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Sensitisation and resistant determinants of cancer cells to death receptor related therapies

Final Report Summary - ONCODEATH (Sensitisation and resistant determinants of cancer cells to death receptor related therapies)

In the last decade, an encouraging decline of the death rate from cancer has been observed, as a result of recent advances in prevention, early diagnosis and therapy. Efforts are made towards the generation of 'smart' anti-cancer drugs that will target specific molecules depending on the molecular phenotyping of the patient's tumour. TNF-related apoptosis-inducing ligand (TRAIL) is currently a promising anti-tumour agent with proven activity on several cell and animal cancer models. A unique feature of TRAIL is its specificity towards malignant cells while sparing normal cells. At present, preclinical studies with recombinant TRAIL are in progress as well as with an anti-TRAIL-R2 monoclonal antibody with no hepatotoxicity. The aim of the present proposal was to identify components in colon and other tumours which confer sensitivity or resistance to cell death induced by TRAIL. The contribution of mutated oncogenes in altering cell death molecules and pathways was examined on priority.

A large number of research tools were produced during: epitope-tagged expression vectors, recombinant retroviruses-lentiviruses, antibodies. Cell lines with stably and/or inducibly overexpressed or silenced by shRNA oncogenes as well of factors involved in the signalling and apoptotic pathways in colon cancer have been generated. Quality assessment of these tools in different applications had been completed. New transgenic mouse models had been generated. New improved forms of TRAIL had been expressed and utilised as well a panel of new small molecule kinase inhibitors had been synthesised and selected for treatment. In addition, improved methods for cell apoptosis and in vivo experiments had been developed and used for the analysis of apoptotic complexes as well a for in vivo assessment of tumourigenicity. In addition, rational combinations of TRAIL with inhibitors have been evaluated. Substantial progress has been made on deciphering the interaction networks of oncogenic with apoptotic signalling pathways and trafficking at the mitochondria level. On the mechanistic part, the network has identified determinants for inhibitors in sensitising to apoptosis as well as has provided detail knowledge of the effect of the inhibitors on the intrinsic apoptotic amplification and execution machinery related to caspases and Bcl-2 family members during TRAIL-induced apoptosis. Assessment of sensitivity of tumours induced by activated oncogenes in transgenic mice and nude mice to inhibitors alone or in the combination with the recombinant mouse TRAIL has been examined.

The results of the network resulted in 12 published and 3 submitted joint publications as well as 138 individual publications in high impact scientific journals like Cell, Cancer Cell, Mol. Cell., Lancet Oncol., Science, EMBO J., Nature Cell Biol., Gastroenterology, Cancer Res. Cell Death Differentiation, Oncogene, Br J Cancer and more. There are eight new individual publications submitted-in preparation. The members of the network also presented the major results in scientific conferences. Interaction between the network participants was intense, which was realised via short-term visits, the annual meetings of the network, bilateral meetings, via ftp site of the network and via electronic communications. The project objectives have been advertised in the press like TV channels, newspapers. The network's web site is at:

Project objective and major achievements

Overall the network aimed:
- to contribute to better understanding of the role of specific activated oncogenes or the expression pattern of certain proteins that can provide sensitivity or resistance of tumour cells to TRAIL induced apoptosis;
- to identify rational combination of molecules inhibiting oncogene pathway components with TRAIL agonists to achieve optimum synergistic effect.

The studies of the network were focussed on TRAIL-induced apoptosis, PI3K, TyK, Aurora kinase and proteasome inhibitors, mitochondrial fission / fusion, caspase-2.

Major achievements

1. We have a collection of an essentially complete set of expression vectors and cell lines overexpressing or lacking individual oncogenes.
2. We have prepared mutant forms of factors necessary to analyse interaction pathways.
3. We have prepared different forms of tagged recombinant TRAIL.
4. We have used newly synthesised PI3K, Hsp90, Aurora and choline kinase inhibitors.
5. We have established a high-throughput method for apoptosis measurement for cell lines using the INCell analyser.
6. Direct orthotopic cell microinjection (OCMI) of human colorectal cancer cell lines in animals has been worked out.
7. We have generated triple transgenic mice allowing concomitant and inducible expression of the K-Ras V12G oncogene and deletion of both p53 alleles specifically in the intestinal epithelium.
8. We have isolated and characterised tumour initiating cells.
9. Improved protocols of DISC complex detection have been worked out.
10. Identification Arf and Rho GAP adapter protein ARAP1 as taking part in the mobilisation of TRAIL-R1 / DR4 to the plasma membrane.
11. Discovery that TRAIL synergises with quercetin to induce cell death in colon cells.
12. Discovery that DNA damage induces two distinct modes of cell death.
13. Biological characterisation of newly synthesised PI3K, Hsp90, Aurora and choline kinase inhibitors.
14. Discovery that Aurora-A regulates nuclear factor-kappaB signalling by phosphorylation of IkappaBalpha.
15. Discovery that genetic interference of choline kinase alpha by siRNA induces cell death in human cancer cells but not in primary cells
16. Discovery that Bax activation and stress-induced apoptosis is delayed by the accumulation of cholesterol in mitochondrial membranes.
17. Efficient protocols for treatment with TRAIL alone or in combination with novel PI3K inhibitors.
18. Identification of novel Aurora kinase inhibitor which synergises with TRAIL by increased cleavage of Caspase 7 and PARP1.
19. Killer TRAIL antitumour activity was demonstrated in colon cancer patient xenograft / SCID mouse models in vivo.
20. Small cell lung carcinoma (SCLC) are resistant to TRAIL. Majority of human SCLC cells are resistant to Death receptor (DR)-mediated apoptosis because of defects in key signalling molecules, such as DRs and / or initiator caspases. Combination of TRAIL and anticancer agents might be beneficial for elimination of caspase-8-containing SCLC cells.
21. FLIP, AKT1 and AKT2 expression or activation were not the responsible of TRAIL-resistance in the colon lines analysed.
22. A list of choline kinase inhibitors cooperating with TRAIL in inducing colon cancer cell apoptosis has been generated.
23. Development and characterisation of intestinal tumour generated in genetically-engineered mice models
24. Establish an assay which evaluates live human tumour-derived cells for anticancer drug response.
25. Role of caspase-2 as initiator caspase was also observed in apoptosis triggered by other chemotherapeutic drugs. The present model for caspases-2 activation includes PIDDosome complex formation.
26. Defining molecules / pathways of the intrinsic apoptotic machinery that are affected by choline inhibitors and can potentially synergise with TRAIL in inducing apoptosis of colon cancer cells.
27. Determination of the sensitivity of tumours implanted in immunosuppressed mice to choline kinase inhibitors and assessment of their optimal dosing and in vivo synergism with the recombinant human TRAIL.

Contribution of ONCODEATH towards personalised cancer therapies:

Conventional cancer treatments have been based mostly on the 'one size fits all' approach. Patients are prescribed medicine in standard doses that are later adjusted by a 'trial and error' process. Patients may suffer severe side effects, and while some respond well to the treatment, others show no improvement. Innovations in the field of genetics have led to a new, personalised treatment approach where understanding the genetic basis of the individual patient and his tumour help determine the patient's eligibility to a proposed treatment and predict its success. The advantages of this approach to patients are self-evident. ONCODEATH has used the approach of identifying two cancer drugs under development such as TRAIL and choline kinase, PI3K and BRAF inhibitors as a novel combinatorial strategy. Our findings where a simultaneous combinatorial treatment of choline kinase inhibitors and TRAIL shows a strong synergistic effect can provide an alternative treatment to those used in the clinic today.

The potential market for new rational combinatorial strategies of combining TRAIL. choline kinase, PI3K, BRAF kinase inhibitors is huge and may have a tremendous economical impact.