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Validation of putative small-molecule antagonists of Ran: an essential regulator of survival in AML, breast and non-small cell lung cancer cells

Summary of project:

We have utilized suppressive subtractive hybridization between mRNAs isolated from the Rama 37 (R37) rat benign non-invasive mammary cell line and a subclone rendered invasive and metastatic by stable transfection with an expression vector for OPN to identify Ran GTPase (Ran) as the next most overexpressed gene, and as such is a possible downstream target for OPN. Ran itself is a small GTPase and is involved in various cellular processes including nucleocytoplasmic transport, apoptosis, mitotic spindle organization and nuclear envelope formation. Moreover it is overexpressed in many human tumours, including stomach, lung, head and neck, pancreas, colon and kidney, but not in their normal cells. Knock-down of Ran in various tumour cell types causes aberrant mitotic spindle formation, mitochondrial dysfunction, and apoptosis. In contrast, loss of Ran in normal cells is well tolerated and does not lead to mitotic defects or loss of cell viability.

This is explained by the idea of a ‘‘cancer-specific’’ utilization of Ran signaling, with the demonstration that Ran is broadly overexpressed in cancer compared with normal tissues in vivo, and its pathway is selectively used by tumor cells to execute mitosis. The connectivity map is a reference collection of gene expression profiles from cultured cells treated with bioactive molecules, linked to pattern matching software to enable data mining. It was originally developed to link disease states, gene perturbations in gene expression, and drug action. In short, if a gene expression signature can be determined for a particular phenotype, the connectivity map is a tool for identifying off-patent/FDA approved therapeutic compounds that can modulate expression of those genes. We have identified a panel of compounds predicted to downregulate Ran. These compounds were identified using SSCMAP, a java based computational tool, and was linked to a gene signature of Ran overexpression generated from a library of over 600 gene expression arrays.

It is hypothesized that these compounds should induce Ran downregulation and mediate cytotoxicity. In so doing, this work aims to validate the connectivity map as a powerful tool with potential for accelerating drug discovery and translation of targeted compounds into the clinic.

The aims of this study are:

  1. Analyse the small molecule compounds identified as potential Ran inhibitors by connectivity mapping
  2. Investigate the effects of the small molecules on Ran mRNA and protein will be assessed in cell culture experiments using AML, breast and non-small cell lung cancer cell line model systems
  3. Determine Ran mRNA and protein expression will be assessed by Q-PCR, and Western blot, respectively
  4. Investigate the effect of small molecules that down-regulate Ran on cell invasion/migration and apoptosis using a range of invasion, cell viability and cell death assays.

Entry requirements:

At least 2:1 Honours degree or equivalent in biological sciences, biochemistry or related subjects.

Supervisors:

Title and name:
Professor Mohammed El-Tanani
Position:
Professor of Molecular Pathology and Cancer Therapeutics
Email address:
Telephone number :
Work+44 (0) 1274 235367
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Title and name:
Professor Richard Morgan
Position:
Director, Institute of Cancer Therapeutics and Professor of Molecular Oncology
Email address:
Telephone number :
Work+44 (0) 1274 233235
View full profile

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