The alarm anti-protease secretory leukocyte protease inhibitor (SLPI) is frequently overexpressed in ovarian cancer cells and has been proposed for inclusion in biomarker panels but function remains unclear. We hypothesized that SLPI overexpression promotes ovarian cancer growth and survival. Low SLPI-expressing Hey-A8 ovarian cancer cells were engineered to produce functional (WT) or protease inhibitor-null (PI-) mutant SLPI; lack of PI activity was confirmed by enzymatic assay. WT/SLPI and PI-mutants stimulated significant proliferation and survival of Hey-A8 ovarian cancer cells under basal culture conditions (P £ 0.02), in soft agar colony number and size (P £ 0.05), and in anoikis resistance (P £ 0.005). SLPI protected the ovarian cancer survival factor, progranulin (PRGN), and HEY-A8 cells from degradation and apoptosis due to neutrophil elastase. PI-/SLPI cells had greater protective activity than WT/SLPI cells. HEY-A8 murine xenografts revealed enhanced solid tumor formation, dissemination, and invasion in WT/SLPI and PI-/SLPI mutants. (1) Modest improvements in survival of ovarian cancer patients have been achieved over the last decade, driving a need for a better understanding of the mechanisms underlying ovarian cancer formation and progression. Secretory leukocyte protease inhibitor (SLPI) has been reported as one of the most commonly amplified genes in epithelial ovarian cancer, (2)(3)(4)(5) an observation that is explained in part by genomic amplification of the SLPI locus, 20q12-13. (6) Significantly elevated serum concentrations of SLPI have been found in ovarian cancer patients compared to healthy women or women with benign ovarian cysts, leading to the inclusion of SLPI in some experimental ovarian cancer biomarker panels. (7) Secretory leukocyte protease inhibitor is a secreted pleiotropic protein with tissue-protective, regenerative, and anti-inflammatory properties. (8) It protects normal mucosal tissues against detrimental proteolytic actions of inflammatory serine proteases, such as neutrophil elastase, through direct inhibition of these enzymes. (9,10) Evidence also points toward direct roles for SLPI in tumor development and progression. (2,11,12) Some of these studies are conflicting depending upon the model used, suggesting context dependence for SLPI function. SLPI was reported to both stimulate and counteract tumor formation, invasion and/or metastasis of cancer cells. (10,(12)(13)(14)(15) The role of the anti-protease properties of SLPI in cancer remains under investigation.Progranulin (PRGN) is a potent growth and survival factor for both normal and cancer cells. (16)(17)(18)(19)(20)(21) Mutations in PRGN are associated with frontotemporal dementia and other neuromuscular degenerative syndromes, (22,23) consistent with our reported prosurvival role. We have shown that anti-sense silencing of PRGN in Hey-A8 ovarian cancer cells reduced in vitro proliferation rates and soft agar colonization. (16) Treatment of ovarian cancer cell lines with neutralizing anti-PRGN antibodies induced a...

BackgroundEpithelial ovarian cancer (EOC) is the deadliest gynecologic malignancy in the United States. Unfortunately, a validated protein biomarker-screening test to detect early stage disease from peripheral blood has not yet been developed. The present investigation assesses the ability to identify tumor relevant proteins from ovarian cancer proximal fluids, including tissue interstitial fluid (TIF) and corresponding ascites, from patients with papillary serous EOC and translates these findings to targeted blood-based immunoassays.Methodology/Principal FindingsPaired TIF and ascites collected from four papillary serous EOC patients at the time of surgery underwent immunodepletion, resolution by 1D gel electrophoresis and in-gel digestion for analysis by liquid chromatography-tandem mass spectrometry, which resulted in an aggregate identification of 569 and 171 proteins from TIF and ascites, respectively. Of these, peroxiredoxin I (PRDX1) was selected for validation in serum by ELISA and demonstrated to be present and significantly elevated (p = 0.0188) in 20 EOC patients with a mean level of 26.0 ng/mL (±9.27 SEM) as compared to 4.19 ng/mL (±2.58 SEM) from 16 patients with normal/benign ovarian pathology.Conclusions/SignificanceWe have utilized a workflow for harvesting EOC-relevant proximal biofluids, including TIF and ascites, for proteomic analysis. Among the differentially abundant proteins identified from these proximal fluids, PRDX1 was demonstrated to be present in serum and shown by ELISA to be elevated by nearly 6-fold in papillary serous EOC patients relative to normal/benign patients. Our findings demonstrate the facile ability to discover potential EOC-relevant proteins in proximal fluids and confirm their presence in peripheral blood serum. In addition, our finding of elevated levels of PRDX1 in the serum of EOC patients versus normal/benign patients warrants further evaluation as a tumor specific biomarker for EOC.

Objectives Secretory leukocyte protease inhibitor (SLPI) is amplified in serous ovarian cancer. We have dissected its function, showing it is a survival factor for ovarian cancer and promotes tumorigenesis and paclitaxel-resistance. We hypothesized that the protease inhibitory function was responsible for modulating SLPI’s invasive capacity. Methods Stable HEYA8 ovarian cancer transfectants expressing vector, wild type SLPI, and protease inhibitor null (F-)SLPI were examined in vitro and in xenografts. Invasion, enzyme activity, and MMP production and function assays were applied. SLPI and MMP immunoexpression were graded on tissue microarray and clinical samples. Statistical comparisons used unpaired T test and ANOVA, where appropriate. Results SLPI and F-SLPI cells caused greater parenchymal and peritoneal dissemination over control cells in xenografts and invasion assays (p<0.001). MMP-9 protease activity was increased in SLPI and F-SLPI cells over control. SLPI, but not F-SLPI, inhibited plasmin activity, necessary for MMP-9 activation and release, and inhibited activation of MMP-9. However, paradoxically, both induced quantitative MMP-9 transcription (p<0.05) and protein (p<0.008), yielding an increased net MMP-9 activity in the face of plasmin inhibition. SLPI and MMP-9 expression were strongly correlated in serous ovarian cancers (r2=0.986) and a set of ovarian cancers (p<0.02). SLPI expression was greater in serous than endometrioid ovarian cancers (p=0.04). Conclusions SLPI stimulates ovarian cancer invasion, modulated in part by its serine protease inhibitory activity attenuating MMP-9 release. However, SLPI induction of MMP-9, independent of protease inhibition activity, is greater yielding a net pro-invasive behavior. These findings further support SLPI as a molecular target for ovarian cancer.