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Reprogramming the Transcriptional Response to Hypoxia with a Chromosomally Encoded Cyclic Peptide HIF-1 Inhibitor.

ABSTRACT: The cellular response to hypoxia is orchestrated by HIF-1, a heterodimeric transcription factor composed of an ? and a ? subunit that enables cell survival under low oxygen conditions by altering the transcription of over 300 genes. There is significant evidence that inhibition of HIF-1 would be beneficial for cancer therapy. We recently reported a cyclic hexapeptide that inhibits the HIF-1?/HIF-1? protein-protein interaction in vitro and prevents HIF-1-mediated hypoxia-response signaling in cells. This cyclic peptide was identified from a library of 3.2 × 10⁶ members generated using SICLOPPS split-intein mediated protein splicing. With a view to demonstrating the potential for encoding the production of a therapeutic agent in response to a disease marker, we have engineered human cells with an additional chromosomal control circuit that conditionally encodes the production of our cyclic peptide HIF-1 inhibitor. We demonstrate the conditional production of our HIF-1 inhibitor in response to hypoxia, and its inhibitory effect on HIF-1 dimerization and downstream hypoxia-response signaling. These engineered cells are used to illustrate the synthetic lethality of inhibiting HIF-1 dimerization and glycolysis in hypoxic cells. Our approach not only eliminates the need for the chemical synthesis and targeted delivery of our HIF-1 inhibitor to cells, it also demonstrates the wider possibility that the production machinery of other bioactive compounds may be incorporated onto the chromosome of human cells. This work demonstrates the potential of sentinel circuits that produce molecular modulators of cellular pathways in response to environmental or cellular disease stimuli.

SUBMITTER: Mistry IN

PROVIDER: S-EPMC6014682 | biostudies-literature | 2017 Mar

REPOSITORIES: biostudies-literature

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Reprogramming the Transcriptional Response to Hypoxia with a Chromosomally Encoded Cyclic Peptide HIF-1 Inhibitor.

Mistry Ishna N IN Tavassoli Ali A

ACS synthetic biology 20161123 3

The cellular response to hypoxia is orchestrated by HIF-1, a heterodimeric transcription factor composed of an α and a β subunit that enables cell survival under low oxygen conditions by altering the transcription of over 300 genes. There is significant evidence that inhibition of HIF-1 would be beneficial for cancer therapy. We recently reported a cyclic hexapeptide that inhibits the HIF-1α/HIF-1β protein-protein interaction in vitro and prevents HIF-1-mediated hypoxia-response signaling in cel ...[more]

PMID: 27978620

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Project description:BACKGROUND:Hypoxia inducible factor-1 (HIF-1) is considered as the most activated transcriptional factor in response to low oxygen level or hypoxia. HIF-1 binds the hypoxia response element (HRE) sequence in the promoter of different genes, mainly through the bHLH domain and activates the transcription of genes, especially those involved in angiogenesis and EMT. Considering the critical role of bHLH in binding HIF-1 to the HRE sequence, we hypothesized that bHLH could be a promising candidate to be targeted in hypoxia condition. METHODS:We inserted an inhibitory bHLH (ibHLH) domain in a pIRES2-EGFP vector and transfected HEK293T cells with either the control vector or the designed construct. The ibHLH domain consisted of bHLH domains of both HIF-1a and Arnt, capable of competing with HIF-1 in binding to HRE sequences. The transfected cells were then treated with 200 µM of cobalt chloride (CoCl2) for 48 h to induce hypoxia. Real-time PCR and western blot were performed to evaluate the effect of ibHLH on the genes and proteins involved in angiogenesis and EMT. RESULTS:Hypoxia was successfully induced in the HEK293T cell line as the gene expression of VEGF, vimentin, and ?-catenin were significantly increased after treatment of untransfected HEK293T cells with 200 µM CoCl2. The gene expression of VEGF, vimentin, and ?-catenin and protein level of ?-catenin were significantly decreased in the cells transfected with either control or ibHLH vectors in hypoxia. However, ibHLH failed to be effective on these genes and the protein level of ?-catenin, when compared to the control vector. We also observed that overexpression of ibHLH had more inhibitory effect on gene and protein expression of N-cadherin compared to the control vector. However, it was not statistically significant. CONCLUSION:bHLH has been reported to be an important domain involved in the DNA binding activity of HIF. However, we found that targeting this domain is not sufficient to inhibit the endogenous HIF-1 transcriptional activity. Further studies about the function of critical domains of HIF-1 are necessary for developing a specific HIF-1 inhibitor.

Dataset Information

Reprogramming the Transcriptional Response to Hypoxia with a Chromosomally Encoded Cyclic Peptide HIF-1 Inhibitor.

Publications

Reprogramming the Transcriptional Response to Hypoxia with a Chromosomally Encoded Cyclic Peptide HIF-1 Inhibitor.

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