Study On The Molecular Mecanismh Of NID1-Promoted Metastasis Via EMT In Ovarian Cancer

Ovarian cancer is the most lethal gynecological malignancy worldwide, characterized by no effective diagnostic markers, early metastasis, and drug resistance. Elucidation of the molecular basis for ovarian cancer progression, especially its metastasis, contributes to the development of effective molecular diagnostic tools and therapeutic targets for the sake of improving its suvival. We have previously conducted a secretory/releasing proteomic study in ovarian cancer, and found that nidogen 1, NID1, is a novel candidate diagnostic biomarker of ovarian cancer. The NID1 protein level in the plasma of ovarian cancer patients was significantly higher than that in the plasma of healthy individual, and a NID1-based panel for the ovarian cancer diagnosis showed a better diagnostic performance. Likewise, its protein level was higher in the plasma and the tumor tissues of ovarian cancer patients with lymph node metastasis than that of patients without lymph node metastasis. As a critical component in basement membrane, however, its detailed function in ovarian cancer remains unknown. Given that there is a close link among tumor invasion, EMT, and the basement membrane and the NID1 protein level was positively correlated with lymph node metastasis, we speculate that NIDI plays an important role in ovarian cancer invasion and metastasis. Our study focus on the migration-promoted effect of NID1 on ovarian cancer cells and its molecular mechanism described below, so as to accelerate the clinical evaluation of NID1 as a candidate prognostic marker and therapeutic target in ovarian cancer.Part Ⅰ:The establishment of cell models with stable NID1 overexpression and transient NID1 reduction. OVCAR-3 cells were transfected with NID1 expression vector from which the stable monoclone (denoted as OVCAR-3-NID1-MC cell) was selected, and then determined by quantitative RT-PCR and Western blot. Meanwhile, HEY cells were separately transfected with two NID1 siRNA (i.e. NID1-si798 and NID1-si2983), and also determined 72 hr post-transfection.Part Ⅱ:Functional role of NID1 in invasion and migration of ovarian cancer cells. OVCAR-3-NED1-MC cells exhibited significantly greater motility and invasiveness comparing with these of the control group, via wound healing assay, Transwell migration and invasion assays. It suggested NID1 overexpression promoted the invasion and migration of OVCAR-3 cells. Concordantly, both of HEY-NID1-si798 and HEY-NID1-si2983 cells showed markedly reduced cell migration through transwell compared to the control group, suggesting depletion of NID1 inhibited the invasion and migration of HEY cells.Part Ⅲ:Functional role of NID1 in EMT of ovarian cancer cells. Different from cube-like epithelial morphology of the control cells, OVCAR-3-NID1-MC cells presented spindle-like mesenchymal morphology. Consistent with the phenotypic change associated with NID1 overexpression was an reduced expression of epithelial marker E-cadherin concomitant with an increased expression of mesenchymal markers (Vimentin and N-cadherin) and transcription factor Twist-2, as detected by both quantitative RT-PCR and Western blot. The change of Vimentin was also verified by immunofluorescent (IF) analysis. Correspondingly, HEY-NID1-si798 cells and HEY-NID1-si2983 cells tended to present cube-like epithelial morphology, not like spindle-like mesenchymal morphology of the control cells. Despite the unchange of Vimentin, N-cadherin and Twist-2, an increased expression of E-cadherin was consistent with the phenotypic change associated with NID1-depletion, as determined by Western blot and IF analyses. The above results indicated that NID1 promote EMT of ovarian cancer cells.Part Ⅳ:ERK/MAPK pathway involved in EMT of ovarian cancer cells induced by NID1. The protein expression of phosphorylated ERK1/2 was increased in OVCAR-3-NID1-MC cells compared with the control cells, as detected by Western blot. Subsequently, we treated OVCAR-3-NID1-MC cells with an ERK/MAPK inhibitor (U0126). Inhibition of ERK1/2 phosphorylation caused the reversal of the expression of these EMT relevant markers. It implied that NID1 probably activated ERK/MAPK signaling pathway to regulate the expression of EMT relevant markers and then propel EMT in ovarian cancer cell. Moreover, FAK was activated in OVCAR-3-NID1-MC cells as well, for which the protein expression of phosphorylated FAK (i.e. FAK pY397) was significantly increased. It implicated FAK may be an upstream regulator of ERK/MAPK pathway.In summary, on the basis of stable NID1-overexpressed and transient NID1-depleted ovarian cancer cells, we first found that NID1 promoted the invasion and migration of ovarian cancer cells with the occurrence of EMT, demonstrating NID1 promoted ovarian cancer metastasis via EMT. Second, ERK/MAPK pathway was activated in NID1-overexpressed ovarian cancer cells and its ablation reversed the expression of most EMT relevant markers, demonstrating NIDI mediated ERK/MAPK pathway to promote the EMT of ovarian cancer cells. Likewise, the activation of FAK in NID1-overexpressed ovarian cancer cells indicated NIDI may activate Integrin-FAK this upstream regulatory signaling to mediate ERK/MAPK pathway, which need to be further verified.