Overexpression of Sprouty 2 in mouse lung epithelium inhibits urethane-induced tumorigenesis

Abstract

Members of the Sprouty family encode novel proteins that are thought to function primarily as intracellular antagonists of the Ras-signaling pathway. Increased Ras signaling is a critical characteristic of human lung adenocarcinoma, the most common type of non-small cell lung cancer. Sprouty 2 is expressed in the lung epithelium, the tissue layer from which lung cancers arise. We hypothesized that overexpression of Sprouty 2 in the distal lung epithelium would inhibit lung tumorigenesis. To test the hypothesis, the consequences of overexpressing Sprouty 2 in the distal lung epithelium on urethane-induced mouse lung tumorigenesis were determined. Urethane is a chemical carcinogen found in tobacco smoke that causes activating mutations in Kras and induces lung tumors in mice. Sprouty 2-overexpressor mice developed significantly fewer lung tumors compared with their littermate controls (13.2 +/- 1.1 versus 18.1 +/- 1.3, P = 0.006). Tumor diameter was also significantly smaller in Sprouty 2 overexpressors (0.85 mm +/- 0.03 versus 0.95 mm +/- 0.02, P = 0.005). Sprouty 2 overexpression did not alter Kras mutational frequencies in urethane-induced tumors, suggesting that the tumor-suppressing effect of Sprouty 2 overexpression acts at a stage after Kras mutation, perhaps by interfering with receptor tyrosine kinase-induced signaling. These results demonstrate that Sprouty 2 overexpression inhibited both tumor initiation and subsequent tumor growth.

Figure 1.

Schematic representation of Spry2 conditional gain-of-function transgene. The unrecombined allele constitutively expresses the β-galactosidase–neomycin fusion cDNA. In the presence of the Cre recombinase, the β-galactosidase–neomycin fusion cDNA is deleted, and the Spry2 IRES HPLAP cDNA is constitutively expressed. “Constitutive promoter” = CMV enhancer element fused to the Chick β actin promoter; P = loxP; IRES = internal ribosomal entry site.

Figure 2.

Overexpression of the recombined Spry2 IRES AP transgene in the lung. SPC-rtTA, Tet-o-Cre, Spry^GOF triple transgenic adult mouse and a littermate control drank water supplemented with doxycycline for 1 week. Mice were then killed, and lungs were processed for alkaline phosphatase histochemical staining and then cryosectioned. (A) Littermate control lungs show no alkaline phosphatase staining. (B) In contrast, triple transgenic lungs show alkaline phosphatase staining in alveoli due to Cre-mediated recombination of the Spry2^GOF transgene, which results in expression of both Spry2 and the AP reporter cDNAs. Magnification = ×200. (C) Western blot demonstrating doublet of Spry2 protein in lung homogenates from two Spry2 overexpressors (lanes 1 and 2) and littermate controls (lanes 3 and 4). Bracket marks doublet.

Figure 3.

Low-power view of alkaline phosphatase histochemical stained lung from littermate control (left panel) and triple transgenic (right panel) mice from Figure 2, demonstrating significant patchiness in staining for alkaline phosphatase, a surrogate for Spry2 expression.

Figure 4.

Mice that overexpress Spry2 (lightly shaded bars) in the lung epithelium developed significantly fewer urethane-induced lung tumors than littermate controls (darkly shaded bars) 16 weeks after injection of urethane (13.2 ± 1.1 versus 18.1 ± 1.3, mean ± SEM; P = 0.006).

Figure 5.

Mice that overexpress Spry2 (lightly shaded bars) in the lung epithelium developed significantly smaller urethane-induced lung tumors than littermate controls (darkly shaded bars) 16 weeks after injection of urethane (0.85 mm ± 0.03 versus 0.95 mm ± 0.02, mean ± SEM; P = 0.005).