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. 2008 Dec;10(12):1362-72, following 1372.
doi: 10.1593/neo.08784.

Neurofibromin 1 (NF1) defects are common in human ovarian serous carcinomas and co-occur with TP53 mutations

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Free PMC article

Neurofibromin 1 (NF1) defects are common in human ovarian serous carcinomas and co-occur with TP53 mutations

Navneet Sangha et al. Neoplasia. 2008 Dec.
Free PMC article

Abstract

Ovarian serous carcinoma (OSC) is the most common and lethal histologic type of ovarian epithelial malignancy. Mutations of TP53 and dysfunction of the Brca1 and/or Brca2 tumor-suppressor proteins have been implicated in the molecular pathogenesis of a large fraction of OSCs, but frequent somatic mutations in other well-established tumor-suppressor genes have not been identified. Using a genome-wide screen of DNA copy number alterations in 36 primary OSCs, we identified two tumors with apparent homozygous deletions of the NF1 gene. Subsequently, 18 ovarian carcinoma-derived cell lines and 41 primary OSCs were evaluated for NF1 alterations. Markedly reduced or absent expression of Nf1 protein was observed in 6 of the 18 cell lines, and using the protein truncation test and sequencing of cDNA and genomic DNA, NF1 mutations resulting in deletion of exons and/or aberrant splicing of NF1 transcripts were detected in 5 of the 6 cell lines with loss of NF1 expression. Similarly, NF1 alterations including homozygous deletions and splicing mutations were identified in 9 (22%) of 41 primary OSCs. As expected, tumors and cell lines with NF1 defects lacked mutations in KRAS or BRAF but showed Ras pathway activation based on immunohistochemical detection of phosphorylated MAPK (primary tumors) or increased levels of GTP-bound Ras (cell lines). The TP53 tumor-suppressor gene was mutated in all OSCs with documented NF1 mutation, suggesting that the pathways regulated by these two tumor-suppressor proteins often cooperate in the development of ovarian carcinomas with serous differentiation.

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Figures

Figure 1
Figure 1
ROMA detects homozygous deletions including NF1 in primary OSCs. Using a moving average algorithm of the window of five data points, the raw DNA copy number ratio of tumor over normal reference was smoothened and plotted against the chromosomal position of all the oligonucleotide probes on chromosome 17 for tumor samples OSC-1 (A) and OSC-11 (B). The arrows point at the apparent homozygous deletions including the NF1 locus. To provide greater resolution of the deleted region, an area of 4 Mb across the NF1 deletion is shown for OSC-1 (C) and OSC-11 (D). Location of NF1 is as indicated in panel (D). Chromosomal positions of ROMA oligonucleotide probes were based on the NCBI build 34 (hg16 assembly).
Figure 2
Figure 2
Southern blot analysis reveals homozygous deletions of NF1 in primary OSCs. (A) Southern blot analysis of EcoRI-digested OSC DNA samples using three probes at different locations within the NF1 gene. To control for loading, the blots were rehybridized with a probe for DHX40, which lies telomeric to NF1 on chromosome 17q. (B) The relative location of each probe on chromosome 17 is shown in the diagram.
Figure 3
Figure 3
Expression of NF1 is reduced or absent in several ovarian carcinoma cell lines. (A) NF1 transcripts in the indicated cell lines were detected by Northern blot analysis, using expression of GAPDH as a loading control. Relative expression of NF1 in each cell line normalized to GAPDH (arbitrary units) is indicated. (B) Nf1 protein levels in the same cell lines were determined by Western blot analysis using an anti-Nf1 antibody. Detection of actin was used as a loading control.
Figure 4
Figure 4
The PTT assay shows truncated Nf1 peptides in ovarian carcinoma cell lines and primary tumors. (A) PTT was performed using five overlapping cDNA segments encompassing the entire NF1 coding region. Representative examples of truncated peptides in segments II and III from the indicated cell lines are shown. The TOV21G cell line expresses normal levels of full-length NF1, and it was used to determine the size of the expected (normal) PTT products in each segment. (B) Representative examples of truncated peptides in Segments III, IV and V from the indicated OSC samples are shown. The TOV21G cell line expresses normal levels of NF1, and it was used to determine the size of the expected (normal) PTT products in each segment.
Figure 5
Figure 5
NF1 cDNA sequence alterations in representative cell lines and primary tumors. Chromatograms showing (A) absence of 17 bp of exon 17 from the cDNA of cell line OVCA429, (B) deletion of exon 4a in cDNA from PEO4, (C) portion of 178 bp insertion from intron 44 between exons 44 and 45 in tumor OSC-18, and (D) deletion of exons 22 and 23-1 in cDNA from tumor OSC-25.
Figure 6
Figure 6
The Ras pathway is activated in ovarian carcinoma cell lines and primary OSCs. (A) Ras-GTP and total Ras levels in the ovarian carcinoma cell lines were detected using a Ras activation assay kit as described in the Materials and Methods section. Representative data are shown. The blot was reprobed with antiactin as a loading control. HCT116, which does not express NF1, and HEY, which expresses mutant KRas (G12D), were used as a positive controls for Ras-GTP levels. Expression of Nf1 in each cell line [low or absent (-) versus readily detectable (+)] is indicated below each lane. (B) Immunohistochemical analysis of pMAPK in primary OSCs; representative examples are shown. OSC-32 (left panel) has a NF1 mutation and shows strong nuclear staining for pMAPK in the tumor cells, with absence of staining in the non-neoplastic stromal cells. No NF1 alterations were detected in OSC-33, which is negative for pMAPK expression (right panel).

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