Figure 5. Loss of Rasal2 promotes metastasis and Ras activation in a genetically engineered mouse model of breast cancer.

(A) Schematic of Rasal2 genomic locus and pNMDi4 genetrap cassette. Un-shaded regions in exons 1 and 18 mark 5′ and 3′ UTRs, respectively. Known domains of Rasal2 are noted (PH, C2, and RasGAP). See Experimental Procedures for detailed description of pNMDi4. The genetrap cassette targets the third intron of Rasal2.

(B) Genotyping of Rasal2 mice to distinguish wild type (WT), heterozygous mutant (het), and homozygous mutant (hom).

(C) Western blot confirming loss of Rasal2 protein in genetrap animals (mammary gland tissue). WT: wild type, het: heterozygous, hom: homozygous mutant.

(D) Top: H&E images of primary mammary adenocarcinomas from MMTVneu; Rasal2 +/+ and MMTVneu; Rasal2 −/− animals. Bottom: H&E images of lung metastases from MMTVneu; Rasal2 +/+ and MMTVneu; Rasal2 −/− animals. M indicates metastases.

(E) Lung metastasis burden in MMTVneu; Rasal2 +/+ and MMTVneu; Rasal2 −/− animals. Lung metastasis incidence: percent of tumor-bearing females with lung metastases at sacrifice (p=0.05; n=24 MMTVneu; Rasal2 +/+, n=23 MMTVneu; Rasal2 −/−). Average number of lung metastases per animal: counted per representative section of lungs for each tumor-bearing female (p=0.04). Average metastasis burden per animal: average total area of metastasis in a representative section of lung for each tumor-bearing female (arbitrary units; p=0.04). See also Figure S2.

(F) H&E images of metastases to brain (a), gut (b), ovary (c), and kidney (d) in compound tumor-bearing females. M indicates regions of metastasis.

(G) Western blot analysis of phospho-ERK (pERK) and phospho-AKT (pAKT) levels in primary mammary tumors from MMTVneu; Rasal2 +/+ animals (numbers 1–9) and MMTVneu; Rasal2 −/− animals (numbers 10–18).