Identification of genes up-regulated in urothelial tumors: the 67-kd laminin receptor and tumor-associated trypsin inhibitor

Abstract

Studies investigating changes in gene expression in urothelial carcinoma have generally compared tumors of different stages and grades but comparisons between low-grade, noninvasive tumors and normal urothelium are needed to identify genes involved in early tumor development. We isolated the urothelium from a low-grade tumor and corresponding normal mucosa by laser capture microdissection on frozen sections. The RNA extracted was amplified to generate suppressive subtractive cDNA libraries. Random sequencing of cDNA clones identified approximately 100 unique species. Of these 83% were known genes, 15% had homology to genes with an unknown function in humans, and 2% did not show homology to any published gene sequence. Two of the known genes, the 67-kd laminin receptor (67LR) and tumor-associated trypsin inhibitor (TATI), had previously been associated with metastatic progression in many tumor types, although 67LR has not been investigated in urothelial tumors. Immunolabeling of the original tissue with antibodies against these two genes confirmed overexpression, validating our strategy: 67LR was not expressed in the normal urothelium but was present in the tumor, whereas TATI expression was confined to umbrella cells in the normal urothelium, but extended to all cell layers in the tumor. We investigated both markers further in a separate series of tumors of different stages and grades. TATI was more consistently overexpressed than 67LR in all tumor grades and stages. Levels of secreted TATI were significantly higher in urine samples from patients with tumors compared to controls. Our strategy, combining laser capture microdissection and cDNA library construction, has identified genes that may be involved in the early phases of urothelial tumor development rather than with disease progression, highlighting the importance of comparing tumor with normal rather than just tumors of different stages and grades.

Figure 1.

PCR analysis of subtraction efficiency. a: GAPDH band is seen after 18 cycles in the unsubtracted cDNA. b: GAPDH band appears five cycles later in the subtracted material, five cycles representing ∼20-fold difference in levels, as described by the PCR Select subtractive hybridization kit method (Clontech).

Figure 2.

67LR expression in normal urothelium and tumors. a: Example of normal urothelium showing occasional positive basal cells (the test case was negative). b: More widespread, but still predominantly basal expression in a noninvasive papillary tumor. c: Membrane and cytoplasmic expression in invasive foci contrasting with the absence of expression in residual Von Brunn’s nests. Original magnifications, ×200.

Figure 3.

TATI expression in normal urothelium and tumors. a: Expression predominantly along the lumenal edge of umbrella cells in normal urothelium. b and c: Full thickness, cytoplasmic expression in papillary tumor (b), and dysplastic urothelium (c). d: Cytoplasmic expression in invasive tumor foci. Original magnifications, ×200.

Figure 4.

Comparison of CK20 and TATI expression. a: The papillary tumor is negative for CK20 although the adjacent normal urothelium shows expression in superficial cells. b: The same tumor shows strong full thickness TATI expression with positivity along the lumenal edge in the normal urothelium. Original magnifications, ×50.

Figure 5.

a: Comparison of urinary total protein/creatinine index between patients and controls. Lines represent median values. Note the logarithmic scale. b: Comparison of urinary TATI/creatinine index between patients and controls. Lines represent median values.