Effects Of Forkhead Box C2 On Carcinogenesis And Lymphatic Metastasis In Endometrial Carcinoma

Endometrial cancer is one of the three most common malignant carcinoma in femal reproductive system. The attack rate of endometrial cancer has become higher in recent 20 years, according to literatuer showing us that the attack rate was 6.5 per 100,000 person, and there were about 20,000 patients all over the world. Up to now, we haven't build any accurate statistics system to calculate the attack rate in China. But a retrospective study of endometrial cancer clinic cases from 1969~2003 revealed that the new attack rate of endometrial cancer was increasing at logarithmic speed every ten years. And after 21th century, average number of this cases was forty times than thirty years ago. From all these data, we could see that the attack rate of endometrial cancer was increasing at amazing speed.Clinical and pathological observations suggest that for endometrial carcinoma, transport of tumor cells via lymphatics is a common pathway of initial dissemination, with patterns of spread via afferent lymphatics following routes of natural drainage. Lymphovascular space invasion (LVSI) was an independence risk factor which could predict bad prognosis. We all know that five years survival rate of patients without lymphatic metastasis was obviously higher than the patients presenting lymphatic metastasis. In the past time, we took it for granted that the malignant cells travelled from inherent lymphatic vessel and planted in local lymph node. But nowadays, we knew the truth that the form of new lymphatic vessel around carcinoma cell was the key step of tumor metastasis. The lymphangiogensis which was derivated and regulated by many growth factors and cytokine network directly participate in cancer metastasis. The recent identification of lymphatic endothelial cell specific markers, such as vascular endothelial growth factor receptor-3 (VEGF-R3), lymphatic endothelial hyaluronan receptor-1 (LYVE-1) and podoplanin , has provided tools to investigate the discrepancies in endometrial lymphatic distribution. Now lymphatic vessel was found in normal endometrium and myometrium across the menstrual cycle with immunohistochemical stain. Lymphatic vessels of the functionalis were small and sparsely distributed whereas the basalis lymphatics were larger and sometimes closely associated with spiral arterioles and there was no difference in LVD during the whole cycle. The lymphangiogensis in tumor was necessary for malignant cell growth, invading and metastasis, and the LVD could affect the invasion of carcinoma cell and survival rate directly. So more and more doctors are studying if controlling the growth of lymphatic vessel is a kind of method to treatment cancer.The FOXC2 gene is a member of the forkhead, or'winged-helix'family of transcription factors, which now number over 30 in mammals. Three groups have previously reported mRNA expression studies of Foxc2 during mouse embryogenesis. The gene was shown to be expressed in the developing mesodermal mesenchyme of the head, kidney, and bones, and to play a role in somite formation. By 8.5 d.p.c., it was also expressed in the developing heart, blood vessels and limbs, and is essential for normal development of the aortic arch and axial skeleton. In adults, Foxc2 is expressed in adipose cells , and polymorphic variants have been associated with diabetes and insulin resistance in some studies.Mutations in the FOXC2 transcription factor cause a major form of hereditary lymphedema, the lymphedema–distichiasis syndrome. Foxc2 is expressed in developing lymphatic vessels and other tissues associated with lymphedema–distichiasis syndrome. Since the process of embryogenesis and malignant genesis had some same characters, we presumed that FOXC2 might take part in the genesis of endometrial carcinima. We would explore the expression mode of FOXC2 in normal endometrium during menstration cycle and endometrial cancer tissue, then find the relationship between FOXC2 mode and the density of lymphatic vessel. Our final purpose was to confirm if FOXC2 gene induce and regulate lymphangiogensis in endometrial cancer. Further more to provide theory for the research of control the growth of lymphatic vessel to cure cancer. PartⅠ: The pattern of lymphatic spread in endometrial carcinomaaiObjective: The purpose is to determine the rate of lymph node metastases in women with endometrial carcinoma and to compare the pattern if lymphatic spread of endometrial carcinoma and cervical carcinoma. Methods: We retrospectively analyzed the lymphatic spread in 104 patients with endometrial carcinoma and 253 patients with cervical carcinoma. All patients underwent a complete pelvic and para-aortic lymphadenectomy from caudal to the median circumflex to the level of renal vessels. Results: The incidence of lymphatic metastases in endometrial carcinoma was higher than that in cervical carcinoma (22.1% vs 16.2 %). The pathologic grade (G1:12.1%;G2:21.4%; G3:34.5%)and depth of myometrial invasion(≤1/2:11.9%>1/2:29%)but not ages and pathologic types was correlated with nodal positivity. Para-aortic and pelvic lymph node involvement alone and both were observed in 4.3%, 34.8% and 60.9% for endometrial carcinoma and 0%, 68.3% and 31.7% for cervical carcinoma. Both of hem exhibited the highest obturator nodes positive rates (73.9% and 70.7%) with suprainguinal nodes positivity being lowest (8.7% and 7.3%). When compaired with cervical carcinoma, endometrial carcinoma showed higher para-aortic and sacral nodes positive rates (65.2%vs31.7% and 21.7%vs17.1%) and lower external iliac nodes positive rates (17.4%vs41.5%). Conclusion: Endometrial carcinoma has a distinct lymphatic spread pattern compared with cervical carcinoma and can directly metastasize to both pelvic lymph nodes and para-arotic lymph nodes with pelvic lymph nodes metastases being dominant. Positive lymph nodes are common in all grades.PartⅡ: Lymphangiogensis of normal endometrium and endometrial adenocarcinomaObjective: Information about lymphatics and lymphangiogenesis in the human endometrium is limited. We investigated the distribution of endometrial lymphatic vessels during the normal menstrual cycle and in association with endometrial adenocarcinoma. Methods: Full thickness uterine samples (n=15 proliferative; n=15 secretory) and endometrial adenocarcinoma samples (n=20 stage IA; n=20 stage IIIC) were collected for the study and analysed by immunohistochemistry using D2-40 as the specific marker of LECs. Results: Lymphatic vessels of normal endometrium were almost located in functionalis across the menstrual cycle with lymphatics of the basalis sometimes intimately associated with spiral arterioles. The LVD of the endometrium was significantly reduced when compared with the myometrium across the cycle (endometrium 4.99±0.84mm–2; secretory 10.70±1.16mm–2 P<0.001). Intra-tumoural LVD was significantly decreased in both stage endometrioid adenocarcinoma when compared with normal endometrium and myometrium (stage IA : 3.49±0.94mm2, stage IIIC: 2.43±1.08mm2, normal endometrium: 4.98±0.84 mm2, normal myometrium: 10.71±1.16mm2, P<0.001). Intra-tumoural LVD had a significantly decreased tendency from stage IA to stage IIIC. Peri-tumoural LVD for stage IA and stage IIIC tumours was significantly increased compared with normal endometrial LVD but decreased compared with normal myometrial LVD (stage IA : 7.09±1.22mm2, stage IIIC: 8.63±2.88mm2, normal endometrium: 4.98±0.84mm2 normal myometrium: 10.71±1.16mm2, P<0.005). There was significant difference in peri-tumoural LVD between stage IA and stage IIIC adenocarcinomas (P=0.012) and stage IIIC had increased peri-tumoural LVD. Conclusion: Endometrial lymphatics are located in normal endometrium and myometrium across the menstrual cycle and located in intra and peri of endometrioid adenocarcinoma, and increases in endometrial adenocarcinoma peri-tumoural lymphatics are associated with increases lymphatic metastasis.PartⅢ: FOXC2 expressed in normal endometrium and endometrioid adenocarcinoma and assosiatied with lymphangiogensis in endometrial adenocarcinomaObjective: to investigated the expression of FOXC2 in normal endometrium during the normal menstrual cycle and in association with endometrial adenocarcinoma. Methods: the same samples with partⅡwere analysed by immunohistochemistry using FOXC2 mouse mAb, and FOXC2 mRNA level was detected by real time PCR. The intensity and distribution patterns of the specific immunohistochemical staining was evaluated using a semi- quantitative method (IRS score). The IRS for epithelium and stroma were recorded respectively. Results: Out of 15 proliferative endometrium, 11 showed positive immunoactivity in epithelium (73.3%), as for secretory endometrium, the positive rate was 80% (12 out of 15). When compared the IRS of epithelium, there was no significant difference between proliferative and secretory endometium (proliferative IRS=1.53±1.30, secretory IRS=2.1±1.68, p=0.284). Normal endometrium showed positive immunoactivity in stromal cells. The secretory endometrium showed significantly increased FOXC2 expression compared with proliferative endometrium (proliferative IRS=1.93±1.03, secretory IRS=3.93±1.53, p < 0.01). Endometrioid adenocarcinoma showed significantly increased FOXC2 expression compared with normal endometrial epithelium both in epithelium and stroma (in epithelium: normal endometrium IRS=1.83±1.51, endometrioid adenocarcinoma IRS=5.18±2.82, p<0.01, in stroma: normal endometrium IRS=2.93±1.64, endometrioid adenocarcinoma IRS=6.78±2.15, p<0.01). The FOXC2 expression in stroma significantly increased when pelvic and/or para-aotic lymph node was involved in (stage IA IRS=5.70±1.66, stage IIIC IRS=7.85±2.08, P=0.01). FOXC2 was immunolocalized in LECs only in endometrioid adenocarcinoma not in normal endometrium. The RT-PCR showed the same results with immunohistochemistry. Conclusion: FOXC2 might associate with the genesis of endometrial carcinoam and the lymphangiogensis in endometrial adenocarcinoma intra and peri-tumoural lymphatics.PartⅣ: Construction of eukaryotic expression plasmid pcDNA3.1(-)-FOXC2Objective: Construct the expression plasmid recombinate with FOXC2 gene for further study of gene function. Methods: FOXC2 gene fragment was obtained with PCR from human genome followed by putting FOXC2 gene fragment into eukaryotic expression vector pcDNA3.1 (-) with double restriction enzyme. We confirmed the plasmid by DNA sequencing. Result: The 1524bp FOXC2 DNA fragment from human genome DNA was obtained and then the recombined eukaryotic expression vector pcDNA3.1(-)-FOXC2 was built succsessfully. It had the same sequence with Genebank by DNA sequencing. Conclusion: we success build the pcDNA3.1 (-)-FOXC2 eukaryotic expression vector for futher study the function of FOXC2 gene.PartⅤ: Effects of FOXC2 on HEC-1B migrationObjective: In order to invest the effects of FOXC2 on human endometrial carcinoma cell line HEC-1, we constructed an FOXC2-overpressing cell modle. Methods: pcDNA3.1(-) -FOXC2 eukaryotic expression vector was transfected into HEC-1B with LipofectamineTM 2000 and dthe positive cell clone was selected by G418. FOXC2 mRNA level among untransfected cell, transfected with empty plasmid and with recombined plasmid was compared using RT-PCR. The growth curve was used to compare cell proliferation rate and transwell cabin was used to compare the invasion ability among three kinds of cells.Results: on the 6th day after transfetion, control cells were totally dead because of 500μg/ml G418, and 250μg/ml G418 was used to cultivate transfeced cells continuously. Cells became the logarithmic growth phase on 3rd days followed by the proliferation rate decreasing lightly on 5th day and the cell growth was inhibited after 6th day. FOXC2 mRNA level was higher in recombined plasmid transfected cells than in the other two kinds of cells (P < 0.01), and there was no different between the control cells and the empty plasmid transfected cells. Cells were put on the upper tier of transwell basement. After 24h, cells could penetrate matrigel. There was no defference among three kinds of cells when the penetrating cells were counted with viola crystalline (F=0.674, p=0.518). Conclusion: The pcDNA3.1-FOXC2 eukaryotic expression vector could express in HEC-1B cell line, but the invasion ability had not increased after transfeced with pcDNA3.1-FOXC2 eukaryotic expression vector. We presumed that the invasion ability of tumor cells might depend on the complicated network in vivo rather than depend on some single factor.