DAB2IP modulates primary cilia formation associated with renal tumorigenesis

Abstract

Primary cilium is a microtubule-based organelle that projects from the surfaces of most mammalian cell types and protrudes into the extracellular milieu as an antenna-like sensor to senses extracellular physical and biochemical signals, and then transmits signals into cytoplasm or nucleus to regulate numerous physical and developmental processes. Therefore, loss of primary cilia is associated to multiple cancer progression, including skin, breast, pancreas, ovarian, prostate, and kidney cancers. Our previous studies demonstrate that high prevalent loss of DAB2 Interacting Protein (DAB2IP) is associated with renal cell carcinoma, and we found a kinesin-like protein, kinesin family member 3A (KIF3a), was significantly increased in DAB2IP-interacting protein fraction. KIF3 is one of the most abundant kinesin-2 family proteins expressed in cells, and it is necessary for ciliogenesis. In this study, we observed that loss of DAB2IP in normal kidney epithelial cell significantly impair primary cilia formation. We unveiled a new mechanism of primary cilia stability via DAB2IP and KIF3a physical interaction at DAB2IP-PH domain. Furthermore, we found that KIF3a also act as a tumor suppressor in renal cell carcinoma, affect tumor development and patient survival.

Keywords: DAB2IP; KIF3a; Primary cilia; Renal cell carcinoma; Tumor suppressor gene.

Fig. 1

The role of DAB2IP in primary cilia formation. (A) HK and (B) MDCK cells were transfected with vector control (Con) or DAB2IP knockdown vector (KD) (C) 769P and 293 cells were transfected with vector control (Vc) or DAB2IP expression vector (OE). All cells were cultured in either nonstarvation (NS, 5% FBS) or starvation condition (S, 1% FBS) for 16 h. All Fixed cell were stained with primary cilia marker ARL13B (Green) and nuclear counterstaining DAPI (Blue) and the number of ciliated cells was calculated (D) Mice kidney sections were collected in DAB2IP wild-type (WT) and knock out (KO) mice. Primary cilia were identified by primary cilia marker Ac-tubulin, and gamma-tubulin. *, P < 0.05; **, P < 0.01; ***, P < 0.001. All experiments were performed at least 3 times. Representative picture was shown. Statistic data represented mean ± standard deviation. DAB2IP, DAB2 Interacting Protein; FBS, fetal bovine serum; MDCK, Madin-Darby Canine Kidney cell.

Fig. 2

KIF3a as an interactive partner of DAB2IP in primary cilia regulation. (A) Primary cilia related protein enriched in DAB2IP binding fraction were listed according to normalized spectral index (SI_N) and listed as heatmap format. (B) The protein-protein association was schemed in STING database according. Unconnected proteins were removed. Colored nodes represent query proteins. Edges represent protein-protein associations, and the density of edges represents the confidence of association. (C) Cells purified from KIF3a WT and KIF3a KO mice were used to analyze primary cilia forming rate. Ac-tubulin (Green) was used to visualize primary cilia, DAPI (Blue) was used to indicate nucleus. (D) The expression of DAB2IP and KIF3a in mouse cells was confirmed by qRT-PCR and western blot. (E and F) DAB2IP was either knocked down (KD) in HK and MDCK cells or overexpressed in (OE) 769P and 293 cells. The protein expression of DAB2IP and KIF3a were examined by western blot. *, P < 0.05; **, P < 0.01; ***, P < 0.001. All experiments were performed at least 3 times. Representative picture was shown. Statistic data represented mean ± standard deviation. DAB2IP, DAB2 Interacting Protein; DAPI, 6-diamidino-2-phenylindole; KIF3a, kinesin family member 3A; MDCK, Madin-Darby Canine Kidney cell; qRT-PCR, Quantitative real-time PCR.

Fig. 3

Interaction of DAB2IP with KIF3a at its N-terminal PH domain. (A) HK cell was immunoprecipitated with KIF3a antibody, and the expression of DAB2IP and KIF3a were then immunoblotted. (B) 293 cells-DAB2IP OE was immunoprecipitated with DAB2IP antibody, and the expression of DAB2IP and KIF3a were then immunoblotted. (C) HK cell was used in immunofluorescence staining with KIF3a (Green), DAB2IP (Red), and Ac-tubulin (White) antibodies. DAPI (Blue) staining represented nucleus. (D) Primary cilia were cut off and isolated from HK and MDCK cells. KIF3a, DAB2IP and Ac-tubulin protein expression were analyzed. (E) ACHN and 293 cells were transfected with various DAB2IP constructs including vector control (Vc), Full length (Full), N terminal (N), and C terminal (C) of DAB2IP, and dot blot assay was used to analyze KIF3a binding efficacy. (F) DAB2IP constructs including Vc, Full, N, PH, C2, and C terminal were overexpressed in 293 cells, and the expression was determined by Flag. Dot blot assay was used to analyze KIF3a binding efficacy. (G) ACHN cells transfected with various DAB2IP domain constructs were used to analyze the degree of primary cilia formation. *, P < 0.05; **, P < 0.01; ***, P < 0.001. All experiments were performed for at least at least 3 times. Representative picture was shown. Statistic data represented mean ± standard deviation. DAB2IP, DAB2 Interacting Protein; KIF3a, kinesin family member 3A; MDCK, Madin-Darby Canine Kidney cell; PH, Pleckstrin homology.

Fig. 4

The effect of DAB2IP on the protein stability of KIF3a leading to primary cilia formation. (A) DAB2IP and KIF3a mRNA expression were detected in HK control (Con) and DAB2IP knock down (KD) cells. (B) 769P wild-type (WT) cells were treated with 2.5 µM MG132 for different time points (0, 12, 24, 48 h) and the expression of KIF3a and DAB2IP protein was analyzed by western blot. (C) HK-Con or -DAB2IP KD cells and ACHN-Vc or -DAB2IP OE cells were treated with 10 nM at different time points (0, 12, 24, 48 h) and the expression of KIF3a and DAB2IP were analyzed by western blot. (D) 769P cells transfected with DAB2IP or/and KIF3a expression vectors and the expression of KIF3a and DAB2IP protein was determined by western blot. (E) Various 769P sublines-DAB2IP or KIF3a OE were used to examine the primary cilia formation. ARL13B (Red) was used to indicate primary cilia. DAPI was used to indicate nucleus. *, P < 0.05; **, P < 0.01; ***, P < 0.001. All experiments were performed at least 3 times. Representative picture was shown. Statistic data represented mean ± standard deviation. DAB2IP, DAB2 Interacting Protein; DAPI, 6-diamidino-2-phenylindole; KIF3a, kinesin family member 3A.

Fig. 5

The effect of KIF3a on renal tumorigenesis. (A) HK Con and KD cells (right panel), mice derived KIF3a WT and KO (middle panel) and (B) 769P Vc and KIF3a OE cells were subjected to colony forming assay with 100 cells plated onto 6-well plate for 1 wk. All colonies were stained with crystal violet and determined fold change compared to their independent control. (C) HK (Con, KD) and 769P (Vc, KIF3a OE) cells were subjected to anchorage independent growth (AIG) assay with 5,000 cells mixed in upper 0.6% agarose soft agar and covered on top of the 1.2 % agarose bottom agar. All cells were cultured for 2 wk and colonies were counted and determined fold change compared to their control. (D) HK Con and KIF3a KD (1 × 10⁶ cells) were subcutaneously injected in SCID mice. Tumor incidence and tumor volumes were measured at Wk 7. (E) KIF3a expression in harvested tumors was analyzed by western blot. Cell lysates from HK Con and KIF3a KD were used as the control. *, P < 0.05; **, P < 0.01; ***, P < 0.001. All experiments were performed at least 3 times. Representative picture was shown. Statistic data represented mean ± standard deviation. KIF3a, kinesin family member 3A.

Fig. 6

Clinical relevance of KIF3a in RCC development. (A) KIF3a gene expression level was obtained from TCGA database. Paired sample (adjacent normal vs. tumor) were selected. (B) KIF3a and DAB2IP gene expression levels were obtained from TCGA database, and Pearson correlation were performed. (C) KIF3a gene expression levels were categorized according to individual tumor grade (normal, grade 1 and 2, grade 3 and 4) or stage (normal, stage 1 and 2, stage 3 and 4). (D) Patients were separated by the mid-value of KIF3a and DAB2IP expression in tumor, groups including DAB2IP^low/ KIF3a^low (Red), DAB2IP^low/ KIF3a^high (Black), DAB2IP^high/ KIF3a^low (Green), and DAB2IP^high/ KIF3a^high (Blue). The overall survival of patients was plotted accordingly. DAB2IP, DAB2 Interacting Protein; KIF3a, kinesin family member 3A; RCC, renal cell carcinoma; TCGA, The Cancer Genome Atlas.