Down-regulation of epidermal growth factor receptor by selective expansion of a 5'-end regulatory dinucleotide repeat in colon cancer with microsatellite instability

Abstract

Purpose: The epidermal growth factor receptor (EGFR) is overexpressed in several tumor types, and its expression is influenced by the length of a 5'-end microsatellite repeat (CA)n: the longer the repeat, the lower the expression. Dinucleotide repeats accumulate insertion/deletion types of mutations in tumors with microsatellite instability. We designed this study to estimate the occurrence of these mutations in EGFR(CA)n and their relevance in carcinogenesis of microsatellite instability-positive colon and gastric tumors.

Experimental design: We analyzed the frequency of EGFR(CA)n mutations in vivo in 55 colorectal and 14 gastric microsatellite instability-positive cancers, and in vitro in single-cell clone cultures of microsatellite instability-positive colon tumor cell line LS174. Single-cell clone cultures with different repeat lengths were analyzed by fluorescent-activated cell sorter for EGFR cell-surface expression. A correlation analysis was done between EGFR(CA)n mutations and mutations in KRAS, BRAF, and p53.

Results: Unlike single-cell clone cultures, which exhibited higher rate of deletions compared with insertions, most of EGFR(CA)n mutations in colon and gastric tumors were insertions. Longer EGFR(CA)n correlated with lower EGFR cell-surface expression in single-cell clone cultures. In colon cancers, the elongation of the repeat was associated negatively with mutations in KRAS and BRAF, but not in p53.

Conclusions: The EGFR(CA)n elongation observed in tumors cannot be explained by an intrinsic property of this repeat favoring insertions versus deletions. Instead, a selection for repeat elongation occurs in microsatellite instability-positive tumors, leading to EGFR down-regulation. These findings suggest that in microsatellite instability-positive tumors current therapies targeting EGFR overexpression may have either no effect or an opposite to the expected effect.

Fig. 1

EGFR (CA)_n mutations in microsatellite instability – positive tumors. A, electrophoregrams of (CA)_n-containing PCR fragments amplified from seven (–7) microsatellite instability – positive colon tumor and matched normal tissues. Arrows, positions of bands that correspond to alleles with 17, 20, 29, and 31CA units. Right, a lower exposure of some of the samples at left from an independent experiment. B, distributionof EGFR allele length in microsatellite instability – positive gastrointestinal (52 colorectal and 14 gastric) tumors and matchednormal tissues. Openbars, normal tissue; closedbars, tumor tissue.

Fig. 2

EGFR cell-surface expression in LS174T subclones with alleles of different (CA)_n lengths. Each dot on the graph, the average value of the expression of at least two independent clones with the same allele length from two independent repeated experiments. The trend line was automatically drawn by Microsoft Excel software using y = α × exp(β × X) formula for approximation of the experimental results. The exact formula is shown inside the chart area.

Fig. 3

Association analysis between insertions in EGFR (CA)_n (top) and deletions in EGFR 3′ UTR (A)_n (bottom), and mutations in KRAS (solid dots) and BRAF (empty dots). Similar trend was observed when analyzing separately microsatellite instability tumors with mutations in K-ras (P = 0.13; n = X) or in B-raf (P = 0.03; n = X) versus microsatellite instability tumors with no mutations in these genes. No cases were found with concomitant K-ras and B-raf mutations.