Fibroblasts in omentum activated by tumor cells promote ovarian cancer growth, adhesion and invasiveness

Omentum metastasis is a common occurrence in epithelial ovarian cancer (EOC), which is often accompanied by ascites that facilitates the spread of EOC cells. A subpopulation of fibroblasts-the cancer-associated fibroblasts (CAFs) are important promoters of tumor progression. We have shown previously that CAFs exist not only in omentum with EOC metastasis but also in omentum without metastasis. In the present study, using primary human fibroblasts isolated from normal omentum (NFs) and omentum with ovarian cancer metastasis (CAFs), we established in vitro coculture models and a 3D culture model mimicking human omentum structure for investigation of interactions between fibroblasts and cancer cells. We demonstrate that EOC cells activate NFs and promote their proliferation via transforming growth factor-β1 (TGF-β1) signaling, and the activated fibroblasts contribute to the invasion and adhesion of EOC cells. Moreover, EOC cells and NFs coculture led to overexpression of hepatocyte growth factor (HGF) and matrix metalloproteinase-2 (MMP-2) and adhesion and invasion of EOC cells could be partially suppressed by blocking the function of HGF or MMP-2. Additionally, mouse peritoneal dissemination models of EOC confirmed the activation of fibroblasts by cancer cells and the tumor growth- and metastasis-promoting effects of activated fibroblasts in vivo. Our findings indicate that activated fibroblasts in omentum form a congenial environment to promote EOC cells implantation. It is an intriguing concept that targeting the activation of omentum fibroblast through the inhibition of TGF-β1 signaling can be used as a new therapeutic strategy against ovarian cancer omentum metastases, which needs further study.     

Metastatic dissemination of human ovarian epithelial carcinoma is promoted by alpha2beta1-integrin-mediated interaction with type I collagen

Metastatic dissemination of epithelial ovarian carcinoma is thought to be mediated via tumor cell exfoliation into the peritoneal cavity, followed by adhesion to and invasion through the mesothelium which overlies the contents of the peritoneal cavity. In this study, we have utilized short-term primary cultures to analyze the effect of specific extracellular matrix proteins on properties of human ovarian epithelial carcinoma cells which contribute to the invasive phenotype. Analysis of cell:matrix adhesive profiles indicated that ovarian carcinoma cells adhere preferentially to type I collagen. Immunoprecipitation analyses demonstrated the presence of the collagen-binding alpha2beta1 integrin in biotin-labeled ovarian carcinoma cell membranes, and cellular adhesion was inhibited by blocking antibodies directed against the alpha2 and beta1 integrin subunits. The alpha2beta1-binding peptide Asp-Gly-Glu-Ala (DGEA) was also moderately effective at blocking adhesion to collagen relative to the control peptide Ala-Gly-Glu-Ala (AGEA). Analysis of cell motility on protein-coated colloidal gold coverslips demonstrated that ovarian carcinoma cells migrate preferentially on type I collagen coated surfaces. Type I collagen promoted migration in a concentration-dependent, saturable manner, with maximal migration observed at a collagen-coating concentration of 50 microg/ml. Migration on collagen was inhibited by antibodies directed against the alpha2 and beta1 integrin subunits and by DGEA peptide, providing evidence for the role of the alpha2beta1 integrin in ovarian carcinoma cell motility. Culturing ovarian carcinoma cells on type I collagen gels led to a significant increase in conversion of the matrix metalloproteinase 2 zymogen to the 66-kD form, suggesting that adhesion to collagen also influences matrix-degrading proteinases. These data suggest that alpha2beta1-integrin-mediated interaction of ovarian carcinoma cells with type I collagen, a protein prevalent both in the mesothelial extracellular matrix and in the peritoneal cavity of ovarian carcinoma patients, may function on multiple levels to promote metastatic dissemination of ovarian carcinoma cells.     

钙黏素和整合素在卵巢上皮性癌中的研究进展

卵巢上皮性癌(EOC)是妇科癌症中最致命的恶性肿瘤。在过去的30年间,虽然诊断和治疗水平取得了进展,但是卵巢上皮性癌的发病率和死亡率却保持不变。卵巢上皮性癌腹膜转移癌晚期发病于附着的小簇癌细胞,这些癌细胞从初始部位脱落并经由腹水携带附着于腹部腹膜或网膜。这种行为表明细胞-细胞或细胞-基质黏附机制调节卵巢上皮性癌的生长和增殖。复杂的下游信号可能受黏附分子和共表达激活的信号蛋白之间的功能性串扰所影响,导致卵巢上皮性癌细胞的增殖/存活和迁移/侵袭。本文旨在阐述细胞与细胞机制的影响,该过程由钙黏素和细胞-细胞外基质黏附,由整合素对膜受体和细胞质蛋白质介导的信号级联放大,这些在卵巢上皮性癌细胞的增殖、迁移和侵袭中起着一定的作用。     

Role of the polypeptide N-acetylgalactosaminyltransferase 3 in ovarian cancer progression: possible implications in abnormal mucin O-glycosylation

Previously, we have identified the polypeptide N-acetylgalactosaminyltransferase 3 (GALNT3) gene as notably hypomethylated in low-malignant potential (LMP) and high-grade (HG) serous epithelial ovarian tumors, compared to normal ovarian tissues. Here we show that GALNT3 is strongly overexpressed in HG serous EOC tumors as compared to normal ovarian tissue. Moreover, the GALNT3 expression significantly correlated with shorter progression-free survival (PFS) intervals in epithelial ovarian cancer (EOC) patients with advanced disease.Knockdown of the GALNT3 expression in EOC cells led to sharp decrease of cell proliferation and induced S-phase cell cycle arrest. Additionally, GALNT3 suppression significantly inhibited EOC cell migration and invasion. Gene expression profiling and consecutive network and pathway analyses confirmed these findings, as numerous genes and pathways known previously to be implicated in ovarian tumorigenesis, including EOC tumor invasion and metastasis, were found to be downregulated upon GALNT3 suppression, while some tumor suppressor genes were induced. Moreover, GALNT3 downregulation was associated with reduced MUC1 protein expression in EOC cells, probably related to destabilization of the MUC1 protein due to lack of GALNT3 glycosylation activity. GALNT3 knockdown was also accompanied with increase of the cell adhesion molecules β-catenin and E-cadherin, which are normally suppressed by MUC1 in cancer, thus supporting the role of the GALNT3-MUC1 axis in EOC invasion.Taken together, our data are indicative for a strong oncogenic potential of the GALNT3 gene in advanced EOC and identify this transferase as a novel EOC biomarker and putative EOC therapeutic target. Our findings also suggest that GALNT3 overexpression might contribute to EOC progression through aberrant mucin O-glycosylation     

SMYD3 promotes implant metastasis of ovarian cancer via H3K4 trimethylation of integrin promoters

Detachment of cancer cells from the primary tumor and formation of spheroids in ascites is required for implantation metastasis in epithelial ovarian cancer (EOC), but the underlying mechanism of this process has not been thoroughly elucidated. To mimic this process, ovarian cancer cells were grown in 3D and 2D culture. Hey and OVCA433 spheroids exhibited decreased cell proliferation and enhanced adhesion and invasion. SMYD3 expression was elevated in ovarian carcinoma spheroids in association with increased H3K4 methylation. Depletion of SMYD3 by transient siRNA, stable shRNA knockdown and the SMYD3 inhibitor BCI-121 all decreased spheroid invasion and adhesion. Gene expression arrays revealed downregulation of integrin family members. Inhibition assays confirmed that invasion and adhesion of spheroids are mediated by ITGB6 and ITGAM. SMYD3-deficient cells regained the ability to invade and adhere after forced overexpression of SMYD3, ITGB6 and ITGAM. However, this biological ability was not restored by forced overexpression of SMYD3 in ITGB6- and/or ITGAM-deficient cancer cells. SMYD3 and H3K4me3 binding at the ITGB6 and ITGAM promoters was increased in spheroids compared to that in monolayer cells, and the binding was decreased when SMYD3 expression was inhibited, consistent with the expression changes in integrins. SMYD3 expression and integrin-mediated adhesion were also activated in an intraperitoneal xenograft model and in EOC patient spheroids. In vivo, SMYD3 knockdown inhibited tumor metastasis and reduced ascites volume in both the intraperitoneal xenograft model and a PDX model. Overall, our results suggest that the SMYD3-H3K4me3-integrin pathway plays a crucial role in ovarian cancer metastasis to the peritoneal surface.     

Novel Axl-driven signaling pathway and molecular signature characterize high-grade ovarian cancer patients with poor clinical outcome

High-grade epithelial ovarian cancer (HGEOC) is a clinically diverse and molecularly heterogeneous disease comprising subtypes with distinct biological features and outcomes. The receptor tyrosine kinases, expressed by EOC cells, and their ligands, present in the microenvironment, activate signaling pathways, which promote EOC cells dissemination. Herein, we established a molecular link between the presence of Gas6 ligand in the ascites of HGEOCs, the expression and activation of its receptor Axl in ovarian cancer cell lines and biopsies, and the progression of these tumors. We demonstrated that Gas6/Axl signalling converges on the integrin β3 pathway in the presence of the adaptor protein p130Cas, thus inducing tumor cell adhesion to the extracellular matrix and invasion. Accordingly, Axl and p130Cas were significantly co-expressed in HGEOC samples. Clinically, we identified an Axl-associated signature of 62 genes able to portray the HGEOCs with the shortest overall survival. These data biologically characterize a group of HGEOCs and could help guide a more effective therapeutic approach to be taken for these patients.     

Involvement of SDF-1alpha/CXCR4 axis in the enhanced peritoneal metastasis of epithelial ovarian carcinoma

Epithelial ovarian carcinoma (EOC) spreads by implantation of tumor cells onto the human peritoneal mesothelial cells (HPMCs) lining the peritoneal cavity. The aim of this study was to determine whether the stromal cell-derived factor-1alpha (SDF-1alpha)/CXCR4 axis is involved in the interaction of EOC cells with HPMCs in peritoneal metastasis. Clinically, we first evaluated CXCR4 expression in sections from 36 primary EOCs using immunohistochemistry. We next examined whether SDF-1alpha played roles in EOC progression, including in proliferation, cell motility, attachment to HPMCs, and the in vivo development of peritoneal metastasis through CXCR4. Of the 36 carcinomas, 16 cases (44.4%) were positive for CXCR4 immunoexpression. Positive CXCR4 expression significantly predicted poorer overall survival compared with negative expression (p = 0.0069). We found CXCR4 expression in both EOC cells and HPMCs. In contrast, the level of production of SDF-1alpha by HPMCs was higher than that by various EOC cells. Functionally, SDF-1alpha induced enhanced attachment between ES-2 cells and HPMCs or extracellular matrix components. The enhancement of adhesion potential by SDF-1alpha was inhibited by AMD3100, a CXCR4 antagonist, and by phosphatidylinositol 3 kinase and p44/42 inhibitors. Furthermore, intraperitoneal treatment with AMD3100 resulted in reduced dissemination in nude mice inoculated with ES-2 cells. The present results suggest that there may be a link between the SDF-1alpha/CXCR4 axis and enhanced intraperitoneal dissemination of EOC and that CXCR4 may be a novel target for the treatment of EOC.     

The hypoxia-related microRNA miR-199a-3p displays tumor suppressor functions in ovarian carcinoma

During the dissemination of ovarian cancer cells, the cells float in the peritoneal cavity without access to a vascular supply and so are exposed to hypoxic conditions, which may cause the ovarian cancer cells to acquire a more aggressive and malignant phenotype. In this study, we screened microRNAs (miRNAs) to identify those that displayed altered expression patterns under hypoxic conditions and then analyzed their functional roles in ovarian cancer progression. miRNA PCR arrays performed on cells from 2 ovarian cancer cell lines (CaOV3 and RMUG-S) revealed miR-199a-3p as one of the miRNAs that are downregulated under hypoxia. In silico analyses indicated that MET is one of the target genes for miR-199a-3p; subsequently, miR-199a-3p expression was found to be inversely correlated with c-Met expression in ovarian cancer. Transfection of precursor miR-199a-3p into ovarian cancer cells reduced c-Met expression and inhibited the phosphorylation of c-Met, extracellular signal-regulated kinase, and AKT; in addition, proliferation, adhesion, and invasiveness were inhibited. Moreover, overexpression of miR-199a-3p in cancer cells significantly suppressed peritoneal dissemination in a xenograft model. In summary, the hypoxia-related microRNA miR-199a-3p drastically inhibits ovarian cancer progression through the downregulation of c-Met expression. Therefore, miR-199a-3p is a potential target for treating ovarian cancer dissemination.     

c-Met overexpression is a prognostic factor in ovarian cancer and an effective target for inhibition of peritoneal dissemination and invasion

The hepatocyte growth factor receptor c-Met is a receptor tyrosine kinase that plays an important role in tumor growth by activating mitogenic signaling pathways. The goal of this study was to evaluate the role of c-Met in the biology of ovarian cancer and to determine its potential as a therapeutic target. c-Met protein expression was detected by immunohistochemistry in 138 advanced-stage ovarian cancers using a tissue microarray annotated with disease-specific patient follow-up. Fifteen of 138 (11%) tissues had c-Met overexpression. Median survival for patients with high c-Met levels was 17 months versus 32 months (P = 0.001) for patients with low c-Met expression. Infection of SKOV-3ip1 cells with an adenovirus expressing a small interfering RNA (siRNA) against c-Met efficiently inhibited c-Met protein and mRNA expression as well as extracellular signal-regulated kinase and phosphatidylinositol 3-kinase signaling. It also inhibited adhesion to different extracellular matrix components, human primary mesothelial cells, and full-thickness human peritoneum and, in vivo, to mouse peritoneum. This was paralleled by a significant reduction in alpha(5) and beta(1) integrin protein and mRNA expression as well as a reduction of urokinase and matrix metalloproteinase (MMP)-2/MMP-9 activity. In SKOV-3ip1 ovarian cancer xenografts, i.p. treatment with the c-Met siRNA significantly reduced tumor burden, ascites formation, protease activity, and the number of peritoneal implants but not tumor size or angiogenesis. These results suggest that c-Met overexpression is a prognostic factor in ovarian cancer and that targeting c-Met in vivo inhibits peritoneal dissemination and invasion through an alpha(5)beta(1) integrin-dependent mechanism. Therefore, c-Met should be explored further as a therapeutic target in ovarian cancer.     

Expression of vascular endothelial growth factor and E-cadherin in human ovarian cancer: association with ascites fluid accumulation and peritoneal dissemination in mouse ascites model.

Ascites formation and peritoneal dissemination are critical problems in patients with advanced ovarian cancer. Vascular endothelial growth factor (VEGF), also known as angiogenic growth factor, is a potent mediator of peritoneal fluid accumulation and angiogenesis of tumors. E-Cadherin is an adhesion molecule that is important for cell-to-cell interaction. To elucidate the molecular mechanism of ascites formation and peritoneal dissemination of ovarian cancer, we examined the expression of VEGF and E-cadherin in different ovarian cancer cell lines and utilized nude mice to compare the biological characteristics of ovarian cancer cells. Three human ovarian cancer cell lines (AMOC-2, HNOA and HTBOA) were used in this study. Expression of genes was analyzed by northern blotting and RT-PCR methods. AMOC-2 expressed E-cadherin, but not VEGF. HNOA expressed VEGF without E-cadherin expression. HTBOA expressed both VEGF and E-cadherin. Each human ovarian cancer model revealed a specific feature. The AMOC-2 mouse had a single large peritoneal tumor without ascites or remarkable peritoneal dissemination. HTBOA and HNOA mice had bloody ascites and marked peritoneal dissemination. Introduction of VEGF antisense into HTBOA cells could inhibit the ascites formation. It is suggested that VEGF is important for the ascites formation via the increased vascular permeability effect. The deregulation of E-cadherin expression might be involved in the peritoneal dissemination. These molecules are important for the formation of specific features of advanced ovarian cancer. Ovarian cancer cell lines that had different gene expression patterns produced nude mouse human ovarian cancer models with different characteristics.     

Expression of Vascular Endothelial Growth Factor and E-Cadherin in Human Ovarian Cancer: Association with Ascites Fluid Accumulation and Peritoneal Dissemination in Mouse Ascites Model

Ascites formation and peritoneal dissemination are critical problems in patients with advanced ovarian cancer. Vascular endothelial growth factor (VEGF), also known as angiogenic growth factor, is a potent mediator of peritoneal fluid accumulation and angiogenesis of tumors. E-Cadherin is an adhesion molecule that is important for cell-to-cell interaction. To elucidate the molecular mechanism of ascites formation and peritoneal dissemination of ovarian cancer, we examined the expression of VEGF and E-cadherin in different ovarian cancer cell lines and utilized nude mice to compare the biological characteristics of ovarian cancer cells. Three human ovarian cancer cell lines (AMOC-2, HNOA and HTBOA) were used in this study. Expression of genes was analyzed by northern blotting and RT-PCR methods. AMOC-2 expressed E-cadherin, but not VEGF. HNOA expressed VEGF without E-cadherin expression. HTBOA expressed both VEGF and E-cadherin. Each human ovarian cancer model revealed a specific feature. The AMOC-2 mouse had a single large peritoneal tumor without ascites or remarkable peritoneal dissemination. HTBOA and HNOA mice had bloody ascites and marked peritoneal dissemination. Introduction of VEGF antisense into HTBOA cells could inhibit the ascites formation. It is suggested that VEGF is important for the ascites formation via the increased vascular permeability effect. The deregulation of E-cadherin expression might be involved in the peritoneal dissemination. These molecules are important for the formation of specific features of advanced ovarian cancer. Ovarian cancer cell lines that had different gene expression patterns produced nude mouse human ovarian cancer models with different characteristics.     

Engagement of collagen-binding integrins promotes matrix metalloproteinase-9-dependent E-cadherin ectodomain shedding in ovarian carcinoma cells

Reversible modulation of cell-cell adhesion, cell-matrix adhesion, and proteolytic activity plays a critical role in remodeling of the neoplastic ovarian epithelium during metastasis, implicating cadherins, integrins, and proteinases in i.p. metastatic dissemination of epithelial ovarian carcinoma (EOC). Aberrant epithelial differentiation is an early event in ovarian carcinogenesis; thus, in contrast to most carcinomas that lose E-cadherin expression with progression, E-cadherin is abundant in primary EOC. Metastasizing EOCs engage in integrin-mediated adhesion to submesothelial interstitial collagens and express matrix metalloproteinases (MMP) that facilitate collagen invasion, thereby anchoring secondary lesions in the submesothelial matrix. As metalloproteinases have also been implicated in E-cadherin ectodomain shedding, the current study was undertaken to model the effects of matrix-induced integrin clustering on proteinase-catalyzed E-cadherin ectodomain shedding. Aggregation of collagen-binding integrins induced shedding of an 80-kDa E-cadherin ectodomain [soluble E-cadherin (sE-cad)] in a MMP- and Src kinase-dependent manner, and sE-cad was prevalent in ascites from ovarian cancer patients. Expression of MMP-9 was elevated by integrin aggregation, integrin-mediated ectodomain shedding was inhibited by a MMP-9 function blocking antibody, and incubation of cells with exogenous MMP-9 catalyzed E-cadherin ectodomain shedding. In contrast to other tumors wherein sE-cad is released into the circulation, EOC tumors maintain direct contact with sE-cad-rich ascites at high concentration, and incubation of EOC cells with physiologically relevant concentrations of recombinant sE-cad disrupted adherens junctions. These data support a novel mechanism for posttranslational modification of E-cadherin function via MMP-9 induction initiated by cell-matrix contact and suggest a mechanism for promotion of EOC metastatic dissemination.     

Binding of endostatin to human ovarian cancer cells inhibits cell attachment

Endostatin, a C-terminal fragment of collagen type XVIII, is one of the well-characterized endogenous inhibitors of angiogenesis. Endostatin is known to bind integrin alpha(5)beta(1), which is upregulated on tumor endothelium. Most of the ovarian cancer cells express significant amounts of alpha(5)beta(1) integrin, which is important for ovarian cancer cells to attach to the peritoneal wall. Therefore we investigated whether endostatin could directly bind ovarian cancer cells and inhibit tumor cell attachment to extracellular matrix. Binding of endostatin to ovarian cancer cells was characterized by preincubation with function blocking antibodies to integrin subunits. These studies showed that ovarian cancer cell attachment to fibronectin-coated wells can be inhibited by alpha(5)beta(1) integrin specific antibodies as well as endostatin. Downregulation of integrin alpha(5) and beta(1) by siRNA abrogated the binding of OVCAR5 and human umbilical vein endothelial cell to endostatin. Although endostatin treatment did not affect ovarian cancer cell migration, treated cells failed to attach mouse peritoneal wall preparations. These studies suggest an extra-antiangiogenic role for endostatin, which can be used prevent peritoneal attachment and dissemination of ovarian cancer cells.     

The inflammatory cytokine tumor necrosis factor-alpha generates an autocrine tumor-promoting network in epithelial ovarian cancer cells

Constitutive expression of the inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) is characteristic of malignant ovarian surface epithelium. We investigated the hypothesis that this autocrine action of TNF-alpha generates and sustains a network of other mediators that promote peritoneal cancer growth and spread. When compared with two ovarian cancer cell lines that did not make TNF-alpha, constitutive production of TNF-alpha was associated with greater release of the chemokines CCL2 and CXCL12, the cytokines interleukin-6 (IL-6) and macrophage migration-inhibitory factor (MIF), and the angiogenic factor vascular endothelial growth factor (VEGF). TNF-alpha production was associated also with increased peritoneal dissemination when the ovarian cancer cells were xenografted. We next used RNA interference to generate stable knockdown of TNF-alpha in ovarian cancer cells. Production of CCL2, CXCL12, VEGF, IL-6, and MIF was decreased significantly in these cells compared with wild-type or mock-transfected cells, but in vitro growth rates were unaltered. Tumor growth and dissemination in vivo were significantly reduced when stable knockdown of TNF-alpha was achieved. Tumors derived from TNF-alpha knockdown cells were noninvasive and well circumscribed and showed high levels of apoptosis, even in the smallest deposits. This was reflected in reduced vascularization of TNF-alpha knockdown tumors. Furthermore, culture supernatants from such cells failed to stimulate endothelial cell growth in vitro. We conclude that autocrine production of TNF-alpha by ovarian cancer cells stimulates a constitutive network of other cytokines, angiogenic factors, and chemokines that may act in an autocrine/paracrine manner to promote colonization of the peritoneum and neovascularization of developing tumor deposits.     

Endostatin binding to ovarian cancer cells inhibits peritoneal attachment and dissemination

Ovarian cancer cells use integrins to attach to the peritoneal wall. Integrin alpha(5)beta(1) is also the target for the angiogenesis inhibitor, endostatin. Therefore, the ability of endostatin to competitively inhibit tumor cell seeding of the peritoneum was investigated. An imaging method was developed to determine early phases of peritoneal dissemination of ovarian cancer cells. Using this method, endostatin was found to bind ovarian cancer cells through integrin alpha(5)beta(1) and inhibit vessel cooption efficiently. Although both angiostatin and endostatin are potent inhibitors of tumor angiogenesis, peritoneal attachment and vessel cooption was blocked only by the endostatin. Knocking down the expression of integrins alpha(5) and beta(1) in ovarian cancer cells interfered with endostatin-mediated inhibition of peritoneal seeding. Furthermore, adenovirus-mediated in situ expression of endostatin either inside the peritoneum or by the ovarian tumor cells inhibited peritoneal seeding and dissemination in vivo. Endostatin treatment also prevented primary ovarian cancer cells from attaching to mouse peritoneal wall. These studies show a paraendothelial mechanism by which endostatin can inhibit peritoneal dissemination of ovarian cancer cells and raises the possibility of intraperitoneal expression of endostatin to reduce recurrence.     

Indoleamine 2,3‐dioxygenase promotes peritoneal metastasis of ovarian cancer by inducing an immunosuppressive environment

Indoleamine 2,3‐dioxygenase (IDO) is a tryptophan‐catabolizing enzyme that has immunoregulatory functions. Our prior study showed that tumoral IDO overexpression is involved in disease progression and impaired patient survival in human ovarian cancer, although its mechanism remains unclear. The purpose of the present study is to clarify the role of IDO during the process of peritoneal dissemination of ovarian cancer. Indoleamine 2,3‐dioxygenase cDNA was transfected into the murine ovarian carcinoma cell line OV2944‐HM‐1, establishing stable clones of IDO‐overexpressing cells (HM‐1‐IDO). Then HM‐1‐IDO or control vector‐transfected cells (HM‐1‐mock) were i.p. transplanted into syngeneic immunocompetent mice. The HM‐1‐IDO‐transplanted mice showed significantly shortened survival compared with HM‐1‐mock‐transplanted (control) mice. On days 11 and 14 following transplantation, the tumor weight of peritoneal dissemination and ascites volume were significantly increased in HM‐1‐IDO‐transplanted mice compared with those of control mice. This tumor‐progressive effect was coincident with significantly reduced numbers of CD8⁺ T cells and natural killer cells within tumors as well as increased levels of transforming growth factor‐β and interleukin‐10 in ascites. Finally, treatment with the IDO inhibitor 1‐methyl‐tryptophan significantly suppressed tumor dissemination and ascites with reduced transforming growth factor‐β secretion. These findings showed that tumor‐derived IDO promotes the peritoneal dissemination of ovarian cancer through suppression of tumor‐infiltrating effector T cell and natural killer cell recruitment and reciprocal enhancement of immunosuppressive cytokines in ascites, creating an immunotolerogenic environment within the peritoneal cavity. Therefore, IDO may be a promising molecular target for the therapeutic strategy of ovarian cancer.     

Evidence for preferential adhesion of ovarian epithelial carcinoma cells to type I collagen mediated by the αA2β1 integrin

Epithelial ovarian carcinoma, the leading cause of gynecologic cancer death, is characterized by widespread intra-abdominal metastases mediated primarily by surface shedding of tumor cells and peritoneal implantation. Whereas hematogenous metastasis is known to involve cellular adhesion, extracellular matrix proteolysis and cell migration, the role of these processes in the intraperitoneal dissemination of ovarian cancer remains unclear. To analyze further the role of adhesion and proteolysis in ovarian carcinoma dissemination, we have characterized the adhesive profiles of 4 primary cultures of ovarian carcinoma cells and 5 ovarian carcinoma cell lines. Our data demonstrate preferential adhesion of ovarian carcinoma cells to interstitial type I collagen. Analysis of adhesion molecule expression demonstrated the presence of the α2 and β1 integrin subunits by cell surface ELISA, immunoprecipitation and immunohistochemistry. Furthermore, antibodies directed against the α2 and β subunits inhibited adhesion of ovarian carcinoma cells to type 1 collagen by 56% and 95%, respectively. Plasminogen activator and matrix metalloproteinase production by adherent cells was not altered as a consequence of adhesion to individual extracellular matrix proteins; however, adhesion to an extracellular matrix comprised primarily of interstitial collagen increased plasminogen activator activity in 5 of 5 cell lines. Since the ovarian carcinoma micro-environment is rich in type 1 collagen, our data suggest that preferential adhesion to type 1 collagen followed by secretion of serine and metalloproteinases may represent a biochemical mechanism by which the intraperitoneal dissemination of ovarian carcinoma is mediated. © 1996 Wiley-Liss, Inc.     

SOX2 is required to maintain cancer stem cells in ovarian cancer

Ovarian cancer cells can form spheroids under serum‐free suspension culture conditions. The spheroids, which are enriched in cancer stem cells, can result in tumor dissemination and relapse. To identify new targetable molecules in ovarian cancer spheroids, we investigated the differential expression of genes in spheroids compared with that under monolayer culture conditions by qPCR microarray. We identified that SOX2 is overexpressed in spheroids. We then proved that SOX2 expression was increased in successive spheroid generations. Besides, knockdown of SOX2 expression in SKOV3 or HO8910 ovarian cancer spheroid cells decreased spheroid formation, cell proliferation, cell migration, resistance to Cisplatin treatment, tumorigenicity, and the expression of stemness‐related genes and epithelial to mesenchymal transition‐related genes, whereas overexpression of SOX2 in SKOV3 or HO8910 ovarian cancer cells showed the opposite effects. In addition, we found that SOX2 expression was closely associated with chemo‐resistance and poor prognosis in EOC patients. These results strongly suggest that SOX2 is required to maintain cancer stem cells in ovarian cancer. Targeting SOX2 in ovarian cancer may be therapeutically beneficial.