Myoferlin: A predictive marker of response to radiotherapy and survival in patients with locally advanced rectal cancer and a poteintal therapeutic target


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Hayley Fowler1, Rachael Clifford1, David Bowden1, Paul Sutton1, Naren Govindarajah1, Matthew Fok1, Jason Parsons1, Dale Vimalachandran2
1University of Liverpool, 2Countess of Chester Hospital NHS Foundation Trust

Abstract

Background

Our proteomic analysis of rectal cancer samples identified myoferlin as the most significantly downregulated protein in patients who responded well to neoadjuvant chemoradiotherapy. Myoferlin plays a role in membrane repair and VEGF signal transduction and its expression correlates with worse prognosis in various other epithelial cancers.

Method

To validate the proteomic findings immunohistochemical (IHC) analysis for myoferlin was performed of matched tumour and lymph node samples from 111 patients with locally advanced rectal cancer. Manipulation of myoferlin was achieved using siRNA and a novel small molecular inhibitor (wj460). Clonogenic assays and spheroid models using immortalised colorectal cells lines and patient derived tumour organoids were used to analyse for radiosensitisation. Underlying mechanisms were investigated using comet assays to evaluate the impact of myoferlin manipulation on DNA double strand break repair kinetics and further investigated using immunofluorescence staining for yH2Ax, RAD51 and 53BP1.

Results

IHC analysis of tumour samples confirmed myoferlin expression correlated with response to neoadjuvant chemoradiotherapy. High myoferlin expression was also associated with spread to both local lymph nodes and the development of distant metastases (p<0.05). These patients also had a significantly worse 5-year survival (p = 0.01, HR 3.5 95% CI [1.27, 10.04]). Quantification of myoferlin using immunoblotting in immortalised colorectal cancer cell lines demonstrated that high myoferlin expression was associated with increased radioresistance. Clonogenic assays and spheroid models following manipulation of myoferlin using siRNA or wj460 demonstrated significantly increased radiosensitivity across all cell lines. Tumour organoids with higher protein levels of myoferlin were the most radioresistant.  Following myoferlin knockdown with siRNA and irradiation, cells had a significantly impaired ability to repair DNA double strand breaks. This appeared to be mediated via non-homologous end-joining via the DNA-PKsc pathway with delayed phosphorylation of DNA-PKsc following irradiation.

Conclusion

Our results demonstrate that high myoferlin expression is a poor prognostic marker of both radiotherapy response and overall survival. Manipulation of myoferlin improves radiosensitivity of cancer cells in-vitro which may be mediated via the DNA-PKsc pathway.  Myoferlin therefore warrants further investigation as a biomarker and therapeutic target in locally advanced colorectal cancer. 

Impact statement

Improving response to radiotherapy and long term survival in patients with rectal cancer