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

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.

A double missense variation of the BUB1 gene and a defective mitotic spindle checkpoint in the pancreatic cancer cell line Hs766T.

To determine sequence variations of the BUB1 and BUB1B genes in pancreatic cancer, the entire coding regions of the BUB1 and BUB1B genes were sequenced in pancreatic cancer cell lines and xenografts. Although only polymorphic alterations were found in the BUB1B gene, the aneuploid pancreatic cell line Hs766T had two novel missense variants (p.[Y259C;H265N]) in the BUB1 gene. These mutations were on the same allele, accompanied by a wild-type BUB1 allele. This change was not found in other samples, the literature, or 110 additional chromosomes from a reference population. Compared to two cell lines having microsatellite instability (MIN), the TP53 wild-type pancreatic cell line Hs766T had a defective mitotic spindle checkpoint, indicative of a cell line with chromosomal instability (CIN). Evidence that this checkpoint pathway can be abrogated by mutations in the BUB1 gene (Cahill et al., 1998) supports the suggestion the missense mutations of the BUB1 gene in the Hs766T cell line may contribute to its observed mitotic checkpoint defect.

Evidence of selection for clones having genetic inactivation of the activin A type II receptor (ACVR2) gene in gastrointestinal cancers.

The activin signaling pathway parallels the transforming growth factor (TGF)-beta pathway. Both use extracellular ligands and cell surface receptors that are structurally and functionally related, as well as the same intracellular mediators (SMADs 2-4) to transmit these signals. Members of both pathways have been characterized previously as tumor suppressor genes on the demonstration of inactivating mutations in human neoplasms, e.g., genetic inactivation of the activin type I receptor was reported recently in pancreatic cancer. Here, we present evidence of selection for mutations of the activin A type II receptor (ACVR2) gene during human gastrointestinal carcinogenesis. Two 8-bp polyadenine tracts of the ACVR2 gene are targets for inactivating frameshift mutations in gastrointestinal neoplasms having microsatellite instability (MSI). These mutations are similar to those of the 10-bp polyadenine tract within the TGF-beta type II receptor (TGFBR2), a well-characterized target of frameshift mutations in the same neoplasms. We identified biallelic mutations of ACVR2 in 25 of 28 MSI colorectal and pancreatic cancers. In addition, a mutation in the ACVR2 gene combined with loss of the wild-type allele was found in a non-MSI pancreatic cancer. This evidence is compatible with a high degree of selection for inactivation of the ACVR2 gene in tumorigenesis, supporting ACVR2 as a candidate tumor suppressor gene in gastrointestinal cancers.

The desmoplastic response to infiltrating breast carcinoma: gene expression at the site of primary invasion and implications for comparisons between tumor types.

The gene expression patterns of desmoplasia are becoming exposed through the application of global gene expression technologies such as cDNA microarrays or serial analysis of gene expression (SAGE). These patterns represent the sum of the many cellular components of the host stromal response to an infiltrating carcinoma. In studies of human neoplasms, it would be useful to identify those prototypical genes that characteristically indicate the recognizable forms of the responses to individual tumor types. Such genes may offer clues to better understand the process of invasion itself, the interactions between tumor and host cells, and tumor-specific differences in invasion. We used SAGE-defined genes and in situ transcript labeling to characterize the desmoplastic stroma induced by infiltrating ductal carcinomas of the breast. Principal component analysis identified 103 SAGE tags as specific for invasive breast carcinomas, in comparison with in situ duct carcinomas or normal breast epithelium. Of these, 68 tags corresponded to known genes. Six of the 68 genes from this breast cancer "invasion-specific" cluster were further characterized by in situ hybridization to breast cancer tissues. Results of in situ hybridization demonstrated that each gene was expressed within one of five distinct regions of the invasive tumors (neoplastic epithelium; angioendothelium; inflammatory, panstromal, and juxtatumoral stroma), reflecting a defined architectural structure to the transcriptome of invasive breast cancers. Two of these 6 genes were specifically expressed by the stromal cells within the invasive carcinoma; however, 1 (collagen 1alpha1) was expressed throughout the stromal response (panstromal expression), whereas the second (osteonectin) was specifically expressed within the juxtatumoral stromal cells, indicating a critical "regionality" of gene expression within the stromal response itself. A comparison of the gene expression profiles of the juxtatumoral stroma in breast and pancreatic carcinomas indicated important differences between the two, suggesting tumor-specific or organ-specific differences in the desmoplastic responses. Some of the genes presented are novel markers of the invasive process, imply communication at the host/tumor interface, and suggest potential therapeutic targets.

Transcriptional regulation of the human polymeric Ig receptor gene: analysis of basal promoter elements.

Secretory Igs provide the first line of adaptive immune defense against ingested, inhaled, and sexually transmitted pathogens at mucosal surfaces. The polymeric Ig receptor regulates transport of dimeric IgA and pentameric IgM into external secretions. The level of expression of polymeric Ig receptor is controlled to a large extent by transcription of the PIGR gene in mucosal epithelial cells. Here we present a detailed analysis of the promoter of the PIGR gene by transient transfection of luciferase reporter plasmids into cultured cell lines. Comparisons of the human and mouse PIGR promoters in human and mouse intestinal and liver cell lines demonstrated that the human PIGR promoter was 4- to 5-fold more active than the mouse PIGR promoter in all cell types, and that both the human and mouse PIGR promoters were more active in intestinal than in liver cell lines. Targeted deletions of 22-bp segments of the human PIGR promoter revealed that the region from nt -63 to -84 is crucial for basal transcription, and that two upstream regions can act as positive or negative regulators. Point mutations within the region from nt -63 to -84 demonstrated that an E box motif, which binds the basic helix-loop-helix protein upstream stimulatory factor, is required for PIGR promoter activity. Two additional regulatory motifs were identified in the proximal promoter region: a binding site for AP2, and an inverted repeat motif that binds an unidentified protein. These findings suggest that cooperative binding of multiple transcription factors regulates basal activity of the human PIGR promoter.