SIP1 induced epithelial to mesenchymal transition (EMT) promotes metastasis and chemoresistance in colorectal cancer.

Bowel cancer is the second most common cause of cancer related death in the UK, and it is spread of the cancer (Metastasis), that is the main cause of this high death rate. Unfortunately, up to a third of patients present with evidence of cancer spread at initial presentation, and up to half of all cases subsequently experience recurrence of disease from previously undetected occult metastases after their operation. While there has been some advances in the management of patients with recurrent bowel cancer, the vast majority of patients in this position are incurable and sadly can expect a median life expectancy of less than two years, even with the latest chemotherapy drugs.

Emerging evidence in recent years has highlighted the role of certain key molecules that normally regulate the development of embryos, to be abnormally expressed in cancer cells, making them not only more likely to invade and spread, but also to become resistant to chemotherapeutic drugs. Little is known about the expression or role of one of these key proteins, known as SIP1, in bowel cancer.

Preliminary work conducted by me so far has highlighted that SIP1, when present in bowel tumors, results in an increased likelihood of cancer recurrence, reduced patient survival, and increased resistance against commonly used chemotherapeutic drugs in the treatment of bowel cancer. Consequently, SIP1 may be a helpful biomarker for clinically useful outcome measures in patients with bowel cancer. I have conducted further work to try to explain the mechanism behind this observation. Our results suggested that one potential explanation for the resistance to chemotherapy observed is due to the increased ability of cancer cells, containing SIP1, to repair the damage brought about by chemotherapy, and thereby avoid cancer cell death.

Based on the above novel observations, the primary goals of my research proposal are two fold.

The first aim of my study is to better understand the underlying mechanisms by which bowel cancer cells increase their capacity to repair cellular injury through the actions of SIP1. This work will not only enhance our understanding of basic biological processes in cells, but it may also enable the creation of targeted drugs to inhibit these repair systems and subsequently increase the sensitivity of cancer cells to chemotherapy drugs, potentially impacting the lives of many sufferers. This work will involve the use of cells grown in culture, but also studies in a carefully calculated number of mice to ensure that my findings are as closely related to physiological circumstances as possible, and therefore more likely to take the next step and enter studies in man.

As a clinician, my goal is to translate new laboratory findings into patient care, consequently my second aim is to more fully develop and test the use of SIP1 as a biomarker that could help in the personalisation of the management of bowel cancer by assisting in risk prediction for cancer recurrence and potentially chemotherapy response. Validating my promising results on a larger and independent group of patients is an essential per-requisite for translating these findings into real-time patient management. For that reason, I have teamed up with colleagues in Italy and we aim to test the generality of my findings in an independent patient group from their unit.

If successful, my research will provide significant insight into mechanisms by which cancer spreads and gains resistance to current drug therapies, and may facilitate better clinical management and drug discovery in future years.

The aim of my study is to investigate the biological consequence of SIP1 induced EMT to metastasis and chemoresistance. My objectives are to 1) Characterise the transcriptional regulation of ERCC1 in epithelial and mesenchymal CRC cells, 2) Investigate the contribution of ERCC1 up regulation by SIP1 to chemoresistance, 3) Perform in-vivo experiments to validate SIP1 induced chemoresistance by modulating ERCC1 expression and 4) Validate SIP1 expression as a predictive biomarker of outcome in an independent patient cohort. Objective 1 will be achieved by cloning the promoter region of ERCC1 in pGL3 (luciferase reporter) plasmid and conducting luciferase assays in induced and un-induced cells. I will also change critical nucleotides in the E-box regulatory sequence by site directed mutagenesis to identify motifs facilitating ERCC1 transactivation. To investigate the influence of ERCC1 up regulation by SIP1 to chemoresistance, I will generate stable knock down and ERCC1 transfected DLD-SIP1 cells. I will then undertake apoptosis assays using Oxaliplatin before and after induction of EMT. I will also use an established orthotopic mouse model to analyse the influence of SIP1 mediated up regulation of ERCC1 and monitor real time tumour growth and chemotherapy response using IVIS Lumina III in vivo system. Additionally, after final treatment I will cull and undertake immunohistochemistry (IHC) to assess for the presence of SIP1 expressing metastatic cells. Finally, I will validate the role of SIP1 as a predictive biomarker in an independent patient cohort. The results of this study will provide scientific and clinical insight into mechanisms mediating chemoresistance in CRC. Demonstrating ERCC1 mediates chemoresistance may highlight the necessity to manufacture specific ERCC1 inhibitors, a potential chemosensitiser. Validating, SIP1 as a biomarker in an independent patient cohort will facilitate its progress towards a prospective study and clinical use in the future.

Metastasis and associated chemoresistance is the ultimate reason for cancer related mortality. This project represents an innovative and unique research strategy to investigate the role of SIP1 induced epithelial to mesenchymal transition (EMT) in mediating chemoresistance and metastasis in colorectal cancer (CRC). The project contains clinical and basic scientific research themes that may result in significant academic and economic/societal impacts by application in the clinical and pharmaceutical environment. My preliminary data has demonstrated SIP1 induced EMT leads to significant up regulation of ERCC1, a nucleotide exchange repair protein involved in response to platinum based chemotherapy. Investigating cellular response to DNA damaging agent's after up/down regulating ERCC1 and monitoring apoptotis response is of great academic interest, due to its potential clinical impact. Improved knowledge of molecules mediating DNA damage repair will further scientific insight and enable development of targeted therapies in the future. Development and application of ERCC1 inhibitors as chemosensitisers, for use in conjunction with platinum based agents could have profound consequences on disease recurrence and patient survival in CRC. Furthermore development of orthotopic murine models with the capability of initiating EMT will be a world first and provide a platform to assess therapeutic efficacy in vivo in the future.

Validating my results highlighting SIP1 expression as a predictive cellular marker in an independent patient cohort, will result in a significant step towards its development as a biomarker. The cancer sciences division of the University of Southampton is uniquely positioned to undertake this study, as the antibody against SIP1 was raised 'in house' and is currently being patented. When fully validated and optimised, SIP1 represents a clinical tool with the ability to sub-classify CRCs and pioneer a molecular pathological approach to improve prediction of micro-metastatic risk after surgical resection. The results would facilitate progression towards a prospective study to validate clinical efficacy and could be of significant commercial interest. SIP1 expression may also help clinicians internationally personalise care by aiding difficult decisions such as optimal surveillance interval, patient selection for adjuvant therapy and guide choice of chemotherapeutic regimen. The results may also guide heath care trusts, and policy makers establish guidelines nationally and internationally in the optimal management and follow up of patients with CRC.

The University of Southampton has a world-class reputation for commercialising academic research. For example, locally established companies such as Karus therapeutics, specialises in developing and marketing small molecule anti-cancer agents and would be invaluable should my research identify therapeutically relevant targets.

Time scale to deliver impact milestones: -
Year 1; I will start actively joining in the public outreach themes organized by the University of Southampton. I hope to undertake and complete validation of SIP1 expression as a predictive biomarker in an independent patient cohort. Year 1-3; I plan to present my data at local, national and international conferences and publish data in peer reviewed journals.Year 2-3 establish an orthotopic mouse model, which will be used as a platform to study chemoresistance in the current and future studies. In the long term, if my work identifies novel targets, I hope to collaborate with companies such as Karaus therapeutics to develop pharmaceutical products or engage with the clinical trials unit to set up prospective trials to commercialise our results.