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. 1997 Jul 22;94(15):8132-7.
doi: 10.1073/pnas.94.15.8132.

Altered expression of the WT1 wilms tumor suppressor gene in human breast cancer

G B Silberstein  1 , K Van Horn, P Strickland, C T Roberts Jr, C W Daniel
Affiliations

Altered expression of the WT1 wilms tumor suppressor gene in human breast cancer

G B Silberstein et al. Proc Natl Acad Sci U S A. .

Abstract

The product of the WT1 Wilms tumor suppressor gene controls the expression of genes encoding components of the insulin-like growth factor and transforming growth factor beta signaling systems. The role of these growth factors in breast tumor growth led us to investigate possible WT1 gene expression in normal and cancerous breast tissue. WT1 was detected by immunohistochemistry in the normal mammary duct and lobule, and the patterns of expression were consistent with developmental regulation. In a survey of 21 infiltrating tumors, 40% lacked immunodetectable WT1 altogether and an additional 28% were primarily WT1-negative. Cytoplasmic, but not nuclear, localization of WT1 was noted in some tumor cells and WT1 was detected, sometimes at high levels, in more-advanced estrogen-receptor-negative tumors. In this highly malignant subset, the tumor suppressor protein p53, which can physically interact with WT1, was also sometimes detected. WT1 mRNA was detected in normal and tumor tissue by reverse transcription-coupled PCR. Alternative splicing of the WT1 mRNA may regulate gene targeting of the WT1 protein through changes either in its regulatory or zinc-finger domains. The relative proportions of WT1 mRNA splice variants were altered in a random sample of breast tumors, providing evidence that different tumors may share a common WT1-related defect resulting in altered regulation of target genes.

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Figures

Figure 1
Figure 1
Photomicrographs illustrating immunolocalization of WT1 protein in the epithelial cells of normal and cancerous human breast tissue. Cells that are positive for WT1 protein stained brown; the blue nuclear stain is hematoxylin. (A) Nuclear morphology and WT1 staining characteristics of normal mammary ductal cells. Cells with oval nuclei and diffuse chromatin were WT1-positive (triangles and box in the center). Cells with polygonal nuclei and compact chromatin were positive (large arrowheads) or negative (small arrowhead) for WT1. The latter stained deeply with hematoxylin. Myoepithelial cells were WT1-positive (open arrow; dotted line delineates the boundary between lumenal and myoepithelial cells). L, ductal lumen. (Inset) Antibody preincubated with cognate peptide shows reduced staining. Myoepithelial cells are on the left of dotted line. (Bar = 15 μm.) (B) Ductal tip partially involved with carcinoma in situ. Normal cellular arrangement occurred on the right side of the duct; a multilayered tumor appears on left side. L, lumen. The top of the L points to transitional zone between the normal and tumorous sides of the duct. Immunostaining of monolayer of normal-appearing ductal cells on the right side of the duct: some ductal cells with oval nuclei and diffuse chromatin were WT1-positive (triangles); fewer were WT1-negative (curved arrow). Numerous cells with polygonal nuclei were WT1-positive (large arrowheads). The tumor element consists primarily of cells with large oval WT1-negative nuclei containing diffuse chromatin. Some cells in this mass had WT1-positive cytoplasm (small arrowheads). Myoepithelial layer lies outside dotted line. (Bar = 15 μm.) (C) Infiltrating ductal carcinoma. Tumor cells have proliferated to form masses (large arrowheads) within which all cells were WT1-negative. A band of infiltrating tumor cells is visible (small arrowhead). This tumor and the previously described tumor in situ are from the same patient. WT1-positive stromal cell is positive control for immunostaining. (Bar = 20 μm.) (D) Nuclear and cytoplasmic immunostaining for WT1 in infiltrating ductal carcinoma. WT1 immunostaining subdivides this population roughly in half on the basis of cytoplasmic versus nuclear localization of the protein. Cells with only cytoplasmic WT1 immunoreactivity (arrowheads) are intermixed with cells having nuclear stain. In addition, there are some cells that have both cytoplasmic and nuclear WT1 (triangles). (Bar = 20 μm.) (E) Normal mammary lobule. Normal-appearing lumenal epithelium stained with moderate intensity for WT1 (arrowheads). The nuclei of the myoepithelial cells stained deeply for WT1 (open arrow; dotted line delineates boundary between myoepithelial and lumenal cells). (Bar = 20 μm.) (F) Infiltrating lobular carcinoma. Section from vicinity of normal lobule (E). The nuclei of tumor cells are uniformly negative for WT1; however, some cells showed cytoplasmic staining (arrowheads). (Bar = 15 μm.)
Figure 2
Figure 2
WT1 gene structure, splice variants, and detection scheme. The 10 exons of WT1 are shown as boxes and the alternatively spliced 51-base and 9-base sequences encoded by exon 5 and the 3′ end of exon 9 (KTS) are shaded. The location of the four zinc-finger motifs are noted above the boxes and the positions of the forward (F) and reverse (B) oligonucleotide primers relative to the transcript are shown.
Figure 3
Figure 3
Detection and analysis of WT1 mRNA splice variants in normal (reduction mammoplasty) and cancerous breast tissue. (A) Reverse transcription-coupled PCR and Southern blot hybridization analysis of KTS splice variants for presence or absence of the 9-base KTS sequence. Upper row of signals, plus KTS; lower row, minus KTS. Normal (control) is kidney and a mixture of plus- or minus-KTS WT1-containing plasmids. Round 1 primers, F3/B3; round 2 primers, F11/B1. Two rounds of 30 and 25 amplification cycles for mammary gland WT1 mRNA; kidney WT1 mRNA could be detected with a single round of 30 cycles. (B) Reverse transcription-coupled PCR and Southern blot hybridization analysis of exon 5 splice variants. Upper row of signals, plus exon 5; lower row, minus exon 5. Normal (control) is kidney. Round 1 primers, F3/B3; round 2 primers, F1/B5.
Figure 4
Figure 4
Reverse transcription-coupled PCR analysis of the relative proportion of KTS and exon 5 splice variants in normal versus cancerous breast tissue. (A) KTS variants. Bars: solid, plus forms; hatched, minus forms. (Total number of PCRs: normal, n = 31; tumor, n = 17). Normal tissue samples (n = 10) consisted of tissue from eight reduction mammoplasty patients and two samples of normal breast tissue in the vicinity of tumors. As a group and based on age (ranging from 23 to 45 years), these noncancer patients were considered premenopausal with the possible exception of one individual (52 years). Six tumor samples were analyzed. Data are the mean ± SD. (B) Exon 5 variants. Bars: solid, plus forms; hatched, minus forms. (Total number of PCRs: normal, n = 26; tumor, n = 15.)
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