid cell lines and results show an up-regulation of the mRNA in 80% of BL cell lines whereas it’s lost in the controls, suggesting an oncogenic role of this protein in BL but additional studies need to be performed to confirm this hypothesis. Since these new findings may highlight a different role of these miRNA in BL compared to their well-known pro-tumorigenic function in epithelial tumors we cross a miR-221/222 KO mouse with the well-known EµMyc transgenic mouse model. The miR-221/222 KO doesn’t show any particular phenotype but when we breed the KO with the transgenic EµMyc we observe an early development of the BL pathogenesis in 5 out of 8 miR-221/222 KO/ EµMyc tg positive and death at 3-4 months of age while the wild type miR-221/222/ EµMyc tg are still alive at 6 months of age without showing any enlarged lymph nodes. Unfortunately, even these preliminary results indicate that the loss of miR-221/222 can play an important role in the pathogenesis of BL, the number of wild type miR-221/222 are not enough for the statistical analysis; for this reason we are increasing the numbers of litters and we need further investigations. Our findings indicate that miR-221/222 can be critical mediators for BL pathogenesis and together with other important genetics alteration such as translocation of MYC can lead to the aggressive phenotype that this B cell malignancy usually shows. These results highlight the potential role of this cluster of microRNAs to be a good tool of diagnosis and prognosis for BL