Expression And Effect Of FOXC2 In Human Ovarian Tumors

Ovarian tumors are common gynecological diseases. Among these tumors, the ovarian cancer occupies the highest mortality rate of all gynecological malignancies.The ovary consists of multiple histological constituents, and malignancies could derive from these different kinds of histology, usually lacking early symptoms and challenges early diagnosis. Recent studies support the existence of GSCs in mammalian ovaries with which ovarian malignancies exhibit their resemblance,indicating an “atavism” of the nature of ovarian malignancies, leading to the corollary of shared regulation mechanism between the development of malignancies and embryos, where regulating factors during embryo development might act as crucial factors in cancer development.FOXC2 is a transcription factor that participates in multiple processes during early embryo development such as tissue differentiation, composition, and the development of blood and lymphatic vessels, whose expression generally cease after birth. In recent years, FOXC2 has been found to be associated with the development,especially the development of blood and lymphatic vessels of cervical cancer,endometrial cancer, breast cancer, colon cancer and melanoma. Other reports have described its critical functions in the development of cancer stem cells.VEGF is a critical factor in the process of neovascularization, cancer growth,invasion and metastasis. VEGF promotes the endothelial cell migration,differentiation and infiltration by inducing BCL-2 expression in the endothelial cells of microvessles, thus inducing cancer development and neovascularization. The mechanism involves the ligation of VEGF with VEGFR2 on the endothelial cell membrane, via P13K-Akt pathway, stimulates BCL-2, enhancing the survival of cancer cells while promoting the expression of CXCL8 and IL-8, which are important downstream targets during intra-lesional blood vessel formation.Up to now, no studies have focuses on the expression and function of FOXC2 in ovarian tumors of different histology. If FOXC2 is involved in the development of ovarian tumors, its detailed function and its association with VEGF-IL-8 signalingduring tumor development and neovascularization is to be clarified. In this studied,we determined the expression, distribution and function of FOXC2 in different types of ovarian tumors, and analyzed the function and mechanism of FOXC2 in tumor growth, invasion and vascularization, and deepened the understanding of ovarian tumorgenisis and metastasis.Purpose:To investigate the expression profile of FOXC2 in different types of ovarian tumors, and its effect and mechanisms on cancer biology and its association with cancer diagnosis and prognosis.Methods:The level and site of expression FOXC2 was determined by immunohistochemistry in collected pathological tissues of ovarian cancers of epithelial, germ cell and sexcord-stromal cell origin. FOXC2 expression was silenced with sh RNA in Human epithelial ovarian cystadenocarcinoma cell line SKOV-3, and its growth was determined by CCK-8 assay, migration by scratch assay, tumorgenisis by cell cloning experiment and invasion by invasion assay. Real-time PCR and indirect immunofluorescence was performed to analyze the interaction between FOXC2 and VEGF and IL-8 signaling.Results:1. The expression of FOXC2 in human ovarian cancers(1)FOXC2 expression in ovarian epithelial cancers The positive rate of FOXC2 s expression is 88.13%(52/59) in epithelial cancers.The expression in serous cancers is lower than that of mucous cancers.In epithelial ovarian cystadenocarcinomas, low levels of FOXC2 expression could be found in the cytoplasm of a portion of germinal epithelial cells. Low to intermediate expression could be detected in borderline serous cystadenoma, and strong expression of FOXC2 and VEGF detected in CD31 positive microvascular endothelial cells. An increasing expression of FOXC2 was detected from stage I C to III C serous papillary cystadenocarcinomas, with strong expression of FOXC2 in microvascular endothelial cells.The expression of FOXC2 in the ovarian mucous cystadenocarcinoma is located in the cytoplasm of mucous epithelial cells and stromal cells. In the borderline mucous cystadenomas, FOXC2 is expressed in both the cytoplasm and the nuclei of the stromal vascular epithelial cells. In mucous cystadenocarcinomas, FOXC2 is mainly expressed in the cytoplasm and the nuclei of the stromal cells and the cytoplasm of microvascular wall.(2)FOXC2 expression in ovarian germ cell cancers The positive rate of FOXC2 expression in ovarian germ cell cancers is 40%(2/5).No expression was observed in the mature cystic teratomas. In the squamous cell carcinoma lesions of mature cystic teratomas with somatic cell malignant transformation, scattered strong nuclear expression was observed in a portion of stromal cells. Low level of expression was observed in the germinal epithelial cells of Grade III ovarian immature teratoma. In ovarian thyroid cyst and ovarian mixed germ cell cancer, no expression was observed.(3)FOXC2 expression in sexcord-stromal cell cancers The expression rate is 60%(3/5). No FOXC2 expression was found in SERTOLI cells and strong nuclear expression was determined in LEYDIG cells in highly differentiated ovarian stromal cell tumors. In SCTAT, strong expression was observed in the cytoplasm. In ovarian adult granulosa cell tumors, scattered cell with karyomitosis showed stong nuclear FOXC2 expression. In fibrothecomas, no expression was found.2. The effect of FOXC2 sh RNA silencing on the biology of SKOV-3.(1)Transient transfection of FOXC2 sh RNA resulted in significantly reduced cell numbers in CCK8 assay.(2)FOXC2 silencing induced wider scratch compared with the control group and sh NC group after 72 h cell culture in the scratch assay.(3)When seeded with 300 or 600 cancer cells, the control group showed a significantly higher tumorogenisis with 87.6% in the 300 group and 90.5% in the 600 group compared with 25.3% and 24.7% in the FOXC2 silenced group.(4)Transwell assay showe significantly higher number of infiltrating cells in the sh NC group comprared with FOXC2 silencing group after 24 h cell culture.Quantification showed 96.8±16.84 in the control group vs 27.4±9.34 in the FOXC2 silencing group.3. The effect of FOXC2 silencing on human ovarian cancer VEGF and IL-8 signaling(1) silencing of FOXC2 m RNA in SKOV-3 cells resulted in 77.8% reduction of VEGF m RNA expression, while no IL-8 m RNA changes was observed.( 2) Indirect immunofluorescence determined significantly lowered VEGF signal in FOXC2 silencing group compared with the control, while no change in the signal of IL-8 was observed.Conclusion1. FOXC2 is highly expressed in the ovarian epithelial cancers, of which the mucous cancers have higher expression compared with the serous cancers. There is positive correlation between FOXC2 expression and the staging of Ovarian serous papillary cystadenocarcinoma, in which the mucous cancers have high expression than the serous cancers.2. FOXC2 is co-expressed with VEGF in the cancer cells and neovasculature of ovarian mucous cystoadenocarcinomas, suggesting an inter-connection between the two factors. FOXC2 might participate in the angiogenisis of ovarian cancers.3. FOXC2 is expressed in the SKOV-3 cells, silencing its expression has a negative effect on cancer growth, migration, invasion and tumorgenesis.4. FOXC2 silencing down regulates VEGF but not IL-8, suggesting its role as an upstream regulator.