Cancer Letters

Cancer Letters

Volume 200, Issue 1, 8 October 2003, Pages 57-67
Cancer Letters

Extracellular matrix protein 1 (ECM1) is over-expressed in malignant epithelial tumors

https://doi.org/10.1016/S0304-3835(03)00350-1Get rights and content

Abstract

The extracellular matrix protein 1 (ECM1) is a secreted protein that has been implicated with cell proliferation, angiogenesis and differentiation. In the present study, we used immunohistochemical staining to examine the expression of ECM1 in a panel of human tumors and found that it was closely correlated with some types of tumors including: invasive breast ductal carcinoma (83%), esophageal squamous carcinoma (73%), gastric cancer (88%) and colorectal cancer (78%). Significantly, ECM1expression was correlated with the metastatic properties of the tumors. Primary breast cancers that had formed metastases were 76% positive while those that had not metastasized were only 33% positive. ECM1 expression was also correlated with PCNA a marker for proliferation, but not with CD34, a marker for endothelial cells. These results indicate that ECM1 tends to be preferentially expressed by metastatic epithelial tumors.

Introduction

The extracellular matrix protein 1 (ECM1) was originally identified as an 85 kDa glycoprotein secreted by a murine osteogenic stromal cell line [1]. Subsequently, two forms of human ECM1 were described: ECM1a (68 kDa) which is the full length form consisting of 540 amino acids; and ECM1b (46 kDa) which is an alternatively spliced form with 415 amino acids [3]. These two forms of ECM1 are coded by a single gene, which has been mapped to 1q21 outside of the epidermal differentiation complex region [3], [4].

The distribution of ECM1 changes as a function of embryonic development. In mouse embryos, ECM1a is present in a number of tissues and is particularly prominent in blood vessels [2], [3], [5], [6]. Indeed, its expression pattern is similar to that of flk-1, a recognized marker for endothelial cells [6]. As development progresses, ECM1b is expressed in the tail, front paws and skin [3]. In adult humans, ECM1a is predominantly expressed by the skin [5], tonsils, heart and placenta [3]. In the case of the skin, ECM1a is restricted to the basal cell layer, while ECM1b is predominantly found in the suprabasal layers [3], [5].

Recent studies have implicated ECM1 with a number of physiological functions. For example, ECM1 appears to help regulate endochondral bone formation. Merregaert and his coworkers found that Ecm1 is expressed in the connective tissues surrounding the developing bones (perichondrium), but not in cartilage itself. At low concentrations, recombinant ECM1 stimulated alkaline phosphatase activity, whereas at higher concentrations, it inhibited alkaline phosphatase activity and mineralization [7]. In addition, ECM1 may be involved in the differentiation of keratinocytes as suggested by the fact that it is expressed in the skin and its gene is located is located close to the epidermal differentiation complex at 1q21 [3], [5]. Recently, Hamada and co-workers identified a homozygous loss-of-function mutation in the ECM1 gene in a family suffering from lipoid proteinosis, a rare, autosomal recessive disorder also called Urbach–Wiete disease [8], [9], [10], [11], [12], [13], [14]. The loss of functional ECM1 appears to be responsible for the classical symptoms of which include thickening of the skin, mucosa and certain viscera the widespread deposition of hyaline (glycoprotein) and disruption of the basement membrane [8], [9], [10], [11], [12], [13], [14]. The secretion of ECM1 appears to be required for the maintenance of the extracellular matrix in the skin and mucosa.

In a recent study, we had found that ECM1 may also promote both cell proliferation and angiogenesis [6]. When a highly purified preparation of ECM1 was added to cultures of Human Umbilical Vein Endothelial Cells (HUVEC), Adult Bovine Aortic Endothelium (ABAE), Bovine Retinal Endothelial Cells (BREC) and Brain Endothelial Cells (BEC) cells in serum-free medium, their growth was significantly stimulated as measured by thymidine incorporation. Under these conditions, the stimulation induced by ECM1 was only slightly less than that of FGF-2 and VEGF. However, the stimulatory effects of ECM1 appeared to be cell line specific in that MDA-435, MDA-468 and TSU cells showed little or no response. In another set of experiments, we found that ECM1 can also promote angiogenesis. A highly purified preparation of ECM1 was applied to small pieces of filter paper that were then applied to the chorioallantoic membranes of chicken eggs and two days later this region was subjected to computerized image analysis. The results of this analysis indicated that ECM1 had a marked stimulatory effect on degree of vascularization. Given the fact that ECM1 has been implicated with cell proliferation and angiogenesis, it also seemed possible that ECM1 may also contribute to tumor progression. Indeed, in this same study we had found that ECM1 could be detected in some breast cancer tissues, including metastases to both the lymph node and bone [6].

In the present study we have examined the expression of ECM1 in a panel of human tumors with affinity-purified antibodies against ECM1. Our goal here was two fold: first to determine if ECM1 expression could be used as a marker for a particular pathological condition; and secondly to test whether it was correlated with markers of tumor proliferation and angiogenesis. The results of this study indicated that several types of human epithelial malignant tumors expressed significant levels of ECM1 and that its expression was indeed correlated with tumor proliferation but not with angiogenesis.

Section snippets

Patients and tissues samples

Both paraffin-embedded (296 sections from 263 cases) and fresh-frozen (10 cases) samples of human tumors were obtained from the tumor bank of Lombardi Cancer Center at Georgetown University, Pathology Departments of Beijing General Hospital and Naval General Hospital in Beijing, Changhai Hospital in Shanghai, and tissue array samples from Cybrdi (Rockville, MD). For the paraffin-embedded samples, 5 μm thick sections were stained with hematoxylin and eosin and then evaluated according to the WHO

Expression of ECM1 in various tumors and tissues

We analyzed a panel of human tissues for ECM1 by immunohistochemical staining. These samples represented a spectrum of conditions ranging from normal (regions adjacent to the tumors) to invasive carcinomas. Representative examples of the staining are shown in Fig. 1.

In the case of normal tissue, the vast majority was negative for ECM1 staining (Fig. 1A, normal colon mucosa, Fig. 1D, normal gastric mucosa and Fig. 1G, lobular mild hyperplasia of the breast). Of the 14 normal tissues examined, 13

Discussion

The major conclusion of this study is that ECM1 is upregulated in a distinct subset of tumors. As shown in Table 2, ECM1 staining was positive in 83% of invasive ductal carcinomas of the breast, 73% of squamous cell carcinomas of the esophagus, 88% of adenocarcinomas of the stomach, 78% of adenocarcinomas of the colon and 75% of lung carcinomas. Overall, the average positive frequency in carcinomas was 75.0%, which was much higher than that of sarcomas (16.6%), epithelial hyperplasia (11.7%)

Acknowledgements

This work was supported mainly by Susan G. Komen Breast Cancer Foundation, in part by NCI/NIH (R29 CA71545), US Army Med. Res. and Mat. Command (DAMD17-98-1-8099, DAMD17-00-1-0081 and DAMD17-01-1-0708) to L.Z. It was also partially supported by the Belgian Fonds voor Wetenschappelijk Onderzoek (onderzoeksprogramma G.0123.96 and G.0085.98). The authors thank Professor E. Van Marck for his insightful comments on the manuscript.

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