Ischemia-Induced K-ras Mutations in Human Colorectal Cancer Cells: Role of Microenvironmental Regulation of MSH2 Expression

Abstract

Mutation of the K-ras gene is one of the most common genetic alterations in solid tumors, including colorectal cancer. The relatively late emergence of K-ras mutations in colorectal cancer is particularly striking in the class of mismatch repair-deficient tumors associated with earlyonset microsatellite instability. We, therefore, tested the hypothesis that the microsatellite instability phenotype itself does not efficiently trigger K-ras mutations in colorectal cancer cells, but rather that tumor-associated microenvironmental conditions (e.g., hypoxia and hypoglycemia) contribute to this event by modulating genetic instability. We examined K-ras G13D mutation using PCR-RFLP analysis in two different microsatellite instability colorectal cancer cell lines (HCT116 and DLD-1) and their variants in which the mutant (but not the wild-type) K-ras allele has been genetically disrupted (Hkh-2 and Dks-8). We found K-ras G13D mutation to occur at far greater incidence in cells derived from xenografted tumors or exposed to conditions of combined hypoxia and hypoglycemia in vitro . Interestingly, this mutagenesis was neither enhanced by induced oxidative damage nor prevented by the antioxidant vitamin E. Moreover, the accumulation of K-ras mutations was paralleled by down-regulation of the key mismatch repair protein MSH2 in xenografted tumors, particularly in hypoperfused areas and under hypoglycemic conditions (in vitro). In contrast, the microsatellite stable colorectal cancer cell line Caco-2 neither accumulated K-ras mutations nor showed down-regulation of MSH2 under these conditions. Thus, our study suggests that ischemia may not simply select for, but can actually trigger, increased mutation rate in crucial colorectal cancer oncoproteins. This finding establishes a novel linkage between genetic instability, tumor ischemia, and genetic tumor progression and carries important implications for applying anticancer therapies involving tumor hypoxia (e.g., antiangiogenesis) in microsatellite instability cancers. (Cancer Res 2005; 65(18): 8134-41)

Low-dose metronomic cyclophosphamide treatment mediates ischemia-dependent K-ras mutation in colorectal carcinoma xenografts

“…While originally reported to be non-tumorigenic (Shirasawa et al, 1993) in our hands these cells demonstrate attenuated tumorigenicity (Shahrzad et al, 2005), and in this study formed palpable tumors in all mice 45 days after subcutaneous injection of 2 Â 10 6 cells into the right flank of RAG-1 null mice. When the average tumor size reached 35 mm 3 mice were randomized into treatment and control groups and subjected to low-dose (metronomic) cyclophosphamide (LDM) for a period of 2 weeks followed by a 2-week break period.…”

“…Since we had previously shown that K-ras wt/null Dks-8 xenografts develop mutations in codon 13 of this oncogene (Shahrzad et al, 2005), we explored the possibility that this event may have been enhanced in treated tumors.…”

“…Thus, a rapid accumulation of mutant cells was facilitated in tumors exposed to LDM-induced hypoxia. Using similar PCR-RFLP approaches (modified from Toyooka et al, 2003;Shahrzad et al, 2005), we further examined K-ras mutations at codon 61 in the xenografts. We found no evidence for de novo development of mutation at this additional codon in either control or treatment group of the xenografted tumors (Supplementary Figure 1).…”

“…In addition to various selective mechanisms (Rak et al, 1996;Yu et al, 2001Yu et al, , 2002, we have recently reported that the ischemic tumor microenvironment may actually induce de novo K-ras mutations in CRC cells, notably those with a defect in DNA mismatch repair (MMR) mechanisms (Shahrzad et al, 2005). In this case, ischemic conditions downregulated one of the MMR proteins (MSH2) leading to an increase in frequency of K-ras mutations and eventual enrichment in mutant CRC cells (Shahrzad et al, 2005).…”

“…In addition to various selective mechanisms (Rak et al, 1996;Yu et al, 2001Yu et al, , 2002, we have recently reported that the ischemic tumor microenvironment may actually induce de novo K-ras mutations in CRC cells, notably those with a defect in DNA mismatch repair (MMR) mechanisms (Shahrzad et al, 2005). In this case, ischemic conditions downregulated one of the MMR proteins (MSH2) leading to an increase in frequency of K-ras mutations and eventual enrichment in mutant CRC cells (Shahrzad et al, 2005). Mutant K-ras occurs in approximately half of all CRC cases, 75-100% of pancreatic cancers and almost 50% of lung adenocarcinomas (Minamoto et al, 2002), and is associated with chemoresistance and radioresistance (Bernhard et al, 2000;Perona and Sa´nchez-Pe´rez, 2004), and the angiogenic phenotype (Rak et al, 1995).…”