Increased expression of stromelysin-3 in basal cell carcinomas

We examined the expression of two groups of matrix metalloproteinases (MMPs), stromelysin and interstitial collagenase, in human skin cancer by northern blot analysis and in situ hybridization. Stromelyins-3 (ST-3) mRNA was overexpressed more than tenfold in 17 of 19 (89%) specimens of basal cell carcinoma (BCC) but in only three of 13 (23%) cutaneous squamous cell carcinomas (SCCs). Stromelysin-1 and -2 (ST-1/2) mRNA was overexpressed in three of 19 (16%) BCC and three of 13 (23%) SCC. Collagenase mRNA was overexpressed in nine of 19 (47%) BCC and three of 13 (23%) SCC. No mRNA for ST-3, ST-1/2, or collagenase was detected by northern analysis in 21 specimens of adjacent normal skin. Because of these findings, we examined the specific location of the ST-3 mRNA in BCC specimens by in situ hybridization. ST-3 mRNA was particular abundant in the characteristic stroma adjacent to the invasive basaloid tumor islands of the BCC and absent in the malignant cells. Moreover, ST-3 mRNA was expressed and induced by phorbol ester treatment in adult dermal fibroblasts but not in keratinocytes. In vitro studies have shown that MMPs are involved in the degradation of extracellular matrix molecules. Our finding of ST-3 mRNA overexpression in 17 of 19 (89%) BCC specimens is consistent with a role for this molecule in local invasion of stroma by BCC. Our in situ hybridization data suggested that while ST-3 is not expressed by malignant basal cells themselves, these tumor cells may induce the expression of ST-3 in adjacent nonmalignant stromal elements such as fibroblasts. © 1994 Wiley-Liss, Inc.     

Correlation between basic fibroblast growth factor immunostaining of stromal cells and stromelysin-3 mRNA expression in human breast carcinoma.

We examined the localization of basic fibroblast growth factor (bFGF) in a series of human breast carcinomas using immunohistochemistry. Staining was observed in tumour cells in 15 out of 54 (28%) tumours and in the adjacent stroma in 34 out of 54 (63%) tumours examined. No correlation was observed between positive staining of these two compartments. The relationship between bFGF staining and expression of the metalloprotease stromelysin-3, and between bFGF and microvessel density, was examined. A statistically significant correlation (P < 0.003) was observed between bFGF staining of the stromal compartment and high expression of stromelysin-3 (ST-3; MMP-11) metalloprotease mRNA by stromal cells. In contrast, no correlation was observed between bFGF and intratumour microvessel density (IMD). These results raise the possibility that bFGF may be involved in the induction of stromelysin-3 mRNA expression in breast cancer stroma.     

Expression of the stromelysin-3 gene in fibroblastic cells of invasive carcinomas of the breast and other human tissues: a review

Summary Stromelysin-3 (ST3) is a putative new matrix metalloproteinase (MMP) which may play a role in the progression of human carcinomas, and exhibits unique structural and functional characteristics among the MMP family. The ST3 gene, which is generally not expressed at significant levels in benign breast tumors, has been found to be expressed in all invasive breast carcinomas tested so far. The gene is also expressed in somein situ breast carcinomas, which have a higher probability to become invasive. ST3 RNA and protein are specifically found in fibroblastic cells immediately surrounding the neoplastic cells, both in invasive andin situ breast carcinomas. The same expression pattern is observed in other types of human carcinomas, and the highest ST3 RNA levels are observed in tumors that exhibit high local invasiveness. The ST3 gene is also expressed in fibroblastic cells during the inflammatory phase of wound healing, which suggests that ST3 gene expression in stromal fibroblasts may be under the control of factors produced by inflammatory cells during wound healing, and by cancer cells during carcinoma progression. ST3 may thus represent a stroma-derived factor necessary for the progression of epithelial malignancies, and its manipulation may possibly be used to develop new anti-cancer agents.     

Characterization of monoclonal antibodies against stromelysin-3 and their use to evaluate stromelysin-3 levels in breast carcinoma by semi-quantitative immunohistochemistry

Stromelysin-3 (ST3) is a matrix metalloproteinase which is expressed in fibroblastic cells of most human invasive carcinomas and represents a potential new prognostic indicator. Expression of recombinant ST3 forms in Escherichia coli from cDNA constructs indicated that high levels of expression were achieved when the ST3 pro-domain was deleted. The putative mature form of ST3 thus produced and recovered from bacterial inclusion bodies was used to prepare monoclonal antibodies (MAbs) against ST3 by immunization of BALB/C mice. Ten hybridomas producing MAbs against ST3 were obtained and analyzed for their ability to detect endogenous ST3 in breast cancer and in conditioned media from human fibroblasts. One of these MAbs (5ST-4A9) was found to be suitable for the routine detection of ST3 on breast cancer tissue sections, thus opening the possibility to evaluate ST3 prognostic value in breast cancer using semi-quantitative immunohistochemistry. © 1995 Wiley-Liss, Inc.     

Expression of variant forms of fibroblast growth factor receptor 2 mRNA in human breast carcinomas

FGF (fibroblast growth factors) and FGF receptors may play a role in stroma-epithelium relationships in the mammary gland. Dysregulation of their interactions may be important in mammary carcinogenesis. Isoforms of the FGFR2 receptor, differing in the structure of the extracellular region, are expressed in the mammary gland and may diversely affect stroma-epithelium relationships. We determined the mRNA variants encoding these isoforms in human cell lines and breast carcinomas. All possible combinations of variants were found. No correlation was observed between the presence of a particular variant and the expression of any FGF gene tested, or the status of any histoclinical parameter.     

Prognostic value of ERBB family mRNA expression in breast carcinomas

The ErbB-driven autocrine growth pathway has been implicated in the development and progression of most common human epithelial malignancies; its blockade is therefore a promising therapeutic strategy, and several candidate drugs are currently undergoing clinical trials. Paradoxically, little is known of the expression pattern of these 4 genes in human tumors, and the clinical significance of the 2 most recently discovered ERBB genes, ERBB3 and ERBB4, is unclear. We used a real-time quantitative RT-PCR assay to quantify ERBB family mRNA copy numbers in a large series of breast tumors from patients with known long-term outcome. ERBB gene expression varied widely, by more than 2 orders of magnitude for ERBB1 and ERBB3, more than 3 orders for ERBB2 and more than 4 orders for ERBB4. We found a positive correlation between ERBB3 and ERBB4 mRNA levels, and a negative correlation between the expression of these 2 latter genes and that of ERBB1. Compared to normal breast tissue, ERBB1 was underexpressed (82.3% of tumors), ERBB2 (16.9%) and ERBB3 (46.2%) were overexpressed and ERBB4 was both underexpressed (24.6%) and overexpressed (29.2%). Links were also found between ERBB status on the one hand and Scarff-Bloom-Richardson (SBR) histopathological grade and estrogen receptor alpha (ERa) status on the other hand. Relapse-free survival (RFS) was shorter among patients with ERBB3-overexpressing tumors (p=0.0092) and longer among those with ERBB4-underexpressing tumors (p=0.0085) relative to patients with normal expression of the respective genes; in contrast, RFS was not significantly influenced by ERBB1 or ERBB2 mRNA status. Only ERBB4 status retained prognostic significance in Cox multivariate regression analysis (p=0.015). Our results point to the involvement of several ErbB-specific ligands (amphiregulin and neuregulin 1) and enzymes or adaptor molecules (PI3K, Src, Shc and Grb7) in the ErbB pathway dysregulation associated with breast cancer. These findings reveal a complex expression pattern of ERBB gene family members in breast tumors and suggest that it is this pattern of expression, rather than the expression of individual family members, that should be taken into account when evaluating antitumoral drugs designed to target these receptors.     

Localization of estrone sulfatase in human breast carcinomas

We generated anti-human El-STS monoclonal antibodies to localize estrone sulfatase (E1-STS) in human breast carcinomas. In particular, we examined the MCF-7 clone E3, ZR-75-1, MDA-MB 231, and MDA-MB-468 breast cancer cell lines and 25 breast carcinomas by either immunohistochemistry or Western blotting analysis. Simultaneously, we analyzed histological data, estrogen receptor (ER) status, progesterone receptor (PgR) status and epidermal growth factor receptor (EGFR) in breast tissue. All were surgical specimens from female patients. Nine of 25 carcinomas were obtained from premenopausal women, and 16 carcinomas were obtained from postmenopausal women. All cell lines demonstrated positive staining for E1-STS. Interestingly, fine granulated staining of E1-STS on the cell membrane was observed. In addition, Western blotting analysis detected a 65 kD protein with an E1-STS specific band in all breast cancer cell lines regardless of the presence or absence of E2. Twenty-two of 25 (88.0%) carcinomas showed positive staining for E1-STS, whereas negative staining was observed in the interstitial tissue surrounding tumors. In the premenopausal patients, 8 of 10 carcinomas (80.0%) showed positive staining for E1-STS, whereas 14 of 15 carcinomas (93.3%) revealed positive staining in the postmenopausal patients. The frequency of E1-STS expression was relatively higher in postmenopausal patients than in premenopausal patients but not statistically significant. The intensity of immunostaining for E1-STS depended upon the size of the tumor (NS). There was no correlation between E1-STS expression and other parameters. This evidence suggests E1-STS expression may be involved in the development of breast cancer. Further studies are necessary to clarify the relationship between E1-STS expression and prognostic factors. Immunoreactive E1-STS may be localized in cancer cells but not in surrounding tissues in breast cancer.     

Cytogenetic studies on human breast carcinomas

Cytogenetic studies were performed on cell material obtained from surgical specimens of 50 human breast carcinomas and from 61 cancerous effusions of 46 patients. Classical cytogenetic analyses of numerical chromosome changes and marker chromosomes revealed the non-random involvement of chromosomes #X and #22 as monosomics, of chromosomes #3, #7, and #19 as trisomics, and chromosome #1 (particularly p 13 to q 12) in marker formation. Karyotypic evolution was followed in vitro and in vivo and showed a highly individualistic pattern of stability and variability.In addition, a systematic screening for the presence of cytogenetic equivalents of gene amplification (double minutes DM, homogeneously staining regions HSR) was carried out. A high incidence of DM-positive cases was detected in primary tumors (48%) as well as in metastatic cells from effusions (40%), with the frequency of DM-containing metaphases ranging from 1 to 100% in the positive cases. This finding supports the assumption of the fundamental biological importance of gene amplification in human solid tumors.Furthermore, chromosome breakage and micronuclei were observed in breast carcinoma cells as an apparent consequence of therapy-independent mutability.     

Cell proliferation and vascularization in human breast carcinomas.

Cell proliferation and vascularization were studied in 10 human breast carcinomas by an immunoenzyme technique. The monoclonal antibody (MAb) Ki-67 was used as a marker for proliferating cells and a polyclonal antibody directed against human von-Willebrand factor to identify blood vessels. The proportion of Ki-67-labelled cells varied from 1% to 20%, the number of small blood vessels from 4.4/mm2 to 57.6/mm2. Within single histological sections of individual tumours the percentage of proliferating cells was not related to the number of small blood vessels. However, after evaluation of 5 sections of each tumour, the average values showed that tumours with a high grade of vascularization had a higher percentage of Ki-67-positive cells than poorly vascularized samples. The influence of vascular density on cell proliferation was investigated in a selected area of one of the tumours (in 2-dimensions) and with regard to the over- and underlying sections (in 3-dimensions). After 2-dimensional evaluation, distances from proliferating cells to the closest blood vessel between 10 and 390 microns were observed, and after 3-dimensional evaluation none of the proliferating cells measured was located more than 130 microns away from the closest vessel.     

Steroid metabolism and oestrogen receptors in human breast carcinomas

Metabolism of7α[³H] testosterone and oestrogen receptor activity have been measured in54 human breast cancers. All tumors converted testosterone to Δ4 androstenedione,5α dihydrotestosterone and5α androstanediol, but unequivocal evidence for production of oestradiol was obtained only29 of the tumours. Thirty-seven tumours were classified as oestrogen receptor ‘positive’ containing levels in excess of5 fmol/mg cytosol protein. Although mean conversions to Δ4 androstenedione,5α dihydrotestosterone and5α androstanediol were all higher in oestrogen receptor negative tumours as compared with receptor positive group, the differences did not reach statistical significance. There was, however, a significant trend for oestradiol synthesis to be associated with oestrogen receptor positive tumours (P < 0.025). All tumours with very high level of receptors converted testosterone to oestradiol.     

Expression of epidermal growth factor receptor mRNA and protein in primary breast carcinomas

The expression of epidermal growth factor receptor (EGFR) mRNA and protein has been determined in a group of breast carcinomas and compared to oestrogen and progesterone receptor (ER, PgR) status, as well as pathological features. In situ hybridization using a digoxigenin-labelled oligonucleotide probe was applied to formalin-fixed paraffin-embedded sections, and immunohistochemistry was used to determine EGFR protein.EGFR mRNA was detected in 66% of carcinomas with a third having labelling similar to normal breast tissue, 22% heterogeneous weak to strong labelling, and 11% strong labelling. EGFR protein was detected in 36% and these tumours had a strong correlation to lack of ER and high histological grade. The presence of EGFR protein was strongly correlated with more intense labelling for EGFR mRNA (p < 0.0001). This contrasted with normal breast in which both EGFR protein and mRNA were present with varying degrees in both tumours and a normal breast control. The ER-/PgR- carcinomas showed the full range of EGFR mRNA labelling. It is postulated that oestrogen or oestrogen regulated proteins are involved in regulation of EGFR mRNA and protein. In a proportion of tumours lacking steroid receptors regulation is lost, leading to EGFR overexpression.     

High levels of stromelysin-3 correlate with poor prognosis in patients with breast carcinoma

Stromelysin 3 (ST3) is a matrix metalloprotease (MMP) expressed in fibroblast-like cells of most human invasive carcinomas. In this investigation, ST3 was measured by semiquantitative immunohistochemistry in III primary breast cancers. ST3 levels showed no correlation with tumor size, axillary-node status or tumor grade (Scarff-Bloom-Richardson system; SBR) but were significantly associated with higher nuclear grade (modified SBR). In addition, ST3 levels were significantly higher in ductal than in lobular cancers. Patients with high scores of ST3 staining had a shorter disease-free interval and shorter overall survival than patients with low scores. ST3 is thus one of the first MMPs to correlate with patient outcome in breast cancer. These findings are consistent with earlier clinical and experimental observations suggesting that ST3 contributes to breast-cancer progression. © 1996 Wiley-Liss, Inc.     

Hormone synthesis by primary breast carcinomas and prognosis in human breast cancer

The ability of human breast carcinomas to convert pregnenolone to progesterone and dehydroepiandrosterone to delta 4-androstene-3,17-dione (delta 4) was investigated as a potential aid for prognosis, and the following observations were recorded. 1. Neither the amounts of progesterone or delta 4 synthesized nor delta 4/progesterone ratios correlated with tumour size or lymph node involvement. 2. delta 4 synthesis was lower in carcinomas from patients who had recurrences within 2 years of mastectomy than in carcinomas from those who remained free of metastases. 3. Life table analysis of the results indicated that these parameters appeared unlikely to be useful aids for prognosis.     

Cumulative regional allelotyping of human breast carcinomas

We have analyzed losses of heterozygosity (LOH) at markers from chromosomes 1, 2, 4 and 5 in a panel of 53 consecutive breast carcinomas. Together with a parallel analysis of LOH at chromosome 3, this allowed the identification of twenty-one regions of LOH. In contrast, in a comparative analysis of chromosome X, no region of loss could be defined. Cumulative regional allelotyping of 38 tumors with 71 markers from the five autosomes (37% of the genome) enabled the study of the respective distribution of regions of frequent loss, to identify associations of regions of LOH, and to distinguish tumors frequently affected by LOH such as lobular carcinomas.