Reversing the Tide of Resistance

Image: Flickr - Joanna Vaughan
The relatively rapid development of drug resistance is a major obstacle to successful chemotherapy. Resistance is frequently attributed to the outgrowth of cells within the tumour which have a genetic survival advantage in the presence of drug treatment such as enhanced drug efflux, impaired drug binding or the ability to use alternative survival pathways. More recently, it has been found that acquired drug resistance does not necessarily need a stable, heritable genetic alteration and, moreover, that response to treatment can be restored following a ‘drug holiday’. Whilst modelling the acute response to a variety of anti-cancer drugs in treatment-sensitive human tumour cell lines, researchers at Massachusetts General Hospital Cancer Center and the Dana-Farber Cancer Institute consistently found a small subpopulation of reversibly ‘drug-tolerant’ cells. They found that whereas the vast majority of EGFR mutant non-small cell lung cancer-derived cells (PC9 cells) were killed by exposure to a high concentration (100 x IC50) of EGFR tyrosine kinase inhibitors (TKIs), a small fraction of cells survived. Similar populations of ‘drug-tolerant persisters’ (DTPs) were found when PC9 cells were treated with cisplatin and also in several other cancer cell lines with established drug sensitivity, suggesting that a drug-tolerant cell subpopulation is broadly present in tumour-derived cell lines.

Although DTPs are largely quiescent, about 20% eventually resume proliferation in the presence of drug to give colonies of cells referred to as ‘drug-tolerant expanded persisters’ (DTEPs) which can propagate indefinitely in the presence of drug. DTPs rapidly regain sensitivity when grown in drug-free media whereas restoration of sensitivity in DTEPs occurs at higher passage number. The reduced drug-sensitivity of both DTPs and DTEPs was linked to increased expression of a gene that encodes a chromatin-modifying enzyme, KDM5A. Although there are, as yet, no inhibitors of KDM5A, its known association with histone deacetylases (HDACs) led the team to test the effect of HDAC inhibitors on DTPs and DTEPs. Trichostatin A, an inhibitor of class I/II HDACs was found to rapidly kill PC9-derived DTPs and DTEPs but to have no effect on parental PC9 cells or TKI-resensitised DTEPs. The team went on to show that continous treatment with HDAC inhibitors, whilst having no effect on growth and survival of parental P9 cells, can prevent the emergence of EGFR TKI resistance. As well as HDAC inhibitors, a selective inhibitor of the insulin-like growth factor 1 receptor (IGF-1R) kinase also virtually eliminated the emergence of EGFR TKI-tolerant DTEPs. IGF-1R signalling was found to be necessary for drug-tolerant phenotypes in other cancer cell lines and to be mediated by the histone-demethylating activity of KDM5A.

The team hope that the results seen in cell culture experiments will extend to cancer patients and have already begun a clinical trial to see whether a combination of a chromatin-modifying agent with the EGFR TKI, erlotinib, may prevent or delay the development of resistance. Although the trial is not yet completed, early data indicate that the inclusion of a chromatin-modifying agent can dramatically improve clinical benefit in a subset of patients demonstrating acquired TKI resistance.

The study is published in the journal Cell.

HIV drugs could treat prostate cancer and CFS – if they are caused by XMRV

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The retrovirus, xenotropic murine leukemia virus-related virus (XMRV), has been – controversially – linked to both prostate cancer and chronic fatigue syndrome (CFS). In an attempt to clarify the association of XMRV with disease, researchers at Emory University are developing a serum-based assay to detect neutralising antibodies to the virus which should begin to answer basic questions about how widespread the virus is, and how it is transmitted. The team found good agreement between their serum based assay and polymerase chain reaction (PCR) and fluorescence in situ hybridization (FISH) tests carried out on prostate samples from cancer patients, and showed that at least some of the patients had been infected with XMRV. The study is published in the April issue of Urology.

Meanwhile, other researchers at Emory University/Atlanta Veterans Affairs Medical Center and the University of Utah have been looking for ways to treat XMRV should it turn out to have a causal role in prostate cancer or chronic fatigue syndrome. The team evaluated 45 compounds, mostly drugs approved for the treatment of HIV/AIDS, and found that four of them were able to inhibit XMRV with EC50 values of < 1µM. XMRV replication was studied in both MCF-7 cells (generated from human breast cancer) and LNCaP cells (generated from human prostate cancer). The most effective compounds were two nucleoside reverse transcriptase inhibitors (zidovudine and tenofovir disoproxil fumarate) and two integrase inhibitors (raltegravir and L-000870812). Despite the lack of homology (only 14% identity) between HIV-1 integrase and XMRV integrase, raltegravir showed particularly good activity against XMRV with EC50 values of 0.005µM and 0.03µM in MCF-7 and LNCaP cells respectively (cf 0.001µM for HIV-1 grown in PBMCs). The EC90/EC50 ratio was significantly higher for XMRV grown in MCF-7 cells than for XMRV grown in LNCaP cells or for HIV-1 grown in PBMCs (700, 15 and 9 respectively). Synergy studies were carried out in LNCaP cells: combinations of raltegravir and any of the other three compounds were found to act synergistically.

The authors hope that if XMRV is established as a cause of prostate cancer or CFS, existing HIV treatments may prove to be effective therapies for these conditions.

The study is published in PLoS ONE.

Proof of Concept for siRNA in Melanoma

Green and Red Lights
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By far the majority of marketed drugs modulate the activity of proteins. Since the discovery of RNA interference (RNAi) over 10 years ago, a major goal has been to develop agents which will selectively block the synthesis of target proteins. Small interfering RNAs (siRNAs) occur naturally and are believed to have evolved as a defence mechanism against viral infection but synthetic siRNAs could potentially be used to silence any target gene. Although the idea is not conceptually difficult and many cancer-associated genes have been silenced in laboratory experiments, there have been a number of obstacles in developing RNAi for clinical use, not least of which is how to deliver the siRNAs to the target cells.

A team led by scientists and clinicians at the California Institute of Technology (Caltech) has now published the results of a small phase I clinical trial in patients with skin cancer showing that targeted nanoparticles can traffic into tumours and deliver siRNAs in a dose-dependent fashion when administered intravenously. The siRNA-containing nanoparticles used in the study are being developed by Calando Pharmaceuticals, Inc. and the technique used for detecting and imaging the nanoparticles inside cells from tumour biopsies was developed at Caltech. Although phase I studies are primarily designed as safety studies, the team was able to demonstrate that, in one patient who received the highest dose of nanoparticles, the target mRNA (M2 subunit of ribonucleotide reductase (RRM2)) had been cleaved at the predicted position and that protein levels had also been reduced.

Although more trials will be needed to show that such treatment is safe and effective, the study provides the first evidence that nanoparticles and RNAi can be combined to reduce expression of cancer-associated genes in human patients.

The results are published in the journal Nature.

Don’t Ignore the Neighbours

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Out of every hundred investigational cancer drugs that enter clinical trials, only eight prove to be sufficiently safe and effective for FDA approval. Scientists at the Dana-Faber Cancer Institute believe that those odds can be shortened by taking into account neighbouring cells in the tumour microenvironment in preclinical testing.

Although both non-malignant cells and secreted proteins from tumour and stromal cells are recognised to be active participants in cancer progression, early laboratory testing is typically carried out using homogeneous populations of tumour cells. The Dana-Faber group have now developed a tumour cell-specific in vitro bioluminescence imaging (CS-BLI) assay that is suitable for high throughput screening. Cell viability can be measured in tumour cells stably expressing luciferase in the presence or absence of non-malignant accessory cells (for example, stromal cells) or drug treatment.

Unlike conventional screening methods, CS-BLI was able to identify compounds with increased activity against tumour cells interacting with stroma. For example, reversine showed more potent activity in an orthotopic model of diffuse myeloma bone lesions than in conventional subcutaneous xenografts. CS-BLI was also able to identify stroma-induced chemo-resistance such as imatinib resistance in leukaemia cells. The use of CS-BLI should help to identify preclinical candidates that overcome stroma-mediated drug resistance as well as those that can act in a synthetic lethal manner in the context of tumour-stroma interactions, thereby increasing the chances of clinical success.

The study is published in Nature Methods.

Role for Tau in ‘Microtentacles’ and Metastasis

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Tau proteins interact with tubulin to stabilise microtubules and are abundant in neurons but less common in non-neuronal cells. Recently, tau proteins have received a bad press because of the association between tangles of hyperphosphorylated tau and Alzheimer’s disease and now researchers at the University of Maryland have reported that tau proteins may also play a role in tumour metastasis. Tau was found to promote formation of extensions of the plasma membrane or ‘microtentacles’ on breast cancer cells which break away from the primary tumour and circulate in the bloodstream. The microtentacles then increase the ability of the cells to attach to the walls of capillaries in the lung and seed new tumours. Tau protein has previously been associated with chemotherapy-resistant breast cancers and a poor prognosis but this is the first time that it has been implicated in metastasis. In the present study involving 102 breast cancer patients, 52% showed tau expression in metastases and 26% showed significantly increased tau expression as the disease progressed.

The team hope that drugs might be identified which will prevent the growth of microtentacles and inhibit tumour metastasis. Although current breast cancer treatments are often effective in treating the primary tumour, they are less effective in treating metastatic cancer which can develop years after the primary tumour is discovered and is the leading cause of death in cancer patients.

The study is published in the journal Oncogene.

New Breast Cancer Subtype

cancer awareness
Image: Flickr - ATKR
Gene expression profiling is used to guide treatment options for women with breast cancer. Endocrine therapies – tamoxifen or aromatase inhibitors – are offered to women whose cancer is oestrogen receptor (ER) positive whilst the monoclonal antibody, trastuzumab (Herceptin®) and the small molecule, lapatinib (Tykerb®) are used to treat women whose cancer overexpresses the HER2 receptor. About 15% of breast cancers – the so-called triple negative breast cancers that don’t have receptors for oestrogen, progesterone or HER2 – don’t respond to hormone therapy or to HER2 blockers and the prognosis for women with these cancers is relatively poor.

Researchers at Washington University University School of Medicine in St. Louis have now identified a gene that is overexpressed mainly in ER-negative, HER2-negative and triple negative breast cancers, leading to the possibility of a new clinical biomarker and potential treatments. Upregulation of Wnt signalling coreceptor, LRP6 (low-density lipoprotein receptor-related protein 6), was found in about a quarter of the breast cancer samples that the researchers examined. Previous studies had shown that the protein Mesd (mesoderm development) blocks LRP6 and was able to slow the growth of cultured breast cancer cells. Mesd also inhibits the development of mammary tumours in mice, without producing known pathway-dependent side-effects such as bone lesions, skin disorders or intestinal malfunctions. A smaller fragment of Mesd was found to be as effective as full length Mesd and to have improved stability.

The study is published in the Proceedings of the National Academy of Sciences.

Although the study offers the prospect of targeted therapy for women with breast cancer that is currently difficult to treat, both screening and prescribing practices need to improve for such discoveries to realise their full potential. A recent news feature in Nature Biotechnology highlights differing views on testing as well as the problems associated with diagnostic tests for HER2 – both of which may be compromising women’s access to appropriate and effective treatment.

Strange Landscape of Cancer Cells

strange landscape
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Compared with the genome of a normal cell, that of a cancer cell has sections that are missing, repeated or scrambled. Although some of the mutations are probably driving the cancer, others may have little relevance to the cancer’s progression. To understand which mutations are important in the development and spread of cancer, a multi-national team including researchers from the Broad Institute and the Dana-Farber Cancer Institute have studied over 3000 primary human cancer specimens representing more than 26 cancer types, including lung, prostate, breast, ovarian, colon, oesophageal, liver, brain, and blood cancers.

Classically, cancers have been classified (and treated) by their tissue of origin but, more recently, it has been realised that oncogenes are not restricted to one type of cancer. In the new study, the team found that amplifications and deletions of regions of the genome, known as somatic copy-number alterations or SCNAs, are not evenly distributed across the cancer genome but are concentrated in less gene-rich regions where they may be better tolerated. As with other mutations seen in cancer cells, many SCNAs may not play an important role in cancer growth but some will identify genes that help to promote and maintain cancers.

The most common SCNAs were found to be either relatively long (the length of an entire chromosome or a single chromosome arm) or short (about 1.8 million base pairs). More than 150 regions of short, or focal, SCNA – which because of their compact size are more likely to pinpoint important cancer-linked genes – were found to be altered at significant frequency across several cancer types. Of these, 122 did not coincide with genes already known to be amplified or deleted in cancer but the team also found copy number changes in BCL2 family genes – which are already linked to cancer and are the target of some cancer drugs – in over half of all samples. Most focal SCNAs were not unique to one type of cancer suggesting that common genetic mutations underlie different types of tumour, a finding which may lead to improved diagnosis and more targeted treatments for cancer.

The study is published in the journal Nature and the researchers have also built an online tool, the Tumorscape portal, to allow other researchers to freely access their data.

Blocking Dab2 Could Stop Cancer Spread

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Around 85% of all tumours are epithelial in origin and have epithelial markers on the cell surfaces. In breast, ovarian, pancreatic and colon-rectal cancers, metastasis – when cells break away from the primary tumour to initiate a new tumour elsewhere in the body – is often fatal. A process known as epithelial-mesenchymal transdifferentiation (EMT) plays a role in releasing epithelial cells from the surface of solid tumours and transforming them into transient mesenchymal cells which have reduced cell-cell adhesion properties and increased ability to migrate and establish new tumours. It was already known that transforming growth factor-β (TGF-β) induces EMT but the downstream events were unclear.

Scientists in the US have now shown that TGF-β triggers the formation of a protein known as disabled-2 (Dab2) which, in turn, activates the EMT process. An increased understanding of the signalling pathway for modulating EMT could lead to the design of drugs to delay or halt EMT and control tumour metastasis. The discovery may also help to understand the progression of other diseases.

The study is published in Nature Cell Biology.

Rational Design of New Type II Kinase Inhibitor

Crystal structure of sorafenib complexed with B-RAF, PDB ID=1UWH
Crystal structure of sorafenib complexed with B-RAF, PDB ID=1UWH
Protein kinases play important roles in regulating most cellular processes and are commonly activated in cancer cells. A number of kinase inhibitors – including antibodies and small molecules –have already been approved for the treatment of cancer and many others are currently being tested. The majority of kinase inhibitors developed so far are ATP mimetics identified by high-throughput screening of catalytic kinase domains at low ATP concentration. Such compounds – so-called type I inhibitors – may lack specificity for individual kinases and/or be less effective when ATP concentrations are high. Crystal structures have revealed that some compounds – the type II inhibitors – occupy an allosteric site accessible only in the inactive conformation of the kinase and researchers at the Moores Cancer Center at the University of California have now designed selective type II inhibitors of PDGFRβ (important for pericyte recruitment) and B-RAF (important for endothelial cell survival).
compound 6
Compound 6

Using the X-ray crystallographic structure of the type II inhibitor, sorafenib, bound to B-RAF, the team designed a small library of compounds based on a constrained amino-triazole scaffold predicted to stabilise kinases in the inactive state. The compounds were then tested for antivascular activity in both cell-based models and a zebrafish embryogenesis model. Compound 6 was found to inhibit both PDGFRβ and B-RAF cellular signalling – which produces a synergistic effect on tumour growth – but to have no effect on a variety of other cellular targets. The compound showed antiangiogenic activity in both zebrafish and murine models of angiogenesis and was also shown to suppress murine orthotopic tumors in both the kidney and pancreas.

The study is published in the Proceedings of the National Academy of Sciences.

MAGL Contributes to Aggressiveness in Cancer

white tiger
Image: Flickr - Tambako the Jaguar
Despite a growing understanding that the conversion of normal cells into cancerous cells is accompanied by metabolic changes, it remains unclear whether many of these changes play crucial roles in malignancy and disease progression. Increased lipid synthesis by fatty acid synthase has been suggested to contribute to cancer growth through both metabolic and signalling pathways. Researchers at the Scripps Institute reasoned that increased lipid synthesis must be accompanied by a lipolytic pathway to liberate stored fatty acids and have now shown that levels of monoacylglycerol lipase (MAGL) are highly elevated in aggressive cancer cells compared with less aggressive cancer cells and that this lipase, through hydrolysis of monoacylglycerols (MAGs), controls free fatty acid (FFA) levels in cancer cells.

JZL184 structure
The resulting MAGL-FFA pathway promotes migration, survival, and in vivo tumour growth. Aggressive cancer cells thus partner lipogenesis with high lipolytic activity to generate an array of pro-tumorigenic signals that support their malignant behaviour. Treatment with the selective MAGL inhibitor, JZL184, significantly reduced FFA levels in aggressive cancer cells, a finding that contrasts with the function of MAGL in normal tissues, where the enzyme does not generally control FFA levels. Knock down of MAGL activity using shRNA probes in aggressive melanoma, ovarian and breast cancer cells reduced MAGL activity by 70%–80%, with corresponding elevations in MAGs and reductions in FFAs. shMAGL cancer cell lines showed reduced in vitro migration, invasion and survival under serum starvation conditions. Treatment with JZL184 also reduced cancer cell migration, but not survival, perhaps indicating that maximal effects on aggressiveness need sustained inhibition of MAGL. shMAGL cancer cells also showed markedly reduced tumour growth rates in subcutaneous xenograft transplantation studies performed in immune-deficient mice.

Daily treatment of mice bearing MAGL-expressing tumours with JZL184 (40 mg/kg po) produced similar impairments in tumour growth rates. Addition of palmitic or stearic acid, two principal FFAs regulated by MAGL in aggressive cancer cells, to cells with genetically or pharmacologically reduced levels of MAGL restored their migratory activity in vitro. Similarly, tumour growth was enhanced in MAGL-deficient xenografts when the mice were fed a high fat diet. Cancer cells engineered to stably over-express MAGL also showed significantly reduced MAGs and elevated FFAs, a profile that was accompanied by increased migration, invasion and survival in vitro and enhanced tumour growth in vivo.

The effects of MAGL on cancer aggressiveness were found not to be mediated by endocannabinoid signalling but are suggested instead to be, at least in part, caused by increased production of bioactive lipids such as LPA and PGE2 that act on GPCRs to promote high migratory activity.

Both in vitro and in vivo studies showed that aggressive cancer cells acquire the ability to liberate FFAs by increased expression of MAGL and that this contributes to the aggressive phenotype. Since MAGL is not required for cell survival, but instead promotes progression to a more aggressive phenotype, if shown to slow tumour progression in people, inhibitors of MAGL may have a better safety profile and offer advantages over existing treatments for cancer.

The study is published in the journal Cell.