Feature: Seeking a miRacle

Peter Leedman has a very clear memory of his “eureka!” moment. In 2006, PhD student Rebecca Webster told him she had positively identified a novel interaction between a microRNA (miRNA) molecule and a key target in cancer, the epidermal growth factor receptor (EGFR).

Webster confidently assured Leedman that the three prime untranslated region (3’ UTR) of the messenger RNA for human EGFR was almost certainly a target for microRNA-7 (miR-7). 3’ UTR is a region on a messenger RNA (mRNA) that is not used in translation into a protein.

“Rebecca had to generate her own algorithm to predict microRNA binding sites, because this was before the birth of TargetScan and other microRNA target prediction web sites that are readily available today,” says Leedman.

It was the news that Leedman, Deputy Director of the Western Australian Institute for Medical Research (WAIMR) and Professor of Medicine at the University of Western Australia, had been hoping for.

At only 22 nucleotides in length, miR-7 may be a molecular minnow, but its potential as a generic weapon against some of the most common and aggressive human cancers is inversely proportionate to its size.

He says epithelial cancers – cancers that arise in the specialised surface tissues of the body, including the mucosal lining of internal organs – constitute one of the most common classes of human cancers. They include head and neck cancers, pancreatic cancer, non-small cell lung cancer, gastric cancer, colorectal carcinoma, squamous cell carcinoma (skin), breast, ovarian and prostate cancer.

A large proportion of epithelial tumour cancer cells over-express EGFRs on their surface. They may also constitutively over-express ligands that activate the receptor, such as EGF or transforming growth factor alpha (TGF-α), creating a positive feedback loop that drives cell growth and division, and tumour progression.

Overexpression of the EGFR is also not limited to epithelial tumours. Glioblastomas, aggressive glial cell-derived tumours of the central nervous system, commonly have elevated EGFR levels on their cell surface.

In many of these cancers, over-expression of EGFR is due to gene-duplication events, but in others – such as in many glioblastomas – over-expression occurs without gene duplication, suggesting that other, post-transcriptional mechanisms are driving aberrant EGFR expression in cancer cells.

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According to Leedman, proliferation of EGFRs on epithelial cancer cells correlates with a tumour’s rate of progression, resistance to chemotherapy and radiotherapy, and poor prognosis for patients.

This observation, and the fact that miR-7 is down-regulated in central nervous system tumours, hinted that the molecule regulates multiple, normal cellular pathways and acts as an endogenous tumour suppressor in others.

After EGF binds and activates its cognate receptor on the cell surface, tyrosine kinase molecules phosphorylate the receptor’s intracellular domain, making docking sites accessible to signalling molecules that activate downstream pathways.

These include the MAPK and PI3 kinase-Akt pathways, which promote cell growth and survival, as well as invasive growth and angiogenesis – the formation of new blood vessels that nourish tumours.

Leedman says the search for new therapies for tumours overexpressing the EGFR has focused recently on monoclonal antibodies that target EGFRs, and kinase inhibitors to block phosphorylation of proteins involved in cell growth and division. Monoclonal antibodies directed against EGFRs should arrest the cell cycle, causing the cell to die by necrosis – not apoptosis.

But, says Leedman, although some patients’ tumours respond dramatically to EGFR-targeting monoclonal antibodies like cetuximab and trastuzumab, and EGFR tyrosine kinase inhibitors like gefitinib and erlotinib, which compete for tyrosine kinase binding sites on the C-terminus of the EGFR receptor, the clinical improvement hasn’t been as significant as initially envisaged.

Some patients show a promising initial response, but their tumours eventually become resistant to such therapies because they strongly select for mutations that constitutively activate other downstream effector genes in the MAPK or PI3 kinase-Akt pathways, even when EGFR expression is inhibited, or phosphorylation of its C-terminus is blocked. Other patients develop serious side-effects, which limits the use of some of these targeted therapies.

What is needed is a therapy that can simultaneously suppress EGFR expression, while repressing downstream effectors in the MAPK and PI3 kinase-Akt pathways. MiR-7 appears to do just that.

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International race

In 2005, the WAIMR research team was one of many international groups involved in a race to identify novel molecules which, in healthy epithelial tissues, keep a tight rein on EGFR expression.

A molecule that could down-regulate expression of EGFR and other downstream oncogenes would potentially provide clinicians with a versatile therapy to arrest the growth of cancerous cells, and force them to differentiate and die.

The challenge would then be to target the therapy to tumours, says Leedman. The pharmaceutical industry is investing millions of dollars in developing targeted delivery systems to deliver molecules safely to tumours. The team hopes that there will be no shortage of suitors interested in a partnership to take the EGFR therapy towards the clinic, if preclinical studies are successful.

“We’re still going to have chemotherapy and radiotherapy as the cornerstones of cancer treatment, but we’re seeing the development of an increasing number of personalised therapies in clinical trials and at the bedside, and these new treatment options are expected to increase,” says Leedman.

MicroRNAs are small (<23 nucleotides) RNA molecules that are typically encoded within the anti-sense strand of chromosomal DNA. They operate post-transcriptionally, binding complementary sequences in mRNA and forming double-stranded segments that prevent ribosomes translating the mRNA from producing protein molecules themselves.

In animals, the microRNA rarely achieves perfect complementarity with its target sequence in the 3’ UTR of a messenger RNA, with some nucleotides remaining unpaired. But ribosomes run on monorails, and the presence of a double-stranded RNA on the leader sequence, however loosely bound, prevents the ribosome engaging and translating the downstream message to produce a protein molecule.

MicroRNAs can also regulate target gene expression by accelerating decay of the target mRNA. Many microRNAs activate both pathways producing maximal effect, with miR-7 and the EGFR being a good example.

The requirement to make only a loose match with a target mRNA cuts the microRNA some slack, permitting it to bind more than one sequence in the same mRNA, or to bind mRNAs from multiple genes. Microarray and proteomics experiments have shown that single microRNAs can coordinately regulate expression of hundreds of genes in multiple cellular pathways.

MicroRNAs may act as tumour suppressors, by coordinately up-regulating expression of tumour-suppressor genes, while repressing oncogenes. But vesting so much regulatory power in a single microRNA like miR-7 could have dire consequences should mutation or some other form of dysregulation disrupt its normal expression.

Then, entire pathways can go awry: previously repressed miRs and oncogenes may be activated and over-expressed, while tumour-suppressor miRs and genes are shut down, tipping the cell into cancerous growth.

Read the second part of this feature, Human focus of miRNA.