Adenomatous polyposis coli gene mutation alters proliferation through its beta-catenin-regulatory function in aggressive fibromatosis (desmoid tumor)

Abstract

Aggressive fibromatosis is a monoclonal proliferation of spindle (fibroblast-like) cells. A subset of lesions contain somatic truncating adenomatous polyposis coli (APC) gene mutations, and all of the lesions contain an elevated beta-catenin protein level. A major function of APC is to regulate beta-catenin protein level. Beta-catenin has a dual function in the cell: it is a member of the adherens junction, and it binds transcription factors in the tcf-lef family, transactivating transcription. Cell cultures from aggressive fibromatoses containing an APC mutation were studied. Transient transfection of the full-length APC gene caused decreased proliferation and beta-catenin protein level in these cultures. To determine whether beta-catenin protein level was responsible for the change in proliferation rate, stable transfections of deltaN89beta-catenin (a stabilized form that is not degraded by APC, but retains all other functions) were achieved in half of the cultures derived from each tumor, whereas the other half were transfected with an empty vector. Transfection of the full-length APC gene in cultures that were stably transfected with deltaN89beta-catenin did not result in a change in proliferation. The type I promotor of p56lck contains an HMG consensus region, to which members of the tcf-lef family can bind. p56lck was expressed in cultures not transfected with the full-length APC gene and in cultures transfected with the full-length APC gene and deltaN89beta-catenin, but not in cultures transfected with only the full-length APC gene. These data show that APC truncating mutations give aggressive fibromatosis cells a proliferative advantage through beta-catenin and suggest that beta-catenin acts to transactivate transcription.

Figure 1.

Demonstration of successful transfection of the full-length APC gene and ΔN89β-catenin. Shown is immunohistochemistry using a C-terminal antibody to APC from cultures transfected with an empty vector (A) and transfected with the full-length APC gene (B). Cytoplasmic staining for the C-terminal portion of APC is visualized in almost every cell in B, with 85% of cells overall showing cytoplasmic staining for APC 4 days after transfection. C: Western blot using a C-terminal antibody to β-catenin. The left lane (C) is loaded with protein from cells transfected with an empty vector, and the right lane (T) is loaded with cells transfected with ΔN89β-catenin. The second, shorter-length, band (ΔN89) in the right lane is ΔN89β-catenin.

Figure 2.

Proliferation, measured using bromodeoxyuridine incorporation and immunohistochemistry. P, positive nuclear staining; N, negative nuclear staining. (A) is from cells transfected with only empty vectors, (B) is from cells transfected with the full-length APC gene, and (C) is from cells transfected with the full-length APC gene and ΔN89β-catenin. Over 10 high-powered fields, 65% of cells from cultures shown in (A), 20% from (B), and 55% from (C) showed bromodeoxyuridine incorporation, thus illustrating that ΔN89β-catenin transfection “rescues” the decrease in proliferation from APC transfection.

Figure 3.

Western analysis for β-catenin and RT-PCR for p56lck. A: Western blot for β-catenin on equal quantities of extracted protein from cells transfected with an empty vector (lane C), and transfected with full-length APC at lower (50%, lane T1) and higher (85%, lane T2) efficiencies. A decrease in protein level with higher efficiency is demonstrated. B: RT-PCR data for p56lck and reduced glyceraldehyde-phosphate dehydrogenase from cultures transfected with the empty vector only (lane A), with full-length APC (lane B) , or with full-length APC and ΔN89β-catenin (lane C). Expression of reduced glyceraldehyde-phosphate dehydrogenase is present in all of the cultures, but p56lck is only expressed in lanes A, and C, which are the cultures with a high β-catenin protein level and high proliferation rate. Lane N is loaded with products from a normal fibroblast explant cell culture.