Oesophageal adenocarcinoma is associated with a deregulation in the MYC/MAX/MAD network

Abstract

Oesophageal adenocarcinoma, which arises from an acquired columnar lesion, Barrett's metaplasia, is rising in incidence more rapidly than any other cancer in the Western world. Elevated expression of c-MYC has been demonstrated in oesophageal adenocarcinoma; however, the expression of other members of the MYC/MAX/MAD network has not been addressed. The aims of this work were to characterise the expression of c-MYC, MAX and the MAD family in adenocarcinoma development and assess the effects of overexpression on cellular behaviour. mRNA expression in samples of Barrett's metaplasia and oesophageal adenocarcinoma were examined by qRT-PCR. Semi-quantitative immunohistochemistry and western blotting were used to examine cellular localisation and protein levels. Cellular proliferation and mRNA expression were determined in SEG1 cells overexpressing c-MYCER or MAD1 using a bromodeoxyuridine assay and qRT-PCR, respectively. Consistent with previous work expression of c-MYC was deregulated in oesophageal adenocarcinoma. Paradoxically, increased expression of putative c-MYC antagonists MAD1 and MXI1 was observed in tumour specimens. Overexpression of c-MYC and MAD proteins in SEG1 cells resulted in differential expression of MYC/MAX/MAD network members and reciprocal changes in proliferation. In conclusion, the expression patterns of c-MYC, MAX and the MAD family were shown to be deregulated in the oesophageal cancer model.

Figure 1

mRNA expression of MYC/MAX/MAD network genes in Barrett's metaplasia and oesophageal adenocarcinoma. qRT–PCR was used to examine expression of genes encoding c-MYC, MAD1, MXI1, MXI1-0 and MAX in Barrett's metaplasia (BM n=25) and oesophageal adenocarcinoma (OAC n=37). Graphs represent mean fold change relative to matched normal gastric control (G, normalised to one) ±1 s.e.m. ^* denotes significant change relative to G, ° denotes significant difference between BM and OAC (P<0.05).

Figure 2

MYC/MAX/MAD network protein expression in Barrett's metaplasia and oesophageal adenocarcinoma. Expression of c-MYC, MAD1 and MXI1 protein was examined in Barrett's metaplasia (BM n=6) and oesophageal adenocarcinoma (OAC n=15) with matched normal gastric mucosa (G) by western blotting. Immunoreactive bands were assessed by semi-quantitative densitometry. Expression in BM and OAC is expressed relative to G (normalised to one); cytokeratin 19 (CK19) was employed for normalisation of protein loading. A representative western blot for each protein is shown alongside densitometry data representing mean expression change ±1 s.e.m. ^* denotes significant change relative to G, ° denotes significant change between BM and OAC.

Figure 3

Immunolocalisation of MYC/MAX/MAD network proteins in Barrett's metaplasia and oesophageal adenocarcinoma. Paraffin sections of normal oesophagus, normal gastric fundus, Barrett's metaplasia and oesophageal adenocarcinoma were subjected to immunohistochemistry using antibodies designed against c-MYC, MAD1, MXI1 and MAX. Magnification × 40.

Figure 4

Confirmation of expression in transfected SEG1 cells. SEG1 cells were transfected with pcDNA3.1/Zeo-MYCER, pcDNA3-MAD1 or the corresponding empty vector (VO); in the case of MYCER the chimeric product was then activated by the addition of 4-OHT. (A) qRT–PCR was utilised to assess MYC () or MXD1 () mRNA expression. (B) Western blotting demonstrated the expression the chimeric protein in SEG1-MYCER or MAD1 in SEG1-MAD1. Densitometric scanning approximated the fold increase in expression; a representative blot is also shown. Values represent the mean of two experiments each performed in triplicate ±1 s.e.m. ^* denotes statistical significance (P<0.05).

Figure 5

c-MYC network expression in SEG1 cells expressing exogenous MYC/MAX/MAD network proteins. (A) qRT–PCR was utilised to evaluate the expression of MXD1 , MXI1 , MXI-0 and MAX mRNA in SEG1 cells transiently overexpressing MYCER. Relative gene expression is expressed as a ratio of SEG1-MYCER not stimulated using 4OHT normalised to one. (B) Expression of MYC , MXI1 , MXI-0 and MAX mRNA was assessed in SEG1 cells transiently overexpressing MAD1. Relative gene expression is expressed as a ratio of mock transfected cells normalised to one. Data represent the mean of two independent experiments each performed in triplicate ±1 s.e.m. ^* denotes statistical significance (P<0.05).