The cell-surface serine protease prostasin is lost during cervical squamous cell carcinogenesis

Abstract

The glycosylphosphatidylinositol (GPI)-anchored serine protease prostasin has been reported to have increased expression with tumor-promoting properties in some cancer types, while expression is lost and prostasin displays tumor-suppressing properties in other cancer types. Due to these context-dependent and opposing expression patterns and functions of prostasin, characterization of each cancer type is important. In the present study, we aimed to determine the expression of prostasin in the normal cervix and in cervical squamous cell carcinoma (CSCC), the most common type of cervical cancer. We found that prostasin protein is expressed in both murine and human cervix and is consistently localized on the cell surface in suprabasal layers of squamous cells in healthy cervical epithelia. To assess whether prostasin protein is differentially expressed during cervical carcinogenesis, we performed a comprehensive immunohistochemical analysis of prostasin protein expression levels and localization in tissue arrays of paraffin-embedded human cervical carcinomas compared to the corresponding normal tissue. Prostasin protein is expressed in the well-differentiated cellular strata with expression patterns similar to pan-keratin and E-cadherin, and is lost during the dedifferentiation of epithelial cells, a hallmark of high-grade CSCC. The prostasin expression profile, with differential expression in cancer, provide valuable information that may give clues to the function(s) of this protease in normal epithelial biology and carcinogenesis.

Keywords: Cell surface serine protease; Cervical squamous cell carcinoma; PRSS8; Prostasin.

Fig. 1

Analysis of prostasin protein expressed in mammalian cells and validation of prostasin antibodies. (a) Schematic representation of the prostasin protein containing a C-terminal GPI-anchor and an N-terminal serine protease (SP) domain. The prostasin antigens used for generation of the two polyclonal antibodies pAb1 and pAb2, and the monoclonal antibody mAb are indicated. Table below indicates working concentrations of antibodies (b) Proteins from whole mouse organ lysates were separated by SDS-PAGE and analyzed by western blotting using the pAb1 antibody. β-tubulin staining of the membranes was performed to ensure equal protein loading. (c) C-33 A cells were transfected with full-length human prostasin cDNA (Pros) or the vector without cDNA insert (empty vector, EV). Endogenous prostasin was silenced in MCF0A cells using two non-overlapping synthetic RNA duplexes (si1 and si2). A %GC-matched non-targeting RNA duplex was used as a negative control (Con). Whole cell lysates were separated by SDS-PAGE and analyzed by western blotting using prostasin antibodies, pAb1 (left), pAb2 (middle), or mAb (right). β-actin staining of the membranes was performed to ensure equal protein loading. The positions of molecular mass markers (kilodaltons) are indicated at the left of all blots.

Fig. 2

Expression of prostasin, E-cadherin, and pan-keratin in cervix and CSCC. Immunohistochemical analysis of prostasin expression in normal human squamous epithelia and CSCC using two independent prostasin antibodies (mAb and pAb2). (a) Representative example of prostasin in normal cervix detected with mAb. Strong epithelial cell surface staining in suprabasal, squamous epithelial cells in normal cervix is observed (white arrowheads) with no significant staining in the basal cells (black arrowheads) or in the mesenchymal compartment (indicated with asterisks) Epi=normal epithelium. (b, c) Staining for the epithelial differentiation markers E-cadherin (b) and pan-keratin (c) showed a similar population of cells as for prostasin. (d-k) Representative examples of serial sections of a grade 2 CSCC focused on areas with squamous epithelial differentiation stained with prostasin antibody mAb (d) or pAb2 (e). (f-g) Corresponding E-cadherin and pan-keratin staining. (h) Primary antibodies were substituted with non-immune rabbit/mouse IgG in serial sections of all samples and no significant staining was observed. (i-k) High magnification photos of the sections shown in (d-f) illustrating near identical staining using two different prostasin antibodies and E-cadherin in cells displaying near identical localization staining is in well-differentiated CSCC cells (black arrowheads) Size bars all panels; 25 μm.

Fig. 3

Expression of prostasin protein in LSIL, HSIL, low, moderate, and high grade cervical squamous cell carcinomas. (a-h) Representative IHC detection of prostasin protein in cervical tissue sections using mAb. (a-b) Prostasin expression in all suprabasal layers with strongest in the most differentiated layers of LSIL (arrowheads, staining intensity 3) with more diffuse and weaker staining in HSIL (arrowhead, staining intensity 1). (c, d) In grade 1 cervical squamous carcinomas prostasin staining was detected in the well-differentiated carcinoma cells (arrowheads, staining intensity 3) with weak and diffuse staining in less differentiated cells. (d) High magnification showing strong cell-surface staining in well-differentiated grade 1 CSCC cells (arrowhead). (e, f) Example of weak, diffuse and intracellular staining in majority of the CSCC cells in a grade 2 cervical tumor (e) (arrowhead, staining intensity 2) and (f) a different grade 2 tumor with only a few cells with detectable prostasin staining (arrowheads, staining intensity 1). (g, h) In high grade poorly differentiated tumors (Grade 3) prostasin was below the detection limit (g) (staining intensity 0) of this assay or the staining intensity was very low and only observed in few cells (staining intensity 1) (h). Epi, epithelial cells, Ca=carcinoma cells. The stromal compartments of the tumors are indicated with asterisks. LSIL=Low-grade Squamous Intraepithelial Lesion; HSIL=High-grade Squamous Intraepithelial Lesion. Size bars all panels; 25 μm.

Fig. 4

Significantly decreased expression of prostasin protein in high grade cervical squamous cell carcinomas. Scatterplots illustrating the intensity of IHC staining of prostasin in tissue array sections. Horizontal bars represent median values. Staining intensities were determined as described in “Materials and Methods”. (**P<0.01, *** P<0.001, ****P<0.0001). Statistical analysis is described in “Materials and Methods”. LSIL=Low-grade Squamous Intraepithelial Lesion; HSIL=High-grade Squamous Intraepithelial Lesion.