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Wnt Signaling Inhibitors

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The April 12 issue of PNAS (Proceedings of the National Academy of Sciences) features a lead article by investigators at NYU, Cornell and Rational Therapeutics, on the identification of three compounds that inhibit the important cell signaling pathway known as WNT (catenation of Wg and Int).

The WNT signaling pathway was originally described in fruit flies as a determinate of wing shape. It was subsequently shown to be an important factor in human stem cell differentiation. Thereafter, its role in cancer was described.

Certain colon cancers associated with a familial syndrome have a mutation in the WNT pathway. This results in an extremely high incidence of colon cancer. It is known that lung cancers, breast cancers, leukemias and lymphomas may share this pathway.

To date, there have been no clinical therapies available to treat WNT-driven tumors. Recognizing the importance of this pathway, the investigators at NYU and Cornell used a technology known as small interfering RNA (SIRNA) to shut down the WNT signal. They then screened 14,000 known chemicals for activity that mimicked the SIRNA effect. Three compounds were identified.

When the compounds showed activity in cell-lines that were WNT addicted, the investigators at NYU provided the compounds to Rational Therapeutics where they applied the EVA-PCD (functional profiling) technique to measure activity in human tumor samples.

The results confirmed activity and showed that several colon cancers, as well as other tumor types, had favorable profiles. The compounds were not uniformly effective, indicating that they were not simply toxins. Instead, they appeared to selectively injure cells that we assume are driven by WNT-related events.

The beauty of this study represents the introduction of a new paradigm of drug development. Following the elegant and highly sophisticated high throughput method employed by investigators at NYU and Cornell, these compounds were put to the very practical test of human relevance.

The identification of activity in human tissues at concentrations similar to those associated with other classes of drugs, suggest that these novel compounds may have promise with these heretofore-untreatable cancers. This highly productive collaboration could prove a new model for the development of effective new therapies.

PNAS April 12, 2011 vol. 108 no. 15 5929-5930


http://robertanagourney.wordpress.com/2 ... velopment/

http://www.rationaltherapeutics.com/dow ... ibitor.pdf

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A green glow from a fruit fly is giving researchers the green light when they are on the right path in their quest to develop compounds that help prevent cancer.

The glow, the result of some tinkering in Drosophila, the workhorse of the genetics world, lets researchers know when powerful cancer-prevention signals similar to those spurred by protective chemicals in broccoli, cabbage, and other foods, have been turned on in the organism.

The chemical signaling system is one of the major ways that the body defends itself against toxic assaults and threats like cigarette smoke, diesel exhaust, and dangerous microbes. A gene known as KEAP1 senses danger and then unleashes NRF2, which triggers rampant anti-oxidant activity in a cell.

Scientists from the University of Rochester Medical Center have discovered that the pathway, long recognized in people and other animals, is active in fruit flies, too, opening the door to faster, less expensive ways to find compounds that spur our natural anti-oxidant activity. The work, funded by the National Cancer Institute, is reported in the January 15, 2008 issue of Developmental Cell.

This is one of the main mechanisms the body uses to fight off the things that give you cancer, said Dirk Bohmann, Ph.D., professor in the Department of Biomedical Genetics and a geneticist who studies fruit flies in an effort ultimately aimed at improving human health.

This puts cells into an anti-oxidant defense mode. Drug development and testing is very, very expensive and time-consuming. This work should speed the development of new drugs aimed at preventing cancer, added Bohmann.

Bohmann did the work along with former postdoctoral Gerasimos P. Sykiotis, M.D., Ph.D., who teamed up with Bohmann to develop novel approaches for the study of the NRF2 pathway after earning his medical and doctoral degrees from the University of Patras in Greece. Sykiotis is now with the Model Organisms Unit of the Novartis Institutes for Biomedical Research in Cambridge, Mass., where he is applying the genetic tools generated in the study to characterize the role of NRF2 signaling in Drosophila models of human diseases.

Scientists have known that the pathway exists in people, rodents, and zebrafish, and so Bohmann and Sykiotis went hunting for it in the fruit fly genome. They found that one form of a gene called CNC, which is widely known to be involved in determining the development of a fruit fly's head, serves like NRF2, turning on cellular defenses on a broad scale.

The defenses include activation of molecules known as thioredoxins and glutathione S-transferases, which are anti-oxidants that help a cell get rid of toxins and damaged molecules in its environment. Unlike popular anti-oxidants in certain foods and vitamins, whose effects in the body are transient, Bohmann points out that a fundamental genetic change like a boost in NRF2 activity throughout an organism would supply an ongoing amplified anti-oxidant response.

While the main application of the work is in boosting the body's ability to resist cancer, the research could also make a difference for patients who have cancer that is resistant to current drugs. In 2006, a team from Johns Hopkins showed how this same signaling pathway allows some cancer cells to fight off drugs intended to kill them. Gaining a foothold on the system in fruit flies gives researchers an added tool as they search for ways to thwart these rogue cancer cells.

In their experiments, Bohmann and Sykiotis modified fruit flies so they would glow green when exposed to ultraviolet light when the signaling pathway is functioning. Sure enough, flies with more active CNC glow more brightly than regular flies, giving the team an easy, visual way to see whether the pathway is activated.

The team demonstrated the technology using a compound called oltipraz, which targets the pathway and has been tested in people as a cancer-prevention agent. The flies that ate food with the compound glowed more strongly, demonstrating that the NRF2 pathway was more active in these flies.

Turning on our natural anti-oxidants is big business for many companies trying to develop compounds to protect us from cancer and to slow the aging process, said Bohmann. The same genetic principles govern many organisms, from flies to rodents to people, and we're hopeful that our tool in fruit flies will speed this work for the benefit of patients.

When Bohmann and Sykiotis boosted the activity of the pathway, fruit flies were three times more likely to survive an exposure to a toxin than regular flies. And flies with a more active signaling system can live 10 percent longer than the other flies.

It's the first time that the system, long known to be an important anti-oxidant and cancer prevention pathway, has also been shown to play a role in giving an organism a longer lifespan. The link gives new insight into the well-established connection between aging and cancer risk.

Two of Bohmann's colleagues at the University of Rochester Medical Center are also studying the NRF2 pathway. Steve Georas, M.D., professor of medicine and chief of the Division of Pulmonary and Critical Care, is looking at the role of NRF2 in people with asthma. And Irfan Rahman, Ph.D., associate professor of Environmental Medicine, has shown how NRF2 protects the lungs of smokers against the assault of cigarette smoke and other pollutants. He has shown that organisms in which NRF2 is weakened or absent have weak lungs and are much more prone to conditions like emphysema.

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Small or short inhibitory RNA (siRNA) is a short sequence of RNA which can be used to silence gene expression. Also known as small interfering RNA, short interfering RNA or silencing RNA. siRNA binds to complementary mRNA molecules and represses translation through degradation.

siRNA is a class of double-stranded RNA molecules, 20-25 nucleotides in length, that play a variety of roles in biology. The most notable role of siRNA is its involvement in the RNA interference (RNAi) pathway, where it interferes with the expression of a specific gene. In addition to its role in the RNAi pathway, siRNA also acts in RNAi-related pathways, e.g., as an antiviral mechanism or in shaping the chromatin structure of a genome; the complexity of these pathways is only now being elucidated.

siRNAs were first discovered by David Baulcombe's group at the Sainsbury Laboratory in Norwich, England, as part of post-transcriptional gene silencing (PTGS) in plants. The group published their findings in Science in a paper titled "A species of small antisense RNA in posttranscriptional gene silencing in plants".[1] Shortly thereafter, in 2001, synthetic siRNAs were shown to be able to induce RNAi in mammalian cells by Thomas Tuschl, and colleagues in a paper published in Nature.[2] This discovery led to a surge in interest in harnessing RNAi for biomedical research and drug development.

1. Hamilton A, Baulcombe D (1999). "A species of small antisense RNA in posttranscriptional gene silencing in plants". Science 286 (5441): 950–2. doi:10.1126/science.286.5441.950. PMID 10542148

2. Elbashir S, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T (2001). "Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells". Nature 411 (6836): 494–988. doi:10.1038/35078107. PMID 11373684

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Non-small cell lung cancer (NSCLC), the most common form of lung cancer, is usually treated with surgery and chemotherapy. However, a small group of patients can also be helped by treatment with tyrosine kinase inhibitors (TKI). New research published in BioMed Central's open access journal BMC Medicine, shows that blocking production of epidermal growth factor receptor (EGFR) using RNAi, alongside TKI (or antibody therapy), could enhance the effect of TKI on NSCLC cell death, and slow cell growth.

There are 1.2 million new cases of lung cancer worldwide every year and about 75-85% of these are NSCLC. 10% of cases with NSCLC have a mutation in the gene responsible for making EGFR, and three quarters of these patients respond well to TKIs. However others are unresponsive, and, even for the people who responded at first, the cancer always ultimately becomes resistant to TKI therapy.

In an attempt to circumvent this problem researchers from Universitair Ziekenhuis Brussel, in Belgium, tested the effect of adding RNAi to standard TKI or antibody therapy in NSCLC cells. Proteins are made in the cell's cytoplasm, but DNA is stuck inside the nucleus. Consequently, in order to produce a protein, a cell must first copy the gene's DNA sequence, producing an intermediary molecule called RNA which is able to travel out of the nucleus to the cytoplasm. RNAi 'interferes' with the mechanism for producing proteins by blocking proper function of the intermediary RNA stage.

A team led by Prof De Grève and his co-worker, Gang Chen, found that the small interfering RNA (siRNA) molecule they designed was able to inhibit EGF protein production in all the NSCLC cell lines tested. It was also able to slow down cell growth and increase cell death (apoptosis) in all the cell lines – although some responded better than others. When they tested siRNA alongside TKI and or monoclonal antibodies they discovered that the siRNA had an additional positive effect on the cell lines than the standard treatment alone.

Prof De Grève explained, "Some EGFR mutations were more sensitive to siRNA than others - we saw especially good results against the mutation in exon 19. We think that this reflects the fact that the growth of cells with this mutation is driven by EGFR, but that other mutations are not as active. A different EGFR mutation produces cells which cannot be inhibited by TKI, however even these cells were sensitive to siRNA."

These results provide hope for NSCLC patients with EGFR mutations, as it could potentially enhance TKI therapy. It may also help those who do not respond to TKI, or who have become resistant to TKI therapy. Additionally this treatment may benefit another group of patients where the gene for EGFR is too active in their cancer and too much EGFR is produced.

More information: Targeting the epidermal growth factor receptor in non-small cell lung cancer cells: the effect of combining RNA interference with Tyrosine Kinase inhibitors or Cetuximab, Gang Chen, Peter Kronenberger, Erik Teugels, Ijeoma Adaku Umelo and Jacques De Grève, BMC Medicine (in press)

Provided by BioMed Central

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Treatment-induced damage to tumor microenvironment promotes chemoresistance

Catharine Paddock PhD.

A new study from the US finds that in the process of targeting and killing off cancer cells, chemotherapy may also spur healthy cells in the neighbourhood to release a compound that stimulates cancer growth, eventually leading to treatment resistance. They hope their finding will lead to better therapies for cancer and buy precious time for patients with advanced cancer.

Senior author Peter S. Nelson, of the Human Biology Division at the Fred Hutchinson Cancer Research Center in Seattle, and colleagues, write about their findings in a paper published online on 6 August in Nature Medicine.

Nelson told the media: "Cancer cells inside the body live in a very complex environment or neighborhood. Where the tumor cell resides and who its neighbors are influence its response and resistance to therapy."

The reason chemotherapy eventually fails when treating advanced cancer, said Nelson, is because the dose you would need to give the patient to wipe out the cancer would also kill the patient.

In the lab, you can "cure" almost any cancer: you just give a huge dose of toxic chemotherapy to the cancer cells in the petri dish.

But you can't do that to patients, because the high dose would not only kill cancer cells but also healthy cells, said Nelson.

So treatment of common solid tumors has to be given as smaller doses paced out in cycles, to give healthy cells time to recover in the intervals.

But the drawback is that this approach may not kill all the cancer cells, and those that survive can become resistant to subsequent cycles of the chemotherapy.

In their study, Nelson and colleagues found one mechanism through which this can happen.

They studied a type of normal, non-cancerous cell, the fibroblast, that lives near cancer tumors.

In animals, fibroblasts help maintain connective tissue, which is found throughout the body and acts like a "scaffolding" that holds other types of cells and tissue. Fibroblasts are also important for healing wounds and producing collagen.

But under other, non-usual circumstances, they can behave in unexpected ways.

When their DNA is damaged, for instance by chemotherapy, fibroblasts can release a broad range of compounds that stimulate cell growth.

Nelson and colleagues examined cancer cells from prostate, breast and ovarian cancer patients who had been treated with chemotherapy, and found specifically, that when the DNA of fibroblasts near the tumor is damaged by chemotherapy, they start producing a protein called WNT16B in the microenvironment of the tumor.

And, they also found, when the protein reaches a high enough level, it causes cancer cells to grow, invade surrounding tissue, and resist chemotherapy.

"The expression of WNT16B in the prostate tumor microenvironment attenuated the effects of cytotoxic chemotherapy in vivo, promoting tumor cell survival and disease progression", they write.

Researchers already knew, that the WNT family of genes and proteins are important for growth of both normal and cancer cells, but this study now reveals they may also have a role in promoting treatment resistance.

The researchers saw some WNT proteins increased 30-fold, which was "completely unexpected", said Nelson.

Cancer treatments are becoming increasingly specific, using precise "sniper" approaches to target key molecules rather than general "scatter gun" approaches such as damaging DNA.

The researchers say their findings suggest the microenvironment of the tumor can also play a role in the success or failure of these more precise approaches.

For example, the same cancer cell may react quite differently to the same treatment, in different microenvironments.

They suggest their discovery could help make treatments more effective, for instance by finding a way to block the tumor microenvironment's response.

Professor Fran Balkwill, a Cancer Research UK expert on microenvironments, told the press the study ties in with other studies that show "cancer treatments don't just affect cancer cells, but can also target cells in and around tumors".

Sometimes the effect can be helpful, said Balkwill, giving the example of when chemotherapy triggers health immune cells to attack nearby tumors.

"But this work confirms that healthy cells surrounding the tumor can also help the tumor to become resistant to treatment. The next step is to find ways to target these resistance mechanisms to help make chemotherapy more effective," he added.

Catharine Paddock PhD. "Chemotherapy Can Inadvertently Encourage Cancer Growth." Medical News Today. MediLexicon, Intl., 6 Aug. 2012

"Treatment-induced damage to the tumor microenvironment promotes prostate cancer therapy resistance through WNT16B"; Yu Sun, Judith Campisi, Celestia Higano, Tomasz M Beer, Peggy Porter, Ilsa Coleman, Lawrence True & Peter S. Nelson; NatureMedicine, Published online 05 August 2012;DOI:10.1038/nm.2890

http://www.nature.com/nm/journal/vaop/n ... .2890.html

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"In the lab, you can cure almost any cancer: you just give a huge dose of toxic chemotherapy to the cancer cells in the petri dish. But you can't do that to patients, because the high dose would not only kill cancer cells but also healthy cells." How many times have I seen this happen with labs using single-endpoint assays (and basic leukemic type assays).

By examining drug-induced cell death events in native-state three-dimensional (3D) microclusters, the functional profiling platform recognizes the interplay between cells, not only stromal, but vascular elements, cytokines, macrophages, lymphocytes, mesenchymal cells, fibroblasts, smooth muscle cells, pericytes and other microenviromental factors known to be crucial for clinical response prediction. The human tumor primary culture microspheroid contains all of these elements.

The (slippery) polypropylene material prevents the attachment of fibroblasts and epithelial cells and encourages the tumor cells to remain in the form of three-dimensional (3D) floating clusters. Our body is already 3D (not 2D) in form, making this novel step better replicate that of the human body. When allowed to grow in vitro, "fresh" living cancer cells develop into these tiny microspheroid clusters that form a complex biosystem in which each malignant cell reacts upon it fellow colonists in subtle but important ways.

The microclusters recapitulate the human tumor environment, while the "3D" advancement recreates the extracellular matrix (metalloproteinases). The functional profiling platform studies cancer response to drugs within this microenvironment, enabling it to provide a snapshot of cancer's behavior within the human body and provides a more accurate representation of how cancer cells are likely to respond to treatment in the clinic.

Researchers at the University of Washington School of Medicine in Seattle have found that when cells become cancerous, they also become 100 times more likely to genetically mutate than regular cells. The findings may explain why cells in a tumor have so many genetic mutations, but could also be bad news for cancer treatments that target a particular gene controlling cancer malignancy.

If cancer cells do indeed become "mutator" cells, traditional chemotherapy and other "targeted" drugs may never be very effective against advanced tumors. This means that cancer cells in a tumor will have mutations that protect them from therapeutics.

A chemotherapy drug may target a particular oncogene, which is a gene that affects the malignancy of a particular cell. But if cancer cells are mutator cells, a single tumor may have cells with many different types of oncogenes and drug-resistant genes.

A chemotherapy drug may kill off some of the cancerous cells, but millions of other cells in the tumor will live on. To be effective, a chemotherapy treatment may have to target more than one oncogene: so-called combination chemotherapy. The more mutations, the further along the tumor may be in its development to malignancy or metastasis.

To lay the foundation for personalized cancer treatment, the ultimate "driver" could be the "functional" profiling platform. Cells speak to each other and the messages they send are interpreted via intracellular pathways. You wouldn't know this using genotype analysis. Phenotype analysis provides the window. It can test various cell-death signaling pathways downstream.

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