ANKHD1, a New Ankyrin-Repeat Protein, Binds to SIVA and May Modulate ROS Generation, Cell Cycle and Apoptosis Signaling in Cancer Cells

Ankyrin-repeat-containing proteins regulate multiple cellular functions including transcriptional and cell-cycle regulation, ion channel, cell survival, cell signaling and participate in protein-protein interactions via their repeat motifs. Ankyrin Repeat and KH Domain Containing 1 (ANKHD1) was first identified in LNCaP, a human prostate cancer cell line. We recently reported a higher expression of AKNHD1 in human acute leukemia cell lines and in samples from patients with acute leukemia, when compared to normal hematopoietic cells, suggesting a role for ANKHD1 in leukemogenesis. Here, we report the association between ANKHD1 and SIVA, its role in Reactive Oxygen Species (ROS) generation, cell cycle and the gene expression profile after ANKHD1 inhibition in cancer cells. To identify ANKHD1 interacting proteins, we used the yeast two-hybrid system for screening a normal human bone marrow cDNA library with ANKHD1 as the bait. We identified the pro-apoptotic SIVA as an ANKHD1-interacting protein. ANKHD1 interacted with the C-terminal region of both SIVA isoforms. The N-terminal and C-terminal regions of SIVA were required for these interactions, as detected through a yeast two-hybrid system using different SIVA constructs (SIVA1, SIVA2, SIVA C-terminal, SIVA N-terminal, SIVA Dead Domain) and ANKHD1 as the bait. Immunoprecipitation-Western blot assay showed that this interaction occurred both in vitro and in vivo. The in vitro interaction was detected by co-transfection of SIVA1-GFP or SIVA2-GFP and ANKHD1-HA in HEK293 cells; the in vivo interaction was detected in the acute lymphoblast leukemia cell line, Jurkat, and in LNCaP cells. Post-transcriptional ANKHD1 gene silencing was carried out using small interfering RNA in LNCaP cells. After 72 hours of transfection, cells were collected for analysis. Western blotting and real time PCR showed an 80% decrease in ANKHD1 expression. Flow cytometry studies revealed that ANKHD1 inhibition resulted in a 40% reduction in ROS generation and induced cell cycle perturbations with a reduced number of cells that entered the G2-M phase, compared with control cells. Microarray analysis using the Codelink™ Human whole genome bioarray (GE Health Care) was performed in cells submitted or not to ANKHD1 inhibition. Gene modulation was analyzed according to HTself (self-self based statistical test for low replication microarray studies). Differentially-expressed genes were observed involved in apoptosis (26 up; 13 down), cell cycle regulation (26 up; 11 down) and proliferation (15 up; 8 down), corroborating the role of ANKHD1 in cellular pathways involved in neoplasia. Genes involved in apoptosis were validated by real time PCR. Upregulated by ANKHD1 inhibition: STAT1, PTEN, SIAH1, PI3KR2, SOCS3, TP53INP1, GADD45B, IHPK2 and BNIP2. Downregulated genes: CEBPG, GLO1, NPM1 and AMIGO2. Previous studies by other authors have demonstrated SIVA1 and SIVA2 to be overexpressed in acute lymphoblast leukemia cell lines, where they bind and inhibit BCL-XL and induce apoptosis. Furthermore, ROS generation is reported to be essential to the aggressive phenotype of cancer cells, including dysregulated growth, colony formation, cell migration, and invasion, suggesting that targeting ROS production might offer a novel mechanism in combating cancers. Thus, our results suggest an important role of ANKHD1 in the pathogenesis of leukemia. The identification of new disease-specific targets for cancer expands treatment options and increases our chances of successful treatment.