BRAF and FBXW7 (CDC4, FBW7, AGO, SEL10) mutations in distinct subsets of pancreatic cancer: potential therapeutic targets

Abstract

The recognition of biologically distinct tumor subsets is fundamental to understanding tumorigenesis. This study investigated the mutational status of the serine/threonine kinase BRAF and the cyclin E regulator FBXW7 (CDC4, FBW7, AGO, SEL10) related to two distinct pancreatic carcinoma subsets: the medullary KRAS2-wild-type and the cyclin E overexpressing tumors, respectively. Among KRAS2-wild-type carcinomas, 33% (3 of 9) contained BRAF V599E mutations; one of which was identified in the pancreatic cancer cell line COLO357. Among 74 KRAS2-mutant carcinomas, no BRAF mutations were identified. Among the KRAS2/BRAF wild-type carcinomas, no mutations within pathway members MEK1, MEK2, ERK1, ERK2, RAP1B, or BAD were found. Using pancreatic cancer microarrays and immunohistochemistry, we determined that 6% (4 of 46 and 5 of 100 in two independent panels) of pancreatic adenocarcinomas overexpress cyclin E. We identified two potential mechanisms for this overexpression including the amplification/gain of CCNE1 gene copies in the Panc-1 and Su86.86 cell lines and a novel somatic homozygous mutation (H460R, in one of 11 pancreatic cancer xenografts having allelic loss) in FBXW7, which was accompanied by cyclin E overexpression by immunohistochemistry. Both BRAF and FBXW7 mutations functionally activate kinase effectors important in pancreatic cancer and extend the potential options for therapeutic targeting of kinases in the treatment of phenotypically distinct pancreatic adenocarcinoma subsets.

Figure 1.

BRAF mutations in pancreatic cancer. Tumors (PX) and the COLO357 cell line display the V599E mutation (diamond). Constitutional DNA samples (N) verify mutation is somatic.

Figure 2.

Cyclin E overexpression in pancreatic adenocarcinoma. Pancreatic carcinoma tissue arrays 1 (A and B) and 2 (C) studied for cyclin E expression. B: Higher magnification of A inset. Normal ductal structures (D) failed to express nuclear cyclin E. E: The pancreatic tumor xenograft, PX221, carries a homozygous CAT to CGT somatic missense mutation within exon 9 (diamond). F: Immunohistochemistry confirms the predicted overexpression of cyclin E in PX221. Magnifications: A, ×40; B-D and F, ×100.

Figure 3.

CCNE1 and AKT2 fluorescence in situ hybridization (FISH). A: Interphase nuclei of the Su86.86 cell line showing a marker of 19p (TCF3/E2A, red) and extra copies of CCNE1 (green). B: Metaphase spread of Su86.86 showing TCF3 (red, 19p) and CCNE1 (green). Ectopic CCNE1 is shown with an arrow. C: Metaphase spread of normal cells showing single-copy signals for TCF3 (red, 19p), CCNE1 (green, 19q) and AKT2 (red, 19q). D: Metaphase spread showing amplification of AKT2 (red, q arm) and its centromeric neighbor, CCNE1 (green), in the Panc-1 cell line. TCF3 (red) is also shown as a marker of 19p. E: Southern blots of CCNE1 in the Su86.86, AsPC-1, and Panc-1 cell lines compared to levels in N57 (normal). The WI-12306 clone served as a loading control.