The Role Of Hypoxia In The Formation Of Vasculogenic Mimicry In Ovarian Cancer

Epithelia ovarian cancer has the highest mortality rate of all gynecological malignancies, which reflects the fact that most patients are diagnosed with advanced cancer. Vasculogenic mimicry describes the ability of aggressive cancer cells to form vasculogenic-like networks in vitro in the absence of endothelial cells or fibroblasts, concomitant with their expressions of vascular cell-associated molecules. Vasculogenic mimicry has now been shown to occur in many different aggressive cancers including ovarian cancers.The hypoxic microenvironment of tumors results in changes in metabolism, angiogenesis and survival of the cells that are orchestrated by HIF-1α, and depending on tissue specificity. Hypoxia contributes to the progression of a variety of cancers by activating adaptive transcriptional programs that promote cell survival, motility and tumor angiogenesis. Given that HIF-1α has been found to be a crucial inducer contributing to tumor EMT, it is of great interest to examine the role of HIF-1α in VM formation in ovarian cancers. Although the importance of hypoxia and subsequent hypoxia-inducible factor-1α (HIF-1α) activation in tumor angiogenesis is well known, their role in the regulation of vasculogenic mimicry is unclear.Purpose:The study aims to investigate the effect of hypoxia on VM formation in ovarian cancer and to explore possible mechanism involved. Exploring the growth-promoting mechanisms of the tumor microenvironment will enhance our understanding of cancer biology and may identify new therapeutic approaches.Methods:1) Through the Tumor Tissue Bank of Tianjin Cancer Hospital,71ovarian cancer patients with detailed pathologic and clinical information were selected. Expression of HIF-1α protein was studied by immunohistochemistry in71specimens of epithelial ovarian cancers. To explore the potential mechanism, we examined the expressions of E-cadherin, vimentin, Twistl, Slug, Snail and VE-cadherin, FLT-1, FLK-1, and studied E-cadherin, vimentin topological correlation with HIF-la in VM-positive human ovarian cancers.2) A well-established model of Three-dimensional Matrigel culture in vitro was used to compare vasculogenic mimicry formation under hypoxia and normoxia using two ovarian cancer cell lines, SKOV3and OVCAR3. Transwell invasion assay and migration assay were performed to evaluate the effect of hypoxia on cell motility and invasion.3) The expression levels of HIF-la and E-cadherin, vimentin following hypoxia and normoxia were assessed using quantitative PCR and western blot. Immunofluorescence microscopy staining was performed to confirm the effect of hypoxia on the protein expressions of E-cadherin and vimentin.4) The expression levels of VE-cadherin following hypoxia and normoxia were assessed using western blot.5) Zymography assays were performed to detect the effect of hypoxia on the activity of MMP-2and MMP-9.Results:1) VM present in Ovarian Cancer associated with HIF-la expression. VM was detected in18out of71Ovarian Cancer samples (25%). HIF-1α nuclear expression could be detected in11of the18(61%) samples in the VM-positive group and in17of the53(32%) samples in the VM-negative group. HIF-1α protein expression was significant between the VM-positive group and the VM-negative group. A Kaplan-Meier survival analysis revealed that the patients with expression of HIF-la had a shorter survival period than those without HIF-la expression.2) Hypoxia facilitates invasiveness, migration, and VM formation of Ovarian Cancer cellWe observed the emergence of more pipe-like structures and cellular plasticity in hypoxic group than normoxic group. We only observed simpler and less pipe-like structures in normoxic group than hypoxic group. Two different types (the patterned matrix type and the tubular type) of VM were observed in vitro under hypoxia. SKOV3cell lines formed looping patterns rich in extracellular matrix in3D cultures containing Matrigel,OVCAR3cell lines formed networks of tubular or sinusoidal channels.The invasive capacity of the ovarian cancer cells was significantly facilitated under hypoxia compared with that under normoxia in both SKOV3(P=0.000) and OVCAR3cells (P=0.000); an increase in cell migration was observed under hypoxic conditions SKOV3and OVCAR3cell lines compared with the control (P<0.05).Zymography assays were performed to detect the effect of hypoxia on the activity of MMP-2and MMP-9.3) Hypoxia leading to VM formation through inducing EMTThe HIF-la expression was topologically correlated with loss of E-cadherin expression and up-regulated the expression of vimentin in11of the18VM-positive patients (61%). EMT-associated molecular Twist and Slug had higher expressions in VM-positive ovarian cancer samples than those in VM-negative ovarian cancer samples.In vitro experiments, we observed that ovarian cancer cell lines had morphological EMT-like changes (a more fibroblastoid morphology, a loss of cellular cohesiveness) after150μM CoCl2hypoxia for48hours. Messenger RNA (mRNA) and protein levels demonstrated induction of EMT after hypoxia, as shown by a shift in expression of epithelial markers (E-cadherin) to mesenchymal markers (vimentin).4) Hypoxic could induce tumor cells highexpression of VE-cadherin, promote the formation of VM. VE-cadherin is exclusively expressed by highly aggressive tumor cells and is critical in tumor VM.VM-associated molecular VE-cadherin had higher expressions in VM-positive ovarian cancer samples than those in VM-negative ovarian cancer samples. In vitro experiments,VM-related characterizing protein VE-cadherin was detected to be highly expressed under hypoxia.Conclusions:1) VM was associated with HIF-la expression in ovarian cancer, hypoxia promoted VM formation.2) Hypoxia inducing EMT was one of the mechanisms leading to VM formation.3) Hypoxia facilitated invasiveness, migration of ovarian cancer cell.4) Hypoxic could induce tumor cells highexpression of VE-cadherin, promote the formation of VM...