The role of miR-31 in regulating response of colorectal cancer to EGFR inhibition - relationship to the detriment observed in the New EPOC study

Bowel cancer is the second most common cause of cancer death in the UK. Death usually results from spread to other parts of the body, of which the liver is the commonest site. Approximately one quarter of patients with bowel cancer that has spread to the liver are able to have an operation to remove the cancer. Of those the majority are treated with chemotherapy as well to try and reduce the likelihood of the cancer coming back. Over the last decade there has been interest in the use of targeted cancer therapies including cetuximab which is a growth factor blocker. This treatment is currently recommended by NICE for people with bowel cancer that has spread to the liver that is not suitable for surgery, at an estimated additional cost of £17,000 per patient.

The New EPOC trial was undertaken to see if people with operable bowel cancer that has spread to the liver would also benefit from this treatment. The study treated patients with routine chemotherapy before and after liver surgery with or without cetuximab. The aim was to see whether the addition of this treatment would reduce the chance of the cancer getting worse or coming back after the operation. Unfortunately the trial produced an unexpected result such that patient's cancer came back sooner in the group receiving cetuximab. It is imperative that we conduct further research in order to understand why this happened.

At the time of agreeing to participate in the trial, patients were also asked to give consent for their tissue to be used for studies in the future. These include redundant formalin fixed samples of their original bowel cancer and samples from their liver operation. This tissue now forms a vital resource for us to conduct laboratory based studies to try and explain this trial result.

MicroRNAs are a recently discovered form of genetic material. They are expressed in healthy tissue, but corrupted in cancer. Our preliminary data shows that the level of a particular micro RNA, miR-31, expressed by the bowel cancers of patients in the New EPOC study may correlate with how they responded to treatment with cetuximab. The aim of my project is to study the mechanism behind this observation, that is the role of miR-31 in the response of a bowel cancer to the growth factor inhibitor cetuximab. Our preliminary work certainly suggests that it is likely to play an important role, but at present we do not know exactly which genes it is controlling. We will explore the mechanism by which miR-31 influences the progression of bowel cancer using tried and tested laboratory techniques which enable us to quantify cancer invasion and metastasis development. We will also use a mouse model to replicate the treatments used in the New EPOC study in order to dissect whether it was a particular combination of treatments, e.g. liver surgery combined with the growth factor inhibitor, that was detrimental.

This project has key implications for the treatment of patients with bowel cancer in the future. The current restriction of the use of cetuximab to patients without a genetic change in their tumours called a KRAS mutation has proven insufficient in a number of studies, including New EPOC, to predict benefit. Determining the functional role of miR-31 in regulating the response of bowel cancer to growth factor blockers would enhance our understanding of which patients would benefit from these treatments, and assist in rationally selecting patients for use of targeted drugs. This, together with validation of miR-31 as a predictor of response in other trials, may permit the development of a test to allow better patient selection in the future thereby sparing those that will not likely benefit from the associated toxicities and loss of opportunity to receive other treatments. In the longer term it may open up new therapeutic opportunities for bowel cancer, and given the substantial costs of this targeted therapy provide health economic advantages also.

Background
The New EPOC trial demonstrated detriment with the use of cetuximab in the setting of operable CRLM. Our preliminary data suggest that miR-31 may be predictive of the effect of cetuximab in this study.

Objectives
To dissect the functional role of miR-31 to enhance our understanding of the effect of EGFR inhibition in CRC and to validate miR-31 as a predictive biomarker.

Methodology
External validation will be provided by analysing miR-31 expression of tissue from the PICCOLO study of panitumumab in advanced CRC. The molecular targets of miR-31 will be identified using in-silico analyses and experimentally verified using western blotting, qPCR, and 3' UTR reporter assays in vitro, and verified in vivo on TMAs I have constructed. Biological significance of altered miR-31 expression will be explored using standard functional assays with CRC cell lines in which miR-31 has been selectively induced or knocked-down. A mouse-metastasis model will be used to determine the effect of altered miR-31 expression on disease progression in vivo. SCID mice will undergo laparotomy with colonic implantation of CRC cell lines with normal, reduced, or overexpression of miR-31. To assess the effect of EGFR inhibition, mice will be treated with chemotherapy+/- cetuximab. Partial hepatectomy at the time of intra-caecal implantation will assess influence of liver regeneration. The IVIS Lumina III in-vivo imager will be used to non-invasively assess disease progression. This will provide a model for the validation and further dissection of putative targets of miR-31.

Scientific and medical opportunities
The New EPOC study is not the only trial in CRC to show detriment with the use of EGFR inhibition. A greater understanding of the molecular profiles associated with these adverse outcomes will permit better patient selection for EGFR inhibition, and may offer new therapeutic targets. Lastly it may enhance understanding of the mechanisms underlying CRC progression.

This proposal aims to further unravel the complexities surrounding treatment resistance to EGFR inhibitors and as such is of great academic interest. There is the opportunity to develop a new biomarker based on our preliminary data suggesting that miR-31 is predictive of response to cetuximab. In addition it may allow us to uncover some of the mechanisms that underlie CRC progression and contribute to therapeutic advancements.

KRAS mutation is an established predictive biomarker of resistance to anti-EGFR therapy in CRC. However, data suggest that one-third to a half of patients with KRAS wild-type CRC treated with EGFR inhibition do not derive any benefit. In many cases this represents a lost opportunity for a patient to be treated with an alternative agent that may have offered them a better outlook. Furthermore at present there are even instances of harm such as that observed in the New EPOC study; the earlier cancer progression in a subset of potentially curable patients requires explanation. More recently data supports the exclusion of patients with any RAS mutation from receiving anti-EGFR therapy, although in the New EPOC study more stringent selection of an all RAS wild-type cohort does not alter the detriment observed with the addition of cetuximab to chemotherapy and surgery. By contrast our preliminary data suggest that if we had selected the New EPOC cohort based on the expression of miR-31 then detriment would not have occurred. This offers the promise of enabling better molecular stratification of patients with CRC in the future for the current therapies available and would have direct patient benefits.

Due to the high cost of anti-EGFR antibody therapies the current NICE guidance limits their use specifically to cetuximab in patients with KRAS wild-type inoperable liver only metastatic disease. The use of cetuximab and panitumumab outside of this is reliant upon the cancer drugs fund which permits their use for a much wider number of indications in advanced CRC provided the tumour is all RAS wild-type. The validation of a biomarker based on miR-31 allowing further subselection of patients should improve the benefit:risk profile of these drugs and would inform both clinicians and policy makers. This may allow NICE to reconsider the licensed indications for these therapies at the same as resulting in substantial cost savings to the NHS.

While this project has arisen from a desire to explore the reasons why EGFR inhibition can fail, the mechanistic dissection of the functional role of miR-31 should enhance our understanding of CRC progression as a whole. This would be of potential interest to a number of researchers including those working on projects quite disparate from our own. At present relatively little research activity is dedicated to understanding the pathogenesis of metastasis or the molecular mechanisms that underlie cancer progression. However, this elaborate process is responsible for the vast burden of cancer associated morbidity and mortality and anything that provides scientific insights to this is valuable.

In the longer term it is hoped that therapeutic opportunities may arise. Identification of the molecular drivers of treatment resistance is critical to improving the clinical benefit of anti-EGFR therapies. Certainly in the short term the development of a biomarker test based on miR-31 would likely reduce the number of patients suitable for anti-EGFR therapies. However, if we can use this observation to deduce the mechanisms by which tumours progress in the presence of EGFR inhibition, new potentially exploitable therapeutic targets may be discovered. This would lead to opportunities to commericialise this research in collaboration with pharmaceutical companies to develop new therapeutic agents. In the future it may be that EGFR inhibitors will be used in combination with other targeted therapies, thereby increasing the number of patients that can benefit from these treatments.