Late diagnosis and metastasis are major causes for the high mortality rate of OvCa [1
]. Chemokines have been shown to play important roles in organ-specific homing of cancer cells to distant organs [12
]. CCL25-CCR9 interactions are key to leukocyte homing to the small bowel [21
], a common and fatal site of OvCa metastasis. In this regard, high levels of CCL25 in the gut mucosa and expression of CCR9 by OvCa cell lines suggest CCL25-CCR9 interactions might play a significant role in the mucosal homing of OvCa cells.
While metastasis of well-differentiated endometrioid adenocarcinoma are typically limited to the uterine body, mildly and poorly differentiated invasive endometroid adenocarcinomas have been associated with cervical invasion and distal metastasis [23
]. Serous papillary carcinoma is an aggressive form of endometrial cancer that is likely to present with deep myometrial invasion and lymph vascular involvement. By the time most affected women are diagnosed, serous papillary carcinoma often spreads outside the uterus. Serous endometrial intra-epithelial carcinoma is a recently recognized entity with the same cytological features and p53 mutations as uterine serous carcinoma, with the former associated with stromal and/or myometrial invasion and extra-uterine metastasis. Interestingly, we found the highest expression of CCR9 in serous papillary and endometroid carcinomas in comparison to non-neoplastic and to a lesser degree in mucinous adenocarcinoma cases. Given the poor prognosis of serous papillary and endometroid carcinoma, our data supports the development of therapies that target the CCL25-CCR9 axis. Indeed, CCR9 blockade inhibited migration, MMP production, and invasion of OvCa cell lines.
CCL25-CCR9 interactions have been previously implicated in the progression of melanoma and prostate cancers [10
]. Other studies concluded CCR9 is highly expressed by melanoma cells and all melanoma cells isolated from small intestine metastases [25
]. Here we show for the first time that normal ovarian epithelial cells and non-neoplastic tissues express low levels of CCR9, while OvCa cell lines and mucinous adenocarcinoma, papillary serous carcinoma, and endometriod carcinoma tissues express high levels of CCR9. While ovulating ovaries express CCR9 and CCL25, which play important role during ovulation [26
], we show that the expression of CCR9 mRNA by OvCa cell lines is significantly higher than levels expressed by normal adult ovarian epithelial cells. Each of the OvCa cell lines exhibited significantly higher CCL25-mediated migration and invasion, which was CCR9-dependent.
Among the numerous OvCa cell lines studied, CAOV-3 and OVCAR-3 have the highest incidence and average metastatic frequency [27
]. CAOV-3 is an invasive human ovarian papillary carcinoma cell line. While the histological phenotype of the OVCAR-3 cell line is unknown, it was established from the malignant ascites of a patient with progressive adenocarcinoma of the ovary. OVCAR-3 xenografts produce either ascites or solid tumors in the peritoneal cavity [28
], but similar grafts using the more invasive CAOV-3 cell line result in lung and other organ metastasis [27
]. Neoplastic cells must penetrate the basement membrane and invade the interstitial stroma to initiate the metastatic process. To this end, many proteinases are capable of degrading extracellular matrix (ECM) components, but MMPs appear to be particularly important for matrix degradation [29
] and cancer cell dissemination [31
Collagenases (MMP-1, MMP-8 and MMP-13) initiate degradation of several naïve fibrillar collagens, including type-I, -II and -III. Higher expression of MMP-1 has been correlated with progression and poor survival in bladder cancer [32
]. In most instances, increased expression of MMP-1 has a significant negative correlation with survival. Similarly, over production of MMP-8 has been shown to contribute to the invasive potential of OvCa [33
]. Indeed, MMP-8 expression significantly correlated with ovarian tumor grade, tumor stage, and poor prognosis [34
]. Even though CAOV-3 cells expressed slightly less CCR9 than OVCAR-3 cells, CCL25 treatment resulted in significant MMP-1 and -8 mRNA and active protein expression by CAOV-3 >> OVCAR-3 cell lines. On the other hand, MMP-13 is important for the degradation of type-I and -II collagens and its presence in ascites fluid has been used to identify patients at risk for early death from OvCa [7
]. MMP-13 has been well documented in many aggressive cancers, but its expression is most often seen only in the invading front of tumors [35
]. While OVCAR-3 cell supernatants expressed more MMP-13 in response to CCL25 treatment, the more invasive CAOV-3 cells did not produce MMP-13 mRNA or active protein in response to this CCR9 ligand. Possibly, MMP-13 is differentially expressed by OvCa cells and not crucial for ovarian tumor invasiveness per se
Gelatinase-A and -B (MMP-2 and -9), also called type IV collagenases, degrade gelatin, collagen and other basement membrane components. High levels of MMP-2 and -9 have been associated with many diseases, including OvCa, and correlate with poor prognosis [37
]. In an immunohistochemical study of malignant ovarian tissues, positive staining of MMP-2 was associated with poor survival [38
]. In the present study, OVCAR-3 and CAOV-3 cell lines expressed MMP-2 mRNA and active protein. Importantly, CCL25 treatment led to a significant increase in MMP-2 expression by both OvCa cell lines. MMP-9 is frequently up regulated by cancer cells and has been shown to affect tumor metastasis and progression. CCL25 treatment induced an increase in MMP-9 expression by OVCAR-3, but not CAOV-3 cells. Perhaps, the lack of active MMP-9 expression by these cell lines is due to the production of high levels of tissue inhibitors of metalloproteinases (TIMPs). TIMPs are major regulators of matrix metalloproteinase activity. Specifically, TIMP-1 preferably binds and inactivates MMP-9 [39
]. In this regard, high circulating TIMP-1 correlated to the aggressive phenotype and unfavorable prognosis of malignant neoplasias [39
]. Therefore, it is possible that the CAOV-3 cell lines express elevated levels of TIMP-1, which would inhibit the generation of active MMP-9 protein.
Stromelysins (MMP-3, -10, and -11) are typically expressed by normal epithelial cells and degrade a variety of substrates, including type IV, V, IX AND X collagens, fibronectin, laminin, elastin, and proteoglycan core proteins. Many carcinomas express stromelysins; for example, MMP-3 and -10 produced by the head and neck carcinomas are higher than in normal-matched tissue [40
]. In this study, MMP-3 and MMP-10 mRNA and protein were expressed at significantly higher levels by OVCAR-3 and CAOV-3 cell lines, after CCL25 treatment. CCR9 activation also led to an elevation of MMP-11 mRNA and active protein expression by OVCAR-3 cells. Indeed, MMP-11 (or stromelysin-3) expression is more frequently observed in malignant ovarian carcinomas than tumors with low malignant potential [41
Our study shows that the OvCa cell lines, OVCAR-3 and CAOV-3, differentially expressed MMPs that are important for OvCa metastasis after CCL25 stimulation. While OVCAR-3 and CAOV-3 cell lines were both established from malignant ascites [42
], these cell lines selectively migrated chamber inserts and invaded Matrigel in response to CCL25. For example, OVCAR-3, but not CAOV-3, cells poorly attached to host-tissue surfaces and express lamin receptor [43
]. While these responses were CCR9-dependent, other factors no doubt contribute to their abilities to migrate and invade tissue extracellular matrix components.