Feng-qiang Wang

Matrilysin (MMP‐7) promotes invasion of ovarian cancer cells by activation of progelatinase

Although matrilysin (MMP‐7) is overexpressed in various malignancies, few studies have evaluated its role in epithelial ovarian cancer (EOC) invasion and metastasis. We report that the secretion of MMP‐7 in EOC is stimulated significantly by vascular endothelial growth factor (VEGF) and interlukin‐8 (IL‐8). We also examined the in vivo expression of MMP‐7 in EOC and its effects on the in vitro invasion and progelatinase activation. We report that MMP‐7 is overexpressed in ovarian cancer cell lines and EOC surgical specimens. DOV13 cells incubated with active rhMMP‐7 significantly increased cellular invasion and proMMP‐2 activation. RhMMP‐7 also showed the ability to activate proMMP‐2 and proMMP‐9 in immortalized ovarian epithelial cell (IOSE‐29) conditioned medium. In addition, rhMMP‐7 was able to activate progelatinase in a concentration‐dependent manner in vitro. TIMP‐2 or the generic MMP inhibitor‐GM6001 inhibited both the activation of proMMP‐2 and the increased invasion of DOV13 cells promoted by rhMMP‐7. By incubation of MMP2‐TIMP‐2 complex with equal molar rhMMP‐7, MMP‐2 was dissociated from the complex and activated in a time‐dependent manner, suggesting that TIMP‐2 helps to keep the latency of MMP‐2. TIMP–2 also showed inhibitory effects on the MMP‐7 induced increase of gelatinolytic activity in DOV13 and IOSE‐29 conditioned media. A strong co‐localization of MMP‐7 and MMP‐2 was observed in DOV13 cells and ovarian carcinoma permanent tissue sections. These results indicate MMP‐7 is overexpressed in malignant ovarian epithelium and suggest MMP‐7 may facilitate tumor cell invasion in vivo partly through the induction of progelatinase activation.

Vascular endothelial growth factor–regulated ovarian cancer invasion and migration involves expression and activation of matrix metalloproteinases

Vascular endothelial growth factor (VEGF) expression is elevated in primary ovarian tumors and metastases. We examined the effect of VEGF on epithelial ovarian cancer (EOC) in vitro invasion and migration and underlying mechanisms. Using the Matrigel invasion assay and colloidal gold phagokinetic track assay, we found that VEGF induced EOC DOV13 invasion and migration in a matrix metalloproteinase (MMP)–dependent manner. Using Western blotting, we show that VEGF, at 20–80 ng/ml, induced secretion of pro‐MMP‐7 and pro‐MMP‐9 and activation of pro‐MMP‐2 in DOV13 conditioned medium in a concentration‐dependent manner. However, gelatinolytic activity and total MMP‐7 protein in DOV13 conditioned medium reached the maximum upon VEGF treatment at 20–40 ng/ml and decreased at higher‐concentration VEGF treatment (80 ng/ml), as shown by DQ‐gelatin degradation assay and ELISA. In addition to the effect on MMP secretion/activation, VEGF stimulated secretion of TIMP‐2; and blocking TIMP‐2 activity by an anti‐TIMP‐2 MAb significantly increased VEGF (80 ng/ml)–induced DOV13 invasion (p < 0.05), suggesting that VEGF may regulate MMP‐2 activity in DOV13 conditioned medium through TIMP‐2. Using real‐time PCR, we found that VEGF, at 20 ng/ml, significantly increased the expression of VEGFR‐1 and VEGFR‐2 by approximately 4‐fold and 31‐fold, respectively, compared to untreated control (p < 0.05). However, the inducing effect of VEGF on VEGFR‐2 expression and the internal expression of VEGF121 in DOV13 cells decreased with increasing of VEGF concentration, suggesting the existence of a negative feedback regulatory mechanism. In summary, our results indicate that VEGF may regulate EOC invasion and migration through VEGFR‐mediated secretion and activation of MMPs.

Torg Syndrome Is Caused by Inactivating Mutations in MMP2 and Is Allelic to NAO and Winchester Syndrome

Torg syndrome is a multicentric osteolysis syndrome of unknown etiology. We identified mutations in the MMP2 gene in a patient with Torg syndrome that resulted in complete loss of MMP2 activity. MMP2 mutations were previously identified in patients with NAO and Winchester syndrome. Our findings suggest that Torg, NAO, and Winchester syndrome are allelic disorders.

Roles of LPA in ovarian cancer development and progression

Lysophosphatidic acid (LPA), a bioactive phospholipid, stimulates survival, proliferation, adhesion, migration and invasion of ovarian cancer cells through the activation of G-protein-coupled plasma membrane receptors. LPA and its receptors are aberrantly expressed in ovarian cancer, with high levels predominantly found in malignant ascites and in the plasma of ovarian cancer patients. LPA signals multiple intracellular pathways, such as Ras/MEKK1-MAPK and PI3K/Akt, to promote growth factors and protease expression, and induce angiogenesis and tumor cell invasion through the extracellular matrix and across the basement membrane. Only a small portion of this intricate lipid-signaling cascade has been characterized thus far. We believe that elucidation of this complex transduction network will provide further opportunities to understand the mechanism of ovarian carcinogenesis, invasion and metastasis.

Biomarkers for ovarian cancer detection and therapy

Despite advances in surgical technologies and the development of more effective chemotherapeutics, epithelial ovarian cancer (EOC) remains the leading cause of death in women with gynecologic malignancies. The high mortality and morbidity associated with EOC is mostly attributed to the inability to detect the disease before it is widely disseminated throughout the abdominal cavity. In the past decade, tremendous efforts have been taken to search for novel biomarkers that will detect EOC at an early stage, which may also serve as new targets for the prevention and control of metastasis. Here, we review recent developments in EOC early detection and targeted therapy, as well as new technologies for the discovery of novel biomarkers.

Inhibition of matrilysin expression by antisense or RNA interference decreases lysophosphatidic acid-induced epithelial ovarian cancer invasion.

Our previous reports show that matrilysin [matrix metalloproteinase (MMP)-7] is overexpressed in epithelial ovarian cancer (EOC) and recombinant MMP-7 promotes EOC invasion in vitro. In the present study, we further evaluated the correlation of MMP-7 expression to EOC invasiveness and examined its role in lysophosphatidic acid (LPA)-induced invasion. By sense and antisense gene transfection in vitro, we show that overexpression of MMP-7 in all MMP-7 stably transfected DOV13 clones significantly enhanced their invasiveness, although MMP-7 antisense transfection caused a 91% decrease of MMP-7 expression (P < 0.01) and 87% decrease of invasion (P < 0.05) in geneticin (G418)-selected DOV13 clone P47-M7As-3 compared with vector-transfected control. As assessed by MMP-7 ELISA, LPA treatment at 10 to 80 μmol/L significantly stimulated the secretion of total MMP-7 in DOV13 conditioned medium (P < 0.01). In addition, LPA apparently induced the activation of MMP-7 in DOV13 cells as detected by gelatin zymography. In the antisense MMP-7-transfected DOV13 clone (P47-M7As-3), LPA-increased invasion was significantly decreased compared with vector control. Moreover, knocking down of MMP-7 by small interfering RNA also suppressed LPA-induced invasion in two EOC cell lines (DOV13 and R182). Altogether, our results show that MMP-7 expression is correlated with EOC invasiveness and LPA-induced MMP-7 secretion/activation may represent a new mechanism that facilitates ovarian cancer invasion besides the well-known induction of MT1-MMP-mediated proMMP-2 activation by LPA. (Mol Cancer Res 2006;4(11):831–41)

New Frontiers for Ovarian Cancer Risk Evaluation: Proteomics and Contrast-Enhanced Ultrasound

OBJECTIVE The grim ovarian cancer statistics are attributed to the fact that most women typically present with widespread disease at the time of initial diagnosis. Our current diagnostic tools, such as pelvic examination and standard ultrasound, are inadequate to detect early-stage epithelial ovarian cancer. In recent years there has been an explosion of important advances in biomedical engineering, proteomic technologies, and computational analyses that has led to the identification of hundreds of previously unknown proteins unique to the pathophysiology of ovarian cancer, some of which are currently under clinical validation. At present, no one biomarker exists with 100% specificity and sensitivity for the accurate detection of early-stage epithelial ovarian cancer. CONCLUSION As the search for a panel of biomarkers detecting cancer, let alone early-stage disease, progresses, diagnostic imaging will continue to play a critical role to confirm or refute these biomarker assays. Interestingly, recent studies using contrast-enhanced ultrasound have shown potential as an early-detection tool by detecting the aberrant vascularity required for tumor growth before the development of a mass. Thus, we propose that the use of proteomic-based biomarker discovery and contrast-enhanced ultrasound may serve as a promising combination to help accurately identify early-stage epithelial ovarian cancer to improve womens health care.

The dire need to develop a clinically validated screening method for the detection of early-stage ovarian cancer

For almost half a century, the WHO has urged the medical community to create screening methods with the primary purpose of detecting disease at an early, treatable stage [1]. This is of particular importance in epithelial ovarian cancer (EOC) given that there are no clinically validated biomarkers or screening protocols available for the accurate detection of early-stage disease. This year, approximately 22,000 women will be newly diagnosed with ovarian cancer, while an additional 15,000 deaths will be attributed to this disease [2]. These statistics make EOC the fifth leading cause of cancer death in women and the most lethal gynecologic malignancy, with more women dying from this disease than all other gynecologic malignancies combined [2]. In addition, during the past 40 years, the overall survival for women with advanced-stage (III/IV) EOC has remained relatively unchanged, at less than 30%, despite surgical advancements and improvements in chemo therapeutics [2]. However, women diagnosed with early-stage EOC (stage I) require less morbid surgical procedures, may not require adjuvant chemotherapy, have an improved quality of life and, most importantly, have an overall survival approaching 93% [2]. Unfortunately, the majority of women (75%) continue to be diagnosed with advanced-stage EOC owing to the rather stealth nature of early-stage disease and the lack of a clinically validated screening method. Therefore, increasing the proportion of women detected with early-stage EOC, particularly stage I, would significantly improve women’s healthcare. The standard screening methods currently in use to detect ovarian cancer include pelvic exams, transvaginal or transabdominal ultrasonography and the serum cancer antigen (CA)125 level. Quantitative CA125 levels have long been the ‘gold standard’ biomarker test for the surveillance of women with EOC; however, it is only elevated in 47% of women with early-stage disease. Moreover, CA125 is nonspecific and is elevated in many disease states, such as other abdominal malignancies, benign diseases and physio logical conditions. Therefore, its effectiveness as an independent screening test is compromised owing to its lack of sensitivity and specificity. Although many other tumor biomarkers have been identified in ovarian cancer during the past decade, few of them have been widely tested for screening purposes. Furthermore, traditional ultra sonography is not an effective population screening test owing to its rather low sensitivity and specificity, both alone and combined with CA125. Given ovarian cancer’s relatively low general population incidence rate (1.8%) [2], a valid screening test must have both a high sensitivity and a high specificity to decrease the number of false-positive test results and the subsequent invasive surgical procedures undoubtedly associated with this result. The ideal screening test for the postmenopausal population (with a prevalence of one in 2500) would require a specificity of 99.6% to yield an approximate positive predictive value of 10%, where a surgeon performs ten operations for each case of ovarian cancer detected. The positive predictive value of most screening tests is determined by specificity and disease incidence – given ovarian cancer’s low frequency, achieving an acceptable positive predictive value requires an extremely high specificity, especially in average-risk populations. Multiple initiatives have been undertaken to discover strategies that detect and diagnose early-stage EOC, including the search for novel serum biomarkers and the development of new technologies, such as contrast-enhanced ultrasonography, with a number of them demonstrating hopeful results. The ideal screening test for ovarian cancer would be a simple measurement of biomolecules in bodily fluids, such as blood,

Lysophosphatidic Acid and Invasion

Lysophosphatidic acid (LPA) is a small, bioactive phospholipid produced by activated platelets, mesothelial cells, macrophage, endothelial cells, fibroblasts, adipocytes, and some cancer cells. It is involved in multiple cellular events of almost every mammalian cell type. Upon binding to G-protein–coupled receptors (GPCRs), LPA exerts a myriad of biological effects, including cell proliferation/survival, induction of neurite retraction, inhibition of gap junctional communication, and cell motility. The estimated concentrations of active, albumin-bound LPA in serum are in the range 1–5 mmol/L. Physiologic and pathophysiologic responses to LPA include wound healing, production of angiogenic factors, chemotaxis, neointima formation, tumor cell invasion, metastasis, and cell cycle progression. A large body of evidence suggests that LPA is relevant to the pathogenesis of epithelial ovarian cancer (EOC). LPA is elevated in the blood and ascites of women with ovarian cancer with levels reaching 80 mmol/L. Ovarian cancer cells also produce LPA to maintain an LPA-rich microenvironment. Accumulating evidence suggest that LPA signaling is involved in the initiation, progression, and metastasis of ovarian cancer and imparts a cytoprotective effect to ovarian cancer cells exposed to cisplatin. Tumor invasion andmetastasis constitute a major problem for the treatment of EOC.Approximately 75%ofwomenwith ovarian cancer already havewidespread peritoneal dissemination and ascites (stage III or IV) at the time of initial diagnosis, witharesulting5-yearsurvivalrateapproaching15%to30%.Metastaticdissemination is the result of a highly organized process starting from the microscopic initial growth of the originating tumor cells ultimately followed by detachment of select invasive tumor cells from the macroscopic primary tumor mass leading to the

Tumor vascular proteins: groundbreaking as potential new ovarian cancer biomarkers

Evaluation of: Buckanovich RJ, Sasaroli D, O’Brien-Jenkins A et al. Tumor vascular proteins as biomarkers in ovarian cancer. J. Clin. Oncol. 25(7), 852–861 (2007).Genome-wide transcriptional profiling has been widely used to identify differentially expressed genes and molecular signatures in many diseases states, including ovarian cancer, in comparison with normal tissues. However, the characteristic expression profile of vascular cells in ovarian cancer has only been minimally investigated. Using immunohistochemistry-guided laser-capture microdissection and tumor vascular cell arrays, Buckanovich and colleagues identified tumor vascular cell profiles in ovarian cancer and validated 12 novel tumor vascular markers (TVMs) by quantitative real-time-PCR, in situ hybridization, immunohistochemistry and western blot analysis. Moreover, they demonstrated that selected TVMs were specifically expressed in tumor vasculature and absent in physiologic angiogenesis. Overexpression of any one of three specific ovarian...