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Estrogen inhibits B-RafV600E-induced senescence in
human primary ovarian epithelial cells
by
Soo Jung Park
Major in Cancer Biology
Department of Biomedical Sciences
The Graduate School, Ajou University
Estrogen inhibits B-RafV600E-induced senescence in
human primary ovarian epithelial cells
by
Soo Jung Park
A Dissertation Submitted to The Graduate School of
Ajou University in Partial Fulfillment of the Requirements
for the Degree of Master of Cancer Biology
Supervised by
Tae Jun Park, M.D., Ph.D.
Major in Cancer Biology
Department of Biomedical Sciences
The Graduate School, Ajou University
i
-ABSTRACT-Estrogen inhibits B-RafV600E-induced senescence in ovarian
epithelial cells
Carcinogenesis is a multistep process in which normal epithelial cells are converted to cancer cells through the sequential acquisition of multiple genetic or epigenetic events. Activating mutations in one of its downstream effectors, B-Raf, have been identified in a variety of human cancers and mutations of B-Raf lead to constitutive activation of mitogen activated protein kinase (MAPK) signaling. About 14% of ovarian primary tumors contain B-Raf point mutation and B-RafV600E in is a major mutation in ovarian primary tumor(Grisham et al., 2013). However, a genetic modification by B-RafV600E in primary ovarian epithelial cells results in oncogene-induced senescence (OIS). In the present study, we observed B-RafV600E induced OIS in ovarian epithelial cells. Furthermore, when we treated estrogen with B-RafV600E lentivirus infection, oncogene induced senescence was significantly downregulated. Estrogen treatment induced p-Erk1/2 translocation from cytoplasm to nucleus via PEA15 phosphorylation.
ii
-
TABLE OF TEXT
ABSTRACT ... i TABLE OF TEXT ... ii LIST OF FIGURES ... iv I. INTRODUCTION ... 1II. MATERIALS AND METHODS ... 4
A. Culture of Human ovarian epithelial cells ... 4
B. Lentiviral system ... 4
C. Real-Time PCR analysis ... 4
D. Immunoblotting ... 5
E. Cell proliferation assay ... 5
F. Senescence-Associated β-Galactosidase Staining ... 5
G. Immunocytochemistry and confocal microscopy ... 6
H. Statistical analysis ... 6
III. RESULTS ... 7
1. Mutation of genes in cancer type ... 7
iii
3. B-RafV600E induces OIS in primary HOSE ... 11
4.Estrogen inhibits B-RafV600E induced senescence in HOSE ... 14
5. Expression of estrogen receptor in B-RafV600E-expressing HOSE ... 17
6. Estrogen regulates translocation of p-Erk1/2 in B-RafV600E-expressing HOSE . ... 19
IV. DISCUSSION ... 22
REFERENCES ... 24
iv
LIST OF FIGURES
Figure 1. Mutation of genes as type of ovarian cancer ... 8
Figure 2. Morphology of Human primary ovarian epithelial cells (HOSE) ... 10
Figure 3. B-RafV600E induces OIS in HOSE ... 12
Figure 4. Inhibition of B-RafV600E-induced senescence by estrogen ... 15
Figure 5. Level of estrogen receptor α and β in B-RafV600E-expressing HOSE ... 18
１
I.INTRODUCTION
Ovarian cancer is the second common gynecologic cancer after cervical cancer. Ovarian epithelial cancer which accounts for approximately 90% of ovarian cancer has less than 40% of 5year survival rate (Cho and Shih Ie, 2009).Traditional classification of ovarian cancer which is derived from the ovarian epithelium is divided into the shape of serous, mucinous, endometrioid and clear cell. But recently,classification of ovarian cancer is divided into type1 and type2.Type1ovarian cancer composed with low grade serous, mucinous, endometrioid and clear cell which are derived from the ovarian epithelium. Feature of type1 ovarian cancershow slow growth rate and undergoes either atypical proliferation or borderline tumor. Type2 ovarian cancer composed withhigh grade serous carcinomas, undifferentiated carcinomas and carcinomas which are derived from fallopian tube and ovarian fimbriae. Feature of type2show high growth rate and frequent p53 mutation (Ng and Barker, 2015).
Molecular genetic study of mutations in the cancer type revealed that B-RafV600E has been reported up to 14% in serous carcinoma (SOC)(Grisham et al., 2013). In addition, B-RafV600E also has been reported up to 71% in serous borderline tumor (SBT) (Malpica and Wong, 2016). B-Raf, participating in theMAPK signaling pathway, regulatescell proliferation, cell cycle, cell survival, angiogenesis, cell migration and connection from cell membrane receptor to nucleus(Cho and Shih Ie, 2009). For example,Ras protein,which is activated by growth factor and hormone signaling, activates multiple downstream pathways like a series of phosphorylation and dephosphorylation events. B-Raf,downstream of Ras, phosphorylates MEK1 and 2. And then, it phosphorylates and activates Erk1 and 2. These phosphorylated molecules affect to several cytoplasmic and nuclear targets, transcription factors such as Ets-1, c-Jun and c-Myc(Michaloglou et al., 2008).
If residue 600 amino acid valine of the Raf is optionally substituted with gluamine, B-Raf has the active form in cell signaling pathway which causes a more than cell growth and
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can be oncogene(Michaloglou et al., 2008). Oncogene is induced by point mutation, insertion mutation, gene amplification andchromosomal translocation. It can lead to conversion of transformed cells if not repaired. Thus, normal cellsare given the opportunity to enter the initial process of tumorigenesis to convert as cancer cell. However, tumorigenesis is occurred not single step but complex step and is blocked by tumor barrier which known as tumor suppressor mechanism(Courtois-Cox et al., 2008). Senescence, one of tumor suppressor mechanism, is induced upon strong mitogenic signals and aberrant expression of several oncogenes including Raf, Akt, cyclin E and others which are known to promote oncogene-induced senescence(OIS). For example, expression of oncogenic Ras in primary human fibroblasts, resulted in a permanent G1 arrest, mediated by tumor suppressor gene such as p53 and p16INK4a. It also caused induction of a phenotype indistinguishable from
replicative senescence(Mooi and Peeper, 2006; Meng et al., 2015).
Ovary secretes sex hormone including estrogen, progesterone and testosterone.Secretion of these hormones is controlled by interacting with releasing hormones from hypothalamus of pituitary gland. Estrogen plays important roles in growth, development and reproduction(Cui et al., 2013). There are 4 different estrogen(ER) signaling pathway: (1) ER-dependent, nuclear-initiated estrogen signaling (2)ER-dependent, membrane-initiated estrogen signaling (3)ER-independent pathway (4)Ligand-independent activation of ER. ER-dependent mechanisms, classified as ‘genomic’ and ‘non-genomic‘, based on whether the end result is transcriptional regulation which can initiate either in the nucleus or at the plasma membrane(Richardson et al., 2012). While, ER-independent mechanisms can initiate by regulating enzymatic activities or interacting with nuclear receptors of non-sex steroid hormone. Pathway 1: nuclear-initiated estrogen signaling, mediated by estrogen receptor α andβ,can stimulate or repress transcription of target gene by binding specific DNA target sequences such as estrogen response elements(EREs) in estrogen-responsive gene promotersor transcription factors such as SP-1, AP-1, Nf-κB and c-Jun without binding DNA directly(Gottsch et al., 2009)(Cui et al., 2013).Pathway 2: membrane-initiated estrogen signaling initiates in the membrane or cytoplasm. It leads to diverse cytoplasmic effects, including the regulation of membrane-based ion channels, regulation of second-messenger systems, and modification of transcription factors or other membrane receptors such as
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tyrosine kinase receptors, insulin-like growth hormone receptors and neurotransmitter receptors(Micevych and Dominguez, 2009). For example, estrogen activates membrane-initiated estrogen receptor to interact with metabotropic glutamate receptors(mGluRs) without glutamate. It also can be mediated via the activation of different protein kinase cascades, such as MAPK/ERK , PI3K/AKT ,cAMP , PKA and PKC(Aronica et al., 1994). Another membrane-initiated receptor, G protein-coupled estrogen receptor 1(GPER), is activated by estrogen and activates several signaling cascades such as PI3K and calcium signaling(Prossnitz et al., 2008). Pathway 3: ER-independent pathway, It does not interact with estrogen- receptor. Estrogen plays a role as antioxidant effects which suppress oxidative stress(Haas et al., 2012).Pathway 4: Ligand-independent activation of ER is activated via signaling cascades such as growth factors, intracellular signaling pathways(PKC, PKA, MAPK and PI3k) through phosphorylation of ERs(Martin et al., 2000; Klotz et al., 2002).
Estrogen iswell known as risk factor that proliferates ovarian epithelial cells through estrogen receptor ,G protein-coupled estrogen receptor 1(GPER), and Receptor Tyrosine kinase(RTK). For patients receiving the post menopause in women and ovarian cancer resection may increase the risk of ovarian cancer by implementing the estrogen replacement therapy(Riman et al., 2002; Morch et al., 2009).
Therefore, in the present study, we investigated the effect whether estrogen inhibits B-RafV600E-induced senescence in human primary ovarian epithelial cells.
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Ⅱ
.MATERIALS AND METHODS
1. Culture of Human ovarian epithelial cells
Human primary ovarian epithelial cells (HOSE) were purchased from ScienCell Research Laboratories (6076 Corte Del Cedro Carlsbad, CA) and were cultured in OEpiCM medium (ScienCell Research Laboratories) supplemented with OEpiCGS (ScienCell Research Laboratories) at 37℃ in a humidified incubator with 5% CO2.
2. Lentiviral system
To generate lentivirus, a control lentiviral vector contained pcDH-CMV-MCS-EF1-Puro sequence (System Biosciences, Mountain View, CA) and Lentiviral system were performed in HEK-293TN cells in present of packaging plasmids (pGagpol, pVSVG, and pCDH-B-RafV600E) adding lipofectamine 2000 (Invitrogen, Carlsbad, CA). Viral supernatants were collected 48 hours after transfection, filtered through 0.45 um syringe filter (Merck Millipore, Billerica, MA, USA). For infection, cell were plated in 60Ø culture dishes and treated with viral supernatant in the presence of 4mg/ml polybrene (Sigma, St Louis, MO) for 8 hours.
3. Real-Time PCR analysis
Total cellular RNAs were isolated from HOSE. cDNA was synthesized by reverse transcription reaction using oligo-dT primers from 1 ug of total cellular RNA. Real-time PCR was performed with Power SYBR Green PCR Master Mix (Applied Biosystems) according to the following conditions: initial activation at 95℃ for 5 minutes, followed by 40 cycles of 95℃ for 15 seconds and 60℃ for 1 minute. The primer sets were produced as follows : B-Raf: 5’-AGA AAG CAC TGA TGA TGA GAG G-3’ , 5’-GGA AAT ATC AGT
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GTC CCA ACC A-3’ ; ESRα: 5’-ATT TGA AGT GGG CAG AGA ACA-3’ , 5’-CAA TAC CAA CAT CAG CCA GAA-3’ ; ESRβ: ATG GAT ATA AAA AAC TCA CCA-3’ , 5’-CGC ATT TCC CCT CAT CC-3’ ; p21: 5’-ATT AGC AGC GGA ACA AGG AGT CAG ACA T-3’ , 5’-CTG TGA AAG ACA CAG AAC AGT ACA GGG T-3’ ; p16: 5’-CTG CCC AAC GCA CCG AAT AG-3’ , 5’-TAC CGT GCG ACA TCG GGA TG-3’ ; 18s: 5’-CGG CTA CCA CAT CCA AGG AA-3’ , 5’-GCT GGA ATT ACC GCG GCT-3’.
4. Immunoblotting
Cells were lysed in RIPA buffer (20 mM Tris pH 7.5, 150 mM NaCl, 1% Nonidet p-40, 0.5% sodium deoxycholate, 1 mM EDTA, and 0.1% SDS) containing protease inhibitor cocktail (K272, Biovision) and phosphatase inhibitor cocktail (K282, Biovision). The isolated proteins were separated on 9-15% SDS-PAGE and transferred onto Poly-vinylidene difluoride membrane (Bio-rad). These membranes were incubated for 12 hours with the following primary antibodies. And then, the membranes were washed with 1X PBST buffer 3 times for 15 minutes each, after that, they were incubated with HRP-conjugated anti-mouse and anti-rabbit (1:3000) for 1 hour and washed with 1X PBST buffer 3 times for 15 minutes each. Antibodies against p21 (sc-6246), cyclinD1 (sc-20044), c-Jun (sc-1694) were purchased from Santacruz Biotechnology, Inc (Santa Cruz, CA). Antibodies against p16 (2D9A12) was purchased from Abcam Ltd (Cambridge, UK). Antibodies against B-Raf (L12G7), p-AKTSer473 (#9271), AKT (#9272), p-Erk1/2 (#9106), Erk1/2 (#9107), ESRα
(#8644), p-PEA-15Ser104 (#2776), PEA-15 (#2780) were purchased from Cell signaling Technology, Inc (Danvers, MA). Antibody against β-actin (Abc-2004) was purchased from AbClon, Inc (Seoul, Korea).
5. Cell proliferation assay
HOSEs (2.0 x 103) were plated in 60Ø dish for 24 hours and treated with 40 nM
17-β-estradiol (E2)(sigma) or ethanol every 2 day. HOSEs were harvested in 3 days after the
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6. Senescence-Associated β-Galactosidase Staining
The cells were fixed with 4% paraformaldehyde for 15 minutes and incubated with SA-β-gal solution (1 mg/ml X-SA-β-gal (5-bromo-4-chloro-3-indolyl-β-D-SA-β-galactopyranoside), 40 mM Citric acid/sodium phosphate pH 5.8, 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 150 mM NaCl, 2 mM MgCl2) for 10 hours at 37℃. After washing with PBS,
SA-β-gal stained cells were counted.
7. Immunocytochemistry and confocal microscopy
The cells were fixed with 4% paraformaldehyde for 15 minutes and washed with 1XPBS 3 times for 15 minutes. And then, 0.1% triton X-100 was added for 15 minutes before Washed 3 times for 15minutes. Next step, 5% BSA is added for 30 minutes. The cells were incubated with primary antibody overnight at 4℃. The primary antibody was given as follows : p-Erk1/2 (1:100). The secondary antibody(1:600) was added for 1 hour. The cells were observed under fluorescence microscope and confocal microscopy.
8. Statistical analysis
Numerical data are presented as mean+ SD of independent determinations. Statistical analysis of differences was performed by student’s t test. P value (p<0.05) was considered as significant.
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Ⅲ
.RESULTS
1. Mutation of genes in cancer type.
To know mutations ratein various type of ovarian cancer, we searched reported reference (Nakayama et al., 2006; Malpica and Wong, 2016). Mutation of B-Raf is involved in low grade serous carcinomas (LGSC) (Fig. 1A). In addition, mutation of B-Raf is showed about 71%in serous borderline tumor (SBT), precursor of low-grade serous carcinomas, and about 14 % in serous ovarian carcinoma (Bosmuller et al., 2013) (Fig. 1B). But, other mutation of genesare not reported in detail and mutation of PIK3CA is rare in SBT (Nakayama et al., 2006). So, we focused on mutation of B-Raf.
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A.
B.
Fig1. Mutation of genesas type of ovarian cancer.
(A) Classification of genes as type of ovarian cancer. (B) Percentage of B-RafV600E in SBT and SOC.
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2. Morphology of primary human ovarian epithelial cells (HOSE).
Before using HOSE, we used primary mouse ovarian epithelial cells. But, MOSE was maintained for 7 days culture. Therefore, we used primary human ovarian epithelial cells (HOSE) and observed morphology of HOSE (Fig. 2A). At day 21, we checked enlarged phenotype of normal HOSE and we performed all experiments within 3 weeks. Next, we generated B-RafV600E lentivirus to overexpress B-RafV600E protein in HOSE and found cellular senescence phenotype such as cellular enlargement and cytoplasmic vacuoles (Fig. 2B).
１０ A.
B.
Fig.2. Morphology of Human primary ovarian epithelial cells (HOSE).
(A) Normal morphology of HOSE. It was observed until 21 days. At 21 days, morphology of HOSE has senescence like phenotype. (B) B-RafV600E-induced OIS in HOSE. HOSE was infected with control or B-RafV600E lentivirus.
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To confirm cellular senescence, we performed senescence analysis including cell proliferation assay (Fig. 3A), SA-β-gal staining (Fig. 3B), mRNA expression of tumor suppressor gene and protein (Fig. 3C). B-RafV600E-expressing HOSE showed that decreasing of cell proliferation, increased of SA-β-gal positive cells and tumor suppressor gene compared with control virus infected cells. We also confirmed the induction of B-Raf by real-time PCR and western blotting.
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１３ C.
Fig3. B-RafV600E induces OIS in primary HOSE.
(A) Cell proliferation assay. After HOSE was infected with control or B-RafV600E lentivirus, we measured cell growth by using hemocytometer at that day. (B) SA-β-gal staining. Expression of SA-β-gal was increased in B-RafV600E-expressing HOSE compared with control in 12days after infection (P<0.005). (C) Measurement of mRNA and Protein. Real-time PCR of B-Raf, p21 and p16 is performed at 16days after infection. In addition, immunoblotting of B-Raf, p-Erk1/2, p21, p16 and Actin is also performed at 16days after infection.
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4. Estrogen inhibits B-RafV600E induced senescence in HOSE.
It is well known that estrogen can stimulate proliferation of cancer and normal ovarian epithelial cells(Ciucci et al., 2016). We assumed that whether estrogen can stimulate proliferation of B-RafV600E-expressing HOSE.HOSE was infected with B-RafV600E lentivirus and then treated with or without 40nM 17-b-estradiol (E2) every 2 day (Fig. 4A).
We determined 40 nM E2that does did not induce HOSE death and 40 nM of E2 could induce
p-Erk1/2 fully. Next, we performed senescence analysis including cell proliferation assay (Fig. 4B), SA-β-gal staining (Fig. 4C) and protein expression of tumor suppressor gene (Fig. 4D) after estrogen treatment. B-RafV600E-expressing HOSEwhich was treated with 40nM E2 increased cells growth rate compared with non-treated B-RafV600E-expressing HOSE. In
addition, estrogen treatment decreased SA-β-gal staining and protein expression of tumor suppressor gene. Furthermore, weanalyzed estrogen-induced protein such as c-jun and cyclin D1(Fig. 4E)(Prall et al., 1997; Garcia et al., 2000), and the results showed that marked induction of c-jun and cyclin D1 protein.
１５ A.
B.
１６ D. E.
Fig4. Level of estrogen receptor α and β in B-RafV600E-expressing HOSE.
(A) Schematization of experiment. HOSE was treated with ethanol as control and with 40nM E2 next day after infection. And then, E2 was treated in Control or
expressing HOSE every 2 day. (B) Cell proliferation assay. After Control and B-RafV600E-expressing HOSE was treated with ethanol or E2 during 6 days, cells (2.0 x 103)were plated
in 60Ø dish for 24 hours and treated with E2 or ethanol every 2day. And then, we measured
cell growth by using hemocytometer at that day. (C) gal staining. Expression of SA-β-gal was increased in B-RafV600E-expressing HOSE compared with control in 13 days after infection and 9 days after treatment of E2 (P<0.005). (D) Measurement of protein.
Immunoblotting of B-Raf, p-Erk1/2, Erk1/2, p16, p21 and Actin is also performed in 16 days after infection and 13 days after treatment of E2. (E) Measurement of estrogen-induced
protein. Immunoblotting of CyclinD1 and C-Jun is performed in16 days after infection and 13 days after treatment of E2.
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5. Expression of estrogen receptorin B-RafV600E-expressing HOSE.
Our group published thatnormal thyrocyte infected with B-RafV600E lentivrus increased in mRNA level of thyroid stimulating hormone(TSHR)(Kim et al., 2014).Accordingly, we assumedthatB-RafV600E-expressing HOSEwould showupregulation of estrogen receptor level. Therefore, we analyzedestrogen receptor alpha and beta expression by real-time PCR and immunoblotting (Fig. 5A and 5B). Unlike our expectation, B-RafV600E-expressing HOSEdid not show the upregulation estrogen receptor α and β.
１８ A.
Fig5.Level of estrogen receptor α and β in B-RafV600E-expressing HOSE.
(A) Measurementof mRNA. At 12 days after infection, we performed Real-time PCR. (B) Measurement of protein. On the same day, we performed immunoblotting.
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6. Estrogen regulates translocation of p-Erk1/2 in B-RafV600E-expressing
HOSE.
We found that estrogen inhibits B-RafV600E induced senescence in HOSE. But, we wondered that how estrogen inhibitedOIS.Estrogen regulated translocation of p-Erk1/2 (Chen et al., 2005).For these reasons, we analyzed localization of p-Erk1/2 by using confocal microscopy and counted by using image of immunocytochemistry (Fig. 6A). p-Erk1/2 is mainly accumulated in cytosol in B-RafV600E-expressing HOSE. But, p-Erk1/2 located in nuclear in estrogen treated B-RafV600E-expressing HOSE. Next, we focused on which mechanism is involved in translocation of p-Erk1/2. It was established that PEA15 regulated location of Erk1/2 (Lee et al., 2015). Therefore, we measured level of PEA15 and p-PEA15S104 (Fig. 6B) (Chen et al., 2008; Lee et al., 2015). We observed that p-PEA15S104 is
increased in estrogen treated B-RafV600E-expressing HOSE. These results suggested that estrogen regulates translocation of p-Erk1/2 via phosphorylation of PEA15.
２０ A.
２１ B.
Fig6. Estrogen mediated translocation of p- Erk1/2.
(A) Localization of p-Erk1/2. We performed confocal microscopy 19 days after infection and 16 days after treatment of E2and counted by using image J. (B) Level of protein.
Immunoblotting of PEA15, p-PEA15S104 and α-Tubulin is performed19 days after infection
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IV.Discussion
If residue 600 amino acid val of the B-Raf is optionally substituted with glu, B-Raf always has the active form in cell signaling pathway which causes a more than cell growth and can be oncogene (Michaloglou et al., 2008). B-RafV600E has been reported up to 14% in serous carcinoma (SOC) (Grisham et al., 2013) and up to 71% in serous borderline tumor (SBT) (Malpica and Wong, 2016). For patients receiving the post menopause in women and ovarian cancer resection may increase the risk of ovarian cancer by implementing the estrogen replacement therapy (Risch, 1996; Riman et al., 2002; Morch et al., 2009).
In this study, we observed whether estrogen regulates B-RafV600E-induced senescence in human primary ovarian epithelial cells (HOSE). Thus, we overexpressed B-Raf protein by using lentivirus system. B-RafV600E-expressing HOSE was decreased in cell growth and increased in SA-B-GAL and expression of p21 but not expression of p16. Next, to identify estrogen effect, 40nM 17-b-estradiol (E2) was treated in normal HOSE and
B-RafV600E-expressing HOSE every 2 day. B-RafV600E-B-RafV600E-expressing HOSE treated E2 increased in cell
growth and level of cyclin D1 and decreased in SA-β-gal compared with B-RafV600E-expressing HOSE. Other hand, no significant difference in normal HOSE and normal HOSE treated E2 better than B-Rafv600e-expressing HOSE. After that, we observed estrogen
receptor α and β whether overexpression of B-Raf affects to expression of estrogen receptors. However, we couldn’t found the difference.To know how estrogen inhibits B-RafV600E-induced senescence and how cell growth was reversed, we focused on relation between estrogen and phosphorylation of Erk1/2. Estrogen can translocate phosphorylation of Erk1/2 into nucleus (Chen et al., 2005). As you know, Erk1/2 must translocate in nucleus to proliferate cells .So, we carried out localization of p-Erk1/2 through Immunofluorescence and confocal microscopy. We proved that p-Erk1/2 was accumulated in cytosol in RafV600E-expressing HOSE. Other hand, p-Erk1/2 was translocated in nucleus in B-RafV600E-expressing HOSE treated E2. It was reported that pea-15 regulates
２３
was involved in translocation of Erk1/2, we observed level of protein including pea-15, p-pea15S104 and p-Erk1/2. As a result, phosphorylation of pea-15 increased in
B-RafV600E-expressing HOSE treated E2.
In further study, we will count amount of p-Erk1/2 in nucleus. And then, we will prove interaction between p-Erk1/2 and PEA15 by performing immunoprecipitation.
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-국문요약-
인간 일차 난소 상피세포에서 에스트로겐에 의한 B-RafV600E
유도 노화극복기전 연구
발암 현상은 정상적인 상피 세포가 여러 유전적 또는 후성적 일들의 연속적인 획득을 통해 암세포로 전환되는 다단계 과정이다. 신호전달과정에 존재하는 효과 기 중 하나인 Raf의 활성형 돌연변이는 다양한 인간 암에서 발견이 되며 B-Raf의 돌연변이는 세포분열을 유발하는 물질에 의해 활성화 되는 단백질 효소신호전달 (mitogen-activated protein kinase signaling)의 지속적인 활성을 유 도한다. 일차 난소 종양의 약 14 %가 Raf 점 돌연변이를 가지고 있으며 B-RafV600E는 일차 난소 종양의 주요 돌연변이다(Grisham et al., 2013). 그러나, 1 차 난소 상피 세포에서 B-RafV600E에 의한 유전자 변형은 암 유전자 유발 성 노화 (OIS)를 초래한다. 본 연구에서는 B-RafV600E 유도 난소 상피 세포 가 난소 상피 세포에서 관찰하였다. 또한, 우리는 폐경기 여성에서 난소 종양 진 행 인자로 알려진 에스트로겐이 난소 상피 세포에서 OIS를 극복 할 수 있는지를 증명할 것이다. 핵심어 : B-RafV600E ; 난소 상피 세포 ; 에스트로겐
