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The first steps of stroma generation are of pivotal importance for carcinogenesis because at this stage are initiated both angiogenesis, the prerequisite for continuous tumour growth, and the proliferation of stromal fibroblasts. These developments contribute to the onset of tumour invasion by secreting several matrix-degrading proteases. Both angiogenesis and the production of proteases are tightly controlled at several levels; of significant importance is transcription. The Ets-1 transcription factor transactivates several genes encoding matrix-degrading proteases and is thought to be involved in both tumour vascularization and invasion. This study therefore investigated, by in situ hybridization and immunohistochemistry, the expression of Ets-1 and of two of its target genes, encoding matrix metalloproteinase (MMP) 1 and MMP 9, in order to demonstrate a topographical in vivo correlation between the expression of these three genes during breast cancer formation. All three genes were first expressed within both endothelial cells and stromal fibroblasts during the onset of stroma generation around intraductal and intralobular in situ carcinomas and they were significantly up-regulated in the stroma of invasive ductal and lobular cancers. The results of this study further support the suggested in vivo role of Ets-1 for both angiogenesis and tumour invasion, via matrix-degrading proteases which are already expressed during the early stages of breast carcinogenesis.

Testicular germ cell tumours (TGCTs) are histologically heterogeneous neoplasms with variable malignant potential. Previously, we demonstrated frequent 3p allele loss in TGCTs, and recently we and others have shown that the 3p21.3 RASSF1A tumour suppressor gene (TSG) is frequently inactivated by promoter hypermethylation in a wide range of cancers including lung, breast, kidney and neuroblastoma. In order to investigate the role of epigenetic events in the pathogenesis of TGCTs, we analysed the promoter methylation status of RASSF1A and nine other genes that may be epigenetically inactivated in cancer (p16 INK4A , APC, MGMT, GSTP1, DAPK, CDH1, CDH13, RARb and FHIT) in 24 primary TGCTs (28 histologically distinct components). RASSF1A methylation was detected in four of 10 (40%) seminomas and 15 of 18 (83%) nonseminoma TGCT (NSTGCT) components (P ¼ 0.0346). None of the other nine candidate genes were methylated in seminomas, but MGMT (44%), APC (29%) and FHIT (29%) were frequently methylated in NSTGCTs. Furthermore, in two mixed germ cell tumours, the NSTGCT component for one demonstrated RASS-F1A, APC and CDH13 promoter methylation, but the seminoma component was unmethylated for all genes analysed. In the second mixed germ cell tumour, the NSTGCT component was methylated for RASSF1A and MGMT, while the seminoma component was methylated only for RASSF1A. In all, 61% NSTGCT components but no seminoma samples demonstrated promoter methylation at two or more genes (P ¼ 0.0016). These findings are consistent with a multistep model for TGCT pathogenesis in which RASSF1A methylation occurs early in tumorigenesis and additional epigenetic events characterize progression from seminoma to NSTGCTs. Oncogene (2003Oncogene ( ) 22, 461-466. doi:10.1038 Keywords: methylation; 3p21.3 tumour suppressor gene; testicular tumours IntroductionTesticular germ cell tumours (TGCTs) are the most frequent malignant tumour in young men and account for 1-2% of all malignancies in males. TGCTs are generally subclassified into seminoma (50%), nonseminoma (40%) or combined (10%) tumours. It is suggested that TGCTs originate from carcinoma in situ (CIS) and that a series of cytogenetic and molecular alterations are associated with progression of CIS to seminoma, which may subsequently develop into a nonseminoma tumour. Thus, seminomas are composed of cells similar to CIS cells and the undifferentiated stem cells of nonseminomas (termed embryonal carcinoma cells) may differentiate into teratomas, yolk sac tumours or choriocarcinomas (Looijenga and Oosterhuis, 1999;Chaganti and Houldsworth, 2000). The most consistent chromosomal alteration in TGCT is an isochromosome 12p(i(12p)), and tumours lacking i(12p) often have other structural abnormalities of 12p including amplification of 12p11.2-pl2.1 consistent with the involvement of a 12p oncogene (Suijkerbuijk et al., 1994;Mostert et al., 1998). Molecular genetic studies of TGCTs have reported allele loss on chromosomes 1, 3, 5, 9, 11, 12q, 13q, 17p and 18q (Looijenga et al., 1994;Mathew et al., 1...

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Hereditary nonpolyposis colorectal cancers (HNPCCs) are an important subgroup of colorectal carcinomas. Compared to sporadic variants, they present several particular features, the most important of which are less invasive and metastatic properties linked to a more favorable prognosis. This contrasts to the generally poor differentiation of the epithelial tumor component. Since matrix-degrading proteases secreted by stromal fibroblasts contribute significantly to tumor invasion, we analyzed the stromal expression of 2 matrix metalloproteinases (MMP-1 and -9) and of one of their regulators, the Ets 1 transcription factor, by both immunohistochemistry and in situ hybridization in sporadic colorectal carcinomas and HNPCC tumors. We found that MMP-1 and -9 as well as Ets 1 are upregulated in the fibroblastic stroma during the development from sporadic adenomas to invasive carcinomas. HNPCC tumors exhibited a significantly lower expression of Ets 1, MMP-1 and -9. These findings on the basis of lower matrix-degrading properties of the fibroblastic tumor stroma in HNPCC tumors might help to explain why, Colorectal carcinoma is among the most common malignancies in industrialized nations. While most cases are sporadic, up to 5% occur as part of hereditary syndromes, among which familiar adenomatous polyposis (FAP) and the hereditary nonpolyposis colorectal cancer (HNPCC) syndrome are the most common. 1-3 Defects of different mismatch repair genes, particularly MSH2 and MLH1, have been identified in recent years to be associated with HNPCC. 4 -6 Compared to sporadic colorectal cancers, HNPCC tumors present several particular features, the most important of which are less invasive and metastatic properties linked to a more favorable prognosis. 7-9 These less invasive and metastatic properties are observed in spite of the less differentiated morphologic phenotype with a lower tendency toward a glandular differentiation in most HNPCC tumors compared to sporadic colorectal cancers. The reasons for these contradictory features of HNPCC tumors are not known. While invasive properties have long been attributed exclusively to the epithelial tumor cells (particularly to poorly differentiated ones), it has become evident that tumor stroma plays a major role for this critical process as well. Stromal fibroblasts or myofibroblasts are a major source of matrix-degrading metalloproteinases (MMPs) necessary for tumor cell invasion. Stromal expression of MMP-2, MMP-9 and stromelysin 1 has been shown to be upregulated during the development of invasive sporadic colorectal cancers 10 and high levels of MMP-1 expression have been directly linked to poor prognosis. 11 In general, the activity of matrix-degrading proteases is tightly regulated at several levels. They include an induction by different cytokines and growth factors (such as EGF, bFGF or TNF␣), the activation of secreted MMP-proenzymes by a proteolytic cleavage and inhibition by several naturally occurring inhibitors such as TIMP1 and -2. 12-14 One of the transcription factors ...