During the last year we have continued to investigate the molecular mechanisms by which transcription factors modulate the action of the progesterone receptor in regulating the ability of the uterus to support embryo development and the molecular factors regulating the progression of lung cancer. Over the last year we have continued to investigate the role of Sox17 and the progesterone receptor PGR in the regulation of the ability of the uterus to support embryo implantation, pregnancy and birth. We then investigate the molecular processes involved in lung cancer using a mouse model. Previously we have demonstrated that ablation of Sox17 in the mouse uterus renders the mouse sterile due to alterations in the signaling of the growth factor Indian hedgehog. We have then investigated the impact of Sox17 on the function of the uterine glands and the the expression of the other major regulator of uterine receptivity leukemia inhibitory factor in mice with uterine ablation of Sox17. Ablation of Sox17 alters gland impacted Lif and Foxa2 gene expression. This indicates that Sox17 may regulate Lif at two levels. First Sox17 ablation results in loss of Foxa2 from the glandular epithelium which may impact Lif expression. Second Sox17 may directly regulate the transcription of Lif. We then identified wher e Sox17 bound the Lif regulatory regions in the genome and identified several peaks of potential regulation. We then conducted in vitro analysis to show that Sox17 works with the estrogen receptor in the regulation of Lif gene expression. Since environmental estrogens impact the womens reproductive tract, this may be a target of environmental estrogen action. Seq analysis we identified several Sox17 binding peaks in the 5 flanking region of the Lif gene. In our analysis of how the transcription factor function in regulating gene expression, we observed that these transcription factors PGR, Gata2 and Sox17 all bind in a similar region 19 kb distal to the Ihh gene promoter, Ihh19. To investigate the importance of this binding site we conducted in vivo deletion of this region using CRISPR-Cas9 technology. Mice homozygous to for this deletion of the binding region a Ihh19 showed significantly reduced Ihh expression specifically in the uterus. This decrease in Ihh expression resulted in altered epithelial-stromal paracrine signaling which impaired pregnancy. Global analysis of genes containing PGR, FOXA2, GATA2 an Sox17 overlapping binding regions identified containing these three overlapping binding sites in 734 genes. When these genes were compared to laser capture dissection and RNA seq of uterine epithelium at day 4.5 of pregnancy, 502 of eh 734 were identified as expressed in this compartment of the uterus. This indicates that regions of the genome that bind these three transcription factors may be critical for uterine epithelial gene expression. B The future aims of this proposal will be to identify the interactions of uterine epithelial enhancers that regulation the changes in dynamic changes in gene expression that occur during the preimplantation period that allows the uterus to allow embryo implantation and the role Sox17 plays in coordinating the activity of the transcription factors at these enhancer regions. This project will also use this information to determine if this cassette can be used to engineer uterine glandular epithelial cells. This work is under review in Nature Communications. We next investigated how Progesterone regulates the smooth muscle of the uterus. The goal of this research is to identify the processes involved in pregnancy and labor. This is important ot understand what process are altered in preterm birth. Preterm birth affects 10 % of human pregnancies and is one of the major causes of infant mortality. The environment has an impact on preterm birth. We accomplished this by ablating the progesterone receptor from the uterus and then altering the expression of the progesterone receptor isoforms PGRA and PGRB in the smooth muscle of the mouse. Ablation of the progesterone receptor on the mouse uterus impacted the ability of the oviduct to transport embryos to the mouse uterus for implantation. This resulted in a decrease in embryo implantation. We also observed an increase n embryo loss in those embryos that did implant showing the importance of progesterone receptor in the uterine smooth muscle. The progesterone receptor has two isoforms, PGRA and PGRB. The delivery of babies at the appropriate time in humans may be due to change in the expression of these isoforms at the time of birth. PGRB seems to prevent early birth and an increase in PGRA appears to promote labor and birth. In order to investigate this possibility, we generated mice in which the PGRA and PGRB isoforms overexpressed. Both PgrA and PgrB female mice exhibited a significant decreased average number of pups per litter compared to control females. PgrB dams demonstrated a significant increase in the length of gestation compared to control mice. PgrB pregnancies were also associated with significant peripartum fetal mortality. Morphological examination of uteri at showed the PgrB uterine tone and loss of the ability for the uterus to maintain constriction. In order to assay the impact of overexpression of PgrB on uterine tone, an intrauterine telemeter pressure catheter was used to compare the in vivo uterine pressure during late pregnancy in control PgrB mice. In vivo uterine pressure measurements demonstrated decreased uterine tone and decreased uterine pressure during late gestation in the PgrB mouse in agreement with the phenotype of labor dystocia, and the morphological gross assessment,. Overall, none of the PgrB parturitions were morphologically similar in appearance to the normal pattern of the control parturitions. This analysis demonstrates that the PGR isoforms have a distinct role in regulation the ability of the smooth muscle of the uterus to support birth and pregnancy. We are now investigating how PGRA and PGRB regulate uterine gene expression. The goal is to extrapolate these findings to human biology to investigate how these isoforms regulate human uterine smooth muscle function to determine what processes may be used to diagnose and treat preterm birth. In a third project we investigated the molecular mechanisms regulating the development of lung cancer in a mouse model. Lung cancer is the leading cause of cancer related deaths worldwide and is directly influenced by the environment. Mutation activating the oncogene KRAS is one of the most frequent mutations found in lung adenocarcinoma. Identifying regulators of KRAS may aid in the development of therapies to treat this disease. The mitogen-induced gene 6, MIG-6, is a small adaptor protein modulating signaling in cells to regulate the growth and differentiation in multiple tissues. We investigated the role of Mig-6 in regulating adenocarcinoma progression in the lungs of genetically engineered mice with activation of Kras. We found that Mig-6 expression is decreased in CCSPCreKrasG12D-induced lung tumors. Ablation of Mig-6 in the KrasG12D background led to enhanced tumorigenesis and reduced life expectancy in mice. During tumor progression, there was increased airway hyperplasia, a heightened inflammatory response, reduced cell death in KrasG12D mouse lungs, and an increase of total and phosphorylated Epidermal growth factor receptor 4 protein levels. Mechanistically, Mig-6 deficiency attenuates the cell apoptosis of lung tumor expressing KRASG12D partially through activating the epidermal growth factor pathway. In summary, Mig-6 may be a target to prevent and treat lung cancer. This work has been published in the journal Lung Cancer.
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