Genetic Evidence for XPC-KRAS Interactions During Lung Cancer Development

Abstract

Lung cancer causes more deaths than breast, colorectal and prostate cancers combined. Despite major advances in targeted therapy in a subset of lung adenocarcinomas, the overall 5-year survival rate for lung cancer worldwide has not significantly changed for the last few decades. DNA repair deficiency is known to contribute to lung cancer development. In fact, human polymorphisms in DNA repair genes such as xeroderma pigmentosum group C (XPC) are highly associated with lung cancer incidence. However, the direct genetic evidence for the role of XPC for lung cancer development is still lacking. Mutations of the Kirsten rat sarcoma viral oncogene homolog (Kras) or its downstream effector genes occur in almost all lung cancer cells, and there are a number of mouse models for lung cancer with these mutations. Using activated Kras, Kras(LA1), as a driver for lung cancer development in mice, we showed for the first time that mice with Kras(LA1) and Xpc knockout had worst outcomes in lung cancer development, and this phenotype was associated with accumulated DNA damage. Using cultured cells, we demonstrated that induced expression of oncogenic KRAS(G12V) led to increased levels of reactive oxygen species (ROS) as well as DNA damage, and both can be suppressed by anti-oxidants. Our results suggest that XPC may help repair DNA damage caused by KRAS-mediated production of ROS.

Keywords: Kras; Lung cancer; ROS; XPC.

Fig. 1. Kaplan-Meier curves of different groups of mice

Mice from four groups (8 mice each for the control, Xpc^−/−, Kras^LA1 groups and 13 mice for Xpc^−/−Kras^LA1 group) were monitored for 60 weeks, and their surviving time was recorded. The data were analyzed using Kaplan-Meier analysis.

Fig. 2. Lung tissue morphology in different groups of mice

Lungs were sectioned and processed for Hematoxylin and eosin staining and photographs were taken at 100× magnification. A shows a normal lung morphology from the control group; B shows lung morphology from Xpc knockout mice; C shows adenoma morphology from Kras^LA1 mice; and D shows adenocarcinoma morphology from Xpc^−/−Kras^LA1 mice.

Fig. 3. Development of ACF in mice with different genetic background

The number of aberrant crypt foci (ACF) was visualized after special staining (see methods for details). No ACFs were observed in Xpc^−/−and the normal control mice, while Xpc^−/−Kras^LA1 mice had a significantly higher number of ACF than those from Xpc^+/+Kras^LA1 mice (P value < 0.05).

Fig. 4. DNA damage analysis in lung tissues from 2-months old mice with different genetic alterations

A: Fresh lung tissues from the control, Xpc^−/−, Kras^LA1 and Xpc^−/−Kras^LA1 mice were processed to measure DNA damage using the COMET assay. Bronchial epithelial cells were isolated and subjected to COMET assay. For each sample, 200 independent cells were evaluated. The difference between the control mice and the other three groups (Xpc^−/−, Kras^LA1 or Xpc^−/−Kras^LA1) were significant (with a P value <0.05; as indicated by *). Data from the Xpc^−/−Kras^LA1 mice were significantly higher than mice with a single gene mutation (Xpc^−/−or Kras^LA1) (with a P value < 0.05, indicated by **) or the sum from two single mutant mice. According to BLISS independence analysis, Xpc loss and Kras^LA1 expression had a more than additive effect (synergy) on induction of DNA damage.

Fig. 5. Detection of relative ROS in lung tissues using the amount of 4-HNE and 8-deoxogunine as markers

Specific antibodies to 8-deoxogunine and 4-HNE were used to detect the relative level of ROS in lung tissues by immunofluorescent staining (A shows 8-deoxogunine, and B shows 4-HNE). Lung tissues from the Xpc^−/−Kras^LA1 mice had the highest levels of 8-deoxogunine and 4-HNE than other groups. The control mice were shown in upper A and B. Representative images from Xpc^−/−Kras^LA1 and the control mice were shown.

Fig. 6. The effect of induced expression of KRAS^G12V on ROS and DNA damage in lung epithelial cells

Lung epithelial BEAS-2B1 cells were engineered to express KRAS^G12V under the control of IPTG. We monitored the relative ROS level by H2DCF-DA after induced expression of KRAS^G12V for 24 hours. NAC (N-acetyl-L-cysteine, 10 mmol/L) was used to decrease the ROS level. A: Changes in the levels of H2DCF-DA in cultured cells under different conditions. B: The levels of DNA damage as shown by COMET assay. Tail moment was used to express level of DNA damage. In the presence of NAC, KRAS^G12V failed to induce tail moment, suggesting that KRAS^G12V induces DNA damage through ROS production. * indicates statistical significance from other groups (P value < 0.05).