The biological consequence of SLPI amplification and overexpression in ovarian cancer is poorly understood. We examined this question through overexpression of SLPI and mutants lacking full protease inhibitory activity in HEY-A8 human ovarian cancer cells with low endogenous SLPI expression. SLPI and its protease inhibitor null mutants stimulated growth and survival under both adherent and density-independent conditions. Further, SLPI and its PI-mutants protected the cells from anoikis. These markers of malignancy translated to enhanced aggressiveness in orthotopic xenografts, resulting in more serosal, omental, and mesenteric disease. Invasion of the serosal implants into organ parenchyma was absent in the mock transfectants at the time of experimental closure, yet progressively greater from WT/SLPI to the PI- mutants. Where the PI- mutants could not inhibit serine protease activity, they remained able to bind the SLPI partner protein and growth factor, PRGN [16
]. SLPI prevented degradation of PRGN and increased its net quantity in vitro and in xenografts, a potential mechanism through which SLPI functions. These cellular and PRGN-protective effects of SLPI on ovarian cancer cells are independent of the ability of SLPI to inhibit elastase. We conclude that SLPI is not be simply a biomarker for presence of ovarian cancer, but its amplification and expression maybe necessary, though not fully sufficient, for ovarian cancer growth and dissemination.
SLPI has been proposed as a potential biomarker for ovarian cancer diagnosis, because of its amplification and overexpression [2
]. Devoogdt and colleagues identified SLPI associated with increased tumorigenic and metastatic potential in a murine Lewis lung cancer model [2
]. SLPI overexpression in a poorly metastatic mouse mammary cancer cell line resulted in augmented growth and spontaneous metastases to lung in vivo [15
]. In contrast, Wang and coworkers found that SLPI overexpression in the same cell background attenuated host proinflammatory response and resulted in significantly fewer hepatic metastases [14
]. It is possible that different functions of SLPI may be dominant in different cancer microenvironments. Anti-inflammatory activities of SLPI include reduction in monocyte production of proteases in response to LPS or concanavalin A stimulation [27
], antimicrobial properties in mucosal fluids, and regulation of TGFß in wound healing [28
mice showed impaired wound healing associated with increased inflammation and neutrophil elastase activity, and enhanced production of TGFß. Our in vivo studies were done in nude mice where the immune system is attenuated and may allow the non-inflammation inhibitory activity of SLPI to be more prominent.
Our findings of increased aggressiveness with increased SLPI are similar to those seen by Devoogdt in the syngeneic lung cancer model. There, SLPI was associated with increased tumorigenicity and pulmonary metastases. However, these effects were attenuated by mutation of Leu72 which abrogates the protease inhibitory activity. In contrast, our model showed equivalent proliferation in vitro and in vivo of WT and the PI-/SLPI transfectants. There was increased aggressiveness in the form of the quality and quantity of metastases with the WT and PI-/SLPI xenografts. Invasion of PI-/SLPI tumors was more single cell and finger-like; these cells also stained more strongly and more consistently for PRGN. Further, we found in vitro that the WT and PI-/SLPI cells were more resistant to anoikis in vitro. Anoikis has been considered a key component of ovarian cancer dissemination. Shed ovarian cancer cells float in the peritoneum and move through the gutters until finding a point of secondary implantation and invasion. Cells must have an intrinsic anoikis survival capacity for successful shedding. Our findings suggest that SLPI may have a differential effect depending upon the cellular background, through the protease inhibition role, and as a putative chaperone and/or growth factor.
SLPI may function either through its protease inhibitory activity as previously reported, or as our current mutation studies show independently of that function, and therefore independently of elastase. The serine protease inhibitory activity has been reported as broader in outreach than regulation of elastase; our demonstration that SLPI is an inhibitor of plasmin serine protease function and overcome elastase apoptosis argues that SLPI may modify multiple serine protease events in ovarian cancer biology. SLPI may interact in a protease-inhibitor independent fashion in ovarian cells with growth stimulating signals, such as PRGN. Our neutralizing and silencing studies suggested SLPI may function with PRGN as a partner or chaperone [10
]. Partnering of SLPI and PRGN was reported by Zhu et al. who found this relationship stimulated wound-healing [24
]. SLPI-binding to PRGN prevented PRGN degradation by inflammatory elastase-like proteases and restored the PRGN anti-inflammatory and growth-promoting properties. They suggested this occur by direct inhibition of elastase enzymatic activity and also through stabilization PRGN to prevent elastase access to PRGN cleavage sites. Our study confirms the indirect blocking role, showing the PI- mutants can partner with PRGN and also that they provide equal or greater protection of PRGN from elastase.
PRGN itself is an important regulator of cell growth, survival and migration [16
]. We previously reported high PRGN expression in various ovarian cancer tumor specimens and cell lines [16
], and we and others showed that antisense and antibody-mediated blockade of PRGN in ovarian cancer cell lines reduced proliferation, agar colony formation, invasion, and triggered apoptosis [16
]. PRGN has been shown to contribute to transformation of ovarian surface epithelium (OSE) or uterine smooth muscle cells through activation of the Ras pathway [20
]. These studies underscore the pro-survival function of PRGN, and through PRGN of its partner SLPI.
We thus advance our initial report of a functional role for amplified SLPI in ovarian cancer. The current findings coupled with activity of exogenously provided SLPI and apoptosis in response to neutralizing anti-SLPI antibodies suggest that SLPI might be successfully abrogated using blocking approaches. SLPI should be targeted for development of molecular therapeutics for ovarian cancer.