Tag Archives: tumour

RNAi: Send in the Nanobots!

The publication of the preliminary results of a small clinical trial of a new therapy called RNA interference (RNAi) online in the scientific journal Nature is causing quite a stir in the scientific community this week.  A team led by Professor Mark E. Davis at Caltech targeted the delivery of a nanoparticle only 70 nanometers in diameter containing small interfering RNA (siRNA) to cancer cells in three patients with metastatic melanoma, which reduced the levels of a protein called RRM2 that is required for the tumour growth.  This trial is the result of over a decade of research in organisms as diverse as nematode worms, mice and monkeys, but why is the result of this trial so noteworthy? And what is RNAi anyway?

Cancer genes in human melanomas have been switched off. Image courtesy of the National Cancer Institute

If you have ever studied biology you will probably be familiar with the “central dogma of molecular biology”; it describes how our genes encode the proteins that are the building blocks, and indeed the builders, of all the cells in our bodies. The very short version is that our genes are made up of sequences of double stranded DNA consisting of the deoxyribonucleotides A,C, G and T, and these sequences are transcribed by a protein called RNA polymerase into matching sequences of the single stranded messenger RNA (mRNA) , made from the ribonucleotides A, C, G and U. Another protein complex known as the ribosome then translates the mRNA sequence into a corresponding sequence of amino acids that when completed make up a brand new protein.  Our new protein almost invariable undergoes further processing but we needn’t concern ourselves with that here.  RNAi is the process where an assembly of proteins named the  RNA-induced silencing complex (RISC) binds short double stranded segments of RNA that in turn target RISC to particular mRNA sequences to which they are complementary.  RISC breaks down the mRNA molecule, preventing production of its associated protein and effectively silencing the targeted gene. The beauty of RNAi is that it allows an organism to target specific mRNA molecules for destruction, and it is a mechanism for regulating the flow of genetic information whose importance we are still only beginning to appreciate.

RNAi was discovered only 12 years ago by Andrew Fire and Craig Mello through their basic research on the regulation of gene expression in the nematode worm Caenorhabditis elegans, a discovery which earned then the Nobel Prize in 2006. C.elegans is a popular model organism for scientists studying gene function and development, its small size and simple structure make it relatively easy to follow the fate of individual cells, while as an animal it shares many of its genes and biological processes with mammals.  This turned out to be the case with when in 2001 it was shown that RNAi helps mice to control hepatitis B infection, and scientists began to examine whether RNAi could be used therapeutically (1). To do this scientists made siRNA, an artificial version of the short double stranded segments of RNA that target RISC to complementary mRNA sequences, and early experiments in mice demonstrated that siRNA induced RNAi could reduce the levels of target proteins in mice.  The first human trials of RNAi began in 2004 for the treatment of wet age related macular degeneration and at first seemed promising, but suffered a setback when further research in mice revealed that the “naked” siRNA injected into the eye in these trials actually stimulated an immune response that was responsible for at least some of the benefits seen in earlier trials (2). This was a worry as an unwanted immune response might lead to an adverse reaction if the siRNA was injected into the bloodstream rather than a small part of the eye.

In recent years scientists have been developing technologies that allow injected siRNA to evade the immune system and target only those tissues where RNAi activity is desired,  reducing the quantity of siRNA that needs to be injected and also the risk of adverse  effects due to RNAi affecting off-target tissues. Mark E Davis, a professor of chemical engineering at Caltech and one of the scientists leading these efforts, uses polymers that assemble with siRNA to form a nanoparticle that resembles a tiny ball with siRNA at its centre.  The nanoparticle shell protects the siRNA from being broken down while it is circulating in the bloodstream, and then interacts with the cell membrane to help the siRNA enter a cell so that it can do its job.  Of course he didn’t want the nanoparticle to release its siRNA payload into any old cell so he attached a protein called transferrin as a targeting ligand to the nanoparticle. Tumour cells express far more of the transferrin receptor on their surfaces than normal cells, and the hope was that the nanoparticles would bind to tumour cells in preference to normal cells.  To test whether this would work Prof. Davis team injected the nanoparticles, containing a siRNA that targeted a cancer gene, into mice that had metastatic Ewing’s sarcoma(3). They observed that the transferrin labelled nanoparticle delivered the siRNA to the tumour cells, knocked down the activity of the target cancer gene and dramatically slowed tumour growth, and when the transferring ligand was removed his effect not seen.  They also observed that the nanoparticle did not stimulate the immune system or affect any of the major organs of the mouse, indicating that their method had solved safety problems seen in earlier RNAi trials.

The targeted nanoparticle used in the study and shown in this schematic is made of a unique polymer and can make its way to human tumor cells in a dose-dependent fashion. Image courtesy of Derek Bartlett and the California Institute of Technology.

Prof. Davis and his colleagues next needed to identify an appropriate target for human trials of their nanoparticle siRNA delivery system, and decided to target the M2 subunit of Ribonuclease reductase (RRM2), a protein that is required for cell division and which has recently been the subject of a lot of research as a target for anti-cancer drugs.   They first used in vitro studies to identify a siRNA sequence that effectively targeted the RRM2 mRNA, which they named siR2B+5, and then demonstrated in mice that this siRNA could block the production of RRM2 and reduce the growth of tumours (4).  As a final safety evaluation prior to human trials they injected different doses of their nanoparticle  containing siR2B+5 and labelled with transferrin to cynomologus monkeys, whose RRM2 mRNA is targeted by siR2B+5 in exactly the same way as in humans,  and found that it was safe and did not produce any unwanted effect on the immune system (5).

The human clinical trial reported this week confirmed that transferrin-labelled nanoparticle injected into the bloodstream were safely delivered siR2B+5 to the tumours of metastatic melanoma patients, and that the siRNA knocked down the production of RRM2 protein by RNAi (6).  Of course this is only a preliminary result, at this stage we don’t know to what extent this experimental treatment will reduce tumour growth in these patients, let alone if it will cure their cancer. If it is a success it will probably need to be combined with other anti-cancer drugs to be fully effective, so it is good to know that thanks to animal research other nanotechnology based drugs such as Lipoplatin are in clinical trials that offer more potent anti-cancer activity with less toxicity than existing anti-cancer drugs. Nonetheless to focus on this uncertainty would be to miss why this small trial is causing such excitement; for the first time scientists have shown that it is possible to target RNAi therapy to a particular tissue type within the body, and that is a breakthrough that opens up a whole new area of medicine. The era of RNAi medicine has begun!

Paul Browne

1)      McCaffrey A.P., et al. “RNA interference in adult mice.” Nature Volume 418, pages 38–39 (2002) DOI: 10.1038/418038a

2)      Castnotto D. and Rossi J.R. “The promises and pitfalls of RNA-interference-based therapeutics” Nature Volume 457(7228), pages 426-433 (2009) DOI:10.1038/nature07758

3)      Hu-Lieskovan S. et al. “Sequence-specific knockdown of EWS-FLI1 by targeted, nonviral delivery of small interfering RNA inhibits tumor growth in a murine model of metastatic Ewing’s sarcoma.” Cancer Re. Volume 65 (19), Pages 8984-8992 (2005) DOI:10.1158/0008-5472.CAN-05-0565

4)      Heidel J. et al. “Potent siRNA inhibitors of ribonucleotide reductase subunit RRM2 reduce cell proliferation in vitro and in vivo” Clin. Cancer Res. Volume 13(7), Pages 2207-2215 (2007) DOI: 10.1158/1078-0432.CCR-06-2218

5)      Heidel J. et al. “Administration in non-human primates of escalating intravenous doses of targeted nanoparticles containing ribonucleotide reductase subunit M2 siRNA.” Proc.Natl Acad. Sci. USA Volume 104(14), Pages 5715-5721 (2007) DOI: 10.1073/pnas.0701458104

6)      Davis M.E. “Evidence of RNAi in humans from systemically administered siRNA via targeted nanoparticles”  Nature Advance Online Publication 21 March 2010 DOI:10.1038/nature08956

Can we protect the brain against tumor metastasis?

Brain metastasis that affect at least 20% of cancer patients are a serious problem for doctors seeking to treat cancer and kill thousands of patients every year, being particularly difficult to treat because many anti-cancer drugs cannot cross the blood-brain barrier and because surgery to remove the tumor can often be difficult and risky.  Patients suffering from breast, lung and skin cancer run a relatively high risk that their cancer will spread to the brain, a worrying fact considering that these are amongst the most common of cancers .  As a consequence of this scientists are very keen to understand how cancer spreads to the brain, with the ultimate aim of preventing that spread.

It has long been thought that brain metastasis is due to interactions between cells that are shed by the primary tumor and the nerve cells of the brain, but real evidence of this from living animals and humans for that theory has been hard to find, and in recent years observations made in animal models of cancer have suggested that blood vessels in the brain rather than nerve cells are the site of the early growth of tumor cells during brain metastasis.  This week a paper in the open access journal PLoS One reports on work done by scientists at Oxford University that confirms that during brain metastasis tumor cells do indeed bind to blood vessels and form tumors before spreading into the surrounding nerve tissue, a result of huge importance to the future treatment and prevention of brain metastasis.

Brain Metastasis From Lung Cancer

Brain Metastasis From Lung Cancer

To demonstrate this Dr Shawn Carbonel and colleagues (1) injected breast and skin tumor cells into the bloodstream or fat tissue of mice and then after several days humanely killed the mice determined where in the brain the micrometastases, small colonies of tumor cells that later grow into tumors, were forming, and found that almost all were associated with the blood vessels. There was no sign of any new blood vessel growth, which indicated that the metastases were associating with the blood vessels, and that it wasn’t simply the case that they were promoting the growth of new blood vessels in the vicinity of the growing tumor. To confirm that this is also true in humans they examined tissue samples that been donated following neurosurgery or autopsy and found that almost all metastases were associated with blood vessels, a finding that supported the results of their experiments in mice.

Now they had to answer a new question; were the micrometastases associated with the blood vessels because they have a preference for interacting with the cells of the blood vessel, or simply because the first part of the brain they come to is that adjecent to the blood vessel?  To answer this the Oxford scientists injected tumor cells that were labelled with green fluorescent protein directly into an area of the brain allowing equal access to both blood vessels and nerve cells, and using a cranial window in the skulls of the mice were able to observe where the GFP-labelled tumor cells ended up.  They observed that the GLF-labelled cells associated almost exclusively with blood vessels, and that the tumors subsequently grow into the surrounding brain tissue.

The tumor cells bind to a blood vessel structure called the vascular basement membrane (VBM), but what the Oxford scientists really wanted to know was what caused the tumor cells to bind to the VBM. Once again using mice with cranial windows fitted they found that an enzyme named focal adhesion kinase was highly active where the tumor cells were interacting with the VBM. Focal adhesion kinase is part of a pathway through which a  class of proteins known as the integrins control the interaction between many cells and either other cells or extracellular proteins such as the components of the vascular basement membrane, an observation which suggested that an integrin plays a key role in the binding of tumor cells to the VBM. They next found that a particular integren named Beta 1 integrin is present on all the tumor cell lines they were studying, and that antibodies blocking it could prevent the tumor cells from binding components of the VBM in vitro and to blood vessels in human brain tissue slices.

But would the anti-Beta 1 integrin blocking antibody prevent tumor metastasis in living animals? The answer was yes, the antibodies greatly reduced the growth tumors from human breast tumor cells that were injected directly into the brains of mice. To further emphasize the importance of Beta 1 integrin in brain metastasis they found that when mouse lymphoma cells that had been genetically engineered to lack Beta 1 integrin were injected into mouse brains they formed far smaller tumors than non-GM lymphoma cells.
This  study changes the way we look at the process of brain metastasis, and more importantly in Beta 1 integrin it identifies a target for new drugs, perhaps monoclonal antibodies, that block the binding of tumor cells to blood vessels and prevent brain metastasis.  With this in mind it is useful to note that studies in mice have found that while Beta 1 integrin is crucial during embryonic development prolonged anti-Beta 1 therapy in adult animals did not produce any overt evidence of toxicity (2), indicating that it should also be possible to inhibit it safely in human patients during anti-cancer chemotherapy.

It’s a very nice paper, my only gripe being that they didn’t examine if anti-Beta 1 integrin blocking antibody therapy could prevent tumor cells injected into the mouse bloodstream from producing micrometastases in the blood vessels of the brain rather than just looking at the growth of tumor cells injected directly into the brain, though I expect that those experiments are now being done and will soon be reported.  There will certainly be a lot of interest in this paper in the cancer research world, and scientists will seek to reproduce these results (a vital part of the scientific process) and then expand on them with their own studies of the safety and efficacy of this approach before clinical trials in humans can begin.

Regards

Paul Browne

1) Carbonell W.S. et al. “The vascular basement membrane as “soil” in brain metastasis.” PLoS ONE Volume 4(6):e5857 (2009) DOI:10.1371/journal.pone.0005857

2) Park C.C. et al. “Beta1 integrin inhibitory antibody induces apoptosis of breast cancer cells, inhibits growth, and distinguishes malignant from normal phenotype in three dimensional cultures and in vivo.”Cancer Res. Volume 66(3), Pages 1526-1535 (2006) DOI:10.1158/0008-5472.CAN-05-3071

Tumor Metastasis: Pieces of the Puzzle

When engaging in discussion about the role of animals in scientific

research I am frequently frustrated by how polarized the debate can be,
with anti-vivisectionists often claiming that animal research has made
little or no contribution to advancing medical science, while
occasionally defenders of animal research seem to imply that animal
research alone was responsible for said advances. The reality is a
little more complex with many approaches, some using animals, some not,
being crucial to the process. A procedure that uses animals might
confirm and extend the findings of an /in vitro/ experiment, and then in
its turn be verified and enlarged upon by a clinical study in man.A study published in /Nature/ this week by Dr Terumi Kohwi-Shigematsu
and colleagues (1), and picked up by the /Independent/ newspaper
http://www.independent.co.uk/news/science/gene-discovery-raises-hopes-of-new-treatment-for-breast-cancer-795002.html,
provides a neat illustration of this process in action. Their work
determined that a protein called SATB1, previously identified as a key
factor in driving the expression of genes required for immune system
T-cell development, was also a key player in breast cancer metastasis.
Breast cancer still kills over 10,000 women every year in the UK, the
majority when the cancer spreads from the breast to other tissues.Dr. Kohwi-Shigematsu’s team started by identifying a protein called
SATB1 that was found in cell lines derived from metastatic breast tumour
cells but not in cell lines derived in non-metastatic cells. A screen
of samples from over 1,000 breast cancer patients found that higher
levels of SATB1 in tumour biopsies were associated with a worse
prognosis. These results indicated that higher levels of SATB1 were
associated with metastasis, but not that these higher levels caused
metastasis. After all the higher SATB1 levels could have been a result
of cells becoming metastatic, so they next used RNA interference (RNAi)
to silence SATB1 gene expression in vitro in a metastatic cell line and
found that this caused the cells to grow more slowly and adopt the
characteristics of non-metastatic cells.At this point they were ready to see what effect different levels of
SATB1 expression had on metastasis in a mouse breast cancer model. In a
series of tests they observed that breast cancer cells expressing SATB1
were far more likely to metastasize and form tumours in other tissues,
and that this metastasis could be blocked by RNAi targeting SATB1,
results that confirmed the key role played by SATB1 in metastasis. They
didn’t stop there though, and returned to /in vitro /microarray studies
which demonstrated that SATB1 affects the activity of over 1,000 genes,
notably increasing expression of metastasis-associated genes while
downregulating tumour-suppressor genes.

So where it goes from here? Perhaps the expression of SATB1 will in
future be used as a criterion when deciding whether a patient would
benefit from more aggressive chemotherapy, or maybe SATB1 will itself
become a target for drugs designed to block metastasis. It may even
turn out that a gene whose expression is altered by SATB1 is a more
tempting target for new anti-cancer drugs. What is certain is that with
this very thorough piece of work Dr Kohwi-Shigematsu’s team has opened a
promising new avenue for cancer research.

Paul Browne

1) Han H.-J. /et al. /”SATB1 reprogrammes gene expression to promote
breast tumour growth and metastasis.” Nature. Vol. 452(7184), Pages
187-93 (2008) PubMed: 18337816.