Analysis of chromosome 10 aberrations in rat endometrial cancer—evidence for a tumor suppressor locus distal to Tp53

Abstract

We have recently shown in the BDII rat model of human endometrial adenocarcinoma (EAC), rat chromosome 10 (RNO10) is frequently involved in chromosomal aberrations. In the present study, we investigated the association between RNO10 deletions, allelic imbalance (AI) at RNO10q24 and Tp53 mutation in 27 rat EAC tumors. We detected chromosomal breakage accompanied by loss of proximal and/or gain of distal parts of RNO10 in approximately 2/3 of the tumors. This finding is suggestive of a tumor suppressor activity encoded from the proximal RNO10. Given the fact that Tp53 is located at RNO10q24-q25, we then performed Tp53 mutation analysis. However, we could not find a strong correlation between AI/deletions at RNO10q24 and Tp53 mutation. Instead, the observed patterns for AI, chromosomal breaks and deletions suggest that major selection was directed against a region located close to, but distal of Tp53. In different human malignancies a similar situation of AI at chromosome band 17p13.3 (HSA17p13.3) unassociated with TP53 mutation has been observed. Although RNO10 is largely homologous to HSA17, the conservation with respect to gene order among them is not extensive. We utilized publicly available draft DNA sequences to study intrachromosomal rearrangement during the divergence between HSA17 and RNO10. By using reciprocal comparison of rat and human genome data, we could substantially narrow down the candidate tumor suppressor region in rat from 3 Mb to a chromosomal segment of about 0.5 Mb in size. These results provide scientific groundwork for identification of the putative tumor suppressor gene(s) at 17p13.3 in human tumors. ' 2006 Wiley-Liss, Inc.Key words: BDII; endometrial adenocarcinoma; RNO10; 17p13.3; allelic imbalance; Tp53 mutation; tumor suppressor gene Endometrial cancer is the most frequently diagnosed female genital tract malignancy in the western world. 1 Endometrial adenocarcinoma (EAC) is the prevalent subtype, accounting for approximately 75% of the reported cases. 2 It has been clearly demonstrated that an inherited genetic predisposition plays a critical role in the development of many cases of EAC, as the risk for a woman to develop EAC is tripled when there is an affected firstdegree relative. 3,4 Molecular genetic analysis of uterine tumor biopsies have revealed alterations in a number of chromosomal regions harboring transforming genes, including tumor suppressor genes (e.g. TP53, PTEN and hMLH1) and oncogenes (e.g. K-RAS and c-ERBB2/neu). 1,5-9 However, the molecular genetic events underlying endometrial cancer tumorigenesis are still poorly understood.Females of the inbred BDII rat strain are genetically prone to spontaneously occurring hormone-related endometrial carcinoma, providing a suitable experimental model system for genetic analysis of inherited EAC in humans. 10,11 Cytogenetic and comparative genome hybridization (CGH) analyses of the tumors pointed to common deletions in the proximal part of rat chromosome 10 (RNO10) in the tumor material. 12 According to Knudson's two-hit theo...

Molecular classification of spontaneous endometrial adenocarcinomas in BDII rats

“…In type I tumors, abnormalities of Tp53 are suggested as late events and are thought to be important step in the progression to high-grade tumors (Ryan et al 2005). Earlier, we found recurrent Tp53 mutations (67%) in BDII rat EAC tumors (Nordlander et al 2007). However, in majority of the mutations resulting in amino acid exchange, the hydrophobicity or hydrophilicity was maintained by equivalent substitutions.…”

“…Frequent Tp53 mutations (Nordlander et al 2007) and lack of K-ras mutation were shown in BDII rat EACs. Additionally, neither microsatellite instability (Behboudi et al 2001, Adamovic et al 2005, Nordlander et al 2005 nor Erbb2 amplifications were previously found among BDII rat tumors.…”

“…PCR amplification products were purified using GFX PCR DNA and Gel Band Purification Kit (Amersham Pharmacia Biotech). The purified fragments were subjected to sequencing using a second set of primers (Table 3) as described previously (Nordlander et al 2007). Briefly, using ABI PRISM BigDye1 Terminator v1.1 Cycle Sequencing Kit (PE Applied Biosystems), the purified DNA fragments were subjected to sequencing according to the protocol provided by the manufacturer.…”

“…Three EAC tumors (RUT3, NUT4, NUT12) displayed variants in the gene sequence, compared with the normal cDNA sequence. RUT3 that contained two copies of proximal RNO10 (Nordlander et al 2007) displayed a deletion of three nucleotides in exon 9 at codon 312 (1131-1133, NM_012591), resulting in deletion of one amino acid, Alanine, from the Irf1 activator domain. Possibly, this change occurred because of nucleotide slippage, since the result of the mutation is that a double CTG triplet (CTGCTG) becomes a single CTG (Fig.…”

“…1A). The cDNA from NUT12 (showing two copies of proximal RNO10 (Nordlander et al 2007)) displayed deletion of 146 nucleotides (nucleotides 349-495, NM_012591) encoding 50 amino acids (codon 49 to 99) of the Irf1 DNA binding domain (Fig. 1B).…”