During the last year we have continued to investigate the molecular mechanisms by which transcription factors modulate the action of the progesterone receptor, PGR, 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 completed our investigation into the role of Sox17 and FOXO1 in the regulation of the ability of the uterus to support embryo implantation. We then investigated to role of the kinase WNK1 in the regulation of uterine physiology and embryo implantation. We also characterized the role of PGR in the regulation of human uterine gene expression. With respect to our investigation of lung cancer, using a mouse model we investigated the molecular mechanism of progression of squamous cell carcinoma in the lung and then investigated the role of the epidermal growth factor rector, ERBB2, in the progression of non Small Cell lung cancer. Below is a summary of our findings. Successful embryo implantation requires a receptive endometrium. Poor uterine receptivity can account for implantation failure in women that experience recurrent pregnancy loss or multiple rounds of unsuccessful in vitro fertilization cycles. Here, we demonstrate that the transcription factors SRY-Box 17 (SOX17), and Forkhead Box O1 (FOXO1) are critical regulators of endometrial receptivity in vivo. Ablation of Sox17 in the uterine epithelium results in altered embryo implantation by impairing critical growth factor signaling pathways including that of Leukemia Inhibitory Factor (LIF) and Indian Hedgehog (IHH). In vivo deletion of the region of the mouse genome where SOX17 binds region upstream of the Ihh gene by CRISPR-Cas technology reduced Ihh expression specifically in the uterus and altered proper endometrial epithelial-stromal interactions, impairing pregnancy. This SOX17 binding peak was also bound by GATA2, FOXA2 and PGR. This cluster of transcription factor binding is common in 737 uterine genes and may represent a key regulatory element essential for uterine epithelial gene expression. This work was published in Nature Communications. Uterine ablation of Foxo1 using the progesterone receptor Cre (PgrCre) mouse model resulted in infertility due to altered uterine epithelial cell polarity and apoptosis, preventing the embryo from penetrating the luminal epithelium. Analysis of the uterine transcriptome after Foxo1 ablation identified alterations in gene expression for transcripts involved in the activation of cell invasion, molecular transport, apoptosis, -catenin (CTNNB1) signaling pathway, and an increase in PGR signaling. Conversely, constitutive expression of epithelial PGR during this receptive period inhibited expression of FOXO1 in the nucleus of the uterine epithelium. The reciprocal expression of PGR and FOXO1 was conserved in human endometrial samples during the proliferative and secretory phase. This demonstrates that expression of FOXO1 and the loss of PGR during the window of receptivity are interrelated and critical for embryo implantation and a maker for uterine receptivity in women. THis work was published in PLoS Genetics. In addition to embryo invasion, the differentiation of endometrial stroma cells, a process called decidualization, is an essential process during pregnancy. WNK lysine deficient protein kinase 1 (Wnk1) was previously shown to play important roles in decidualization in vitro. In this study, we examined the in vivo role of Wnk1 in uterine homeostasis and pregnancy using a uterine Wnk1 deletion mouse model. The impact of uterine deletion of Wnk1 (Wnk1d/d) was evaluated with respect to the ability of the uterus to support pregnancy and uterine morphology. Wnk1d/d mice were severely subfertile due to decreased pregnancy rate, fewer litters, smaller litter size, and increased neonatal lethality. In non-pregnant mice, we observed adenomyotic-like uterine morphology associated with Wnk1 deletion whereby the glandular epithelium is frequently adjacent to or present in the myometrial layer. In addition, this phenotype is aggravated by pregnancy and delivery, as observed in mice after the breeding trial. Transcriptomic analysis demonstrated an increased in estrogen signaling in the uteri of these mice. These results demonstrate a crucial role of Wnk1 in the uterus, where a disruption in this pathway impairs both the uterine glands and smooth muscle, failing the maintenance of normal uterine homeostasis and may be a signaling pathway that protects the uterus for environmental estrogens. The major mechanistic analysis or PGR signaling is conducted in animal models and cells in culture. In order to determine the clinical relevance of these findings we conducted cistromic and transcriptomic analysis of PGR by examining global PR binding (ChIP-seq) in parallel to gene transcriptional network (RNA-seq) in the endometrium of fertile women during the proliferative (P) and mid-secretory (MS) phases. Overlaying the genes with altered PR binding and differentially expressed genes (DEG) during the phase transition identified 653 genes, which are involved in inflammatory response signaling, xenobiotic metabolism, epithelial-mesenchymal transition (EMT), cell death regulation, interleukin/STAT signaling, estrogen response, MTORC1 response as well as KRAS signaling. Selected genes from the xenobiotic metabolism, EMT and apoptosis pathways were validated by ChIP-qPCR and qPCR and found to be consistent with the ChIP-seq and RNA-seq results. In summary, we present here the endometrial landscape during the implantation window in two aspects; firstly, the meaningful binding of PR which is associated with altered gene expression - this identified numerous PR targets as well as PR regulated biological pathways during the implantation. Lung cancer is the leading cause of cancer related death in the world. The environment plays a major role in lung cancer development. Over the last year we found that the activities of the stress related kinases, JNK1/2 are attenuate in a Lkb1-deficiency-driven lung squamous cell carcinoma mouse model. We found that JNK1/2 activities are positively regulated by MKK7 during LSCC development. Pharmaceutically elevated JNK1/2 activities attenuates Lkb1 dependent LSCC formation while compound mutations of Jnk1/2 and Lkb1 further accelerate LSCC progression. JNK1/2 is inactivated in a substantial proportion of human LSCC and JNK1/2 activities positively correlates with survival rates of lung, cervical and head and neck squamous cell carcinoma patients. These findings in Nature Communications. Members of the Epidermal Growth Factor family, acting through the EGFR2 receptor, ERBB2, are oncogenic drivera with frequent gene mutations and amplification in human lung tumors and is an attractive target for lung cancer target therapy. However, the impact of ERBB2 on the pulmonary transcriptome and the in vivo mechanisms regulated by ERBB2 during lung tumor development remains unclear. Here we generated genetic mouse models to show Erbb2 loss inhibited lung tumor development induced by deletion of Pten and Smad4. Transcriptome analysis showed that Erbb2 loss suppressed the significant changes of most of the induced genes by ablation of Pten and Smad4. Overlapping with ERBB2-associated human lung cancer genes further identified those ERBB2 downstream players, potentially conserved in human and mouse lung tumors. Furthermore, MED24 was identified as a crucial oncogenic target of ERBB2 in lung tumor development. Taken together, ERBB2 is required for the dysregulation of cancer-related genes during lung tumor development, such as MED24. This work was published in Cells.
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