Repression of KIAA1199 attenuates Wnt-signalling and decreases the proliferation of colon cancer cells

Abstract

Background: The KIAA1199 transcript is upregulated in colon adenomas and downregulated upon β-catenin knockdown.

Methods: Transcript profiling was performed on >500 colon biopsies, methylation profiling data were compared with transcript data. Immunohistochemistry assessed KIAA1199 protein expression in 270 stage II/III tumours (>3 years follow-up). The effects of stable KIAA1199 knockdown in SW480 cells (three different constructs) were studied using transcriptional profiling, proliferation and protein analysis.

Results: The KIAA1199 transcript was strongly upregulated in 95% of adenocarcinomas. Absent expression in normal mucosa correlated with KIAA1199 promotor methylation. Nuclear and cytoplasmic KIAA1199 protein expression was identified in colon adenocarcinomas and other types of cancers. A subpopulation of patients with tumours strongly expressing KIAA1199 in the nucleus showed a better outcome with regard to recurrence as lung or liver metastases. The KIAA1199 knockdown affected the cell cycle and the Wnt-signalling pathway. Reduced cellular proliferation and decreased KI67, phosphorylated retinoblastoma, β-catenin and ASCL2 protein expression supported these findings. Eighteen Wnt-signalling genes differentially expressed upon KIAA1199 knockdown correlated with the KIAA1199 expression profile in clinical specimens.

Conclusion: The KIAA1199 knockdown attenuates the effects of the Wnt/β-catenin signalling and it may thus be regarded as a regulatory part of this pathway.

Figure 1

The KIAA1199 transcript and protein expression and correlation to clinical outcome. (A) The KIAA1199 transcript profiling of 511 colon samples (normal mucosas, MSS microsatellite stable and MSI instable and adenocarcinomas). (a) The U133A GeneChips (n=122); (b) U133plus2.0 GeneChips (n=389); (details shown in Supplementary Figure 1). (B) Normal colon mucosa showed a very weak expression in the cytoplasm of single cells located in the bottom of the crypts. (C) Western Blot of SW480 cells overexpressing KIAA1199-V5-HIS protein (157 kDa). Lane 1 – Marker Bio-Rad All Blue; lane 2 – the monospecific rabbit polyclonal anti-KIAA1199 antibody was specific for KIAA1199 (specificity is shown in Supplementary Figures 2a and b). (D) Immunohistochemical analysis of 270 stage II adenocarcinomas (>3 years follow-up) using the monospecific anti-KIAA1199 antibody: group 1 showed strong nuclear but weak cytoplasmic KIAA1199 expression, group 2 showed moderate to strong cytoplasmic KIAA1199 expression, no nuclear expression. (E) In all 245 of the 270 samples were informative (118 female, 127 male). Patients without disease recurrence (n=205, median follow-up time 2198 days, range 1099–4368); patients with metastatic recurrence (n=40, median time to recurrence 830 days, range 95–2449). Kaplan–Meier survival estimates for disease-free survival showed that patients with group 1 tumours (blue line) had a significant (log-rank test P-value 0.02) better outcome than group 2 patients (red line). Green ticks: patients censored at death or end of follow-up.

Figure 2

KIAA1199 knockdown affects the Wnt/β-catenin signalling pathway. (A) The IPA showed that a number of genes involved in canonical Wnt/β-catenin signalling were up (yellow) or downregulated (blue) upon KIAA1199 knockdown. (B) Summary of relations between KIAA1199, β-catenin, ASCL2 and LGR5: (1) knockdown from β-catenin shuts off the Wnt/β-catenin signalling resulting in a decreased KIAA1199 transcript expression (Sabates-Bellver et al, 2007) as well as a downregulation of the ASCL2 transcript (Jubb et al, 2006). (2) Knockdown from ASCL2 results in the silencing of LGR5 (Van der Flier et al, 2009). Our data show that knockdown from KIAA1199 resulted in a decreased expression of (3) β-catenin, (4) ASCL2 and (5) LGR5 and to lesser extent some genes targeted by ASCL2 (not shown). A decreased β-catenin expression upon KIAA1199 knockdown may suggest a regulatory loop probably further affecting β-catenin signalling. (C) Immunofluorescence analysis confirmed the downregulation of β-catenin and ASCL2 upon KIAA1199 knockdown in SW480 cells compared with control cells (see also Supplementary Figure 4). Blue: DAPI nuclear stain; green: Alexa488 secondary antibody; × 630 magnification, Zeiss Axiovision, Carl Zeiss, Brock & Michelsen A/S, Birkerød, Denmark). (D) Western blot with extracts from SW480 cells stably transfected with an empty vector (lanes 2 and 4) or KIAA1199-sh3303 knockdown (lane 3, 5) was incubated with anti-β-catenin antibody in a 1 : 4000 (lane 2, 3) or 1 : 2000 dilution (lane 4, 5). β-catenin protein expression was markedly decreased upon KIAA1199 knockdown compared with control cells. β-actin was used as loading control. Lane 1=Marker Bio-Rad All Blue.

Figure 3

KIAA1199 knockdown affects proliferation of SW480 cells. SW480-KIAA1199-sh3303 colon cancer cells depleted of KIAA1199 (•) and an empty vector control (○) were seeded on E-plates on a RTCA-instrument (x-CELLigence, Roche). Experiments were performed at least in triplicates, values are shown as medians and s.d.'s for each group at selected times for a representative experiment. (A) Seeding of 4000 cells per well yielded control cells in the log phase from 36–96 h, cells were monitored in 1 min intervals. The proliferation rate of the KIAA1199-depleted cells was decreased by about 50% compared with control cells. (B) MTT-assays at selected time points confirmed a significant (P<0.05) decrease of proliferation of the KIAA1199-sh3303 depleted SW480 cells compared with control cells. (C) IF showed less nuclear expression of proliferation marker KI67 in KIAA1199-depleted SW480 cells compared with control cells using an 1 : 400 dilution of the anti-KI67 antibody (green: Alexa488-labelled secondary antibody; blue: Hoechst nuclear stain); × 630 (D) IF using a 1 : 250 dilution of the monoclonal anti-phosphoretinoblastoma antibody Rb-pS⁷⁹⁵ reacting specifically with Rb phosphorylated at serine 795 and not with non-phosphorylated Rb showed a dramatical decrease of phosphorylated Rb protein in KIAA1199-depleted SW480 cells compared with control cells. (E) Western blot with SW480 cells stably transfected with empty vector (lanes 2, 4) or KIAA1199-sh3303 knockdown (lane 3, 5), incubated with anti- RB-pS795 antibody (1 : 2000, lane 2, 3; 1 : 1000 lane 4, 5). Expression of the phosphorylated RB-pS795 protein was markedly decreased upon KIAA1199 knockdown compared with control cells. β-actin was used as loading control. Lane 1=Marker Bio-Rad All Blue.