For the first time, we have recently shown that camptothecin, a DNA topoisomerase I poison with effective antitumor activity, can activate the transcription of low-abundance antisense RNAs at the HIF-1a gene locus in human cancer cells. The activation of antisense transcription is likely due to sustained drug interference with transcription regulation mechanisms leading to a more open chromatin conformation and de-repression/activation of antisense transcription. Camptothecin readily inhibits Topoisomerase I in cells, and the enzyme inhibition activates transcriptional Cdk (Cdk9 and/or Cdk7) activity leading to the hyperphosphorylation of the CTD of the largest subunit of RNA polymerase II (RNAP II). This results in an alteration of RNAP II regulation with specific effects at transcription levels. Thus, our work has documented that camptothecin can interfere with specific transcription regulatory steps, impairing the balance of cellular antisense and sense transcripts at the HIF-1a gene locus. That may have a considerable impact on cancer therapy development particularly for non responsive human tumors. Topoisomerase I relaxes supercoiled DNA coupling DNA cleaving and rejoining activities. Normally, the Top1 cleavage complex (Top1cc) is very transient, however, very effective antitumor drugs, such as camptothecin, inhibit the religation step of the catalytic reaction, increasing the levels of Top1cc in cells. Top1 is the sole target of camptothecin, which stabilize Top1ccs leading to irreversible double-strand breaks (DSB) when a Top1cc meets a DNA replication fork. The replicative DSB are considered the cytotoxic lesions as they can efficiently trigger apoptosis of sensitive cancer cells. Thus, the drug has been helpful to dissect checkpoint pathways activated by DNA breakage at replication forks. The established cellular effects of camptothecin are indeed peculiar of DNA damage responses, nevertheless Top1cc occurs primarily in actively transcribed regions, and transcription-dependent effects of Top1cc remain to be established fully. Whether transcriptional effects of camptothecin play any role in the antitumor activity of the drug has remained unexplored. Interestingly, previous reports demonstrated that camptothecin can have an anti-angiogenic activity in animal tumor models and can abolish HIF-1a accumulation in hypoxic cells in a manner independent from the VHL pathway and from replicative DNA damage, but dependent on transcription activity. HIF-1a is a master regulator of the cell response to hypoxia and a promising target of anti-angiogenesis and anticancer therapies. With this project, we intend to test the hypothesis that de-repression/activation of antisense RNAs by camptothecin can regulate HIF-1a activity through a transcriptional and/or post-transcriptional mechanism. Our general hypothesis is that camptothecin interference with Topoisomerase I can result in specific alterations of gene expression patterns that are relevant for the antitumor activity of the drug. In particular, an altered balance of antisense/sense transcripts may affect HIF-1a activity contributing to the control of tumor progression in human patients. Moreover, the new mechanism of the drug antitumor action may set a different rational basis for the development of novel therapeutic strategies for drug-unresponsive human cancers.

Transcriptional Topoisomerase I functions: implications for tumor resistance to enzyme poisons.

CAPRANICO, GIOVANNI
2010

Abstract

For the first time, we have recently shown that camptothecin, a DNA topoisomerase I poison with effective antitumor activity, can activate the transcription of low-abundance antisense RNAs at the HIF-1a gene locus in human cancer cells. The activation of antisense transcription is likely due to sustained drug interference with transcription regulation mechanisms leading to a more open chromatin conformation and de-repression/activation of antisense transcription. Camptothecin readily inhibits Topoisomerase I in cells, and the enzyme inhibition activates transcriptional Cdk (Cdk9 and/or Cdk7) activity leading to the hyperphosphorylation of the CTD of the largest subunit of RNA polymerase II (RNAP II). This results in an alteration of RNAP II regulation with specific effects at transcription levels. Thus, our work has documented that camptothecin can interfere with specific transcription regulatory steps, impairing the balance of cellular antisense and sense transcripts at the HIF-1a gene locus. That may have a considerable impact on cancer therapy development particularly for non responsive human tumors. Topoisomerase I relaxes supercoiled DNA coupling DNA cleaving and rejoining activities. Normally, the Top1 cleavage complex (Top1cc) is very transient, however, very effective antitumor drugs, such as camptothecin, inhibit the religation step of the catalytic reaction, increasing the levels of Top1cc in cells. Top1 is the sole target of camptothecin, which stabilize Top1ccs leading to irreversible double-strand breaks (DSB) when a Top1cc meets a DNA replication fork. The replicative DSB are considered the cytotoxic lesions as they can efficiently trigger apoptosis of sensitive cancer cells. Thus, the drug has been helpful to dissect checkpoint pathways activated by DNA breakage at replication forks. The established cellular effects of camptothecin are indeed peculiar of DNA damage responses, nevertheless Top1cc occurs primarily in actively transcribed regions, and transcription-dependent effects of Top1cc remain to be established fully. Whether transcriptional effects of camptothecin play any role in the antitumor activity of the drug has remained unexplored. Interestingly, previous reports demonstrated that camptothecin can have an anti-angiogenic activity in animal tumor models and can abolish HIF-1a accumulation in hypoxic cells in a manner independent from the VHL pathway and from replicative DNA damage, but dependent on transcription activity. HIF-1a is a master regulator of the cell response to hypoxia and a promising target of anti-angiogenesis and anticancer therapies. With this project, we intend to test the hypothesis that de-repression/activation of antisense RNAs by camptothecin can regulate HIF-1a activity through a transcriptional and/or post-transcriptional mechanism. Our general hypothesis is that camptothecin interference with Topoisomerase I can result in specific alterations of gene expression patterns that are relevant for the antitumor activity of the drug. In particular, an altered balance of antisense/sense transcripts may affect HIF-1a activity contributing to the control of tumor progression in human patients. Moreover, the new mechanism of the drug antitumor action may set a different rational basis for the development of novel therapeutic strategies for drug-unresponsive human cancers.
G. Capranico
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11585/94933
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