The serine protease prostasin (PRSS8) is a potential biomarker for early detection of ovarian cancer

Abstract

Background: Ovarian cancer (OVC) is the deadliest of all gynecologic cancers, primarily as a consequence of asymptomatic progression. The complex nature of OVC creates challenges for early detection, and there is a lack of specific and sensitive biomarkers suitable for screening and detecting early stage OVC.

Methods: Potential OVC biomarkers were identified by bioinformatic analysis. Candidates were further screened for differential expression in a library of OVC cell lines. OVC-specific overexpression of a candidate gene, PRSS8, which encodes prostasin, was confirmed against 18 major human cancer types from 390 cancer samples by qRT-PCR. PRSS8 expression profiles stratified by OVC tumor stage-, grade- and subtype were generated using cDNA samples from 159 OVC samples. Cell-specific expression and localization of prostasin was determined by immunohistological tissue array analysis of more than 500 normal, benign, and cancerous ovarian tissues. The presence of prostasin in normal, benign, and OVC serum samples was also determined.

Results: Gene expression analysis indicated that PRSS8 was expressed in OVC at levels more than 100 fold greater than found in normal or benign ovarian lesions. This overexpression signature was found in early stages of OVC and was maintained in higher stages and grades of OVC. The PRSS8 overexpression signature was specific for OVC and urinary bladder cancer among 18 human cancer types. The majority of ovarian cell lines overexpressed PRSS8. In situ hybridization and histopathology studies of OVC tissues indicated that overexpression of prostasin was largely localized to tumor epithelium and was absent in neighboring stroma. Significantly higher levels of prostasin were found in early stage OVC serum samples compared to benign ovarian and normal donor samples.

Conclusions: The abundant amounts of secreted prostasin found in sera of early stage OVC can potentially be used as a minimally invasive screening biomarker for early stage OVC. Overexpression of PRSS8 mRNA and high levels of prostasin in multiple subtypes of early stage ovarian tumors may provide clinical biomarkers for early detection of OVC, which can potentially be used with CA125 and HE4.

Keywords: Biomarkers; Diagnostic; Early detection; Ovarian cancer; PRSS8; Prostasin; Serum.

Fig. 1

Expression of PRSS8 is specific to OVC cells. Expression of PRSS8 in normal and OVC cell lines (a), in tissue of normal ovary, OVC, and other types of cancer (b), and as detected in ISH (C – left) and IHC (C – right) in normal (N – in bottom left corner of ISH and IHC) and OVC tissues (magnification 20X and 40X). a Gene expression of PRSS8 in OVC cell lines was analyzed by qRT-PCR and normalized. Fold change represents the level of gene expression in OVC cell lines normalized against a normal ovarian cell line IOSE523. b Fold change represents the level of gene expression in cancer tissue samples normalized against the corresponding normal tissue; here, p < 0.001, PRSS8 expression in OVC vs. other cancer types; one-way ANOVA (SigmaStat). c (ISH) left, mRNA of tissues of ovarian tumors in normal individuals and OVC patients were hybridized In situ (20X magnification). (IHC) right, Detection of prostasin levels by immunohistochemistry. Nuclear stain is indicated with solid arrows

Fig. 2

PRSS8 expression is upregulated throughout all stages of OVC. a PRSS8 gene expression levels were measured in tumor tissues of OVC patients at different stages of the disease, and were plotted as individual fold increases. b Average levels of PRSS8 in tumor tissues of OVC patients presented as a function of disease grade

Fig. 3

PRSS8 expression is elevated in different types of OVC. a Levels of PRSS8 were measured in all stage groups of OVC patients and are presented as fold increase over expression in normal individuals. b Expression of PRSS8 in early stage patients (stages I and II) in groups representing 5 different OVC subtypes. Results are presented as fold change compared to expression in normal individuals. OVC subtypes are as follows: papillary serous (PSR), serous (SR), endometriod (ENDM), borderline (BRDLINE), clear cell (CLRC)

Fig. 4

Localization of prostasin in OVC, benign, and normal ovarian tissues. Tissue arrays of OVC, benign, and normal cases were stained for prostasin by immunohistochemistry (magnification of 10-20X). Tissue arrays were generated from normal ovary (N), benign ovary tissue (B), endometriod adenocarcinoma (E), papillary serous adenocarcinoma (PS), clear cell carcinoma (CC), serous adenocarcinoma (S), mucinous adenocarcinoma (M), and borderline carcinoma (BL)

Fig. 5

PRSS8 is upregulated in tissues of OVC patients compared to benign and normal tissues. Bar plots of PRSS8 immunostaining score by OVC stage (a) and OVC grade (b); n = number of stained arrays in each group. Immunostaining of all tissue arrays used in this study was score (range: 0–3) according to levels of staining, where score of (0) means negative staining, (1)-weak positive staining, (2)-positive staining, (3)-strong positive staining. Serum samples from OVC (early-stage), benign, and normal subjects (7 in each group) were subjected to western blot for the appearance of PRSS8 and densitometry values for each group were plotted (c). Primary PRSS8 antibody used in this study is a custom-made antibody (see details in Additional file 1)