Differential expression of matrix metalloproteinases in activated c-rasHa-transfected immortalized human keratinocytes

Abstract

Summary Elevated expression of matrix metalloproteinases (MMPs), a family of secreted proteinases that degrade matrix components of basement membranes and connective tissues, is strongly correlated with malignant expression in various human epithelial cancers and epithelial cancer cell lines. We have tested whether elevated levels of MMP expression are also associated with malignant progression in human cutaneous squamous cell carcinoma. Constitutive levels of expression of steady-state mRNA and of secreted protein encoded by three MMP genes (matrilysin, gelatinases A and B) were compared in a unique in vitro model of human skin carcinogenesis. This model is composed of the parental immortalized non-tumorigenic human keratinocyte line (HaCaT), and three activated c-Harvey-ras-oncogene transfected variants (A-4, 1-7 and 11-4). Although clone A-4 is non-tumorigenic, clones 1-7 and 11-4 exhibit benign and malignant tumorigenic phenotypes, respectively, after subcutaneous injection into athymic nude mice. Northern blot, Western blot, and zymogram analyses revealed three MMP-specific patterns of expression. Constitutive matrilysin mRNA expression was markedly increased in the 1-7 cells compared with HaCaT, A-4 or 11-4 cells. Secreted promatrilysin was distinctly increased in the tumorigenic 1-7 and 11-4 cells compared with the nontumorigenic HaCaT and A-4 cells. Gelatinase A mRNA and secreted gelatinase A protein levels were increased in each transfectant compared with HaCaT. Both active and inactive forms of gelatinase A were detected. Gelatinase B transcripts were not detected, but an EDTA-inhibitable gelatinase activity comigrating with gelatinase B was moderately enhanced in both tumorigenic variants compared with the non-tumorigenic cells. Because promatrilysin and 92-kDa gelatinase secretion were increased in both benign and malignant tumorigenic cells, and not related to invasiveness in this model, it is concluded that enhanced constitutive expression of these two MMPs is associated with acquisition of the tumorigenic phenotype, before acquisition of the malignant phenotype.Keywords: matrilysin; gelatinase; matrix metalloproteinase; human; keratinocyte; HaCaT-ras; malignant progressionThe predominant cause of cancer morbidity and mortality is metastasis of malignant cells from the primary tumour to secondary sites. Therefore, a critical goal in cancer treatment is prevention of metastasis, a process that is considered to involve repeated proteinasemediated degradation and cancer cell invasion of basement membranes and underlying connective tissues. Members of the matrix metalloproteinase (MMP) family of zinc-containing endopeptidases may play a pivotal role in in vivo invasion and metastasis because they can degrade matrix components of the basement membrane. At least 15 different MMPs from human sources have been characterized (Pendas et al, 1997). MMPs are secreted as proenzymes, and are subsequently activated. Furthermore, modulation of MMP activity by endogenous tissue inhibitors of metalloproteinases (TIM...

Matrix Metalloproteinases-2, -3, -7, -9 and -10, But Not MMP-11, Are Differentially Expressed in Normal, Benign Tumorigenic and Malignant Human Keratinocyte Cell Lines

“…It is interesting to note that our clone A-5 synthesizes significantly less p21, the gene product of Ha-ras, than the corresponding I-7 clone (Boukamp et al, 1990) used by Meade-Tollin et al (1998). But in spite of this lower p21 expression, A-5 shows higher expression of MMP-2, -7 and -9 than HaCaT, just like the I-7 clone.…”

“…Meade- Tollin et al (1998) reported on the expression of MMP-2, -7 and -9 in HaCaT and the cell lines A-4 of class I (see above; no corresponding clone was included in the present study), I-7 of class II (like A-5 of our study), and the class III clone II-4 (see above). By Northern blot analysis they detected expression of MMP-2 and MMP-7 mRNAs in the cells.…”

“…When Meade-Tollin et al (1998) investigated secretion into the medium of MMP-2 and -9 by zymography and of MMP-7 by Western blotting, they found that, in comparison to the non-tumorigenic HaCaT and A-4 cells, secretion of MMP-7 and -9 was equally enhanced in the two tumorigenic variants I-7 and II-4. Thus, contrasting our results which clearly demonstrate that MMP-2, -7 and -9 secretion increases with the degree of tumorigenicity, they did not observe a difference in the secretion of these MMPs between the benign tumorigenic I-7 and the malignant II-4 cells.…”

Silencing of the Metastasis Suppressor RECK by RAS Oncogene Is Mediated by DNA Methyltransferase 3b–Induced Promoter Methylation

“…This family of proteins consists of at least 26 enzymes and can be grouped into different subtypes based on the substrate specificity and sequence characteristic (18,19). Previous studies showed that RAS oncogene up-regulated MMP-2 and MMP-9, two MMPs that played critical roles in tumor metastasis, in transformed cells (20,21). Therefore, MMPs are important mediators for RAS-induced metastasis.…”

Farnesyltransferase inhibitor SCH‐66336 downregulates secretion of matrix proteinases and inhibits carcinoma cell migration

“…22 In addition, cell transfection with an activated H-ras increases MMP-9 gelatinolytic activity. 23,24 Since MMP activity is stimulated by uPA and plasmin, 13,14,16,17 plasmin, uPA, uPAR and MMPs could be regulated by FTIs through inhibition of Ras prenylation and activity.…”