Lorne special: Mortality, metastasis and miRNAs

This feature appeared in the January/February 2010 issue of Australian Life Scientist. To subscribe to the magazine, go here.

A decade ago medical science was galvanised by the discovery that the non-protein coding DNA of gene introns – and the vast tracts of DNA between genes – conceal a cryptic RNA-based operating system for the genome. This complex system codes for myriad small RNA molecules – microRNAs – that regulate the activity of the genome’s 23,000-odd protein-coding genes.

The cell nucleus swarms with microRNAs, tiny (21-23 mer) RNA molecules, cleaved from larger RNA precursors by the endonucleases Drosha and Dicer. Because cancer is a disease of gene dysregulation, cancer researchers took an immediate interest and began mining the human genome for associations between miRNAs and oncogenesis, and hit pay dirt.

Associate Professor Greg Goodall, of the Centre for Cancer Biology, was an early player, developing new microarray techniques for quantifying miRNA expression, although he credits his colleagues, such as Dr Philip Gregory and Dr Yeesim Khew-Goodall – who is married to Goodall – for contributing to many of his achievements.

Amongst these achievements has been an investigation into the mechanisms underlying metastasis in epithelial cells and, in particular, the role played by certain miRNAs.

Slipping through

Many solid tumours arise from epithelial cells that line the body's internal plumbing, where they take the brunt of exposure to environmental carcinogens. Epithelial cells can transform into cancerous mesenchymal cells that can break out of the primary tumour and establish deadly secondary tumours in other organs or tissues.

Epithelial cells stick together through thick and thin, until programmed cell death parts them. But within the dysfunctional cellular communities of breast tumours and other epithelial cancers, mutation-damaged cells can evade apoptosis and achieve a kind of immortality by slipping their communal bonds and transforming into mesenchymal stem cells.

As Goodall points out, primary tumours account for only a minority of cancer deaths; most patients succumb to the effects of secondary, metastatic tumours after the primary tumour has been surgically removed or treated with radiation or chemotherapy. This makes the epithelial-mesenchymal transition a promising new frontier in cancer research. A detailed understanding of the mechanisms involved could yield new therapies to suppress metastasis, greatly enhancing survival rates. Goodall believes best prospects may lie not in conventional cancer drugs, but in microRNA-based therapies.

Goodall has shown that the epithelial-mesenchymal transition is marked by loss of E-cadherin – the protein ‘adhesive’ that binds and organises epithelial cells into layered communities. Then, free to roam the lymphatic and blood networks, nomadic new mesenchymal cells can lodge in other organs and spawn metastatic tumours. This places the epithelial-mesenchymal transition (EMT) as a critical phase in the progression of cancer.

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Goodall’s quantitative microarray analysis showed that microRNAs in the miR200-family, including miR-200a, miR-200b and a relative, miR-249, are strongly expressed in healthy mammary epithelial cells, but decline to very low levels during the transformation to mesenchymal cells in cancerous cell lines. Their decline correlates strongly with reduced expression of the epithelial cell marker E-cadherin. The findings show that miR-200 family members suppress the E-M transition and metastasis by maintaining E-cadherin expression.

Epithelial cells are also strongly polarised: they line up in the same orientation and, linked by E-cadherin, form the characteristic ordered layers of epithelial tissues. For as long as they remain bound by E-cadherin, mammary epithelial cells remain mortal and expendable; they are inhibited from transforming into mesenchymal cells and detaching from the tumour. Thus, it appears E-cadherin plays a critical role in preventing metastasis.

Goodall’s findings establish that E-cadherin is both necessary and sufficient for maintaining the epithelial phenotype. This was supported by experiments on a panel of invasive breast cancer cell lines that yielded similar results. His team also has evidence implicating loss of miR-200 microRNAs and E-cadherin expression in bowel cancer metastasis. Other researchers have found a similar association in ovarian cancer, and Goodall believes it may be common to all metastatic epithelial tumours. This raises the exciting possibility of developing RNA-based therapies to reinstate normal miRNA expression patterns and restore E-cadherin expression.

Staying put

Together the Goodall and Khew-Goodall groups have confirmed the potential of RNA-based therapies by engineering cancerous mesenchymal cells to over-express the three miR-200 genes. Goodall found that when cultured epithelial cell are exposed to the cytokine Transforming Growth Factor-beta (TGF-β) – a known agent of metastasis – they will transform into mesenchymal cells.

According to Goodall, cells in breast tumours and other epithelial tumours are commonly exposed in vivo to TGF-β from two sources: their own, internally synthesised supply, and TGF-β signalling from the tumour’s supporting cast of stromal cells.

“Even when we engineer epithelial cells to constitutively express TGF-β, as long as they still express miR-200 micro RNAs, they cannot undergo a TGF-β-induced transformation to mesenchymal cells,” he says.

Goodall and Khew-Goodall described their findings in a co-authored paper in Nature Cell Biology in 2008. Goodall says positional cues (cellular setting) and environmental cues (TGF-β signalling) can down-regulate miR-200 expression, inducing the EM transition and metastasis.

In a paper published later in 2008 in Cancer Research, Goodall and his colleagues, along with Dr Frances Shannon’s group at the Australian National University’s John Curtin School of Medical Research, describe how a double feedback loop regulates E-cadherin expression and the EM transition. They describe how miRNAs regulate E-cadherin expression indirectly, by regulating two intermediary genes coding for the transcriptional repressor proteins ZEB1 and ZEB2.

The miR200a, miR200b and miR-429 microRNAs are all specified by a primary miRNA (pri-miRNA) gene on chromosome 1. A second pri-RNA gene on chromosome 12 specifies another pri-RNA that yields two other microRNAs, miR 200c and mir-141, that collaborate in regulating ZEB expression. In mesenchymal cells, the ZEB proteins bind to the pri-RNA gene’s promoter, preventing the gene from replicating its 7.5 kilobase transcript.

By halting production of the mir200-family microRNAs, the ZEB proteins abolish E-cadherin synthesis, and the epithelial cells transform into mesenchymal cells. The newly transformed mesenchymal cells detach from the primary tumour and are free to move through the blood and lymphatic systems to other organs, where they can spawn multiple secondary tumours. But in healthy epithelial cells, the pre-miR gene’s promoter is left open and active, because production of the ZEB proteins is blocked by the miR-200 microRNAs, which recognise and bind the ZEB mRNA to prevent its translation into protein.

As long as the mir-200 microRNAs have the upper hand in the reciprocal feedback loop, there are no ZEB proteins to repress E-cadherin synthesis. The epithelial cells stay put in the tumour, unable to transform and escape to spawn secondary tumours.

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Unlocking, and locking, metastasis

The Khew-Goodall and Goodall groups are now using animal models of breast cancer to determine whether the microRNAs are capable of suppressing metastasis.

“We are also looking for evidence of loss or down-regulation of the microRNAs in invasive regions of human colon cancers,” says Goodall. “So far we’ve focused on breast and colon cancer, partly because we have good collaborative access to tissue pathologists working on these cancers here in SA Pathology.”

He also believe it’s highly likely that loss of miR-200 expression might be common to most invasive epithelial tumours. “Other groups are looking in prostate and pancreatic cancers, and our collaborators in the U.S. have recently published evidence miR-200 can control lung cancer metastasis.”

According to Goodall, very little is known about the role of this particular genetic axis in normal development. “People are just beginning to look at it, and nobody has published yet. But it’s clearly very important in the epithelial mesenchymal transition,” he says.

“We believe the microRNAs contribute to establishing and maintaining the strong polarity of normal epithelial cells. In terms of the cancer process, and metastasis in general, we think it involves a combination of autonomous events in the cell that either actively drive it towards the invasive mesenchymal form, or permit it to do so.

“The process may involve inductive events from the cell’s environment, like TGF-β signalling by stromal tissues, which is activated in response to the development of the primary tumour. But it may also involve cell-autonomous events, like amplification of ZEB repressor protein expression blocking microRNA expression.

“There has been an explosion of information since we first realised these microRNAs were doing something in humans. The dream is that the microRNAs themselves, or modified forms of naturally occurring microRNAs, might be used as therapeutics, because they would block metastasis at its earliest stages.

“They could force cells to give up their invasive or migratory behaviour. There are also prospects that the targets of the microRNAs could also be targets for new therapies, and that altered patterns of microRNA expression could be used as early diagnostic markers for certain cancers.”

The Centre for Cancer Biology was notified in November that it had been awarded a $3.5 million grant from the Australian Cancer Research Foundation to establish a next-generation DNA sequencing facility, which will further accelerate discovery.

This feature appeared in the January/February 2010 issue of Australian Life Scientist. To subscribe to the magazine, go here.