Nuclear IKK activity leads to dysregulated notch-dependent gene expression in colorectal cancer

Abstract

Nuclear functions for IkappaB kinase (IKK), including phosphorylation of histone H3 and nuclear corepressors, have been recently described. Here, we show that IKK is activated in colorectal tumors concomitant with the presence of phosphorylated SMRT (silencing mediator of retinoic acid and thyroid hormone receptor) corepressor that is aberrantly localized in the cytoplasm. In these tumors, IKKalpha associates to the chromatin of specific Notch targets, leading to the release of SMRT. Abrogation of IKK activity by BAY11-7082 or by expressing dominant negative IKKalpha restores the association of SMRT with Notch target genes, resulting in specific gene repression. Finally, BAY11-7082 significantly reduces tumor size in colorectal cancer xenografts (CRC-Xs) implanted in nude mice.

Fig. 1.

IKK activation in colorectal and pancreatic cancer cell lines. (A) Western blot analysis of P-IKKα/β and total IKKα from the different cancer cell lines compared with HEK-293 and HS27. α-tubulin is used as a loading control. (B) Different cell lines were tested for NFκB-binding activity by EMSA. p65-containing complexes are indicated as NFκB. Arrowhead indicates the supershift with α-p65 antibody. (C) IHC staining of three representative primary colon tumor sections and normal adjacent tissue with α-P-IKKα/β. Images were obtained at ×100 and ×600. (D) IHC staining of serial sections from a representative primary colorectal tumor with the indicated antibodies. Images were obtained at ×400.

Fig. 2.

Nuclear IKKα phosphorylates SMRT and associates with its cytoplasmic translocation in colorectal tumors. (A) Western blot analysis of IKKα in cytoplasmic and nuclear extracts from the different cell lines. α-HDAC1 and α-tubulin are used as fractionation and loading controls. (B) Kinase activity assay of precipitated nuclear IKKα tested on GST-SMRT (amino acids 2321–2525) detected by [³²P]ATP incorporation. Nuclear levels of IKKα in the different cell lines and HDAC1 as a nuclear input control are shown. (C) Kinase activity assay of total extracts from cells transfected with or without DN-IKKα tested on GST-SMRT (amino acids 2321–2525) detected by [³²P]ATP incorporation. Levels of transfected DN-IKKα are detected with α-HA antibody, and α-tubulin is used as a loading control. (D) Indicated cell lines were cotransfected with hes1-luc and the indicated constructs. The graph represents the activity of hes1-luc in the different conditions. The average and standard deviation from duplicates of one representative of three experiments are shown. (E) IHC of sequential sections containing both normal (N) and adenoma (T) tissue (see dotted line) with α-P-IKKα/β, α-P-SMRT, and α-SMRT antibodies. Images were obtained at ×100 and ×600.

Fig. 3.

IKKα is recruited to different Notch target gene promoters correlating with transcriptional activation in colorectal tumors. (A) ChIPs with α-IKKα or α-SMRT and nonrelevant IgG from CRC-X compared with normal colon. PCR detection of the indicated promoters is shown. (B) Semiquantitative RT-PCR from patient-derived CRC-X. (C) Hes1 staining by immunofluorescence of frozen sections from normal colon tissue (×400 to ×600) and two different CRC-X (×600). (D) Semiquantitative RT-PCR from paired normal adjacent and tumor colon samples. (E) IHC of colorectal sections from normal and adenoma tissues with α-Hes1 antibody. Images were obtained at ×100 and ×600. Arrows indicate the presence of nuclear Hes1 in few cells in the bottom of the normal crypt.

Fig. 4.

IKK-target genes are up-regulated in primary tumors and inhibition of IKK reduces tumor volume in vivo. (A) ChIP with α-SMRT antibody and PCR detection of the hes1 and hes5 gene and mRNA expression by semiquantitative RT-PCR from HCT-116, HT29M6, and SW480 treated with DMSO or BAY11-7082 for 48 h. (B) Semiquantitative RT-PCR showing the expression levels of hes1 and hrt1/herp2 in SW480 cells transfected with control vector or DN-IKKα. (C) Representative culture of SW480 cells after 3-day treatment with DMSO or BAY11-7082. (Left) Graphs represent daily cell counts from cultures treated with the indicated concentrations of BAY11-7082. (Center) Photographs show a representative image of the cultures at day 3 of treatment. Images were obtained in an Olympus IX-10 at ×100. (Right) Flow cytometry analysis of AnnexinV staining. (D) CRC-X8 and CRC-X34 tumors were implanted s.c. in nude mice (n = 40). Ten days after transplantation, the animals were treated with BAY11-7082 or DMSO as a control. Tumors were measured weekly, and the results are presented as mean ± SEM. Student's t test for independent analysis was applied to evaluate differences in tumor volume [A(B²)π/6] between treated and control mice.