Kras regulatory elements and exon 4A determine mutation specificity in lung cancer

Abstract

Kras is the most frequently mutated ras family member in lung carcinomas, whereas Hras mutations are common in tumors from stratified epithelia such as the skin. Using a Hras knock-in mouse model, we demonstrate that specificity for Kras mutations in lung and Hras mutations in skin tumors is determined by local regulatory elements in the target ras genes. Although the Kras 4A isoform is dispensable for mouse development, it is the most important isoform for lung carcinogenesis in vivo and for the inhibitory effect of wild-type (WT) Kras on the mutant allele. Kras 4A expression is detected in a subpopulation of normal lung epithelial cells, but at very low levels in lung tumors, suggesting that it may not be required for tumor progression. The two Kras isoforms undergo different post-translational modifications; therefore, these findings can have implications for the design of therapeutic strategies for inhibiting oncogenic Kras activity in human cancers.

Figure 1. Ras protein levels and effects on downstream signaling effectors in HrasKI and KrasKI mice

Analysis of Ras protein levels in lungs of untreated mice showed elevated levels of Hras protein and complete absence of Kras protein in HrasKI homozygous mice. Mice homozygous and heterozygous for the KrasKI allele express similar Kras protein levels to WT mice. No apparent differences in activation of Akt and Erk, as measured by protein phosphorylation, and in level of p38α were observed among mice of the indicated genotypes.

Figure 2. HrasKI mice are highly susceptible to urethane induced lung tumours

HrasKI homozygous mice developed significantly more lung tumours than WT littermates. The increase in lung tumour number correlated with the number of HrasKI alleles. Error bars indicate standard deviations, and statistics were performed using the Wilcoxon Mann-Whitney test.

Figure 3. Lung tumours from HrasKI mice display papillary, solid and mixed growth patterns, and contain cells with an epithelioid morphology

WT and HrasKI mice developed a spectrum of papillary (a), mixed (b) and solid adenomas (c). Whereas WT animals predominantly developed papillary adenomas with occasional mixed and solid tumours, the HrasKI mice tended to develop more mixed and solid adenomas, sometimes with prominent intrabronchiolar extension (c, inset, marked by asterisk). The dashed line in (b) marks the boundary between the papillary (left) and solid (right) portions of the mixed adenoma. Several solid adenomas from HrasKI mice contained epithelioid cells (d, inset enlarged in e) which stained positive for (f) cytokeratin 8/18 and negative for PAS (g). Occasional macrophage clusters (f, keratin-negative cells) were also present in the epithelioid adenomas. Goblet cell metaplasia served as a positive control for PAS/mucin (g, inset). These adenomas displayed features of alveolar type II pneumocytes like their papillary counterparts (not shown) as they stain positively for SPC (h) and negatively for CCA/CC10 (i). The adjacent bronchiolar epithelium stained positive for CCA/CC10 as expected (i, inset).

Figure 4. The KrasKI allele renders mice resistant to urethane induced lung carcinogenesis, and is deficient in suppression of lung tumour development in Kras^LA2 animals

(a) KrasKI homozygous mice were almost free of lung tumours, while KrasKI heterozygous mice remained susceptible to lung carcinogenesis, and developed significantly more lung tumours than WT mice. Mutations in these tumours were found exclusively in the endogenous Kras gene and not the KrasKI allele. (b) Kras^LA2 mice harboring the KrasKI allele, which generates only the 4B isoform, developed almost 3-fold more lung tumours than those carrying an intact Kras gene capable of generating both 4A and 4B isoforms. The HrasKI allele in Kras^LA2 displayed an inhibitory effect on tumor number similar to WT mice. Error bars indicate standard deviations, and statistics were performed using the Wilcoxon Mann-Whitney test.

Figure 5. Kras4A is expressed in normal lung epithelium but not significantly in tumours

Kras-4A staining was observed in a sub-population of Clara cells in WT129/Sv (a) and FVB/N mice (b) but absent in homozygous KrasKI mice (expressing only Kras-4B) (c). The staining was observed throughout the bronchial tree, terminating at bronchio-alveolar duct junctions, locations of the BASCs. In contrast, papillary adenomas from 129/Sv mice do not show significant staining for Kras4A (d), although adjacent bronchial structure showed positive staining (d, inset).