Temporal Dissection of K-rasG12D Mutant In Vitro and In Vivo Using a Regulatable K-rasG12D Mouse Allele

et al. (2012) Temporal Dissection of K-rasG12D Mutant In Vitro and In Vivo Using a Regulatable K-rasG12D Mouse Allele. PLoS ONE 7(5): e37308. doi:10.1371/journal.pone.0037308 G12D Temporal Dissection of K-ras Mutant In Vitro and In G12D Vivo Using a Regulatable K-ras Mouse Allele . Zuoyun Wang 0 Yan Feng 0 Nabeel Bardessy 0 Kwok-Kin Wong 0 Xin-Yuan Liu 0 Hongbin Ji 0 Vladimir V. Kalinichenko, Cincinnati Children's Hospital Medical Center, United States of America 0 1 State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences , Shanghai , China , 2 Massachusetts General Hospital Cancer Center, Massachusetts General Hospital , Boston , Massachusetts, United States of America, 3 Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School , Boston, Massachusetts , United States of America Animal models which allow the temporal regulation of gene activities are valuable for dissecting gene function in tumorigenesis. Here we have constructed a conditional inducible estrogen receptor-K-rasG12D (ER-K-rasG12D) knock-in mice allele that allows us to temporally switch on or off the activity of K-ras oncogenic mutant through tamoxifen administration. In vitro studies using mice embryonic fibroblast (MEF) showed that a dose of tamoxifen at 0.05 mM works optimally for activation of ER-K-rasG12D independent of the gender status. Furthermore, tamoxifen-inducible activation of K-rasG12D promotes cell proliferation, anchor-independent growth, transformation as well as invasion, potentially via activation of downstream MAPK pathway and cell cycle progression. Continuous activation of K-rasG12D in vivo by tamoxifen treatment is sufficient to drive the neoplastic transformation of normal lung epithelial cells in mice. Tamoxifen withdrawal after the tumor formation results in apoptosis and tumor regression in mouse lungs. Taken together, these data have convincingly demonstrated that K-ras mutant is essential for neoplastic transformation and this animal model may provide an ideal platform for further detailed characterization of the role of K-ras oncogenic mutant during different stages of lung tumorigenesis. - . These authors contributed equally to this work. Animal models which allows temporal regulation of gene activity have been proved very valuable for gaining insights into the gene function involved in different tumor formation stages. Previous studies have demonstrated that knock-in of ER region into certain genes including P53 and Myc to generate fusion proteins allow for precise and temporal regulation of gene activity via ectopic provision of 4-hydroxytamoxifen [1,2]. The Trp53ER KI/KI mouse model is the first example of genetic models that allows specific, rapid and reversible perturbation of the biological function of a single endogenous gene in vivo. Studies based on this mouse model have dissected the precise role of P53 engaged in different stages of tumorigenesis as well as tumor regression [2,3,4,5,6,7,8]. Similarly, studies using the MycER mouse allelesignificantly contribute to our understanding about the role of Myc in cell proliferation, differentiation as well as the relationship between stem cells and cancer [1,6,7,9,10,11,12]. Oncogenic mutations including K-RASG12D is frequently observed in approximately 20% of all types of human cancers including carcinomas of the lung, colon, and pancreas [13,14,15,16,17,18]. K-RAS is a small GTPase which works as a binary molecular switch between a GDP-bound inactive form and a GTP-bound active form. When mutated in codon 12, 13, or 61, the K-RAS mutants remain active and constitutively transduce signals through MAPK pathway and PI3K pathway [19,20,21,22]. Previous studies have demonstrated that proteins such Ras or Raf fused to ER could be activated by tamoxifen treatment [23,24,25,26,27]. However, there is no animal model has been established based on this knowledge yet. We have here engineered a knock-in allele encoding a 4hydroxy tamoxifen (4-OHT) inducible estrogen receptor-K-rasG12D (ER-K-rasG12D) with placing the Loxp-Stop-Loxp fragment in front of the exon 1 of K-ras coding region. Using the mouse embryonic fibroblast (MEF), we have shown that ER-K-rasG12D expression is induced by Adeno-Cre treatment and the activity of ER-KrasG12D mutant is regulated by an optimal dose of tamoxifen administration. We further demonstrated that the ER-K-rasG12D mutant is essential for neoplastic transformation as well as tumor maintenance. Materials and Methods Mouse cohorts and Treatment The K-rasG12D and p53 L/L mice were originally generously provided by T. Jacks (Cambridge, MA) and R. Depinho (Boston, MA), respectively. The LSL-ER-K-rasG12D mice allele was constructed by placing the estrogen receptor cDNA in front of the KrasG12D coding region as shown in Figure 1. The targeting vector carried a negative selection marker for diptheria toxin (DT), a positive selection marker for neomycin acetyltransferase (Neo) and loxP sites (black triangles). The restriction sites were BamH I (B); Kpn I (K); Not I (N); Xba I (Xb); Sph I (S). We electroporated embryonic stem cells and selected transformed cells by 39 arm and 59 arm PCR screening to identify 3 recombinants. Blastocyst injections were carried out with these different targeted clones and germline transmission was achieved. The LSL-ER-K-rasG12D mice were then crossed to p53 L/L mice to obtain LSL-ER-K-rasG12D, p53L/L mice. All mice were housed in a specific pathogen-free environment at Shanghai Institute of Biochemistry and Cell Biology and treated in strict accordance with protocols approved by the Institutional Animal Care and Use Committee of the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. These mice were treated with Adeno-Cre through nasal inhalation as described before [28]. After three weeks of nasal inhalation, we treated these mice with 4-hydroxytamoxifen with different doses daily via intraperitoneal injection. Mice were then sacrificed for pathological inspection. Some mice were stopped for tamoxifen treatment for one or two weeks and then sacrificed for pathological inspection. Genotyping primers for p53L/L, LSL-K-rasG12D and LSL-ER-K-rasG12D was listed as following: For p53L/L, forward primer: 59-CACAAAAACAGGTTAAACCCAG-39; reverse primer: 59-AGCACATAGGAGGCAGAGAC-39. For LSL-K-rasG12D and LSL-ER-K-rasG12D, same primers were used for genotyping. Forward primer: 59-CTAGCCACCATGGCTTGAGT-39; reverse primer:59-TCCGAATTCAGTGACTACAGATG-39. Cell culture and cell proliferation assay We generated MEFs from 13.5 post-coitum embryos and grew them in DMEM medium plus 10% fetal bovine serum (Biochrom, AG), penicillin and streptomycin. MEFs (,46105) at 6-well plate were virally infected with Adeno-Cre (46106 CFU) overnight and then changed with fresh medium. The MEFs were then cultured for at least two more passages to get (...truncated)