ZKSCAN3, a Novel Zinc Finger Transcription Factor, Regulates Cyclin D2 Expression in Multiple Myeloma (MM) Cells

Background: Dysregulation of cyclin D has been proposed to represent an early oncogenic event in MM, and often occurs as a result of chromosomal translocations. Cyclin D2 can be induced as a result of translocations involving c-maf, mafB, and FGFR3/MMSET, but its expression is also increased in hyperdiploid and nonhyperdiploid cells without these translocations through unknown mechanisms. We previously identified a novel zinc finger transcription factor, ZKSCAN3, as a new “driver” of colon cancer progression, and found that the cyclin D2 promoter harbored several ZKSCAN3 binding sites, prompting us to investigate a possible role of ZKSCAN3 in MM pathogenesis. Methods: ZKSCAN3 expression was studied in patient-derived primary tumors and normal cells, as well as in human MM cell lines. Results: Tissue microarray studies showed that ZKSCAN3 was strongly expressed in the neoplastic cells of 7/10 MM patients by immunohistochemistry, while in all samples the surrounding normal bone marrow cells showed only background staining. Real-time polymerase chain reaction (qPCR) analysis revealed some level of ZKSCAN mRNA expression in 5/5 MM cell lines studied (RPMI 8226, U266, KAS-6/1, INA-6, ANBL-6), but not in pooled CD19+ normal B-cells. By Western blotting, ZKSCAN3 was most highly expressed in RPMI 8226 and KAS-6/1 cells, which notably do not bear cyclin D dysregulating translocations, while U266 and ANBL-6, which have such translocations, showed the lowest levels of ZKSCAN3 expression. In RPMI 8226 cells, but not the other MM lines, six copies of the ZKSCAN3 gene were detected, suggesting the presence of a previous amplification event. cisRED analysis predicted that ZKSCAN3 may be transcriptionally regulated by paired box gene 5 (Pax5), an essential factor for maintaining the commitment of the B cell lineage which is inactivated during plasma cell differentiation. Consistent with a role for Pax5 in controlling ZKSCAN3 level, a reporter assay showed that Pax5 inhibited ZKSCAN3 promoter activity, and Pax5 expression negatively correlated with ZKSCAN3 expression. Electrophoretic mobility shift and chromatin immunoprecipitation assays showed direct binding of ZKSCAN3 to the endogenous cyclin D2 promoter in these MM cell lines. Knockdown of ZKSCAN3 using short hairpin RNAs reduced Cyclin D2 mRNA and protein expression in RPMI 8226 and KAS-6/1 cells, and also decreased their proliferative rates. Conclusions: These findings support the possibility that ZKSCAN3 contributes to myeloma pathogenesis by dysregulation of cyclin D2, and that events impacting upon ZKSCAN3 may provide MM cells with an alternative pathway for induction of Cyclin D2 in the absence of an activating translocation. ZKSCAN3 may therefore be an attractive candidate for MM therapy.