Feature: Human focus of miRNA

Read part I of this feature, Seeking a miRacle.

Professor Peter Leedman was acting on a hunch in 2005 when he suggested that PhD student Rebecca Webster search among microRNA molecules for the unknown regulator of epidermal growth factor receptor (EGFR) expression.

He suspected most research groups searching for putative targets in the three prime untranslated region (3’ UTR) of the EGFR gene would be looking for sequences that were highly conserved in mammals, including humans.

At the time, sequence conservation across species was considered one of the most important determinants of target identification. “The lack of species conservation for putative miR-7 binding sites in the human EGFR mRNA 3 prime untranslated region didn’t really concern us that much because the human sequence was the key target,” Leedman says.

Once miR-7 had been identified, the team led by Dr Keith Giles, working with Rebecca Webster and Karina Price, set about validating the interaction with the EGFR mRNA. In 2006 they showed that miR-7 significantly decreased EGFR mRNA and protein expression in multiple different cancer cell lines, including cancerous lung, breast, prostate and glioblastoma cell lines.

They went on to use microarray technology and show that miR-7 regulated as many as 300 other genes in cancer cells. Many of the 3’ UTR of these mRNAs contained at least one target site capable of binding miR-7, suggesting that miR-7 may be a master regulator of multiple signalling pathways. The mRNA that specifies EGFR features three miR-7 binding sites, two of which appear to be directly targeted by miR-7, with additive effect.

Leedman says Giles led the subsequent characterisation of additional miR-7 targets and the work leading to the team’s understanding of the multiple signalling pathways regulated by miR-7. This included the discovery that RAF1, another oncogene active in epithelial tumours, was among the roll-call of hundreds of genes with miR-7 target sequences. RAF1 is a direct target of miR-7; its expression is downregulated when miR-7 is added to cancer cells.

MiR-7 also represses expression of the phosphorylated form of protein kinase B (Akt), and extracellular signal-regulated kinase I/II, which are both critical downstream effectors of EGFR signalling.

Herein lies the exceptional promise of miR-7, says Leedman: not only does it reduce EGFR expression in cancerous cells, it represses the expression of other genes, and aberrant protein phosphorylation, in multiple pathways downstream of the receptor (for example phospho-Akt).

The WAIMR team, together with University of Western Australia (UWA) researchers, quietly filed provisional patents on the discovery in 2006, and with funding from the NHMRC, continued working on understanding the biology of miR-7 for several years before publishing. Webster was lead author on the paper, “Regulation of Epidermal Growth Factor Receptor Signalling in Human Cancer Cells” when it was eventually published in the Journal of Biological Chemistry, in February 2009.

In its abstract, it states: “[Our] data establish an important role for miR-7 in controlling mRNA expression, and indicate that miR-7 has the ability to coordinately regulate EGFR signaling in multiple human cancer cell types.”

The paper reported evidence that miR-7 coordinately regulates EGFR signalling at multiple levels, as well as regulating a number of other cellular pathways relevant to normal and tumour cells.

Reports of miR-7 being down-regulated in brain and spinal-cord tumours, and miR-7’s ability to regulate oncogenic EGFR signalling in multiple cancer cell line models, suggested that therapeutic up-regulation of the microRNA in tumours may not only inhibit the growth of primary tumours, but could prevent metastasis.

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IP ‘solid’

The WAIMR together with UWA used the interval between discovery and publication to perform due-diligence checks in the intellectual property – Leedman says the IP is “very solid.” Last year the institute and UWA secured a $650,000 grant from the Medical Research Commercial Fund (MCRF). Brandon Capital Partners manages the $30 million fund, which was established in 2007 to assist the development of early-stage biomedical discoveries.

In October last year, WAIMR, UWA and the MRCF announced it had spun out a new Australian venture company, MiReven, to conduct further preclinical testing and validation of miR-7 as a novel therapy for epithelial cancers, with MRCF Director, Dr Stephen Thompson, on the board.

Commercial interest in microRNA-based medical therapies has grown rapidly in the past year. Sanofi-Aventis signed a broad $750 million tie-up with Regulus Therapeutics in June, and a few days later, Santaris Pharma and miRagen Therapeutics announced a partnership to develop microRNA products in the cardiovascular field. GlaxoSmithKline has taken a development option on Santaris' miR-122 product, miraversen, which entered a Phase II trial for hepatitis C last September.

“We hope the preclinical studies will validate further development of miR-7 as a potential therapy to arrest growth of tumours that overexpress EGFRs,” says Leedman. “We are also very interested in restoring the sensitivity of tumours to other therapies, including targeted therapies, chemotherapy and radiotherapy.

“MiReven is currently investigating the effects of miR-7 in preclinical models, which if successful may eventually lead to a phase I clinical trial in human cancer patients with poor prognostic EGFR over-expressing tumours.

For example, patients with advanced glioblastomas often have a very unfavourable outlook. These tumours respond poorly to conventional therapies, and are extremely resistant to current treatments. If we could restore sensitivity to chemotherapy or EGFR-targeted treatments, it may significantly enhance the therapeutic outcome in these patients.

“There’s a lot of interest around the world in microRNA-based therapies for cancer. MiReven will be looking to increase expression of miR-7 in solid tumours, through targeted systemic delivery of a mimic of the endogenously expressed microRNA.

“If the current studies prove successful, miReven will be looking for a commercial partner. It’s a very hot area, and the pharmaceutical companies are investing heavily to develop efficient and effective systems for targeted delivery of microRNA and siRNA therapeutics into specific clinical areas of need.

“We hope by then they will have well developed delivery systems, and that they will be looking to license-in therapeutic agents, such as miR-7, that have significant clinical potential.”