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

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 µM 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

Continuous activation of ER-K-ras^G12D by 500 µg tamoxifen treatment induced lung hyperplasia and adenoma formation in P53 L/L, LSL-ER-K-ras mice.

<p>A) Continuous activation of ER-K-ras^G12D by 500 µg tamoxifen I.P. daily injection for 12 weeks is sufficient to drive lung adenoma formation in P53 L/L, LSL-ER-K-ras mice after adeno-Cre treatment. The adenoma were positive for Ki67 immunostaining. B) The incidence of lung hyperplasia and/or adenoma formation in <i>P53L/L</i>, <i>LSL-ER-K-ras^G12D</i> mice was shown after adeno-Cre treatment in the absence or presence of tamoxifen treatment.</p

Sustained activation of ER-K-ras^G12D induced by tamoxifen treatment is important for cell proliferation, anchorage-independent cell growth, cell invasiveness and tumor maintenance.

<p>A) Cell number counting of indicated MEFs after withdrawal of tamoxifen after 6 passages in the presence of tamoxifen. The PEK-C MEFs always without tamoxifen treatment and the PEK-C MEFs always kept in tamoxifen were used as control. B) Tamoxifen were withdrawn from the PEK-C MEFs at day 5, 10 and 28 after continuous tamoxifen treatment in soft agar. C) Tamoxifen withdrawal from the PEK-C MEFs after indicated days of tamoxifen treatment in cell invasiveness assay in matrigel. D) Tamoxifen withdrawal for indicated time in the <i>P53L/L</i>, <i>LSL-ER-K-ras^G12D</i> mice previously treated for 12 weeks of tamoxifen. The typical lung pathology and cleaved caspase-3 immunostaining were shown.</p

Treatment of tamoxifen at an optimal dose induced the activation of ER-K- ras^G12D and downstream signaling.

<p>A) The <i>P53−/−</i>, <i>ER-K-ras^G12D</i> MEFs (PEK-C) derived from male embryo was counted for cell number after indicated dosage of tamoxifen treatment after 5 passages. B) Relative expression level of <i>K-ras</i> in indicated MEFs with or without tamoxifen treatment. PEK: <i>P53L/L</i>, <i>Loxp-Stop-Loxp ER-K-ras^G12D</i>; PEK-C: <i>P53−/−</i>, <i>ER-K-ras^G12D</i>; PK: <i>P53L/L</i>, <i>Loxp-Stop-Loxp-K- ras^G12D</i>; PKC: <i>P53−/−</i>, <i>K- ras^G12D</i>. (C) Detection of the ER-K-ras^G12D expression in PEK-C cells with or without 0.05 µM tamoxifen treatment. D) Detection of K-ras and ER-K-ras protein level in MEFs infected with or without adeno-Cre in the presence or absence of 0.05 µM tamoxifen treatment in indicated MEFs. β-actin serves as internal control. E) The activation of Ras effector PI3K pathway signaling was confirmed by western blot.</p