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Adenovirus E1A represses protease gene expression and inhibits metastasis of human tumor cells
S M Frisch1, R Reich, I E Collier
1Division of Dermatology, Washington University School of Medicine, St. Louis, Missouri 63110.
Abstract:
Stable transfection of human tumor cell lines with the adenovirus-5 E1A gene repressed the expression of the secreted proteases, type IV collagenase, interstitial collagenase and urokinase. In addition, E1A blocked the 12-O-tetradecanoyl phorbol acetate (TPA) induction of interstitial collagenase transcription in HT1080 fibrosarcoma cells. Plasmids bearing the interstitial collagenase or type IV collagenase 5' flanking regions linked to a chloramphenicol acetyl transferase coding sequence were constructed and analysed for expression by transient cotransfections into HT1080 cells. Cotransfection with a plasmid bearing a functional E1A gene repressed transcription of the type IV collagenase promoter and blocked the TPA induction of the interstitial collagenase promoter. Furthermore, E1A repressed transcription from a TK promoter driven by AP-1 complex binding sites (TRE), suggesting that E1A interferes with the AP-1 trans-activation pathway. This effect was not, however, due to the repression of c-jun gene transcription by E1A. In fact, the expression of E1A rendered the c-jun gene hypersensitive to TPA induction. Concomitant with reduction in expression levels of secreted proteases, stable E1A transfectants showed reduced metastatic activity in vivo and reduced ability to traverse a reconstituted basement membrane in vitro. Monospecific anti-type IV collagenase antibodies inhibited invasive activity of parental tumor cell lines in the in vitro assay, suggesting a possible causal relationship between the repression of secreted proteases and loss of metastatic properties of the transformants.
Insights
The adenovirus-5 E1A gene suppresses tumor cell proteases, reducing cancer cell invasion and metastasis. This E1A gene expression inhibits key enzymes involved in tumor spread, offering potential therapeutic targets.
Area of Science:
- Molecular Biology
- Oncology
- Virology
Background:
- Tumor cell invasion and metastasis are complex processes often involving secreted proteases.
- The adenovirus-5 E1A gene is known to regulate cellular gene expression.
- Understanding the role of viral genes in modulating tumor cell behavior is crucial for cancer research.
Purpose of the Study:
- To investigate the effect of adenovirus-5 E1A gene expression on secreted protease expression in human tumor cell lines.
- To determine if E1A influences the transcriptional regulation of collagenases and urokinase.
- To assess the impact of E1A-mediated protease repression on tumor cell metastatic potential.
Main Methods:
- Stable transfection of human tumor cell lines with the adenovirus-5 E1A gene.
- Analysis of secreted protease expression (type IV collagenase, interstitial collagenase, urokinase) via molecular assays.
- Reporter gene assays using chloramphenicol acetyl transferase (CAT) linked to protease promoter regions.
- In vitro invasion assays using reconstituted basement membranes.
- In vivo metastatic activity assessment.
Main Results:
- Stable E1A expression repressed the secretion of type IV collagenase, interstitial collagenase, and urokinase.
- E1A blocked the induction of interstitial collagenase transcription by 12-O-tetradecanoyl phorbol acetate (TPA).
- E1A repressed transcription from the type IV collagenase promoter and interfered with the AP-1 trans-activation pathway.
- E1A-expressing cells exhibited reduced in vitro invasion and in vivo metastatic activity.
- Inhibition of type IV collagenase activity reduced the invasive potential of parental tumor cells.
Conclusions:
- Adenovirus-5 E1A gene expression significantly represses key secreted proteases involved in tumor invasion.
- The E1A gene's ability to inhibit protease expression correlates with reduced metastatic potential in tumor cells.
- E1A likely interferes with transcriptional pathways regulating protease genes, including the AP-1 pathway.
- These findings suggest a potential therapeutic strategy targeting protease activity to inhibit cancer metastasis.