The 'omics' of cancer

Novel synthetic molecules that represent a potential combination treatment for late-stage basal breast cancer are moving towards being trialled in animals.

It’s 42 years since President Richard Nixon declared war on cancer, hoping to trump President John F Kennedy’s successful 1962 promise to put an American on the Moon by 1970.

A cure-all for cancer remains as remote a prospect as the first human colony on the Moon. Survival times for many cancers have increased because of earlier diagnosis, and the advent of life-prolonging drugs. But oncology’s greatest need - and greatest challenge - is to develop fast-acting therapies that might save the lives of patients at imminent risk of succumbing to end-stage, drug-resistant cancers.

Associate Professor Pilar Blancafort’s research group at the University of Western Australia has provided a glimpse of the future, in the form of two novel therapies for potential use in combination in late-stage basal breast carcinomas. These new synthetic molecules are already moving towards being trialled in animals.

Triple negative carcinoma

Dr Blancafort, a plenary speaker at the Lorne Genome Conference, will describe her group’s progress towards developing effective treatments for late-stage basal carcinomas of the breast.

Unlike most breast cancers, so-called ‘triple-negative’ carcinomas do not express estrogen, progesterone or human epidermal growth-factor 2 (Her2-neu) receptors, so they are not amenable to treatment with front-line endocrine-receptor antagonists like tamoxifen and herceptin.

Blancafort and her colleagues hope their new molecules will provide a lifeline for women diagnosed with an uncommon and highly aggressive form of triple-negative breast carcinoma, known as late-stage inflammatory breast cancer.

Direct promoter interaction as therapy

Blancafort says a cell’s transformation from a normal to a cancerous state involves wholesale changes in the epigenetic deregulation of key genes that are transmitted to successive generations of daughter cells.

In cancerous cells, demethylases reactivate repressed transcription factors that maintain the multipotency of progenitor cells that build specialised tissues and organs.

However, in cancerous cells the direction of development is reversed: daughter cells regress to a relatively primitive, unspecialised state that, in time, becomes resistant to chemotherapy. With nothing to restrain their rapid growth and proliferation, the malignant cells spread to other organs, where they give rise to aggressive, metastatic tumours.

Meanwhile, methyltransferases progressively methylate large segments of chromosomes, inactivating vital tumour-suppressor genes like P53. Without these sentries to perform quality-control checks on newly replicated DNA, daughter cells accumulate dangerous new mutations and malignant new methylation states that drive them towards uncontrolled cancerous growth.

Modern breast cancer drugs typically target endocrine receptors that study the surface of cancerous cells. Blancafort’s group has taken a completely different tack, by designing synthetic molecules capable of being transported into the cell to interact directly with the promoters of the key genes driving cancerous growth.

Their prime target in triple-negative breast cancers is the gene for the transcription factor engrailed (EN1), an early player in patterning the development of the central nervous system.

Synthetic interference peptides

In a paper published in Oncogene this year, Blancafort’s group reported that EN1 is selectively overexpressed in triple-negative breast carcinomas.

Normally, it is expressed in neural progenitor cells. Later in life it appears to maintain the brain’s pool of long-lived dopaminergic neurons through its pro-survival, anti-apoptotic activity.

Blancafort’s team showed that short interfering RNAs targeted to EN1 triggered potent, selective death of basal breast tumour cells, explaining the cancer’s propensity to develop drug resistance, and to re-emerge and metastastise, after surgery and chemotherapy.

They engineered synthetic interference peptides (iPeps), containing a peptide sequence that mediates the EN1 protein’s interactions with other proteins. The iPeps also contained a cell-penetrating sequence that causes it to ‘home’ to the cell nucleus.

MALDI-TOF mass spectrometry confirmed that the synthetic peptide captured proteins involved in transcriptional and post-transcriptional regulation of proteins involved in inflammatory pathways.

The finding established EN1’s role as an activator of intrinsic inflammatory pathways associated with pro-survival in basal-like breast cancer.

Cancer-specific combination therapies

Blancafort’s team has confirmed the potential of a similar synthetic peptide to knock down EN1, blocking its pro-inflammatory, pro-survival activity.

“It works at micromolar concentrations, which is still a bit high. We want to chemically optimise the peptide to be effective at lower concentrations, and stabilise it to improve its half-life, and to avoid the body’s clearance systems,” Blancafort said.

Blancafort’s team has also encapsulated the iPep in nanoparticles and targeted it to cancerous cells using ligands, including cell-killing drug molecules, to produce synergistic activity.

“Using small amounts of the drug, you get the double benefit of knocking down the transcription factor that drives the cancer, and resensitising resistant cells to the drug’s cell-killing activity.

“We’re looking at extending this approach to other transcription factors and developing cancer-specific combination therapies, so you get an interplay that modulates several signalling pathways, so you kill the cancerous cells at lower dosages.

Animal trials

Blancafort says her team is planning animal trials to optimise the pharmacokinetics of their molecules, to ensure they combine high specificity with low toxicity.

They are developing another novel therapeutic molecule that targets SOX-2, an oncogenic transcription factor overexpressed in a variety of malignancies characterised by a high recurrence rate and poor patient prognosis.

Blancafort says an explosion of studies 2012 and 2013 implicated SOX-2 in the initiation, progression and recurrence of highly aggressive forms of breast and ovarian cancer, prostate cancer, glioblastomas, hepatocellular carcinoma and small cell lung cancer.

In normal breast ductal cells, SOX-2 is epigenetically silenced by methylation of its promoter, but it is hypomethylated and overexpressed in nearly 50% of basal-like triple-negative breast carcinomas.

Blancafort’s team identified SOX-2 as a potential key target for an experimental therapy with a synthetic zinc-finger molecule that will bind to its promoter, replicating the natural silencing effect of methylation.

The suppressor molecule consists of a sequence C2H2 zinc-finger domains; each recognises and binds one unit of a repeated three base-pair DNA motif in the SOX-2 promoter region.

While a three-unit ZF ‘zipper’ is probably sufficient to inactivate SOX-2, Blancafort says the six-unit molecule actually outcompetes the endogenous transcription factors and potently regulates SOX-2. With its exceptional avidity for the SOX-2 promoter, the synthetic ZF ‘off switch’ should work at safe, picomolar concentrations and provide ample time for drugs to kill the resensitised cells.

She says the ZF ‘silencer’ system provides a model for experiments with other synthetic peptide complexes that will outcompete native ligands to occupy the promoters of other oncogenic transcription factors.

A whole genome perspective

Blancafort says such promoter-targeting synthetic complexes look particularly promising as prospective therapies for some of the most aggressive forms of cancer, involving cells in which wholesale disruption of normal epigenetic controls have regressed to a more primitive, stem-like state, including the capacity to grow and proliferate rapidly.

Blancafort says the amplification and overexpression of oncogenes like Myc and Ras in aggressive cancers is a challenge for conventional therapies - the new synthetic silencing complexes offer the advantage that they can be designed to precisely target key oncogenes that drive these refractory cancers and displace the native factors driving their overexpression.

Blancafort says cancers offer many targets for new therapeutic drugs and the future of the new synthetic molecules lies in developing combination therapies to hit multiple targets simultaneously.

“We have so much ‘omics’ information now, so it’s a matter of identifying the important targets in different forms of cancers.

“A model system like ours helps us to understand how these molecules work, what happens over time and how they interact.”

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Lorne conference line-up

Here’s the line-up for the Lorne conferences for 2014, to be held at Mantra Lorne on the Victorian south coast.
19th Lorne Proteomics Symposium
February 6-9
http://www.australasianproteomics.org/lorne-proteomics-symposium-2014/
39th Lorne Conference on Protein Structure and Function
February 9-13
http://www.lorneproteins.org/
26th Lorne Cancer Conference
February 13-15
http://www.lornecancer.org/
35th Lorne Genome Conference
February 16-19
http://www.lornegenome.org/
Lorne Infection and Immunity
February19-21
www.lorneinfectionimmunity.org

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Dr Pilar Blancafort is Associate Professor in the School of Anatomy, Physiology and Human Biology at the University of Western Australia.  Born in Santa Eugenia de Berga, 100 km from Barcelona, in Spain, she studied physics and biology at the University of Barcelona, specialising in biochemistry, genetics and molecular biology. In the last year of her undergraduate studies, she was awarded an ERASMUS (European Community Action Scheme for Mobility of University Students) Fellowship to study at the Free University of Brussels where she did an Honours project with Dr Alain Ghysen, studying neural transcription factors in Drosophila - her first contact with transcription factors and development, the field she specialises in today.

Image credit: ©iStockphoto.com/vitanovski