Cysteine oxidation as a regulator of cancer cell motility and invasion

Cells normally move through tissues during important stages of development. However, some of the same processes that regulate cell movement in a healthy individual also contribute to how cancer cells move away from a primary tumour and spread throughout the body. Understanding how cell movement is regulated not only informs us about normal biology but also helps us to know more about cancer. A product of this knowledge is establishing links between normal biology and cancer that could help to improve diagnosis and to identify potential targets that might be blocked by cancer therapies.

Proteins in cells are often modified by the attachment of small chemical modifications, which may range from small chains of amino acids to molecules as small as oxygen. These modifications may change many aspects of a protein's function, such as how active it is, where it is located or even the protein stability. The addition of oxygen to cysteine amino acid residues has previously been found to be an important regulator of some specific proteins. However, studying cysteine-oxygen modification is technically difficult, both because the tools are not straight-forward to use and because once a protein is removed from its natural context then it may be spontaneously oxidized on cysteine residues. As a result, the field of protein oxidation has not progressed as rapidly as other types of protein modification.

We have found that the process of cell movement increases protein oxidation on cysteine residues, and have identified over 200 proteins that undergo this modification. By studying one protein in particular, we found that its oxidation was increased during migration and that the oxidation of two cysteines in particular blocked its normal activity. When a version of this protein that could not be oxidised was expressed in cells, the ability of cells to stick to a surface and to move in a straight line were reduced, indicating that blocking the protein function through oxidation is important for how cells move efficiently.

In this proposal, we will identify proteins modified by cysteine oxidation in a number of cancer cell lines, and will determine how this modification changes protein function for a selected subset. We will also identify the mechanism for the generation of the reactive oxygen production that causes this modification, and the contribution of a specific protein called Rac1 to this process. Finally, by imaging transplanted tumour cells, we will establish the link between cell invasion and oxygen modification of protein targets in live tissue. We will also examine how blocking Rac1 activity, which has been proposed as a possible anti-cancer drug target, affects tissue invasion and reactive oxygen generation to determine whether this mechanism contributes to the therapeutic effects of these compounds.

Through these studies, we aim to discover novel aspects of the fundamental biology regulating cell movement through tissues. This knowledge will have direct relevance to cancer biology, and may help to establish links that improve diagnosis or identify future cancer drug targets.

Cell motility is regulated by a number of signalling pathways that influence the activity and localization of proteins, often via post-translational modifications (PTM) such as phosphorylation. Although oxidation of cysteine residues has been shown to be an important regulator of some proteins, for example protein tyrosine phosphatases, there is little information regarding the identities of proteins modified by cysteine oxidation that contribute to cell migration. We found that during cell migration, hydrogen peroxide (H2O2) is elevated close to the plasma membrane and throughout the cell cytoplasm by fluorescent lifetime imaging microscopy (FLIM). Migration also increases protein oxidation as determined by the incorporation of the sulfenic acid reactive compound dimedone. Using mass spectrometry (MS) we identified over 200 dimedone labelled proteins and determined that oxidation of Cofilin1 in particular is increased in migrating cells. By biochemical and cell biological analysis, we determined that the oxidation of Cofilin1 inhibits its filamentous actin severing activity and facilitates directional cell migration.

Using mass spectrometry, we will identify proteins undergoing cysteine oxidation during cell migration across a number of tumour cell lines, and in specific instances will determine how these modifications affect protein function. By siRNA mediated knockdown, we will examine how NADPH oxidase complexes contribute to H2O2 production during cell migration and determine the role of the Rac1 GTPase. Finally, using intravital microscopy to do FLIM measurements of fluorescent biosensor proteins, we will determine how H2O2 production and Rac1 activation are associated with tumour cell invasion in living tissue. These assays will also be used to ascertain whether blocking H2O2 generation is part of the mechanism of action of Rac1 inhibitors in reducing tumour cell invasion.

The fundamental nature of the research in this proposal means that the academic researchers are the largest group of potential beneficiaries. To realize maximal benefit, it will be necessary to establish contact with potential collaborators, which will require communication of results followed by direct communication with interested parties by email/telephone. In most instances, we will act proactively to identify potential collaborators who would complement our research plans. However, experience also revealed that some of the most interesting collaborations come from researchers that might not obviously be working in a complementary field, but have come across findings in scientific publications or presentations.

It is also anticipated that the research in this proposal will aid the development and refinement of technologies and methodologies to study H2O2 imaging and identification of cysteine oxidized proteins by mass spectrometry. Protocols will be shared with the research community to aid further research in this field. In addition, reagents developed will be shared, subject to completion of appropriate Material Transfer Agreements.

The outputs from this research project that will benefit academic researchers include the fundamental knowledge obtained. The mass spectrometry experiments will produce large amounts of data about protein oxidation that will be deposited on publicly accessible databases such as PRIDE (PRoteomics IDEntifications database; http://www.ebi.ac.uk/pride/) and PeptideAtlas (http://www.peptideatlas.org/).

The research proposed may generate exploitable intellectual property, which will be evaluated by Cancer Research Technology (CRT), an oncology-focused technology transfer and development company wholly owned by Cancer Research UK (CRUK) that is responsible for the development of exploitation of knowledge arising from research at the Beatson Institute. Its key account project managers ensure that the right partners are identified to take on a development license through CRT's extensive network of contacts and supported by attending multiple oncology focused meetings a year.

A further impact of this research proposal will be the training of a researcher in several advanced technologies, which will contribute to research excellence in the UK. The researcher will be given training in experimental methods by experienced postdoctoral researchers and staff scientists as well as other members of the group. Experienced technicians will also provide support, supervision and training in the use of equipment in the advanced technology facilities. Regular oral and laboratory meetings help to develop communications skills. The PI holds weekly laboratory meetings to discuss research progress and all lab members are expected to present at these meetings with data being reviewed and scrutinised by peers and senior colleagues. Writing of reports and manuscripts will further improve organisational and time management skills. Training in experimental design and data handling and analysis will be provided throughout the fellowship on a regular basis in the laboratory and weekly review meetings. The University of Glasgow and the Beatson Institute have active seminar programmes with national as well as international speakers and fellows are encouraged to attend and to participate by questioning the speakers. Students have the opportunity to attend the University of Glasgow's annual Research Staff Conference, as well as the Beatson's annual International Cancer Conference. Trainees are encouraged to submit abstracts and to present their data at national and international meetings, as appropriate to their subject areas and progress.