Gene expression relationship between prostate cancer cells of Gleason 3, 4 and normal epithelial cells as revealed by cell type-specific transcriptomes

Abstract

Background: Prostate cancer cells in primary tumors have been typed CD10-/CD13-/CD24hi/CD26+/CD38lo/CD44-/CD104-. This CD phenotype suggests a lineage relationship between cancer cells and luminal cells. The Gleason grade of tumors is a descriptive of tumor glandular differentiation. Higher Gleason scores are associated with treatment failure.

Methods: CD26+ cancer cells were isolated from Gleason 3+3 (G3) and Gleason 4+4 (G4) tumors by cell sorting, and their gene expression or transcriptome was determined by Affymetrix DNA array analysis. Dataset analysis was used to determine gene expression similarities and differences between G3 and G4 as well as to prostate cancer cell lines and histologically normal prostate luminal cells.

Results: The G3 and G4 transcriptomes were compared to those of prostatic cell types of non-cancer, which included luminal, basal, stromal fibromuscular, and endothelial. A principal components analysis of the various transcriptome datasets indicated a closer relationship between luminal and G3 than luminal and G4. Dataset comparison also showed that the cancer transcriptomes differed substantially from those of prostate cancer cell lines.

Conclusions: Genes differentially expressed in cancer are potential biomarkers for cancer detection, and those differentially expressed between G3 and G4 are potential biomarkers for disease stratification given that G4 cancer is associated with poor outcomes. Differentially expressed genes likely contribute to the prostate cancer phenotype and constitute the signatures of these particular cancer cell types.

Figure 1

Prostate cancer cell gene expression. A: Cancer CD phenotype. Serial sections of human prostate from specimen 05-179 were stained for CD10, CD13, and CD26. Cancer glands in these serial sections are not stained for CD10 (top left) and CD13 (top right), whereas a single benign gland (lower left in the photomicrographs) is; original magnification 200×. Cells in the cancer glands, like luminal cells of benign glands, are positive for CD26 (bottom left). In the higher magnification (400×, bottom right), the cancer cells show the characteristic prominent nucleoli. B: Dataset clustering. The relationship of the CD26⁺ cancer cell transcriptome to that of CD49a stromal, CD31 endothelial, CD26 luminal or CD104 basal is shown. Whole transcriptomes were used to generate Euclidean distances. The self replicates of CD26⁺ cancer (blue) are in the middle. C: Gene expression in virtual Northern blot format. Affymetrix signal values are represented on a gray scale. The cell type-specific transcriptomes are listed on the top with red arrowheads to identify CD26⁺ cancer and luminal. C4-2, CL1, CL1.1, CL1.31, DU145, LNCaP, PC3 are cancer cell lines.

Figure 2

PCA projections of Gleason 3 and Gleason 4 cancer transcriptomes. A: Three-dimensional projection of G3 and G4 transcriptomes with respect to those of prostate cell transcriptomes for stromal cells (S), luminal cells (L), endothelial cells (E), and basal cells (B) in a PCA-derived subspace. B: Two other orientations from a different point of perspective of the PCA space. The 3D coordinate system was obtained by performing the usual PCA, defining the rotation matrix related to the top three principal components and applying it to all datasets to create a subspace that highlights the expression particularities of each prostate cell type.

Figure 3

Cancer cell vs. cancer tissue transcriptomes. Profile of genes with ≥ 8-fold difference in expression in cancer compared to normal luminal cells as determined by analysis of sorted CD26⁺ (NP and CP) cells and whole tissue transcriptomes (NP and CP). Positive log2(Cancer/Normal) on the y-axis indicates increased cancer expression while negative log2 indicates decreased cancer expression.

Figure 4

Prostate cancer genes. A: Cancer expression of DLX1. Immunohistochemistry shows positive nuclei (three examples indicated by red arrows, left panel) of cancer cells (case 04-030E) strongly stained. Cancer glands are negative for basal CD104 (top right) and luminal CD10 (bottom right); original magnification 200×. B: Candidate prostate cancer biomarkers. Genes with increased expression (based on array signal values as used in Fig. 1C) in CD26⁺ cancer vs. luminal (arrowheads) that encode secreted - AGR2, CEACAM5, CRISP3, NEO1, membrane anchored - LOX, BCMP11 or transmembrane - KCNH8, KCNG3 proteins are shown. C: Western blot verification of AGR2 expression. In nearly all cases of CP except 08-032 (indicated by arrow), AGR2 protein was detected at higher levels. AGR2 in any matched NP samples may be derived from diffusion from the cancer foci. D: Candidate biomarkers from LNCaP. Through dataset analysis, these genes were found differentially expressed between CD10^-/CD26⁺ cancer and CD10⁺/CD26^- LNCaP.

Figure 5

Differential gene expression between Gleason 3 and Gleason 4 transcriptomes. A: Expression levels of selected genes in G3 and G4 relative to luminal cells. B: Expression levels of these G3 and G4 genes in G3+4 vs. G4+5 tumors. C: Differential expression of epithelial genes in G3 and G4 relative to luminal cells. D: Display for LNCaP genes in primary tumors. Positive log2(Cancer/Normal) on the y-axis indicates increased cancer expression and negative log2 indicates decreased cancer expression.

Figure 6

Dataset comparison to 05-179 Gleason 3 transcriptome. A: Dataset correlation. Each node represents a single dataset with the first data point being the 05-179 CD26⁺ cancer transcriptome (in red). In the GSE6099 display, the flanking lowest correlates are those of non-cancer and stromal cells (see B). B: Single-gene query displays. Shown are those for MME/CD10 in both GSE6099 (top), where the laser-captured Gleason grade 3's are indicted by black dots, and GSE 5132 (bottom); FZD8 and AGR2 in GSE6099. Positive log2(Cancer/Normal) on the y-axis indicates increased cancer expression and negative log2 indicates decreased cancer expression.