MMP-1-PAR1 axis mediates LPA-induced epithelial ovarian cancer (EOC) invasion☆
Research Highlights
► MMP-1 promotes epithelial ovarian cancer (EOC) invasion. ► MMP-1-PAR1 axis mediates LPA-induced EOC invasion. ► MMP-1 and PAR1 may represent new therapeutic targets for metastatic ovarian cancer.
Introduction
Despite recent advances in surgical intervention and biologically based chemotherapeutics, epithelial ovarian cancer (EOC) remains the leading cause of death in women with gynecologic malignancies in the United States. In 2009, there were 21,550 estimated new cases and 14,600 estimated new deaths from this disease [1]. The high morbidity and mortality associated with EOC is mainly attributed to the fact that the majority of women (70–75%) present with widespread intra-abdominal metastases at the time of initial diagnosis and ultimate resistance to chemotherapy [2]. Therefore, mechanistic studies on how EOC metastasizes are of great importance and represent a major strategy in the efforts to identify novel targets for the development of more effective therapeutics.
Tumor metastasis is a multi-step process that requires the local invasion of surrounding tissues, followed by blood or lymphatic vessel infiltration, survival in the circulation, extravasation, and secondary growth within secondary host tissue. In order for cancer cells to spread locally, they must be able to penetrate connective tissue barriers such as the basement membrane and interstitial stroma that surround tumors [3], [4]. Abundant evidence has indicated the involvement of matrix metalloproteinases (MMPs), a family of 25 structurally related zinc-dependent endopeptidases, in this process [5], [6], [7]. Among the MMPs, MMP-1 (interstitial collagenase) degrades the most abundant class of extracellular matrix proteins in interstitial connective tissue-fibrillar collagens. Over expression of MMP-1 has been observed in various types of tumors such as gastric [8], colorectal [9], [10], esophageal [11], and breast cancer [12], [13]. In nearly all those cancers, the high expression of MMP-1 was associated with poor survival [14]. In ovarian cancer, MMP-1 together with other MMPs are located in the matrix-rich networks surrounding spheroids of tumor cells called molecular vasculogenic mimicry [15], also co-localized with other MMPs and proteases in malignant effusion cells [16], [17], [18], primary tumors and metastatic lesions of ovarian cancer [19]. In ovarian cancer cell lines, a correlation between MMP-1 activity and cell invasiveness was observed previously [20], [21], suggesting that it plays a role in cell invasion. An early study by Kanamori et al. also demonstrated a correlation between MMP-1 gene expression and the insertion or deletion of a guanine nucleotide in the promoter region of MMP-1 in ovarian cancer [22]. The commonly occurring G/2G promoter polymorphism, which causes enhanced MMP-1 transcription, was shown to be associated with a shortened disease free and overall 5-year survival in 151 Caucasian ovarian cancer patients in a later study [23], although such association was not observed in studies done in north China and Korea population [24], [25]. Despite the clinical correlation observed between high MMP-1 expression and poor survival, few studies have provided mechanistic insight on how MMP-1 affects ovarian cancer progression other than through the degradation of fibrillar collagens.
Within the extracellular milieu, MMP's cleave not only ECM molecules, but also various other cellular components including protease inhibitors, adhesion molecules, growth factor binding proteins, and several cell surface receptor molecules [6]. In 2005, Boire et al. identified a novel MMP-1 signaling axis through the activation of protease activated receptor (PAR)-1 [26] to promote invasion and tumorigenesis of breast cancer cells [27]. PARs are G-protein coupled receptors that contain an internal ligand masked in the resting state. Upon activation by proteolysis, the ligand is exposed and a signal transduction cascade is initiated to regulate cell morphology, proliferation, migration, and adhesion [28], [29]. PAR1 is up-regulated in a wide range of tumor cells such as breast and prostate and plays a role in tumor cell invasion [29], [30]. PAR1 is also over-expressed in malignant ovarian carcinomas as compared to normal ovarian epithelium [31]. Recently, Agarwal et al. reported that targeting PAR1 signaling effectively inhibited PAR1-dependent angiogenesis, ascites formation, and metastasis in ovarian cancer xenograft models [32]. They also showed a strong correlation between proMMP-1 levels in patient fluids samples and malignancy, as well as their ability to induce migration of high PAR1-expressing OVCAR-4 cells, indicating the involvement of MMP-1-PAR1 signaling in ovarian cancer progression [32]. However, the roles of MMP-1-PAR1 signaling axis in EOC invasion and metastasis remain to be extensively explored as only a few studies have investigated this topic, among which we are the first to demonstrate a correlation between MMP-1-PAR-1 expression and EOC cell invasiveness in vitro [33]. In the present study, we examined the direct effect of MMP-1 on cell invasion using DOV13 and R182 cells. To correlate MMP-1 expression with clinical outcome, we also analyzed its mRNA expression in normal and ovarian carcinoma tissues of various stage and histological grade. Furthermore, we evaluated the role of MMP-1-PAR1 signaling in EOC invasion by silencing MMP-1 and/or PAR1 using small interference RNA (siRNA). In addition, since lysophosphatidic acid (LPA) has been shown as an important mediator of EOC invasion and metastasis through the regulation of multiple growth factors, cytokines, and proteases [34], [35], [36], [37], we further investigated whether the LPA-induced EOC invasion is partially mediated by the MMP-1-PAR1 signaling axis.
Section snippets
Cell lines and culture conditions
The ovarian carcinoma cell line DOV13 was provided by Dr. Robert Bast, Jr. (M.D. Anderson Cancer Center, Houston, TX) and maintained under standard conditions in 75 cm2 cell culture flasks37. The human ovarian cancer cell line OVCA429 was maintained in the same culture medium as DOV13 without the addition of insulin. The human ovarian cancer cell line R182 was provided by Dr. Gil Mor (Yale University) and was grown in RPMI 1640 supplemented with 20% FBS (Life Technologies Inc., Carlsbad, CA), 10
MMP1 expression and/or secretion correlates with EOC cell invasion
Previous studies have indicated that MMP-1 might play a role in ovarian cancer invasion [20], [40], however, no direct link between the level of MMP-1 expression and cell invasiveness has been observed. To determine if MMP-1 expression and/or secretion correlates with EOC cell invasion, we first analyzed the levels of MMP-1 mRNA expression (Fig. 1A) in three EOC cell lines (OVCA429, DOV13 and R182) and one immortalized normal ovarian epithelium (IOSE-29) using real-time qPCR, and then measured
Discussion
As the first vertebrate collagenase purified and cloned [41], [42], MMP-1 is mainly expressed during physiological and pathological tissue remodeling processes in vivo, such as wound healing, arthritis, and cancer. Although the roles of MMP-1 have been extensively investigated in a wide variety of cancers [14], its expression profile is relatively less-well studied in ovarian cancer. Most prior studies regarding MMP-1 and ovarian cancer focused on a MMP-1 1G/2G polymorphism and disease
Conflict of interest statement
The authors of this article declare no conflicts of interest.
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Grant Support: The study is supported by funds from the Varadi Ovarian Initiative for Cancer Education, and Mount Sinai School of Medicine.