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R. Jimenez-P. et al.
non-transformed cells.3 Epstein-Barr virus (EBV) infection of normal lymphocytes generates immortalized lym- phoblastoid B-cell lines (LCLs) unable to form tumors in immunodeficient mice but capable to replicate indefinite- ly in liquid culture.4 In contrast, cell lines derived from Burkitt’s lymphoma (BL), a B-lymphocyte tumor strongly associated with EBV in some regions of Africa,5 not only display replicative immortality, but are also tumorigenic in immunodeficient mice.4
Another trait of tumor cells is their capacity to replicate and grow independently of their attachment to a rigid sur- face. Growth of normal tissue cells requires the signals transmitted by plasma membrane receptors that bind extracellular matrix components and transmembrane pro- teins from neighboring cells of the proper microenviron- ment. Most normal tissue cells are not viable when sus- pended in liquid or soft medium, and require adhesion to the surface of a culture vessel. Similarly, immortal, but non-tumoral cells, including NIH-3T3 fibroblasts and LCLs, cannot grow in semi-solid media such as soft agar,4,6,7 and are considered as anchorage-dependent. On the contrary, tumor cells do not need to adhere to a rigid surface for growth and are said to be anchorage-indepen- dent.6 Numerous genes that mediate tumorigenesis have been identified, but very limited information is available regarding genes that specifically mediate anchorage-inde- pendent growth. While anchorage-dependence has been extensively studied in fibroblasts and epithelial cells, it is unknown whether normal lymphoid cells require anchor- age for proliferation. Soft agar not only limits cell binding to the culture vessel surface but also intercellular interac- tions. The incapacity of LCLs to grow in soft agar could therefore be attributed to lack of anchorage to a rigid sub- strate or to neighboring cells. It should be noted that nor- mal lymphoid cells proliferate only in lymphoid organs in vivo, in close contact with the extracellular matrix and with other cells; with appropriate stimuli, these cells can also proliferate in vitro under conditions that permit their attachment to the culture vessel surface and to other cells.
MYC deregulation is one of the most common aberra- tions in human tumors. The characteristic genetic marker of BL cells is a reciprocal translocation involving the MYC gene and one of three immunoglobulin gene loci that leads to deregulated MYC expression.8 MYC encodes a tran- scription factor and chromatin remodeler that regulates the expression of numerous genes involved in various cel- lular processes, including cell differentiation, proliferation, and apoptosis.9-14 Tumorigenesis by MYC (also known as C-MYC) can take place as a consequence of its overex- pression, even in the absence of mutations in its coding region.15 Em-Myc transgenic mice, where Myc overexpres- sion is targeted to B lymphocytes, give rise to lymphomas, but only after a mean latency period of about 6 months, these lymphomas being monoclonal.16 In addition, MYC overexpression in normal cells either arrests them in the G2 phase of the cell cycle17 or induces apoptosis.18 Together, these results suggest that MYC alone cannot elicit tumoral transformation of normal cells and that addi- tional factors might cooperate with MYC in tumorigene- sis.
MYC regulates about 15 percent of the genes in the human genome, and it is expected that some of them par- ticipate in tumor formation. However, it remains unknown which of these genes are critical for MYC- induced transformation. CDCA7, also named JPO1, is a
MYC-induced gene19 whose mRNA is deregulated in sev- eral tumor types relative to the corresponding non-prolif- erative control tissues.20 Little is known about the molecu- lar function of CDCA7. Phosphorylation by AKT regu- lates CDCA7 subcellular localization and its association with MYC.21 There are two alternatively spliced CDCA7 isoforms which contain a zinc finger domain at the C-ter- minus22,23 and associate with the Helicase, Lymphoid-spe- cific (HELLS) SNF2 family member.24 In fact, CDCA7 is required for nucleosome remodeling by HELLS and for DNA methylation maintenance.23,24 However, no function- al differences between CDCA7 isoforms have been reported.
CDCA7 overexpression in tumors could potentially be just a consequence of the presence of more cycling cells in the tumor tissues. Indeed, its expression is induced in the S-phase of the cell cycle25 and it is a transcriptional target not only of MYC,19 but also of E2F1-E2F4,22 factors that are active exclusively in proliferating cells. Alternatively, CDCA7 could potentially play a causative role in tumori- genesis. In this regard, CDCA7 has been assumed to par- ticipate in neoplastic transformation of B cells (http://www.uniprot.org/uniprot/Q9BWT1; https://www. ncbi.nlm.nih.gov/gene/83879), in spite of insufficient evi- dence supporting this assumption. Indeed, previous obser- vations showed that forced expression of CDCA7 barely increased colony formation in a B-cell line already capable of growing in soft agar.19 In addition, transgenic mice over- expressing CDCA7 in the B-cell compartment generated lymphoid malignancies as frequently as control mice (4 out of 45 transgenic mice vs. 3 out of 28 control mice).20 Moreover, CDCA7 forced expression inhibited the induc- tion of anchorage-independent growth induced by MYC in immortal fibroblasts.21 Therefore, a tumorigenic role for CDCA7 has not been experimentally demonstrated and it is, at minimum, controversial.
The design of anti-tumor therapies that do not affect normal cells has proved to be a very difficult task. To iden- tify potential therapeutic targets specific to tumor cells, we used a transcriptomic approach looking for genes specifically involved in anchorage-independent growth, a capacity that correlates extremely well with tumorigene- sis. Here we show that CDCA7 was one of the most sig- nificantly up-regulated genes, that its encoded protein is overexpressed in lymphoid tumors, and that its silencing greatly impairs lymphomagenesis without inhibiting the proliferation of normal cells.
Methods
Detailed Methods can be found in the Online Supplementary Appendix.
Patients
Cases consisted of existing de-identified anonymous biopsy specimens obtained from Hospital Infanta Sofia and from the Spanish Tumor Bank Network in Centro Nacional de Investigaciones Oncológicas (Madrid, Spain). The study was approved by the CSIC Ethics Committee and by the Ethics Committee of Hospital Universitario La Paz (ref. HULP: PI-1658).
Microarray Experiment Data Analysis
RNA from BL and LCL cell lines was extracted and labelled as described.26 The RNA from X50-7, IB-4, and Dana LCL cell lines
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haematologica | 2018; 103(10)