| dc.description.abstract | Colorectal cancer (CRC) is the third most common cancer globally, accounting for approximately 10% of all cancer diagnoses. A third of CRCs occur in the rectum. The majority of CRCs are diagnosed at advanced stages and treatment for locally-advanced rectal cancer (LARC) is neo-adjuvant chemoradiation therapy (neo-CRT) followed by surgical removal of the tumour. Unfortunately, response rates to neo-CRT are modest, with less than 30% of patients achieving a complete pathological response (pCR). Therefore, there is a global unmet need to urgently elucidate the molecular factors influencing response to neo-CRT in rectal cancer to identify new therapeutic targets to boost response to therapy in this setting. Furthermore, the identification of novel predictive biomarkers of response to neo-CRT in rectal cancer would enable improved patient stratification prior to the initiation of treatment. The complement system is an essential arm of innate immunity which is becoming increasingly recognised in the context of cancer. Complement system components have been demonstrated to promote tumourigenesis and alter response to therapy in a number of human cancers. In this thesis, the role of the complement system in the radioresistance of rectal cancer was investigated, to assess the potential for complement as a therapeutic target and a predictive biomarker of response to neo-CRT in rectal cancer.
The inherent radiosensitivity of a panel of colon and rectal cancer cell lines was characterised, identifying HCT116 cells as an inherently radiosensitive cell line, while SW837, HRA-19 and SW1463 cells were inherently radioresistant. Expression of the complement system was characterised in these cells, demonstrating that colon and rectal cancer cells express the central complement components C3 and C5, which are present intracellularly and secreted. Complement was activated in CRC cells, with C3a and C5a anaphylatoxins secreted and retained intracellularly. The total intracellular concentration of C3 and C5 and their respective anaphylatoxins was increased in cells with increased radioresistance, suggesting a role for complement in the response to radiation in CRC. Furthermore, expression of C3 and C5 positively correlated with the surviving fraction of cells at a clinically-relevant dose of radiation. Expression of complement factor B (CFB), an alternative complement activation pathway component, suggested that complement activation may occur via this pathway. CRC cells were demonstrated to express the C3aR and C5aR complement receptors, and membrane-bound complement regulatory proteins, suggesting that they can respond to complement signalling and modulate complement activation, respectively.
In radiosensitive HCT116 cells and radioresistant HRA-19 cells, C3 was demonstrated to functionally modulate the response to radiation at a clinically-relevant dose of radiation. In HCT116 cells, C3 overexpression was not associated with alterations in viability, apoptosis, cell cycle distribution or DNA damage induction and repair, suggesting that C3 may modulate the response to radiation in these cells by another mechanism. In HRA-19 cells, C3 silencing and enhanced radiosensitivity was associated with increased levels of basal DNA damage and altered cell cycle distribution to a more radiosensitive phenotype. Immunoprecipitation of C3 from HRA-19 and HCT116 cells and analysis by mass spectrometry demonstrated that the C3 interactome is significantly altered between these two cell lines, supporting the hypothesis that C3 may engage in different intracellular roles.
Investigation of the effect of tumour cell-derived C3 and recombinant C3a on T cell phenotype demonstrated that complement can maintain T cells in a naïve state. While this would suggest that complement does not boost effector function, assessment of T cell cytokine expression demonstrated that complement potentially shifts T cell responses away from T helper (Th)2-like towards an IFN-g producing, Th1-like phenotype. No effect on T cell proliferation was demonstrated by either tumour cell-derived or recombinant complement.
Investigation of complement expression in pre-treatment rectal tumour biopsies demonstrated that central complement cascade and complement activation pathway components are expressed in rectal tissue at higher levels relative to non-cancer tissue. Expression of C5 mRNA was elevated in the tumour tissue of obese patients suggesting a relationship between obesity status and complement expression in rectal cancer. Complement activation was detected within the circulation in pre-treatment rectal cancer patient sera. Sera levels of C3a were associated with clinical tumour stage. For the first time, pre-treatment sera levels of C3a and C5b-9 were demonstrated to correlate with subsequently poor patient responses to neo-CRT, suggesting that complement may have potential as a circulating predictive biomarker of response to neo-CRT. C5b-9 was also elevated in the sera of patients with worse recurrence-free and overall survival.
Together this thesis demonstrates a functional role for C3 in the response to radiation in vitro, and highlights a potential role for circulating C3a and C5b-9 as predictive and prognostic biomarkers in rectal cancer. | en |
