Investigation into the mechanism of the loss of laminin 5 (alpha3beta3gamma2) expression in prostate cancer

Abstract

Laminin 5 is a pivotal hemidesmosomal protein involved in cell stability, migration, and anchoring filament formation. Protein and gene expression of the alpha3, beta3, and gamma2 chains of laminin 5 were investigated in normal and invasive prostate carcinoma using immunohistochemistry, Northern analysis, and in situ hybridization. Laser capture microdissection of normal and carcinomatous glands, in conjunction with RNA amplification and reverse Northern analysis, were used to confirm the gene expression data. Protein and mRNA expression of all three laminin 5 chains were detected in the basal cells of normal glands. In contrast, invasive prostate carcinoma showed a loss of beta3 and gamma2 protein expression with variable expression of alpha3 chains. Despite the loss of protein expression, there was retention of beta3 and gamma2 mRNA expression as detected by in situ hybridization, Northern and reverse Northern analysis. Our findings imply that an altered mechanism of translation of beta3 or gamma2 mRNAs into functional proteins contributes to failure of anchoring filaments and hemidesmosomal formation. The resultant hemidesmosome instability or loss would suggest a less stable epithelial-stromal junction, increased invasion and migration of malignant cells, and disruption of normal integrin signaling pathways.

Figure 1.

Differential expression of laminin 5 chains by human prostate. A–C: Immunohistochemical staining using mAb BM165 (α3), BM140 (β3), and polyclonal antibody J20 (γ2). Note in A the protein for the α3 chain of laminin 5 is retained by both normal prostate epithelium (n) and variably by prostate carcinoma (c), but the β3 and the γ2 chains were not detected in invasive carcinoma (B and C). Original magnification, ×100.

Figure 2.

In situ hybridization using chain-specific laminin 5 probes. In situ hybridization was performed using digoxigenin-labeled antisense RNA probes of the laminin 5. A: α3. B: β3. C: γ2. The mRNAs of the three chains are present in normal glands (n) as well as in the invasive carcinoma (c). D: Higher magnification of the area of C designated by the black box showing that the level of expression of the γ2 message appears to be higher in the carcinoma cells than in normal glandular epithelium. Original magnifications, ×100 (A–C) and ×400 (D).

Figure 3.

Northern analysis of laminin 5 β3 and γ2 chain expression in human prostate. Total RNA was isolated from the normal prostate epithelial cell line, PrEC 4428, and from three cases of prostate carcinoma and adjacent normal tissue. The Northern blot was probed with ³²P-dCTP-labeled cDNA probes specific for the β3 and γ2 chains of laminin 5, and GADPH. Total RNA from normal prostate epithelial cell line, PrEC 4428, was used as positive control, and GAPDH hybridization was used as a loading control. Northern analysis confirmed the presence of the β3 and γ2 mRNAs of laminin 5 in both normal prostate and prostate carcinomas.

Figure 4.

LCM of prostate carcinoma. Laser capture of target cells was performed using the PixCell II image archiving system on 5-μm cryostat sections of snap-frozen prostate tissue stained by H&E. A: H&E-stained section before LCM. B: Target areas identified as carcinoma by H&E staining are designated by black boxes. Normal glands targeted for capture are surrounded by white boxes. C: White areas of H&E section are the holes left after carcinoma glands have been laser-captured and then transferred to cap surface (D). Original magnification, ×100.

Figure 5.

Reverse Northern blot of laser-captured prostate glands. A: Reverse Northern slot blot of one representative case of malignant prostate tissue. Probe samples were obtained by laser-captured microdissection of ∼1500 cells from sections of snap-frozen prostate tissue. GAPDH and antisense laminin 5 β3 sequences served as positive and negative controls, respectively. Band intensity serves as a relative indicator of the amount of message RNA present in the laser-captured sample before amplification. B: Summary of reverse Northern blot data showing comparative quantitation of cDNA probe phosphor intensities of normal (NL) and carcinoma (Ca). Two representative normal samples (N1 and N2) and five malignant samples (C1 to C5) are arranged by individual (rows) and type of target cDNA (columns). Individual band intensities are expressed as a percentage of the intensity of all seven possible bands on the blot from which it came.