Endogenous expression of Hras(G12V) induces developmental defects and neoplasms with copy number imbalances of the oncogene

Abstract

We developed mice with germline endogenous expression of oncogenic Hras to study effects on development and mechanisms of tumor initiation. They had high perinatal mortality, abnormal cranial dimensions, defective dental ameloblasts, and nasal septal deviation, consistent with some of the features of human Costello syndrome. These mice developed papillomas and angiosarcomas, which were associated with Hras(G12V) allelic imbalance and augmented Hras signaling. Endogenous expression of Hras(G12V) was also associated with a higher mutation rate in vivo. Tumor initiation by Hras(G12V) likely requires augmentation of signal output, which in papillomas and angiosarcomas is achieved via increased Hras-gene copy number, which may be favored by a higher mutation frequency in cells expressing the oncoprotein.

Fig. 1.

Development of mice with a conditional knock-in activating mutation of Hras. (A) Diagram of Hras targeted allele. (Top) Targeting vector consists of a 5′arm containing the WT Hras gene and a 3′arm containing the mutant Hras gene, which are separated by an Frt-flanked Neomycin minigene. (Middle) Targeted allele after crossing with β actin-Flp mice to remove the Neomycin minigene. (Bottom) Targeted allele after crossing with Caggs-Cre mice to remove the WT Hras copy. (B) Southern blot of tail DNA isolated from WT, FR-Hras^G12V, or CC/FR-Hras^G12V± mice cut with XbaI and EcoRV and probed with a genomic fragment containing exons 1–4 of Hras. Wt, wild-type allele; Targ, targeted allele. (C) Sequence trace of products generated by RT-PCR of RNA isolated from MEFs from FR-Hras^G12V or CC/FR-Hras^G12V embryos. (D) Western blot of activated Hras in MEFs from CC/FR-Hras^G12V and control mice. Activated Ras proteins were pulled down with an agarose-conjugated Raf-1 Ras-binding domain, followed by SDS/PAGE gel electrophoresis and immunoblotting with a specific anti-Hras antibody. Western blot of total lysate with Hras IgG is shown below. (E) RT-PCR products of RNA isolated from MEFs from WT or CC/FR-Hras^G12V embryos were incubated with or without Gsu I, which digests WT but not mutant Hras cDNA. Total Hras cDNA in the absence of Gsu I was normalized to 100%. The right panel shows that the mutant Hras cDNA is ≈50% of total Hras in CC/FR-Hras^G12V MEFs.

Fig. 2.

Increased neonatal mortality and cranio-facial deformities in CC/FR-Hras^G12V± mice. (A) Kaplan-Meier survival plot of CC/FR-Hras^G12V and control mice. (B) Decreased body weight in CC/FR-Hras^G12V mice at weaning. Weight of CC/FR-Hras^G12V mice and control littermates at 3 and 20 weeks of age. Bars represent mean ± SE percent-change in body weight versus controls (n = 8; P = 7.7 ×10⁻⁷ at 3 weeks, P = 0.25 at 20 weeks). (C) Coronal sections (4×) across the nasal cavity of a representative WT (a) and CC/FR-Hras^G12V (b) mouse. The mutant mouse exhibits marked nasal septal deviation. Low power magnification (20×) of an incisor of a WT (C) and a mutant (D) mouse. The tooth of the CC/FR-Hras^G12V± mice has an abnormal ameloblast cell lining and defective enamel formation. (e) Higher magnification (40×) demonstrates detachment of the ameloblasts from the adjacent dentin, loss of polarity, and stratification of ameloblasts, as well as areas of enamel sequestration. (f) Mouse with misalignment of incisors and malocclusion. (D) (Left) Whole-body CT scan (sagittal view) illustrating the rectangular region of interest used to determine the overall cephalo-caudal and ventro-dorsal dimensions of the cranial bony structures. (Right) Abnormal cranial dimensions of CC/FR-Hras^G12V mice. Mean cephalo-caudal to ventro-dorsal ratio in aged-matched control and CC/FR-Hras^G12V mice. All mice studied in this article are Hras^G12V heterozygous mice.

Fig. 3.

Squamous papilloma and angiosarcoma development in CC/FR-Hras^G12V mice is associated with augmented Hras signaling. (A) (Upper) Representative papilloma in a CC/FR-Hras^G12V mouse. Papillomas frequently formed in areas exposed to friction. (Lower) Stomach from a 28-week-old CC/FR-Hras^G12V mouse showing multiple papillomas in the stomach fundus, but not in the cardia or antrum. (B) Representative IHC staining for Ki67, pAKT, pS6, and pERK1/2 in sections of papilloma and adjacent non-neoplastic skin from CC/FR-Hras^G12V mice. (C) H&E-stained sections (10×) of the external auditory canal of a CC/FR-Hras^G12V mice with epidermal and sebaceous hyperplasia. (D) Representative H&E section (100×) of angiosarcoma developing in CC/FR-Hras^G12V mice.

Fig. 4.

Hras allelic imbalance in papillomas and angiosarcoma from CC/FR-Hras^G12V mice. (A) PCR of genomic DNA of papillomas with primers that distinguish mutant from WT Hras alleles (see Fig. 1A). W: 622 bp WT allele; M: 666 bp targeted allele because of insertion of loxP site. P1-P7: DNA from papillomas, or indicated nontumoral tissues. (B) PCR of DNA from forestomach papillomas (FP1–FP3), angiosarcomas (A1–A4), or indicated nontumoral tissues. (C) FISH of a representative section from papilloma tissue (P2) using a mouse BAC containing the Hras gene. Papilloma nuclei have 3 to 5 fluorescent signals corresponding to Hras (red), whereas adjacent skin is diploid. Mouse chromosome 7 centromeres are labeled in green. (D) (Left) Increased mutation rate in thyroid cells from FR-Hras^G12V/TPO-Cre mice. Plasmids rescued from DNA extracts of thyroid glands of TPO-Cre/pUR288-LacZ or FR-Hras^G12V/TPO-Cre/pUR288-LacZ were screened for alterations in LacZ, as described in Methods. Bars represent the mean mutation frequency ± SE of pooled samples from TPO-Cre/pUR288-LacZ (n = 3; 10 thyroids per pool) and FR-Hras^G12V/TPO-Cre/pUR288-LacZ (n = 4; 10 thyroids per pool). P < 0.05. (Right) Representative Southern blot of LacZ-negative clones. The type of mutation was determined by PCR amplification and restriction digestion of LacZ-negative clones. Clone 1 contained an inactivating point mutation of LacZ, whereas the other 3 show distinct restriction profiles consistent with recombination events, insertions, or deletions.