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

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

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

VGLL4 plays a critical role in heart valve development and homeostasis.

Heart valve disease is a major clinical problem worldwide. Cardiac valve development and homeostasis need to be precisely controlled. Hippo signaling is essential for organ development and tissue homeostasis, while its role in valve formation and morphology maintenance remains unknown. VGLL4 is a transcription cofactor in vertebrates and we found it was mainly expressed in valve interstitial cells at the post-EMT stage and was maintained till the adult stage. Tissue specific knockout of VGLL4 in different cell lineages revealed that only loss of VGLL4 in endothelial cell lineage led to valve malformation with expanded expression of YAP targets. We further semi-knockout YAP in VGLL4 ablated hearts, and found hyper proliferation of arterial valve interstitial cells was significantly constrained. These findings suggest that VGLL4 is important for valve development and manipulation of Hippo components would be a potential therapy for preventing the progression of congenital valve disease

CBX4 deletion promotes tumorigenesis under KrasG12D background by inducing genomic instability

Abstract Chromobox protein homolog 4 (CBX4) is a component of the Polycomb group (PcG) multiprotein Polycomb repressive complexes 1 (PRC1), which is participated in several processes including growth, senescence, immunity, and tissue repair. CBX4 has been shown to have diverse, even opposite functions in different types of tissue and malignancy in previous studies. In this study, we found that CBX4 deletion promoted lung adenocarcinoma (LUAD) proliferation and progression in KrasG12D mutated background. In vitro, over 50% Cbx4 L/L , Kras G12D mouse embryonic fibroblasts (MEFs) underwent apoptosis in the initial period after Adeno-Cre virus treatment, while a small portion of survival cells got increased proliferation and transformation abilities, which we called selected Cbx4 −/− , Kras G12D cells. Karyotype analysis and RNA-seq data revealed chromosome instability and genome changes in selected Cbx4 −/− , Kras G12D cells compared with Kras G12D cells. Further study showed that P15, P16 and other apoptosis-related genes were upregulated in the primary Cbx4 −/− , Kras G12D cells due to chromosome instability, which led to the large population of cell apoptosis. In addition, multiple pathways including Hippo pathway and basal cell cancer-related signatures were altered in selected Cbx4 −/− , Kras G12D cells, ultimately leading to cancer. We also found that low expression of CBX4 in LUAD was associated with poorer prognosis under Kras mutation background from the human clinical data. To sum up, CBX4 deletion causes genomic instability to induce tumorigenesis under KrasG12D background. Our study demonstrates that CBX4 plays an emerging role in tumorigenesis, which is of great importance in guiding the clinical treatment of lung adenocarcinoma

VGLL4 plays a critical role in heart valve development and homeostasis

Heart valve disease is a major clinical problem worldwide. Cardiac valve development and homeostasis need to be precisely controlled. Hippo signaling is essential for organ development and tissue homeostasis, while its role in valve formation and morphology maintenance remains unknown. VGLL4 is a transcription cofactor in vertebrates and we found it was mainly expressed in valve interstitial cells at the post-EMT stage and was maintained till the adult stage. Tissue specific knockout of VGLL4 in different cell lineages revealed that only loss of VGLL4 in endothelial cell lineage led to valve malformation with expanded expression of YAP targets. We further semi-knockout YAP in VGLL4 ablated hearts, and found hyper proliferation of arterial valve interstitial cells was significantly constrained. These findings suggest that VGLL4 is important for valve development and manipulation of Hippo components would be a potential therapy for preventing the progression of congenital valve disease