Ankyrin repeat and single KH domain 1 (ANKHD1) drives renal cancer cell proliferation via binding to and altering a subset of miRNAs

Maria Fragiadaki,Martin P Zeidler

doi:10.1074/jbc.ra117.000975

Abstract

Clear cell renal cell carcinoma (ccRCC) represents the most common kidney cancer worldwide. Increased cell proliferation associated with abnormal microRNA (miRNA) regulation are hallmarks of carcinogenesis. Ankyrin repeat and single KH domain 1 (ANKHD1) is a highly conserved protein found to interact with core cancer pathways in Drosophila; however, its involvement in RCC is completely unexplored. Quantitative PCR studies coupled with large-scale genomics data sets demonstrated that ANKHD1 is significantly up-regulated in kidneys of RCC patients when compared with healthy controls. Cell cycle analysis revealed that ANKHD1 is an essential factor for RCC cell division. To understand the molecular mechanism(s) utilized by ANKHD1 to drive proliferation, we performed bioinformatics analyses that revealed that ANKHD1 contains a putative miRNA-binding motif. We screened 48 miRNAs with tumor-enhancing or -suppressing activities and found that ANKHD1 binds to and regulates three tumor-suppressing miRNAs (i.e. miR-29a, miR-205, and miR-196a). RNA-immunoprecipitation assays demonstrated that ANKHD1 physically interacts with its target miRNAs via a single K-homology domain, located in the C terminus of the protein. Functionally, we discovered that ANKHD1 positively drives ccRCC cell mitosis via binding to and suppressing mainly miR-29a and to a lesser degree via miR-196a/205, leading to up-regulation in proliferative genes such as CCDN1. Collectively, these data identify ANKHD1 as a new regulator of ccRCC proliferation via specific miRNA interactions.

Highlights

Clear cell renal cell carcinoma represents the most common kidney cancer worldwide
We showed that ANKHD1 controls renal cancer cell proliferation by engaging with and altering the levels of a subset of tumor-suppressing miRNAs
We demonstrated that ANKHD1 physically forms a complex with three target miRNAs via its KH domain, which leads to a significant reduction in the levels of these miRNAs

Summary

Results

Consistent with previous knockdown experiments, forced expression of FL-WT was sufficient to increase the proportion of cells undergoing mitosis, whereas transfection with the FL-Mut reduced proliferation compared with empty vector (Fig. 4F, EV), suggesting that ANKHD1 mediates its pro-proliferative actions via its GXXG loop Taken together, these data demonstrate that ANKHD1 physically associates with its target miRNAs via its single KH domain and that mutations in this region are able to abolish miRNAs binding and in turn reduce the ability of ANKHD1 to enhance proliferation of ccRCC cells. We found that ANKHD1 loss of function led to a statistically significant reduction of each of these genes (apart from CDC42, Fig. S1E), a phenomenon that could be restored by inhibiting miR-29a-3p (Fig. 5D) These data together suggest that ANKHD1 is a positive regulator of proliferation in renal cancer cells via suppressing primarily miR29a-3p and to a lesser degree via miR-205/miR-196a, altering a number of known proliferation altering genes such as CCDN1, leading to increased RCC proliferation

Discussion

Cell culture and transfections

RNA immunoprecipitation

Flow cytometry

Quantitative PCR