DNA mismatch repair deficiency accelerates lung neoplasm development in K-rasLA1/+ mice: a brief report

Inherited as well as acquired deficiencies in specific DNA mismatch repair (MMR) components are associated with the development of a wide range of benign and malignant neoplasms. Loss of key members such as MSH2 and MLH1 severely cripples the ability of the cell to recognize and correct such lesions as base:base mismatches and replicative DNA polymerase errors such as slippages at repetitive sequences. Genomic instability resulting from MMR deficiency not only predisposes cells to malignant transformation but may also promote tumor progression. To test the latter, we interbred Msh2−/− mice with the K-rasLA1/+ transgenic line that spontaneously develops a range of premalignant and malignant lung lesions. Compared to K-rasLA1/+mice, K-rasLA1/+; Msh2−/− mice developed lung adenomas and adenocarcinomas at an increased frequency and also demonstrated evidence of accelerated adenocarcinoma growth. Since MMR defects have been identified in some human lung cancers, the mutant mice may not only be of preclinical utility but they will also be useful in identifying gene alterations able to act in concert with Kras mutants to promote tumor progression.


Introduction
Non-small-cell lung cancer (NSCLC) is the leading cause of cancer-related mortality in the world. One of the most common oncogenic mutations in NSCLC involves activation of the KRAS proto-oncogene, an event seen in 25-30% of these human cancers [1][2][3]. Mirroring this, a Kras-mutant mouse (designated K-ras LA1/+ ) demonstrates stochastic acquisition of an activating Kras G12D mutation as a result of random DNA recombination events in vivo [2]. This leads primarily to the development of spontaneous multifocal lung epithelial hyperplasias, adenomas, and adenocarcinomas that are present at various stages of development in older animals [2,4].
Loss of MSH2, a key component of the DNA mismatch repair (MMR) system results in cancer predisposition in both mice and humans due to the ensuing increase in genomic instability [5,6]. For example, using a transgenic mutational reporter system, we found that Msh2 À/À mice demonstrated up to a 15-fold elevation in mutation frequency in specific tissues [6]. Humans with germline mutations of genes encoding MMR components also show increased mutagenesis, as shown by increased microsatellite instability (MSI), as exemplified by hereditary

Cancer Medicine
Open Access nonpolyposis colon cancer [7]. Interestingly, reduced expression of MSH2 or MLH1 genes has been observed in over 50% of lung adenocarcinomas, and was correlated with decreased overall survival due to increases in MSI and a generalized "mutator" phenotype [8][9][10]. Herein, we report that MMR deficiency accelerates lung tumor development in K-ras LA1/+ mice.

A B C D
sectioning. One section at a depth of 100 lm was stained with hematoxylin and eosin stain (H&E). Tumors were quantified and histopathology determined [1].

Results
Increased tumor number in K-ras LA1/+ ; Msh2 À/À mice A count of grossly visible (macroscopic) tumors on the surfaces of the lungs (>1 mm in diameter) indicated that both K-ras LA1/+ and K-ras LA1/+ ; Msh2 À/À mice had significantly increased tumor burden compared to wt or Msh2 À/À genotypes alone at both 60-90 and 90-120 days old time points, while Msh2 À/À mice exhibited a very low frequency of lung tumor development at both time points  ( Fig. 1A). However, the double mutant K-ras LA1/+ ; Msh2 À/À mice had a significantly greater tumor burden than K-ras LA1/+ mice at 90-120 days (mean age = 99 days). In addition, the K-ras LA1/+ ; Msh2 À/À mice were the only group that demonstrated significant increases in tumor burden between 60-90 and 90-120 days old time points surveyed within the same genotype (Fig. 1A). A representative slide from the center of the lung lobes from each mouse was used to assess microscopic tumor numbers (Figs. 1B and 2). Again, a significant increase in tumor formation was present in the 90-120 days old Kras LA1/+ ; Msh2 À/À mice as compared to all other groups, including the 60-90 days old K-ras LA1/+ ; Msh2 À/À mice.
Accelerated NSCLC development in K-ras LA1/+ ; Msh2 À/À mice Tumor diameters, measured as an indicator of tumor growth and development, revealed that 90-120 days old K-ras LA1/+ ; Msh2 À/À mice showed a trend toward increased tumor size (although no statistical linear trend was observed), compared with K-ras LA1/+ mice, indicative of accelerated NSCLC growth (Fig. 1C). K-ras LA1/+ ; Msh2 À/À mice showed similar pathology to K-ras LA1/+ mice [1], from mild hyperplasia, progressing to adenoma and adenocarcinoma, with the exception of invasive adenocarcinomas present at 100 days in the K-ras LA1/+ ; Msh2 À/À mice (Fig. 3), a feature not commonly seen in K-ras LA1/+ until the latter were closer to 180 days of age (data not shown). Tumor quantification of K-ras LA1/+ ; Msh2 À/À mice demonstrated a significant acceleration in lung tumor development (Table 1), with a higher incidence of adenocarcinomas and a statistically significant increase in adenoma formation as compared to all other groups of mice. There was an increase in Ki67-positive cells in tumors of 90-120 days old Kras LA1/+ ; Msh2 À/À mice, as compared with tumors in mice of the same genotype when these were analyzed at an earlier age (60-90 days) (Fig. 4).

Discussion
Although K-ras LA1/+ mice rapidly develop a range of lung tumors, we found an acceleration of lung adenocarcinoma development in K-ras LA1/+ ; Msh2 À/À mice by~120 days of age. Comparable to our results, deficiencies of two DNA repair genes (Myh À/À and Ogg1 À/À ) predisposed mice to lung tumors, and this was attributed largely to acquired K-ras G12D mutations [12]. The complex nature of the genomic instability that characterizes Msh2-null mice might suggest that in the double mutant K-ras LA1/+ ; Msh2 À/À mice, either K-ras LA1/ + recombination events were occurring at a higher frequency than in mice with K-ras LA1/+ alone, or that addi-tional mutations in key growth control genes were stimulating lung tumor development and possibly progression. The cause of the tumor acceleration was likely due to acquisition of additional pro-oncogenic mutations within the tumor. While we cannot confirm this possibility, we observed an increase in Ki67-positive cells in the 90-120 days old K-ras LA1/+ ; Msh2 À/À tumors, perhaps indicative of a cell cycle checkpoint defect. For example, it is plausible that Msh2 À/À deficiency in the K-ras LA1/+ mice accelerated the acquisition of mutations within the p53 gene, or other genes such as the TGF-b receptor [6], that could potentially play a role in promoting adenocarcinoma development.
Herein, we present evidence that DNA MMR deficiency can act in concert with one of the most frequently activated oncogenes in NSCLC, K-ras, to enhance lung tumor development. In humans, reports of reduced hMLH1 and hMSH2 expression in NSCLC have varied from~18% to 61% and these decreases have typically been attributed to epigenetic silencing. Furthermore, losses of hMSH2 (and other MMR proteins, such as hMLH1), have correlated with an overall poor prognosis [13,14]. This mouse model may provide the impetus required for addressing lung cancer formation in this population, in addition to providing a means for studying the tumor progression and targeted therapeutics.