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A selective role for CDCA7 in lymphomagenesis
The comparison of the transcriptome of 5 BL cell lines (DG-75, Ramos, BL2, Mutu-I and Akata) with a pool of 3 LCL cell lines (X50-7, IB-4 and Dana) using whole-genome microarrays showed >1,600 genes similarly expressed in BL cells that were up- (630 genes) or down-regulated (1,033 genes) significantly (P<0.05) in BL cell lines (Figures 1A-1B) (GEO accession number GSE41865). Among the genes that were regulated >3-fold (484 genes), we selected the 100 genes showing the highest statistical significance. Only 7 of these genes were up-regulated in BL cells (Figure 1C). The information known about the proteins encoded by these genes (http://www.uniprot.org/) suggested that two of them (ID3 and CDCA7) might function as transcrip- tional regulators (Figure 1C) and, as such, they may have the potential to regulate gene expression programs related with malignant transformation. Indeed, ID3 is a critical mediator of BL formation.33,34 Since the role of CDCA7 in tumorigenesis was controversial, we investigated whether the elevated expression of CDCA7 might specifically play a critical role in the malignant transformation of lymphoid cells.
CDCA7 expression is deregulated in Burkitt’s Lymphoma
There are two alternatively spliced CDCA7 isoforms (CDCA7-1 and CDCA7-2) that differ only in the presence of an additional internal exon in CDCA7-1 (Online Supplementary Figure S3A). CDCA7 mRNA levels, meas- ured by qPCR using primers that amplify both isoforms, were markedly higher in BL than in LCL cells (Figure 2A). qPCR analysis with gene expression assays specific for each isoform and Northern blotting showed that CDCA7- 2 was more abundant in both cell types and that levels of both isoforms were markedly higher in BL cells (Figure 2B and Online Supplementary Figure S3B).
CDCA7 protein levels were also compared between lymphoma and immortalized cells. Since we encountered reproducibility difficulties with commercially available antibodies (Abs), we generated a rabbit polyclonal Ab (S99) using a CDCA7 peptide present in both isoforms (Online Supplementary Figure S4A). To determine the speci- ficity of this Ab, we confirmed that it recognized CDCA7- 1 and CDCA7-2 ectopically expressed in HEK-293T cells (Online Supplementary Figure S4B). Immunoblot analysis of BL and LCL cells using anti-CDCA7 S99 showed that CDCA7-2 levels were markedly higher in BL than in LCLs, whereas those of CDCA7-1 were barely detectable in both cell types (Figure 2C). CDCA7-2 was not detected in BL cell lysates if the Ab was previously neutralized with the peptide used during immunization (Online Supplementary Figure 3C), further demonstrating the speci- ficity of the S99 anti-CDCA7 Ab.
To ascertain that the high expression of CDCA7 in BL cell lines was not just a consequence of their in vitro growth, we used qPCR to compare CDCA7 expression in biopsy specimens from BL patients with that in control tissues derived from reactive tonsils, which are enriched in germinal centre B cells. All BL samples expressed 5 to 20 times higher CDCA7 mRNA levels than a pool of 3 reac- tive tonsils (Figure 3A). Although reactive tonsils do not only contain B cells, the absence of additional cells is not expected to account for a >5-fold increase in CDCA7 mRNA expression in the tumor. In fact, CDCA7 protein immunostaining was also higher in BL samples than in the germinal centre of reactive tonsils (Figure 3B), a region
enriched in B cells. Quantification of the CDCA7-positive area in immunohistochemistry-stained sections confirmed a marked expression increase in BL samples (Figure 3C). Together, our results indicate that CDCA7 expression is
Figure 4. CDCA7 mediates anchorage-independent growth of BL cells. DG-75, BL2 and Ramos cells were transduced with lentivirus encoding sh-Ctl, sh-25 or sh-83 and selected in the presence of puromycin >5 days. A. Representative CDCA7 immunoblot analysis of DG-75 (n=4), Ramos (n=5) and BL2 (n=4) cells expressing the indicated shRNAs. Tubulin is shown as loading control. B. Representative images of wells containing these cells seeded in soft agar and C. colony formation quantification relative to cells expressing sh-Ctl shown as mean±s.e.m (n=4 independent experiments, DG-75 and BL2; n=5 independent experiments, Ramos). **P<0.01, ***P<0.001; one-way ANOVA with Bonferroni post-test.
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