Immunohistochemical demonstration of the zinc metalloprotease insulin-degrading enzyme in normal and malignant human breast: Correlation with tissue insulin levels

Abstract

Insulin is a hormone crucial to metabolism and an essential growth factor for normal and neoplastic tissues. We have now determined insulin in extracts of 23 primary breast cancer specimens and of non-neoplastic breast tissues by a chemiluminescent immunoassay. Remarkably, insulin was measured only in grade 3 tumors, whereas grade 2 carcinomas and the normal mammary gland were each insulin-negative. We also performed immunohistochemistry for insulin-degrading enzyme (IDE), a cytoplasmic zinc metalloprotease belonging to the inverzincin family and participating in insulin cleavage. IDE was detected in most insulin-positive grade 3 carcinomas, indicating that it might be dysfunctional in these anaplastic tumors. IDE was equally present in the insulin-negative grade 2 carcinomas. Moreover, five grade 3 carcinomas and one grade 2 carcinoma displayed a loss of heterozygosity in the 10q chromosomal region harboring the IDE gene, but, despite these alterations, IDE was detected immunohistochemically, indicating a retention of the second allele. Compared to the expression of IDE in 92% of the tumors examined, only 57% of 21 normal breast specimens stained positively for IDE. In contrast to this increase in IDE-positive epithelial cells in breast cancer vs. normal breast, additional immunohistochemical analysis of 17 node-positive breast carcinomas and corresponding tumor-bearing lymph nodes showed that IDE expression decreases from primary tumor to lymph node metastasis. Altogether, this study represents the first demonstration of IDE in normal and neoplastic human mammary tissues. Our present report should also provide an experimental starting point towards exploring a potential role of IDE in the control of tumor progression.

Immunohistochemical evidence of ubiquitous distribution of the metalloendoprotease insulin-degrading enzyme (IDE; insulysin) in human non-malignant tissues and tumor cell lines

“…In addition, we confirm the findings of Chang et al (1997) who reported expression of IDE protein by the colon cancer cell line Caco-2. Interestingly, the breast cancer cell lines investigated showed moderate to strong IDE expression, a finding linked to results obtained by immunostaining of high-grade breast cancer specimens for presence of IDE protein (Radulescu et al, 2007). …”

“…Since cells are formalin-fixed and paraffin-embedded, the procedure for construction of cell microarrays was identical to that for tissue microarrays (see legend to Table 1). Cell microarrays were stained for IDE protein expression using polyclonal rabbit antibody UCG 43/6 and a streptavidin-biotin detection kit (LSAB, K5003; Dako) as described by Radulescu et al (2007).Punch cores of a cell clot of the colon cancer cell line Caco-2 known to express IDE protein (Chang et al, 1997) were used as an internal positive control; for negative controls the primary antibody was omitted. a TUM, Department of Obstetrics and Gynecology, Technical University of Munich. …”

“…Kuo et al (1993) examined presence of IDE in rat tissues at the gene and protein level; expression was demonstrated in the testis, tongue, brain, kidney, prostate, heart, muscle, liver, intestine, skin, spleen, lung, thymus, and uterus. Less, however, is known about distribution of IDE protein in human tissues which was demonstrated to be present in the liver, adipocytes (Valera Mora et al, 2003), muscle cells (Neal and Kitabchi, 1982), erythrocytes (Shii et al, 1986), breast and kidney (Duckworth 1998; Radulescu et al, 2007). …”

“…We recently, for the first time, explored protein expression of IDE in human normal and neoplastic breast tissues by immunohistochemistry (Radulescu et al 2007) and reported that IDE expression is elevated in human breast cancer tissues compared to normal breast glands. Furthermore, we detected insulin protein in high-grade breast cancer employing an immunoassay, a result that is in line with early findings by other groups (Castro et al, 1980; Spring-Mills et al, 1984).…”

Intracellular Insulin in Physiology and Disease: Past, Present and Future

“…Based on the finding by Jarett et al according to which the inhibition of IDE activity leads to an increase in the nuclear translocation of insulin (13), I have hypothesized many years ago that IDE may be a (cytoplasmic) tumor suppressor that physiologically prevents the binding and inactivation of (nuclear) RB by insulin by degrading this hormone in the cytosol (Razvan T Radulescu, unpublished observation, 1994). Subsequently, this conjecture of mine has received structural (14,15) and experimental support (16)(17)(18).…”