The Study On The Molecular Mechanisms Involved In Follicle Stimulating Hormone-mediated The Upregulation Of Angiogenesis In Breast Cancer By VEGF

During perimenopausal and menopausal period, women have low serum estrogen and high follicle stimulating hormone (FSH) levels. Although estrogen could promote development of estrogen-dependent breast cancers, low estrogen level in the perimenopausal and menopausal women, who have higher incidence and severity of breast cancer than young women, suggests that another endocrinogical factor, apart from estrogen, could promote breast cancer development.A recent study published on the New England Journal of Medicine reported that FSH receptor was highly expressed in vascular endothelial cells of a wide range of tumors of all grades. The expression of FSH receptor decreased dramatically with the blood vessel getting far away the tumor. The tumor vessels provide adequate nutrition and oxygen for tumor and an easy path for tumor cells to metastasis. In recent years, the role of tumor angiogenesis in the development, metastasis and prognosis of cancers has become a hot area of cancer research. With high level of FSH in menopausal women and high expression of FSH receptor in tumor vessel endothelium, we suppose that FSH enhances the development of breast cancer by promoting the blood vessel growth through the highly expressed FSH receptor of vessel endothelium.Our study applied three methods:clinical research, cell experiment and animal model. We explored the difference and the mechanism underlying of the expression and distribution of the FSH receptor on endothelium from normal breast and breast cancer vessel. By immunohistochemical staining, we found that FSH receptor expressed higher on endothelium of breast cancer vessel than normal breast vessel. We detected a low expression of FSH receptor in HUVEC (human umbilical vein cell line, a widely used vascular endothelial cell line). We also found that the cell-culture medium of breast cancer and VEGF (vascular endothelial growth factor) increased the expression of FSH receptor in HUVEC. VEGF promoted the translocation of FSH receptor from cytoplasm to cell membrane. FSH had no effect on the proliferation of HUVEC. After24-hour pretreatment of VEGF on HUVEC or over-expression of FSH receptor in HUVEC via lentiviruses, FSH affected on HUVEC in the following aspects:1) promoted the proliferation, migration, invasion and angiogenesis of HUVEC;2) decreased the cell junctions of HUVEC and increased expression of matrix metalloproteinase;3) activated calcium influx and the phosphorylation of a wide range of kinases and transcription factors. In animal model, we divided the nude mice into three groups, sham (sham operation), OVX (ovariectomized) and OVX+FSH (ovariectomized mice treated with FSH). We injected all the animals with Bcap37(a estrogen receptor negative breast cancer cell line) and found that the tumor size of the OVX group were larger than sham group, and OVX+FSH showed the significant larger tumor size than other two groups. FSH had no effects on the proliferation of Bcap37. However, the immunohistochemical staining analysis of the MVD (microvessel density) showed that the MVD of OVX group was higher than sham group, and the highest level of MVD was detected in OVX+FSH group. In the conclusion, FSH enhances angiogenesis induced by VEGF, via upregulating activity of VEGF signal pathway. These results provide new insights into the molecular mechanisms underpinning the angiogenic effects of VEGF, and inhibition of FSH-FSHR may reduce tumor growth by reducing angiogenesis in breast cancers.Part one:The expression of FSH receptor on vascular endothelial cell from human breast cancer and normal breastObject:To study the difference and the mechanism underlying of the expression of FSHR on vascular endothelial cells in human breast cancer and normal breast tissue.Method.1. Immunohistochemical staining was used to detect the expression of FSHR on vascular endothelial cells of breast cancer and adjacent normal tissue from biopsies of ten breast cancer patients.2. Real time-PCR, Western-blot, immunofluorescence stain and DNA sequencing were used to detect the expression and distribution of FSH receptor on HUVEC (umbilical vein cell line, a widely used vascular endothelial cell line).3. Explore the expression differences of FSH receptor after treating HUVEC with cell-culture medium of Bcap37and gradient concentrations of VEGF for various periods.4. Rotary confocal microscopy was used to real-time quantitatively detect the distribution changes of FSHR on HUVEC with VEGF treatment within1hour, and HUVEC was transfected with FSHR-GFP plasmids.Results:1. Immunohistochemical staining of the biopsies of ten breast cancer patients showed an obvious higher expression of FSHR on vascular endothelial cells of breast cancer than adjacent normal tissue.2. FSH receptor was expressed in HUVEC, but not very much.3. Cell-culture medium of Bcap37and gradient concentrations of VEGF increased the expression of FSH receptor in HUVEC. VEGF mediated the expression of FSH receptor via ERK pathway.4. VEGF promoted the translocation of FSH receptor from cytoplasm to cell membrane of HUVEC.Conclusions:FSHR expression level in vascular endothelial cells of breast cancer was higher than adjacent normal tissue, which was induced by VEGF produced in cancer cells. Meanwhile, VEGF promoted the translocation of FSH receptor from cytoplasm to cell membrane of HUVEC, which might result in the activation of some pathway through which FSH affected HUVEC. Part Two:The effect of FSH on the angiogenesis of breast cancerObject:To explore the role of FSH in angiogenesis of breast cancer with cell experiment and nude mouse model.Method:Cell experiments:This experiment includes three groups:1) control group,2) HUVEC pretreated with VEGF for24hours followed by FSH treatment,3) FSHR overexpression in HUVEC. The following analyses were performed:1) MTT assay was performed to analyze the effect of FSH on the proliferation of HUVEC.2) Transwell assay was used to examine the role of FSH in the invasion of HUVEC.3) Wound-healing assay was used to study the effect of FSH on the migration of HUVEC.4) Western-blot was used to explore the effect of FSH on the expression of matrix metalloproteinase in HUVEC.5) Immunofluorescence was used to study the effect of FSH on the reconstruction of cytoskeleton protein F-actin.6) Tube formation assay was used to study the effect of FSH on the vessel lumen formation of HUVEC.7) Electron microscopy and immunofluorescence was used to study the effect of FSH on the cell junction of HUVEC.Animal model experiments:1) Divide the animals into three groups:Sham (sham operation);OVX (ovariectomized group); OVX+FSH (ovariectomized with FSH supplementary group). We injected all the animals with Bcap37and examined the size of the tumors once every3days for60days.2) MTT assay was used to study the effect of FSH on the proliferation of Bcap37.3) Immunohistochemical staining with CD31(a biomark of vascular endothelium) was used to explore the difference of the MVD(microvessel density) among three groups.Clinical data analysis:The clinical data of FSH and estrogen levels from17patients with breast cancer were collected. Their breast cancer tissue paraffin section was used for immunohistochemical staining with CD31. Image pluo plus was used to calculate the microvessel density. Spss16.0software was used to explore the relationship between FSH level and MVD.Result:1. Cell experiment:FSH alone had no effect on the proliferation, migration, invasion, matrix metalloproteinase secretion, cell junction or vascular lumen formation of HUVEC.After24-hour pretreatment of VEGF on HUVEC or over-expression of FSH receptor in HUVEC, FSH obviously promoted the proliferation,migration, angiopoiesis and reconstruction of cytoskeleton protein F-actin of HUVEC. After lentivirus interfering the expression of FSH receptor of HUVEC,FSH lost its effects on the proliferation of HUVEC with24-hour treatment of VEGF.2. Animal modelAfter the animals grew their tumors, we kept them for60days and found the tumor size of the OVX group to be larger than Sham group and even larger when supplemented with FSH.FSH had no effects on the proliferation of Bcap37.The immunohistochemical staining of the MVD (microvessel density)of all the tumors showed a higher MVD of OVX group than Sham group,and the highest of all was the OVX group with FSH supplement.FSH has no effect on the synthase of VEGF by Bcap37.2. clinical investigationOur preliminary result showed a linear positive correlation between FSH and MVDConclusion:VEGF promoted the expression and membrane translocation of FSH receptor in vascular endothelium, through which FSH promoting the blood vessel growth and leading to tumor growth promotion. Part three:Study on the mechanism underlying the effect of FSH on angiopoiesisObject:Study the pathway through which FSH promotes the angiopoiesis.Method:Real-time fluorescent quantitative calcium measurement system was used to detect the calcium influx of HUVEC treated with FSH after VEGF pretreatment or FSHR over-expression.Western-blot was used to test the effect of FSH on the phosphorylation of several important signaling pathway proteins, like AKT, Erk and so on.Results:1.FSH promoted the calcium influx of HUVEC after VEGF pretreatment or FSHR over-expression.FSH had no effect on HUVEC transfected with mock-vehicle or without VEGF treatment.2. FSH strongly activated some important kinases and transcription factors in HUVEC with24-hour VEGF pretreatment.Without VEGF, FSH had no or little effect on HUVEC.Conclusion:FSH promoted angiogenesis through multiple signaling pathway mediated by With VEGF...