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Protein aggregation in cancer development: novel heat shock inducing compounds to reduce tumor growth.

Targeting protein aggregation during cancer development

1.1 Mutant p53 protein can exert gain-of-function activity by protein aggregation

Cancer remains one of the leading causes of morbidity and mortality worldwide, and understanding the underlying molecular mechanisms of the disease still forms a major challenge1-4. The p53 tumor suppressor protein, a master regulator of cell cycle arrest and apoptosis, is a cardinal player in the protection against malignant tumor transformation5-7. Given its pivotal role in tumor suppression, p53 is one of the most consistently altered genes in human cancer and is as such intensively studied in cancer research8,9. However, the most fundamental properties of mutant p53 during cancer development remain poorly explained.

A rising number of studies show that mutant p53 can, in addition to the loss of function of the protein by mutation, exert a dominant negative (DN) activity, i.e. one copy of mutant p53 inhibits the tumor suppressor function of the wild-type p53 allele in heterozygous cancer cells, and/or exert oncogenic gain-of-function (GOF) activity, i.e. expression of mutant p53 in tumor cells can promote to genomic instability, metastasis, angiogenesis, metabolic changes and drug resistance to a larger extent than the mere loss of p5310-12. The SWICH lab and others have shown that mutant p53 proteins tend to aggregate, i.e. forms conglomerates of misfolded p53 proteins that entrap functional, wild type p53, as well as other tumor suppressors such as p63 and p73, explaining its GOF/DN activity13,9,14. The impact and prevalence of p53 aggregation during tumor development has yet to be elucidated.

 1.2 p53 protein aggregation is linked to a worse clinical outcome

Studies done by the SWITCH lab on biopsies from various tumor types indicate that the presence of p53 inclusion body formation is prevalent in primary tumors of 6 different human cancer types, and that these inclusion bodies are present in up to 55% of tumors investigated. Importantly, data obtained from a cohort of colorectal tumor samples with clinical follow-up, indicates a correlation of these p53 inclusions with a worse clinical outcome. Strikingly, when classifying the tumors by their p53 genotype, no stratification could be observed for relapse or survival, emphasizing the importance of epigenetic factors during tumor progression. These findings indicate that not only intrinsic properties of the protein, but the combination of the conformational status of p53 with the potency of cells to cope with aggregates, is a determining factor in tumor formation.

 1.3 The protein quality control: role for p53 aggregation in malignancy?

The conformational status of proteins depends on the cellular protein quality control machinery (PQC), which monitors protein folding and aggregation in cells. The PQC is driven by the heat shock (HS) response, which involves the upregulation of heat shock proteins (HSPs) by the heat shock transcription factor 1 (HSF1)15,16. The PQC declines upon aging, meaning cells slowly lose the ability to clear misfolded proteins, making aging an important factor for aggregation diseases17,18. In our patient studies, we also observe a lower expression of proteasomal proteins in samples that show p53 aggregation than in equivalent samples that contain soluble p53. Because of its protective function in the cell, it is not surprising that the HS response can also be exploited by tumor cells19. However, it remains unclear how tumor cells attain this state of elevated heat shock, and if p53 aggregation could be a driver for this oncogenic response.

We have observed that protein aggregation is an important hallmark for cancer progression and prognostic for patient survival. The general aim of my research is therefore to understand the mechanisms how aggregated proteins can contribute to cell transformation and proliferation. Moreover, giving the importance of aggregation in cancer in the clinic, I also want to identify novel therapeutics interfering with protein aggregation. I want to achieve this goal by setting up a key method tool for my research and pursuing following aims:

Key method: Constructing a panel of cell lines with various p53 phenotypes

Little is known about aggregation phenotypes in cancer. Since the goal is to understand the molecular mechanisms that are linked to different p53 phenotypes and identify putative therapeutics, I am constructing a cancer cell line library containing, for each cell line, information on a broad array of parameters including aggregational, mutational and cellular status of p53. Endogenous p53 aggregates characteristics will be compared with amyloid inclusions, found in aggregation-associated diseases such as neurodegenerative diseases including Alzheimer’s and Parkinson. This collection of cell lines will be instrumental in studying the molecular mechanisms (Aim 1) and for the identification of novel therapeutics targeting p53 aggregation (Aim 2).

Aim 1. Protein aggregation: impact during oncogenesis?

As outlined above, while the activation of an oncogenic heat shock response has now been widely observed and accepted19, it still remains unknown why and how the tumor cells attain activated heat shock. We observed heat shock induction in cell lines containing aggregated p53, indicating that aggregated p53 can be a driver of the for tumor cells advantageous heat shock response. Therefore, I want to investigate the role of p53 aggregation as a potential driver/modifier of the heat shock response. I will also compare my observation with studies on p53 aggregation in patient samples, which are currently being evaluated by others at the SWITCH lab.

Aim 2. Evaluate proteostatic regulators in vitro and in vivo

Studies that target the proteostasis mechanism in cancer are mostly focusing on the inhibition of the heat shock response, but have not made it to the clinic due to toxic side effects20,21. However, current approaches for typical aggregation diseases such as Alzheimer’s Disease (AD), are most promising for drugs that induce a HS response to boost the clearance of toxic aggregates22,23. Based on our current model and preliminary insights, in this project I want to evaluate a similar approach of inducing a cellular heat shock response using small chemical compounds that act as proteostatic regulators. Using this approach, I want to achieve clearance of misfolded proteins and aggregates. Studies will be done initially in vitro on the constructed panel of cell lines. The most promising compounds will be tested in vivo by xenograft experiments using various cell lines identified in the first aim, in combination with or without chemotherapeutics.

 

1          Ferlay J, S. H., Bray F, Forman D, Mathers C and Parkin DM. GLOBOCAN 2008, Cancer Incidence and Mortality Worldwide: IARC CancerBase No. 10 (2008).

2          N.C.I. The website of the National Cancer Institute, (2013).

3          Hanahan, D. & Weinberg, R. A. Hallmarks of cancer: the next generation. Cell 144, 646-674, doi:10.1016/j.cell.2011.02.013 (2011).

4          Hanahan, D. & Weinberg, R. A. The hallmarks of cancer. Cell 100, 57-70 (2000).

5          Rivlin, N., Brosh, R., Oren, M. & Rotter, V. Mutations in the p53 Tumor Suppressor Gene: Important Milestones at the Various Steps of Tumorigenesis. Genes Cancer 2, 466-474, doi:10.1177/1947601911408889 (2011).

6          Freed-Pastor, W. A. & Prives, C. Mutant p53: one name, many proteins. Genes Dev 26, 1268-1286, doi:10.1101/gad.190678.112 (2012).

7          Brady, C. A. & Attardi, L. D. p53 at a glance. J Cell Sci 123, 2527-2532, doi:10.1242/jcs.064501 (2010).

8          Petitjean A, M. E., Kato S, Ishioka C, Tavtigian SV, Hainaut P, Olivier M. I. Impact of mutant p53 functional properties on TP53 mutation patterns and tumor phenotype: lessons from recent developments in the IARC TP53 database. Hum Mutat.Jun;28(6):622-9. (2007).

9          Muller, P. A. & Vousden, K. H. Mutant p53 in cancer: new functions and therapeutic opportunities. Cancer Cell 25, 304-317, doi:10.1016/j.ccr.2014.01.021 (2014).

10       Lang, G. A. et al. Gain of function of a p53 hot spot mutation in a mouse model of Li-Fraumeni syndrome. Cell 119, 861-872, doi:10.1016/j.cell.2004.11.006 (2004).

11       Hanel, W. et al. Two hot spot mutant p53 mouse models display differential gain of function in tumorigenesis. Cell Death Differ 20, 898-909, doi:10.1038/cdd.2013.17 (2013).

12       Oren, M. & Rotter, V. Mutant p53 gain-of-function in cancer. Cold Spring Harb Perspect Biol 2, a001107, doi:10.1101/cshperspect.a001107 (2010).

13       Xu, J. et al. Gain of function of mutant p53 by coaggregation with multiple tumor suppressors. Nat Chem Biol 7, 285-295, doi:10.1038/nchembio.546 (2011).

14       Wilcken, R., Wang, G., Boeckler, F. M. & Fersht, A. R. Kinetic mechanism of p53 oncogenic mutant aggregation and its inhibition. Proc Natl Acad Sci U S A 109, 13584-13589, doi:10.1073/pnas.1211550109 (2012).

15       Hartl, F. U., Bracher, A. & Hayer-Hartl, M. Molecular chaperones in protein folding and proteostasis. Nature 475, 324-332, doi:10.1038/nature10317 (2011).

16       Vabulas, R. M., Raychaudhuri, S., Hayer-Hartl, M. & Hartl, F. U. Protein folding in the cytoplasm and the heat shock response. Cold Spring Harb Perspect Biol 2, a004390, doi:10.1101/cshperspect.a004390 (2010).

17       David, D. C. et al. Widespread protein aggregation as an inherent part of aging in C. elegans. PLoS Biol 8, e1000450, doi:10.1371/journal.pbio.1000450 (2010).

18       Lindner, A. B. & Demarez, A. Protein aggregation as a paradigm of aging. Biochim Biophys Acta 1790, 980-996, doi:10.1016/j.bbagen.2009.06.005 (2009).

19       Ciocca, D. R. & Calderwood, S. K. Heat shock proteins in cancer: diagnostic, prognostic, predictive, and treatment implications. Cell Stress Chaperones 10, 86-103 (2005).

20       Brock, W. J. et al. Ocular toxicity assessment from systemically administered xenobiotics: considerations in drug development. Int J Toxicol 32, 171-188, doi:10.1177/1091581813484500 (2013).

21       Neckers, L. & Workman, P. Hsp90 molecular chaperone inhibitors: are we there yet? Clin Cancer Res 18, 64-76, doi:10.1158/1078-0432.CCR-11-1000 (2012).

22       Leak, R. K. Heat shock proteins in neurodegenerative disorders and aging. J Cell Commun Signal 8, 293-310, doi:10.1007/s12079-014-0243-9 (2014).

 

Date:1 Oct 2012 →  31 Dec 2017
Keywords:Reduce tumor growth, Protein aggregation in cancer
Disciplines:Laboratory medicine, Palliative care and end-of-life care, Regenerative medicine, Other basic sciences, Other health sciences, Nursing, Other paramedical sciences, Other translational sciences, Other medical and health sciences
Project type:PhD project