Prostate stem cell antigen: a cell surface marker overexpressed in prostate cancer

Abstract

The identification of cell surface antigens is critical to the development of new diagnostic and therapeutic modalities for the management of prostate cancer. Prostate stem cell antigen (PSCA) is a prostate-specific gene with 30% homology to stem cell antigen 2, a member of the Thy-1/Ly-6 family of glycosylphosphatidylinositol (GPI)-anchored cell surface antigens. PSCA encodes a 123-aa protein with an amino-terminal signal sequence, a carboxyl-terminal GPI-anchoring sequence, and multiple N-glycosylation sites. PSCA mRNA expression is prostate-specific in normal male tissues and is highly up-regulated in both androgen-dependent and -independent prostate cancer xenografts. In situ mRNA analysis localizes PSCA expression in normal prostate to the basal cell epithelium, the putative stem cell compartment of the prostate. There is moderate to strong PSCA expression in 111 of 126 (88%) prostate cancer specimens examined by in situ analysis, including high-grade prostatic intraepithelial neoplasia and androgen-dependent and androgen-independent tumors. Flow cytometric analysis demonstrates that PSCA is expressed predominantly on the cell surface and is anchored by a GPI linkage. Fluorescent in situ hybridization analysis localizes the PSCA gene to chromosome 8q24.2, a region of allelic gain in more than 80% of prostate cancers. A mouse homologue with 70% amino acid identity and similar genomic organization to human PSCA has also been identified. These results support PSCA as a target for prostate cancer diagnosis and therapy.

Figure 1

Northern blot analysis of PSCA expression. (A) Total RNA from normal prostate and LAPC-4 androgen-dependent (AD) and -independent (AI) prostate cancer xenografts were analyzed by using PSCA- or PSA-specific probes. Equivalent RNA loading and RNA integrity were demonstrated separately by ethidium staining for 18S and 28S RNA and are not shown. (B) Human multiple tissue Northern blot analysis of PSCA. The filter was obtained from CLONTECH and contains 2 μg of poly(A) RNA in each lane.

Figure 2

Amino acid sequences of human SCA-2 (hSCA-2), human PSCA (hPSCA), and mouse PSCA (mPSCA). Shaded regions highlight conserved amino acids, and conserved cysteines are indicated by boldface type. Four predicted N-glycosylation sites in PSCA are indicated by asterisks. The underlined amino acids at the beginning and end of the protein represent amino-terminal hydrophobic signal sequences and C-terminal GPI-anchoring sequences, respectively.

Figure 3

In situ hybridization with antisense RNA probe for human PSCA on normal and malignant prostate specimens. (A) PSCA is expressed by a subset of basal cells within the basal cell epithelium (black arrows) but not by the terminally differentiated secretory cells lining the prostatic ducts. (×400.) (B) PSCA is expressed strongly by a high-grade prostatic intraepithelial neoplasia (black arrow) and by invasive prostate cancer glands (yellow arrows) but is not detectable in normal epithelium (green arrow) at ×40 magnification. (C) Strong expression of PSCA in high-grade carcinoma. (×200.)

Figure 4

Biochemical analysis of PSCA. (A) PSCA was immunoprecipitated from 293T cells transiently transfected with a PSCA construct and then digested with either N-glycosidase F or O-glycosidase. (B) PSCA was immunoprecipitated from 293T transfected cells and from conditioned medium from these cells. Cell-associated PSCA migrates higher than secreted PSCA on a 15% polyacrylamide gel. (C) Flow cytometry analysis of mock-transfected 293T cells, PSCA-transfected 293T cells, and LAPC-4 prostate cancer xenograft cells by using an affinity-purified polyclonal anti-PSCA antibody. Cells were not permeabilized to detect only surface expression. The y axis represents relative cell number and the x axis represents fluorescent staining intensity on a logarithmic scale.

Figure 5

In situ hybridization of biotin-labeled PSCA probes to human metaphase cells from phytohemagglutinin-stimulated peripheral blood lymphocytes. The chromosome 8 homologues are identified with arrows; specific labeling was observed at 8q24.2. (Inset) Partial karyotypes of two chromosome 8 homologues illustrating specific labeling at 8q24.2 (arrowheads). Images were obtained by using a Zeiss Axiophot microscope coupled to a cooled charge-coupled device camera. Separate images of 4′,6-diamidino-2-phenylindole-stained chromosomes and the hybridization signal were merged by using image analysis software (nu200 and image 1.57).