Differential expression of DHHC9 in microsatellite stable and instable human colorectal cancer subgroups

Abstract

Microarray analysis on pooled samples has previously identified ZDHHC9 (DHHC9) to be upregulated in colon adenocarcinoma compared to normal colon mucosa. Analyses of 168 samples from proximal and distal adenocarcinomas using U133plus2.0 microarrays validated these findings, showing a significant two-fold (log 2) upregulation of DHHC9 transcript (P<10(-6)). The upregulation was more striking in microsatellite stable (MSS), than in microsatellite instable (MSI), tumours. Genes known to interact with DHHC9 as H-Ras or N-Ras did not show expression differences between MSS and MSI. Immunohistochemical analysis was performed on 60 colon adenocarcinomas, previously analysed on microarrays, as well as on tissue microarrays with 40 stage I-IV tumours and 46 tumours from different organ sites. DHHC9 protein was strongly expressed in MSS compared to MSI tumours, readily detectable in premalignant lesions, compared to the rare expression seen in normal mucosa. DHHC9 was specific for tumours of the gastrointestinal tract and localised to the Golgi apparatus, in vitro and in vivo. Overexpression of DHHC9 decreased the proliferation of SW480 and CaCo2 MSS cell lines significantly. In conclusion, DHHC9 is a gastrointestinal-related protein highly expressed in MSS colon tumours. The palmitoyl transferase activity, modifying N-Ras and H-Ras, suggests DHHC9 as a target for anticancer drug design.

Figure 1

Microarray analysis of 168 samples representing molecular subgroups of CRCs. Transcript expression level of (A) DHHC9 and (B) H-ras on the U133plus2.0 Gene Chip. The black bars correspond to CRC samples, the grey bars to normal mucosa. Expression values are given as log 2 values and all data are normalised. (C) MSS/MSI study. Median log 2 values and standard deviations of normal biopsies (median normal, n=10), CRC patients (median cancer, n=168), MSI (CRC patients with MSI, n=35), MSS (CRC patients with MSS, n=118).

Figure 2

Immunohistochemical analysis of DHHC9 expression. (A) normal mucosa, (B) Dukes A adenocarcinoma adjacent to premalignant tissue, (C) Dukes A adenocarcinoma ( × 200), (D) Dukes D adenocarcinoma, (E) lymph node metastasis and (F) liver metastasis ( × 400). DHHC9 protein expression in MSS vs MSI colon adenocarcinomas. Microsatellite stable tumours (G–L), MSI-H tumours (M–R) ( × 200).

Figure 3

Immunostaining and Western blotting of transiently transfected COS7 cells and immunofluorescence applied to an MSS adenocarcinoma. (A–C) Confocal microscope, (D) Zeiss Microscope: × 100. (A) DHHC9, (B) endogenous 58K protein, (C) merged DHHC9 and 58K protein, (D) DHHC9 Golgi localisation detail. DAPI-stained nuclei are blue. (E) Western blotting of extracts from DHHC9-transfected COS7 cells (MW, molecular weight marker; 1, cells transfected with an empty vector; 2, cells transfected with wild-type DHHC9). (F–K) IF, (F) DHHC9, (G) 58K protein, (H) DAPI, (I) merge (F) and (G); (J) merge (F), (G) and (H); (K) DAB staining of the same tumour.

Figure 4

Proliferation assay. Several different cell lines with MSS (SW480, CaCo2) or MSI (HCT15, HCT116, LS174 TR4, DLD-1) were transfected with DHHC9 or a mock empty vector. Forty-eight hours post-transfection cells were added to 1 × dye binding solution, and fluorescence was measured. Only MSS cell lines SW480 and CaCo2 showed a significantly decreased proliferation rate. Interestingly, also DLD-1 cells, with the wnt pathway switched off, showed a similar decreased proliferation rate.