臺灣博碩士論文加值系統

神經調節肽U (neuromedin U，簡稱NMU) 已知可透過其受體 (NMUR1和NMUR2) 調控多種生理系統的功能。然而，在女性生殖系統中的生理功能則尚不清楚。在卵巢方面，NMU不但表現在顆粒細胞 (granulosa cells) 中，也與NMUR2共同表現在泡膜細胞 (theca cells) 中，並會受到促性腺激素(gonadotropins) 的調控呈現波動起伏變化。此表現形式是透過cAMP訊息傳遞的活化進行調控：在短時間的活化可提高NMU的表現，但在長時間活化下則會提高Zfp36l1的表現，進而透過Nmu mRNA的3端非編碼區 (untranslated region，簡稱為UTR) 促使NMU mRNA的分解。此外，NMU的表現能夠促使發育中的泡膜/間質細胞 (theca/interstitial cells) 或是成熟的泡膜/間質細胞分泌黃體素(progesterone)，但不影響雄烯二酮 (androstenedione) 的分泌。表示在濾泡發育過程中，受促性腺激素調控表現的NMU能透過NMUR2傳遞訊息，並可能參與在與黃體素相關的生理功能中。在子宮方面，我們發現NMU及NMUR2在高分級 (grade) 的人類子宮內膜癌表現量顯著增加。進一步發現在高分級癌細胞株中，NMU可透過促進間質細胞標記分子 (mesenchymal marker) 的表現或往細胞核轉移，或是維持細胞黏附訊息 (adhesion signaling) ，進而調控移動能力或是非貼附性生長 (anchorage-independent growth) 。此外，NMU也能協助表皮生長因子 (epidermal growth factor，簡稱為EGF) 以及轉型生長因子-β (transforming growth factor β，簡稱為TGFβ) 所調控的間質細胞分子表現，也能維持癌細胞在活體內分裂生長的能力。表示在較高分級子宮內膜癌中提高的NMU訊息，具有能促進癌細胞的侵襲以及在活體內生長的能力。

Neuromedin U (NMU) is a neuropeptide that regulates multi-physiological functions through activation of NMUR1 or NMUR2. However, its function has not been elucidated in the female reproductive system. In the ovary, NMU expression was detected moderately in granulosa cells and co-expressed mainly with NMUR2 in theca cells. The fluctuation of NMU was regulated by gonadotropin-driven cAMP signaling and cAMP-mediated RNA-binding protein Zfp36l1, a cis-acting mRNA degradation mechanism. Furthermore, NMU treatment promoted the production of progesterone but not androstenedione in the developing and mature theca/interstitial cells. Thus, these results suggest that gonadotropin-stimulated NMU production controls the progesterone-related physiological roles through NMUR2 in the ovary. In the uterus, NMU and NMUR2 were not only expressed in the endometrial epithelial cells in mouse but also upregulated in the high-graded endometrial cancers in human. In the cell lines isolated from patients with high-graded endometrial cancer, NMU signaling could promote the expression and/or the nuclear translocation of mesenchymal markers. It also could sustain the level of adhesion signaling and this further regulated the cell motility and anchorage-independent growth. Further, NMU signaling also enhanced the EGF- and TGFβ1-mediated expression of mesenchymal markers. It also helped the maintenance of cell division of endometrial cancer cells in vivo. Together, these results suggest that the upregulated NMU signaling may be involved in the metastasis and tumor growth of high-graded endometrial cancer.

致謝 I
摘要 III
ABSTRACT IV
CONTENTS V
ABBREVIATION LIST X
1. INTRODUCTION 1
1.1 NEUROMEDIN U (NMU) 1
1.2 NMU RECEPTORS 2
1.3 THE PHYSIOLOGICAL ROLES OF NMU SIGNALING 5
1.3.1 Smooth muscle contraction 5
1.3.2 Energy homeostasis and feeding behavior 5
1.3.3 Stress responses 7
1.3.4 Nociception 8
1.3.5 Inflammation 8
1.3.6 Hormone secretion 10
1.3.7 Cancer progression 11
1.4 OVARIAN CYCLE 12
1.5 UTERINE CYCLE 14
1.6 ENDOMETRIAL CANCER 14
1.6.1 Types of endometrial cancer 14
1.6.2 The molecular mechanisms involved in the aggressive types of endometrial cancer 15
1.7 OBJECTIVE 17
2. MATERIALS AND METHODS 19
2.1 ANIMALS AND ETHICS 19
2.2 REAGENTS, CHEMICALS AND ANTIBODIES 20
2.3 CDNA SYNTHESIS AND GENE QUANTIFICATION 21
2.4 IHC ANALYSIS AND QUANTIFICATION 22
2.5 PLASMID CONSTRUCTION 23
2.6 CELL LINES 24
2.7 PREPARATION OF PROTEIN SAMPLES AND WESTERN BLOT 24
2.8 ISOLATION AND CULTURE OF PRIMARY RAT GRANULOSA CELLS AND THECA/INTERSTITIAL CELLS 25
2.9 REPORTER GENE ASSAY 26
2.10 QUANTIFICATION OF STEROID HORMONES IN THE CULTURE MEDIA 27
2.11 CLINICAL MATERIALS AND ETHICAL STATEMENTS 28
2.12 MIGRATION AND INVASION ASSAYS 28
2.13 PROLIFERATION ASSAY, ANCHORAGE-INDEPENDENT GROWTH AND MORPHOLOGY OBSERVATION 29
2.14 RHO GTPASE ACTIVATION ASSAY 30
2.15 TUMOR GROWTH IN VIVO AND THE DETERMINATION OF SURVIVAL RATES 30
2.16 DATA ANALYSIS 31
3. RESULTS 32
3.1 THE REGULATORY MECHANISMS AND ROLES OF NMU SIGNALING DURING OVARIAN FOLLICULOGENESIS 32
3.1.1 The expression profile of NMU during the ovarian folliculogenesis 32
3.1.2 Identification of the major NMU receptor and its expression profile during the ovarian folliculogenesis 33
3.1.3 The regulatory mechanisms of NMU during ovarian folliculogenesis 35
3.1.4 Identification of the degradation mechanism of Nmu regulated by cAMP/PKA pathway 36
3.1.5 Identification of the roles of NMU signaling during ovarian folliculogenesis 38
3.2 THE FUNCTIONS OF NMU SIGNALING DURING THE PROGRESSION OF ENDOMETRIAL CANCER 40
3.2.1 The expression profiles of NMU and NMU receptors in the normal endometrium and endometrial cancer 40
3.2.2 The roles of NMU signaling in the regulation of cell motility 41
3.2.3 The roles of NMU signaling in the proliferation and morphological change 44
3.2.4 Identification of the adhesion signaling-related molecules regulated by NMU signaling 45
3.2.5 Characterization of the roles of NMU signaling in the downstream factors of adhesion signaling 46
3.2.6 The regulation of growth factor-mediated EMT by NMU signaling 48
3.2.7 Characterization of NMU signaling in the tumor growth in vivo and the survivorship of mice 49
4. DISCUSSION 51
4.1 THE REGULATORY MECHANISMS AND ROLES OF NMU SIGNALING DURING OVARIAN FOLLICULOGENESIS 51
4.1.1 The positively regulatory mechanisms of Nmu during folliculogenesis 51
4.1.2 The negatively regulatory mechanisms of Nmu during folliculogenesis 52
4.1.3 The roles of NMU and NMS in the ovary 53
4.1.4 The roles of NMU signaling during folliculogenesis 54
4.1.5 Proposed model 56
4.2 THE FUNCTIONS OF NMU SIGNALING DURING THE PROGRESSION OF ENDOMETRIAL CANCER 58
4.2.1 The expression profiles of NMU in the endometrial cancer 58
4.2.2 The possible regulatory mechanisms of NMU in the aggressive endometrial cancer 59
4.2.3 The importance of NMU in the type-I endometrial cancer 60
4.2.4 NMU signaling regulates cell motility of endometrial cancer through adhesion signaling 61
4.2.5 NMU signaling could cooperate with other signals to regulate the metastasis of endometrial cancer 63
4.2.6 Proposed model 65
5. REFERENCES 67
6. FIGURES AND LEGENDS 83
FIGURE 1. THE EXPRESSION PROFILES OF NMU DURING RAT OVARIAN FOLLICULOGENESIS. 84
FIGURE 2. DISTRIBUTION OF THE NMU PROTEIN IN THE OVARIAN FOLLICLES. 86
FIGURE 3. THE TRANSCRIPT PROFILES OF NMUR1 AND NMUR2 DURING THE RAT OVARIAN FOLLICULOGENESIS. 88
FIGURE 4. DISTRIBUTION OF THE NMUR2 PROTEIN IN THE OVARIAN FOLLICLES. 90
FIGURE 5. THE EXPRESSION OF NMU WAS REGULATED BY CAMP/PKA PATHWAY. 92
FIGURE 6. 3’-UTR OF RAT NMU FACILITATED THE DOWNREGULATION OF LUCIFERASE GENE. 94
FIGURE 7. THE TRANSCRIPT PROFILE OF ZFP36L1 IN THE OVARY AND THE REGULATORY ROLES OF ZFP36L1 IN THE MRNA STABILITY. 96
FIGURE 8. THE FUNCTIONAL NMU RECEPTORS WERE EXPRESSED IN THECA/INTERSTITIAL CELLS. 98
FIGURE 9. NMU SIGNALING PROMOTED THE PROGESTERONE PRODUCTION IN THECA/INTERSTITIAL CELLS. 100
FIGURE 10. A PROPOSED MODEL FOR THE REGULATORY MECHANISMS AND FUNCTIONS OF NMU IN THE OVARY. 102
FIGURE 11. THE EXPRESSION PROFILES OF NMU AND NMU RECEPTORS DURING THE UTERINE CYCLE. 104
FIGURE 12. THE EXPRESSION PROFILES OF NMU, NMUR1 AND NMUR2 IN THE ENDOMETRIAL CANCER. 106
FIGURE 13. THE PROFILES OF NMU AND NMUR2 PROTEINS IN THE ENDOMETRIAL CANCER. 108
FIGURE 14. THE TRANSCRIPT PROFILES OF NMU, NMUR1 AND NMUR2 IN THE ENDOMETRIAL CANCER CELL LINES. 110
FIGURE 15. THE ROLES OF NMU SIGNALING IN THE CELL MOTILITY OF ENDOMETRIAL CANCER CELL LINES. 112
FIGURE 16. THE ROLES OF NMU SIGNALING IN THE EXPRESSION OF EMT MARKERS IN RL95-2. 114
FIGURE 17. THE ROLES OF NMU SIGNALING IN THE EXPRESSION OF EMT MARKERS IN HEC1A. 116
FIGURE 18. THE ROLES OF NMU SIGNALING IN THE REGULATION OF PROLIFERATION AND MORPHOLOGY IN ENDOMETRIAL CANCER CELL LINES. 118
FIGURE 19. THE ROLES OF NMU SIGNALING IN THE EXPRESSION OF ADHESION MOLECULES IN THE ENDOMETRIAL CANCER CELL LINES. 120
FIGURE 20. THE ROLES OF NMU SIGNALING IN THE EXPRESSION OF COLLAGEN IV AND HYALURONIC ACID IN RL95-2. 122
FIGURE 21. MATRIGEL RESCUED THE DEFICIENCY OF PROLIFERATION AND ABNORMAL MORPHOLOGY IN NMU-KNOCKDOWN RL95-2. 124
FIGURE 22. THE ROLES OF SRC KINASE IN NMU-MEDIATED CELL PROLIFERATION AND MORPHOLOGY IN RL95-2. 126
FIGURE 23. NMU SIGNALING CHANGED THE ACTIVATION PROFILES OF RHO GTPASES IN RL95-2. 128
FIGURE 24. NMU SIGNALING POSITIVELY REGULATED THE GROWTH FACTOR-MEDIATED MESENCHYMAL MARKER EXPRESSIONS IN ENDOMETRIAL CANCER CELL LINES. 130
FIGURE 25. THE ROLES OF NMU SIGNALING IN THE CANCER PROGRESSION OF RL95-2 IN VIVO. 132
7. TABLES 135
TABLE 1. THE PRIMER PAIRS FOR GENE QUANTIFICATION. 135
TABLE 2. THE PRIMER PAIRS FOR PLASMID CONSTRUCTION. 138

1 Minamino, N., Kangawa, K. &; Matsuo, H. Neuromedin U-8 and U-25: novel uterus stimulating and hypertensive peptides identified in porcine spinal cord. Biochemical and biophysical research communications 130, 1078-1085 (1985).
2 O'Harte, F., Bockman, C. S., Abel, P. W. &; Conlon, J. M. Isolation, structural characterization and pharmacological activity of dog neuromedin U. Peptides 12, 11-15 (1991).
3 Brighton, P. J., Szekeres, P. G. &; Willars, G. B. Neuromedin U and its receptors: structure, function, and physiological roles. Pharmacological reviews 56, 231-248, doi:10.1124/pr.56.2.3 (2004).
4 Hashimoto, T., Masui, H., Uchida, Y., Sakura, N. &; Okimura, K. Agonistic and antagonistic activities of neuromedin U-8 analogs substituted with glycine or D-amino acid on contractile activity of chicken crop smooth muscle preparations. Chemical &; pharmaceutical bulletin 39, 2319-2322 (1991).
5 Okimura, K., Sakura, N., Ohta, S., Kurosawa, K. &; Hashimoto, T. Contractile activity of porcine neuromedin U-25 and various neuromedin U-related peptide fragments on isolated chicken crop smooth muscle. Chemical &; pharmaceutical bulletin 40, 1500-1503 (1992).
6 Fujii, R. et al. Identification of neuromedin U as the cognate ligand of the orphan G protein-coupled receptor FM-3. The Journal of biological chemistry 275, 21068-21074, doi:10.1074/jbc.M001546200 (2000).
7 Domin, J., Ghatei, M. A., Chohan, P. &; Bloom, S. R. Neuromedin U--a study of its distribution in the rat. Peptides 8, 779-784 (1987).
8 Mori, K. et al. Identification of neuromedin S and its possible role in the mammalian circadian oscillator system. The EMBO journal 24, 325-335, doi:10.1038/sj.emboj.7600526 (2005).
9 Tan, C. P. et al. Cloning and characterization of a human and murine T-cell orphan G-protein-coupled receptor similar to the growth hormone secretagogue and neurotensin receptors. Genomics 52, 223-229, doi:10.1006/geno.1998.5441 (1998).
10 Szekeres, P. G. et al. Neuromedin U is a potent agonist at the orphan G protein-coupled receptor FM3. The Journal of biological chemistry 275, 20247-20250, doi:10.1074/jbc.C000244200 (2000).
11 Raddatz, R. et al. Identification and characterization of two neuromedin U receptors differentially expressed in peripheral tissues and the central nervous system. The Journal of biological chemistry 275, 32452-32459, doi:10.1074/jbc.M004613200 (2000).
12 Hedrick, J. A. et al. Identification of a human gastrointestinal tract and immune system receptor for the peptide neuromedin U. Molecular pharmacology 58, 870-875 (2000).
13 Howard, A. D. et al. Identification of receptors for neuromedin U and its role in feeding. Nature 406, 70-74, doi:10.1038/35017610 (2000).
14 Shan, L. et al. Identification of a novel neuromedin U receptor subtype expressed in the central nervous system. The Journal of biological chemistry 275, 39482-39486, doi:10.1074/jbc.C000522200 (2000).
15 Hosoya, M. et al. Identification and functional characterization of a novel subtype of neuromedin U receptor. The Journal of biological chemistry 275, 29528-29532, doi:10.1074/jbc.M004261200 (2000).
16 Lin, T. Y., Huang, W. L., Lee, W. Y. &; Luo, C. W. Identifying a Neuromedin U Receptor 2 Splice Variant and Determining Its Roles in the Regulation of Signaling and Tumorigenesis In Vitro. PloS one 10, e0136836, doi:10.1371/journal.pone.0136836 (2015).
17 Brighton, P. J., Szekeres, P. G., Wise, A. &; Willars, G. B. Signaling and ligand binding by recombinant neuromedin U receptors: evidence for dual coupling to Galphaq/11 and Galphai and an irreversible ligand-receptor interaction. Molecular pharmacology 66, 1544-1556, doi:10.1124/mol.104.002337 (2004).
18 Aiyar, N. et al. Radioligand binding and functional characterization of recombinant human NmU1 and NmU2 receptors stably expressed in clonal human embryonic kidney-293 cells. Pharmacology 72, 33-41, doi:10.1159/000078630 (2004).
19 Wang, C. J., Hsu, S. H., Hung, W. T. &; Luo, C. W. Establishment of a chimeric reporting system for the universal detection and high-throughput screening of G protein-coupled receptors. Biosensors &; bioelectronics 24, 2298-2304, doi:10.1016/j.bios.2008.11.023 (2009).
20 Benito-Orfila, M. A., Domin, J., Nandha, K. A. &; Bloom, S. R. The motor effect of neuromedin U on rat stomach in vitro. European journal of pharmacology 193, 329-333 (1991).
21 Prendergast, C. E., Morton, M. F., Figueroa, K. W., Wu, X. &; Shankley, N. P. Species-dependent smooth muscle contraction to Neuromedin U and determination of the receptor subtypes mediating contraction using NMU1 receptor knockout mice. British journal of pharmacology 147, 886-896, doi:10.1038/sj.bjp.0706677 (2006).
22 Maggi, C. A. et al. Motor response of the human isolated small intestine and urinary bladder to porcine neuromedin U-8. British journal of pharmacology 99, 186-188 (1990).
23 Westfall, T. D. et al. Characterization of neuromedin U effects in canine smooth muscle. The Journal of pharmacology and experimental therapeutics 301, 987-992 (2002).
24 Dass, N. B. et al. Neuromedin U can exert colon-specific, enteric nerve-mediated prokinetic activity, via a pathway involving NMU1 receptor activation. British journal of pharmacology 150, 502-508, doi:10.1038/sj.bjp.0707004 (2007).
25 Kojima, M. et al. Purification and identification of neuromedin U as an endogenous ligand for an orphan receptor GPR66 (FM3). Biochemical and biophysical research communications 276, 435-438, doi:10.1006/bbrc.2000.3502 (2000).
26 Jethwa, P. H. et al. Neuromedin U has a physiological role in the regulation of food intake and partially mediates the effects of leptin. American journal of physiology. Endocrinology and metabolism 289, E301-305, doi:10.1152/ajpendo.00404.2004 (2005).
27 Peier, A. M. et al. Effects of peripherally administered neuromedin U on energy and glucose homeostasis. Endocrinology 152, 2644-2654, doi:10.1210/en.2010-1463 (2011).
28 Shousha, S., Nakahara, K., Miyazato, M., Kangawa, K. &; Murakami, N. Endogenous neuromedin U has anorectic effects in the Japanese quail. General and comparative endocrinology 140, 156-163, doi:10.1016/j.ygcen.2004.11.002 (2005).
29 Kamisoyama, H., Honda, K., Saneyasu, T., Sugahara, K. &; Hasegawa, S. Central administration of neuromedin U suppresses food intake in chicks. Neuroscience letters 420, 1-5, doi:10.1016/j.neulet.2007.03.062 (2007).
30 Maruyama, K. et al. Isolation and characterisation of four cDNAs encoding neuromedin U (NMU) from the brain and gut of goldfish, and the inhibitory effect of a deduced NMU on food intake and locomotor activity. Journal of neuroendocrinology 20, 71-78, doi:10.1111/j.1365-2826.2007.01615.x (2008).
31 Melcher, C., Bader, R., Walther, S., Simakov, O. &; Pankratz, M. J. Neuromedin U and its putative Drosophila homolog hugin. PLoS biology 4, e68, doi:10.1371/journal.pbio.0040068 (2006).
32 Lindemans, M. et al. A neuromedin-pyrokinin-like neuropeptide signaling system in Caenorhabditis elegans. Biochemical and biophysical research communications 379, 760-764, doi:10.1016/j.bbrc.2008.12.121 (2009).
33 Kawai, T. et al. Identification of functionally important residues of the silkmoth pheromone biosynthesis-activating neuropeptide receptor, an insect ortholog of the vertebrate neuromedin U receptor. The Journal of biological chemistry 289, 19150-19163, doi:10.1074/jbc.M113.488999 (2014).
34 Hanada, R. et al. Neuromedin U has a novel anorexigenic effect independent of the leptin signaling pathway. Nature medicine 10, 1067-1073, doi:10.1038/nm1106 (2004).
35 Kowalski, T. J. et al. Transgenic overexpression of neuromedin U promotes leanness and hypophagia in mice. The Journal of endocrinology 185, 151-164, doi:10.1677/joe.1.05948 (2005).
36 Zeng, H. et al. Neuromedin U receptor 2-deficient mice display differential responses in sensory perception, stress, and feeding. Molecular and cellular biology 26, 9352-9363, doi:10.1128/MCB.01148-06 (2006).
37 Benzon, C. R. et al. Neuromedin U receptor 2 knockdown in the paraventricular nucleus modifies behavioral responses to obesogenic high-fat food and leads to increased body weight. Neuroscience 258, 270-279, doi:10.1016/j.neuroscience.2013.11.023 (2014).
38 Hanada, R. et al. A role for neuromedin U in stress response. Biochemical and biophysical research communications 289, 225-228, doi:10.1006/bbrc.2001.5945 (2001).
39 Thompson, E. L. et al. Chronic administration of NMU into the paraventricular nucleus stimulates the HPA axis but does not influence food intake or body weight. Biochemical and biophysical research communications 323, 65-71, doi:10.1016/j.bbrc.2004.08.058 (2004).
40 Wren, A. M. et al. Hypothalamic actions of neuromedin U. Endocrinology 143, 4227-4234, doi:10.1210/en.2002-220308 (2002).
41 Malendowicz, L. K., Nussdorfer, G. G., Nowak, K. W. &; Mazzocchi, G. Effects of neuromedin U-8 on the rat pituitary-adrenocortical axis. In vivo 7, 419-422 (1993).
42 Rucinski, M. et al. Expression of neuromedins S and U and their receptors in the hypothalamus and endocrine glands of the rat. International journal of molecular medicine 20, 255-259 (2007).
43 Trejter, M. et al. Neuromedin-U stimulates enucleation-induced adrenocortical regeneration in the rat. International journal of molecular medicine 21, 683-687 (2008).
44 Ziolkowska, A. et al. Effects of neuromedin-U on immature rat adrenocortical cells: in vitro and in vivo studies. International journal of molecular medicine 21, 303-307 (2008).
45 Cao, C. Q., Yu, X. H., Dray, A., Filosa, A. &; Perkins, M. N. A pro-nociceptive role of neuromedin U in adult mice. Pain 104, 609-616 (2003).
46 Yu, X. H. et al. Pro-nociceptive effects of neuromedin U in rat. Neuroscience 120, 467-474 (2003).
47 Nakahara, K. et al. Neuromedin U is involved in nociceptive reflexes and adaptation to environmental stimuli in mice. Biochemical and biophysical research communications 323, 615-620, doi:10.1016/j.bbrc.2004.08.136 (2004).
48 Torres, R. et al. Mice genetically deficient in neuromedin U receptor 2, but not neuromedin U receptor 1, have impaired nociceptive responses. Pain 130, 267-278, doi:10.1016/j.pain.2007.01.036 (2007).
49 Johnson, E. N. et al. Neuromedin U elicits cytokine release in murine Th2-type T cell clone D10.G4.1. Journal of immunology 173, 7230-7238 (2004).
50 Moriyama, M. et al. The neuropeptide neuromedin U promotes IL-6 production from macrophages and endotoxin shock. Biochemical and biophysical research communications 341, 1149-1154, doi:10.1016/j.bbrc.2006.01.075 (2006).
51 Moriyama, M. et al. The neuropeptide neuromedin U activates eosinophils and is involved in allergen-induced eosinophilia. American journal of physiology. Lung cellular and molecular physiology 290, L971-977, doi:10.1152/ajplung.00345.2005 (2006).
52 Moriyama, M. et al. The neuropeptide neuromedin U promotes inflammation by direct activation of mast cells. The Journal of experimental medicine 202, 217-224, doi:10.1084/jem.20050248 (2005).
53 Rao, S. M. et al. The neuropeptide neuromedin U promotes autoantibody-mediated arthritis. Arthritis research &; therapy 14, R29, doi:10.1186/ar3732 (2012).
54 Abbondanzo, S. J. et al. Nmur1-/- mice are not protected from cutaneous inflammation. Biochemical and biophysical research communications 378, 777-782, doi:10.1016/j.bbrc.2008.11.148 (2009).
55 Fukue, Y. et al. Regulation of gonadotropin secretion and puberty onset by neuromedin U. FEBS letters 580, 3485-3488, doi:10.1016/j.febslet.2006.05.025 (2006).
56 Vigo, E. et al. Novel role of the anorexigenic peptide neuromedin U in the control of LH secretion and its regulation by gonadal hormones and photoperiod. American journal of physiology. Endocrinology and metabolism 293, E1265-1273, doi:10.1152/ajpendo.00425.2007 (2007).
57 Quan, H., Funabashi, T., Furuta, M. &; Kimura, F. Effects of neuromedin U on the pulsatile LH secretion in ovariectomized rats in association with feeding conditions. Biochemical and biophysical research communications 311, 721-727 (2003).
58 Shetzline, S. E. et al. Neuromedin U: a Myb-regulated autocrine growth factor for human myeloid leukemias. Blood 104, 1833-1840, doi:10.1182/blood-2003-10-3577 (2004).
59 Wu, Y. et al. Neuromedin U is regulated by the metastasis suppressor RhoGDI2 and is a novel promoter of tumor formation, lung metastasis and cancer cachexia. Oncogene 26, 765-773, doi:10.1038/sj.onc.1209835 (2007).
60 Takahashi, K. et al. The neuromedin U-growth hormone secretagogue receptor 1b/neurotensin receptor 1 oncogenic signaling pathway as a therapeutic target for lung cancer. Cancer research 66, 9408-9419, doi:10.1158/0008-5472.CAN-06-1349 (2006).
61 Rani, S. et al. Neuromedin U: a candidate biomarker and therapeutic target to predict and overcome resistance to HER-tyrosine kinase inhibitors. Cancer research 74, 3821-3833, doi:10.1158/0008-5472.CAN-13-2053 (2014).
62 Ketterer, K. et al. Neuromedin U is overexpressed in pancreatic cancer and increases invasiveness via the hepatocyte growth factor c-Met pathway. Cancer letters 277, 72-81, doi:10.1016/j.canlet.2008.11.028 (2009).
63 Harten, S. K., Esteban, M. A., Shukla, D., Ashcroft, M. &; Maxwell, P. H. Inactivation of the von Hippel-Lindau tumour suppressor gene induces Neuromedin U expression in renal cancer cells. Molecular cancer 10, 89, doi:10.1186/1476-4598-10-89 (2011).
64 Tokumaru, Y. et al. Inverse correlation between cyclin A1 hypermethylation and p53 mutation in head and neck cancer identified by reversal of epigenetic silencing. Cancer research 64, 5982-5987, doi:10.1158/0008-5472.CAN-04-0993 (2004).
65 Yamashita, K. et al. Pharmacologic unmasking of epigenetically silenced tumor suppressor genes in esophageal squamous cell carcinoma. Cancer cell 2, 485-495 (2002).
66 Hawkins, S. M. &; Matzuk, M. M. The menstrual cycle: basic biology. Annals of the New York Academy of Sciences 1135, 10-18, doi:10.1196/annals.1429.018 (2008).
67 Edson, M. A., Nagaraja, A. K. &; Matzuk, M. M. The mammalian ovary from genesis to revelation. Endocrine reviews 30, 624-712, doi:10.1210/er.2009-0012 (2009).
68 Torre, L. A. et al. Global cancer statistics, 2012. CA: a cancer journal for clinicians 65, 87-108, doi:10.3322/caac.21262 (2015).
69 Bokhman, J. V. Two pathogenetic types of endometrial carcinoma. Gynecologic oncology 15, 10-17 (1983).
70 Abeler, V. M. &; Kjorstad, K. E. Clear cell carcinoma of the endometrium: a histopathological and clinical study of 97 cases. Gynecologic oncology 40, 207-217 (1991).
71 Goff, B. A. et al. Uterine papillary serous carcinoma: patterns of metastatic spread. Gynecologic oncology 54, 264-268, doi:10.1006/gyno.1994.1208 (1994).
72 Konecny, G. E. et al. HER2 gene amplification and EGFR expression in a large cohort of surgically staged patients with nonendometrioid (type II) endometrial cancer. British journal of cancer 100, 89-95, doi:10.1038/sj.bjc.6604814 (2009).
73 Salvesen, H. B. et al. Integrated genomic profiling of endometrial carcinoma associates aggressive tumors with indicators of PI3 kinase activation. Proceedings of the National Academy of Sciences of the United States of America 106, 4834-4839, doi:10.1073/pnas.0806514106 (2009).
74 Djordjevic, B. et al. Clinical assessment of PTEN loss in endometrial carcinoma: immunohistochemistry outperforms gene sequencing. Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc 25, 699-708, doi:10.1038/modpathol.2011.208 (2012).
75 Muinelo-Romay, L. et al. High-risk endometrial carcinoma profiling identifies TGF-beta1 as a key factor in the initiation of tumor invasion. Molecular cancer therapeutics 10, 1357-1366, doi:10.1158/1535-7163.MCT-10-1019 (2011).
76 Lei, X., Wang, L., Yang, J. &; Sun, L. Z. TGFbeta signaling supports survival and metastasis of endometrial cancer cells. Cancer management and research 2009, 15-24 (2009).
77 Park, J. H. et al. Hypermethylation of E-cadherin in endometrial carcinoma. Journal of gynecologic oncology 19, 241-245, doi:10.3802/jgo.2008.19.4.241 (2008).
78 Mell, L. K. et al. Prognostic significance of E-cadherin protein expression in pathological stage I-III endometrial cancer. Clinical cancer research : an official journal of the American Association for Cancer Research 10, 5546-5553, doi:10.1158/1078-0432.CCR-0943-03 (2004).
79 Feng, Z. et al. Aberrant expression of hypoxia-inducible factor 1alpha, TWIST and E-cadherin is associated with aggressive tumor phenotypes in endometrioid endometrial carcinoma. Japanese journal of clinical oncology 43, 396-403, doi:10.1093/jjco/hys237 (2013).
80 Saegusa, M., Hashimura, M., Kuwata, T. &; Okayasu, I. Requirement of the Akt/beta-catenin pathway for uterine carcinosarcoma genesis, modulating E-cadherin expression through the transactivation of slug. The American journal of pathology 174, 2107-2115, doi:10.2353/ajpath.2009.081018 (2009).
81 Spoelstra, N. S. et al. The transcription factor ZEB1 is aberrantly expressed in aggressive uterine cancers. Cancer research 66, 3893-3902, doi:10.1158/0008-5472.CAN-05-2881 (2006).
82 Singh, M. et al. ZEB1 expression in type I vs type II endometrial cancers: a marker of aggressive disease. Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc 21, 912-923, doi:10.1038/modpathol.2008.82 (2008).
83 Tanaka, Y. et al. Prognostic impact of EMT (epithelial-mesenchymal-transition)-related protein expression in endometrial cancer. Cancer biology &; therapy 14, 13-19, doi:10.4161/cbt.22625 (2013).
84 Bellone, M. et al. Expression of alphaV-integrins in uterine serous papillary carcinomas; implications for targeted therapy with intetumumab (CNTO 95), a fully human antagonist anti-alphaV-integrin antibody. International journal of gynecological cancer : official journal of the International Gynecological Cancer Society 21, 1084-1090, doi:10.1097/IGC.0b013e3182187324 (2011).
85 El-Sahwi, K. et al. Overexpression of EpCAM in uterine serous papillary carcinoma: implications for EpCAM-specific immunotherapy with human monoclonal antibody adecatumumab (MT201). Molecular cancer therapeutics 9, 57-66, doi:10.1158/1535-7163.MCT-09-0675 (2010).
86 Huszar, M. et al. Up-regulation of L1CAM is linked to loss of hormone receptors and E-cadherin in aggressive subtypes of endometrial carcinomas. The Journal of pathology 220, 551-561, doi:10.1002/path.2673 (2010).
87 Karahan, N., Guney, M., Oral, B., Kapucuoglu, N. &; Mungan, T. CD24 expression is a poor prognostic marker in endometrial carcinoma. European journal of gynaecological oncology 27, 500-504 (2006).
88 Byers, S. L., Wiles, M. V., Dunn, S. L. &; Taft, R. A. Mouse estrous cycle identification tool and images. PLoS One 7, e35538, doi:10.1371/journal.pone.0035538 (2012).
89 Chern, S. R., Li, S. H., Lu, C. H. &; Chen, E. I. Spatiotemporal expression of the serine protease inhibitor, SERPINE2, in the mouse placenta and uterus during the estrous cycle, pregnancy, and lactation. Reproductive biology and endocrinology : RB&;E 8, 127, doi:10.1186/1477-7827-8-127 (2010).
90 Du, Y. et al. Syntaxin 6-mediated Golgi translocation plays an important role in nuclear functions of EGFR through microtubule-dependent trafficking. Oncogene 33, 756-770, doi:10.1038/onc.2013.1 (2014).
91 Luo, C. W., Kawamura, K., Klein, C. &; Hsueh, A. J. Paracrine regulation of ovarian granulosa cell differentiation by stanniocalcin (STC) 1: mediation through specific STC1 receptors. Molecular endocrinology 18, 2085-2096, doi:10.1210/me.2004-0066 (2004).
92 Hung, W. T., Wu, F. J., Wang, C. J. &; Luo, C. W. DAN (NBL1) specifically antagonizes BMP2 and BMP4 and modulates the actions of GDF9, BMP2, and BMP4 in the rat ovary. Biology of reproduction 86, 158, 151-159, doi:10.1095/biolreprod.111.096172 (2012).
93 Magoffin, D. A. &; Weitsman, S. R. Insulin-like growth factor-I regulation of luteinizing hormone (LH) receptor messenger ribonucleic acid expression and LH-stimulated signal transduction in rat ovarian theca-interstitial cells. Biol Reprod 51, 766-775 (1994).
94 Sun, S. C. et al. Thyrostimulin, but not thyroid-stimulating hormone (TSH), acts as a paracrine regulator to activate the TSH receptor in mammalian ovary. The Journal of biological chemistry 285, 3758-3765, doi:10.1074/jbc.M109.066266 (2010).
95 Pecorelli, S. Revised FIGO staging for carcinoma of the vulva, cervix, and endometrium. International journal of gynaecology and obstetrics: the official organ of the International Federation of Gynaecology and Obstetrics 105, 103-104 (2009).
96 Kuroda, Y. et al. Isolation, culture and evaluation of multilineage-differentiating stress-enduring (Muse) cells. Nature protocols 8, 1391-1415, doi:10.1038/nprot.2013.076 (2013).
97 Barreau, C., Paillard, L. &; Osborne, H. B. AU-rich elements and associated factors: are there unifying principles? Nucleic acids research 33, 7138-7150, doi:10.1093/nar/gki1012 (2005).
98 Duan, H., Cherradi, N., Feige, J. J. &; Jefcoate, C. cAMP-dependent posttranscriptional regulation of steroidogenic acute regulatory (STAR) protein by the zinc finger protein ZFP36L1/TIS11b. Molecular endocrinology 23, 497-509, doi:10.1210/me.2008-0296 (2009).
99 Jefcoate, C. R., Lee, J., Cherradi, N., Takemori, H. &; Duan, H. cAMP stimulation of StAR expression and cholesterol metabolism is modulated by co-expression of labile suppressors of transcription and mRNA turnover. Molecular and cellular endocrinology 336, 53-62, doi:10.1016/j.mce.2010.12.006 (2011).
100 Aso, K. et al. The expression of integrins is decreased in colon cancer cells treated with polysaccharide K. International journal of oncology 42, 1175-1180, doi:10.3892/ijo.2013.1832 (2013).
101 Monier-Gavelle, F. &; Duband, J. L. Cross talk between adhesion molecules: control of N-cadherin activity by intracellular signals elicited by beta1 and beta3 integrins in migrating neural crest cells. The Journal of cell biology 137, 1663-1681 (1997).
102 Barretina, J. et al. The Cancer Cell Line Encyclopedia enables predictive modelling of anticancer drug sensitivity. Nature 483, 603-607, doi:10.1038/nature11003 (2012).
103 Screaton, G. R. et al. Genomic structure of DNA encoding the lymphocyte homing receptor CD44 reveals at least 12 alternatively spliced exons. Proceedings of the National Academy of Sciences of the United States of America 89, 12160-12164 (1992).
104 Peterson, R. M., Yu, Q., Stamenkovic, I. &; Toole, B. P. Perturbation of hyaluronan interactions by soluble CD44 inhibits growth of murine mammary carcinoma cells in ascites. The American journal of pathology 156, 2159-2167, doi:10.1016/S0002-9440(10)65086-9 (2000).
105 Paoli, P., Giannoni, E. &; Chiarugi, P. Anoikis molecular pathways and its role in cancer progression. Biochimica et biophysica acta 1833, 3481-3498, doi:10.1016/j.bbamcr.2013.06.026 (2013).
106 Huveneers, S. &; Danen, E. H. Adhesion signaling - crosstalk between integrins, Src and Rho. Journal of cell science 122, 1059-1069, doi:10.1242/jcs.039446 (2009).
107 Bourguignon, L. Y., Zhu, H., Shao, L. &; Chen, Y. W. CD44 interaction with c-Src kinase promotes cortactin-mediated cytoskeleton function and hyaluronic acid-dependent ovarian tumor cell migration. The Journal of biological chemistry 276, 7327-7336, doi:10.1074/jbc.M006498200 (2001).
108 Bourguignon, L. Y., Wong, G., Earle, C., Krueger, K. &; Spevak, C. C. Hyaluronan-CD44 interaction promotes c-Src-mediated twist signaling, microRNA-10b expression, and RhoA/RhoC up-regulation, leading to Rho-kinase-associated cytoskeleton activation and breast tumor cell invasion. The Journal of biological chemistry 285, 36721-36735, doi:10.1074/jbc.M110.162305 (2010).
109 Ponta, H., Sherman, L. &; Herrlich, P. A. CD44: from adhesion molecules to signalling regulators. Nature reviews. Molecular cell biology 4, 33-45, doi:10.1038/nrm1004 (2003).
110 Galliher, A. J. &; Schiemann, W. P. Src phosphorylates Tyr284 in TGF-beta type II receptor and regulates TGF-beta stimulation of p38 MAPK during breast cancer cell proliferation and invasion. Cancer Res 67, 3752-3758, doi:10.1158/0008-5472.CAN-06-3851 (2007).
111 Wheeler, D. L. et al. Epidermal growth factor receptor cooperates with Src family kinases in acquired resistance to cetuximab. Cancer biology &; therapy 8, 696-703 (2009).
112 Samarakoon, R. et al. Redox-induced Src kinase and caveolin-1 signaling in TGF-beta1-initiated SMAD2/3 activation and PAI-1 expression. PloS one 6, e22896, doi:10.1371/journal.pone.0022896 (2011).
113 Sandhoff, T. W. &; McLean, M. P. Hormonal regulation of steroidogenic acute regulatory (StAR) protein messenger ribonucleic acid expression in the rat ovary. Endocrine 4, 259-267, doi:10.1007/BF02738692 (1996).
114 Manna, P. R., Wang, X. J. &; Stocco, D. M. Involvement of multiple transcription factors in the regulation of steroidogenic acute regulatory protein gene expression. Steroids 68, 1125-1134 (2003).
115 Alberts, A. S., Arias, J., Hagiwara, M., Montminy, M. R. &; Feramisco, J. R. Recombinant cyclic AMP response element binding protein (CREB) phosphorylated on Ser-133 is transcriptionally active upon its introduction into fibroblast nuclei. The Journal of biological chemistry 269, 7623-7630 (1994).
116 Wadzinski, B. E. et al. Nuclear protein phosphatase 2A dephosphorylates protein kinase A-phosphorylated CREB and regulates CREB transcriptional stimulation. Molecular and cellular biology 13, 2822-2834 (1993).
117 Burkart, A. D., Mukherjee, A. &; Mayo, K. E. Mechanism of repression of the inhibin alpha-subunit gene by inducible 3',5'-cyclic adenosine monophosphate early repressor. Molecular endocrinology 20, 584-597, doi:10.1210/me.2005-0204 (2006).
118 Altarejos, J. Y. &; Montminy, M. CREB and the CRTC co-activators: sensors for hormonal and metabolic signals. Nature reviews. Molecular cell biology 12, 141-151, doi:10.1038/nrm3072 (2011).
119 Chinn, A. M. et al. Identification of two novel ACTH-responsive genes encoding manganese-dependent superoxide dismutase (SOD2) and the zinc finger protein TIS11b [tetradecanoyl phorbol acetate (TPA)-inducible sequence 11b]. Molecular endocrinology 16, 1417-1427, doi:10.1210/mend.16.6.0844 (2002).
120 Ida, T. et al. Neuromedin s is a novel anorexigenic hormone. Endocrinology 146, 4217-4223, doi:10.1210/en.2005-0107 (2005).
121 Vigo, E. et al. Neuromedin s as novel putative regulator of luteinizing hormone secretion. Endocrinology 148, 813-823, doi:10.1210/en.2006-0636 (2007).
122 Yang, G. et al. The regulatory mechanism of neuromedin S on luteinizing hormone in pigs. Animal reproduction science 122, 367-374, doi:10.1016/j.anireprosci.2010.10.011 (2010).
123 Yang, G. et al. Expression of NMS and NMU2R in the pig reproductive axis during the estrus cycle and the effect of NMS on the reproductive axis in vitro. Peptides 30, 2206-2212, doi:10.1016/j.peptides.2009.09.024 (2009).
124 New, D. C. &; Wong, Y. H. Molecular mechanisms mediating the G protein-coupled receptor regulation of cell cycle progression. Journal of molecular signaling 2, 2, doi:10.1186/1750-2187-2-2 (2007).
125 Lydon, J. P. et al. Mice lacking progesterone receptor exhibit pleiotropic reproductive abnormalities. Genes &; development 9, 2266-2278 (1995).
126 Robker, R. L. et al. Progesterone-regulated genes in the ovulation process: ADAMTS-1 and cathepsin L proteases. Proceedings of the National Academy of Sciences of the United States of America 97, 4689-4694, doi:10.1073/pnas.080073497 (2000).
127 Russell, D. L., Doyle, K. M., Ochsner, S. A., Sandy, J. D. &; Richards, J. S. Processing and localization of ADAMTS-1 and proteolytic cleavage of versican during cumulus matrix expansion and ovulation. The Journal of biological chemistry 278, 42330-42339, doi:10.1074/jbc.M300519200 (2003).
128 Espey, L. L. et al. Ovarian expression of a disintegrin and metalloproteinase with thrombospondin motifs during ovulation in the gonadotropin-primed immature rat. Biology of reproduction 62, 1090-1095 (2000).
129 Doyle, K. M., Russell, D. L., Sriraman, V. &; Richards, J. S. Coordinate transcription of the ADAMTS-1 gene by luteinizing hormone and progesterone receptor. Molecular endocrinology 18, 2463-2478, doi:10.1210/me.2003-0380 (2004).
130 Brown, H. M. et al. ADAMTS1 cleavage of versican mediates essential structural remodeling of the ovarian follicle and cumulus-oocyte matrix during ovulation in mice. Biology of reproduction 83, 549-557, doi:10.1095/biolreprod.110.084434 (2010).
131 Mittaz, L. et al. Adamts-1 is essential for the development and function of the urogenital system. Biology of reproduction 70, 1096-1105, doi:10.1095/biolreprod.103.023911 (2004).
132 Bridges, P. J., Cho, J. &; Ko, C. Endothelins in regulating ovarian and oviductal function. Frontiers in bioscience 3, 145-155 (2011).
133 Bridges, P. J. et al. Production and binding of endothelin-2 (EDN2) in the rat ovary: endothelin receptor subtype A (EDNRA)-mediated contraction. Reproduction, fertility, and development 22, 780-787, doi:10.1071/RD09194 (2010).
134 Sriraman, V., Sinha, M. &; Richards, J. S. Progesterone receptor-induced gene expression in primary mouse granulosa cell cultures. Biology of reproduction 82, 402-412, doi:10.1095/biolreprod.109.077610 (2010).
135 Palanisamy, G. S. et al. A novel pathway involving progesterone receptor, endothelin-2, and endothelin receptor B controls ovulation in mice. Molecular endocrinology 20, 2784-2795, doi:10.1210/me.2006-0093 (2006).
136 Ko, C. et al. Endothelin-2 in ovarian follicle rupture. Endocrinology 147, 1770-1779, doi:10.1210/en.2005-1228 (2006).
137 Mhawech-Fauceglia, P. et al. Microarray analysis reveals distinct gene expression profiles among different tumor histology, stage and disease outcomes in endometrial adenocarcinoma. PloS one 5, e15415, doi:10.1371/journal.pone.0015415.s001 (2010).
138 Rose, P. G. Endometrial carcinoma. The New England journal of medicine 335, 640-649, doi:10.1056/NEJM199608293350907 (1996).
139 Nam, J. S., Ino, Y., Sakamoto, M. &; Hirohashi, S. Src family kinase inhibitor PP2 restores the E-cadherin/catenin cell adhesion system in human cancer cells and reduces cancer metastasis. Clinical cancer research : an official journal of the American Association for Cancer Research 8, 2430-2436 (2002).
140 Funakoshi, S. et al. Intestine-specific transcription factor Cdx2 induces E-cadherin function by enhancing the trafficking of E-cadherin to the cell membrane. American journal of physiology. Gastrointestinal and liver physiology 299, G1054-1067, doi:10.1152/ajpgi.00297.2010 (2010).
141 Chen, S. Y. et al. Dependence of fibroblast infiltration in tumor stroma on type IV collagen-initiated integrin signal through induction of platelet-derived growth factor. Biochimica et biophysica acta 1853, 929-939, doi:10.1016/j.bbamcr.2015.02.004 (2015).
142 Zaytseva, Y. Y. et al. Inhibition of fatty acid synthase attenuates CD44-associated signaling and reduces metastasis in colorectal cancer. Cancer research 72, 1504-1517, doi:10.1158/0008-5472.CAN-11-4057 (2012).
143 Slomovitz, B. M. et al. Her-2/neu overexpression and amplification in uterine papillary serous carcinoma. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 22, 3126-3132, doi:10.1200/JCO.2004.11.154 (2004).
144 Cancer Genome Atlas Research, N. et al. Integrated genomic characterization of endometrial carcinoma. Nature 497, 67-73, doi:10.1038/nature12113 (2013).
145 Catasus, L., Gallardo, A., Cuatrecasas, M. &; Prat, J. PIK3CA mutations in the kinase domain (exon 20) of uterine endometrial adenocarcinomas are associated with adverse prognostic parameters. Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc 21, 131-139, doi:10.1038/modpathol.3800992 (2008).
146 Gold, L. I. et al. Increased expression of transforming growth factor beta isoforms and basic fibroblast growth factor in complex hyperplasia and adenocarcinoma of the endometrium: evidence for paracrine and autocrine action. Cancer research 54, 2347-2358 (1994).
147 Zakrzewski, P. K. et al. Dysregulation of betaglycan expression in primary human endometrial carcinomas. Cancer investigation 29, 137-144, doi:10.3109/07357907.2010.543213 (2011).
148 Van Themsche, C., Mathieu, I., Parent, S. &; Asselin, E. Transforming growth factor-beta3 increases the invasiveness of endometrial carcinoma cells through phosphatidylinositol 3-kinase-dependent up-regulation of X-linked inhibitor of apoptosis and protein kinase c-dependent induction of matrix metalloproteinase-9. The Journal of biological chemistry 282, 4794-4802, doi:10.1074/jbc.M608497200 (2007).
149 Wheeler, D. L., Dunn, E. F. &; Harari, P. M. Understanding resistance to EGFR inhibitors-impact on future treatment strategies. Nature reviews. Clinical oncology 7, 493-507, doi:10.1038/nrclinonc.2010.97 (2010).
150 Mueller, K. L., Powell, K., Madden, J. M., Eblen, S. T. &; Boerner, J. L. EGFR Tyrosine 845 Phosphorylation-Dependent Proliferation and Transformation of Breast Cancer Cells Require Activation of p38 MAPK. Translational oncology 5, 327-334 (2012).
151 Kong, L., Deng, Z., Shen, H. &; Zhang, Y. Src family kinase inhibitor PP2 efficiently inhibits cervical cancer cell proliferation through down-regulating phospho-Src-Y416 and phospho-EGFR-Y1173. Molecular and cellular biochemistry 348, 11-19, doi:10.1007/s11010-010-0632-1 (2011).