BCL3 and radiation sensitivity in rectal cancer


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Adam Chambers1,Alex Greenhough1,Rhys Morgan1,Danny Legge1,Eleanor Mortensson1,Tracey Collard1,Chris Paraskeva1,Michael Thomas2,Ann Williams1
1University of Bristol,2University Hospital Bristol

Abstract

Background

BCL3 is an important pro-survival protein; upregulated in a subset of colorectal cancers (1, 2). Furthermore, it has been shown that BCL3 promotes survival in colorectal cancer (3). Locally advanced rectal cancer (LARC) is managed pre-operatively with chemo-radiotherapy. Determining why around 40% patients fail to respond to this neoadjuvant chemoradiation is important in the context of improving local recurrence rates, sphincter-saving surgery rates and overall disease-free survival for patients with LARC. The aim of this study is to investigate whether BCL3 promotes survival in the context of irradiation.

Method

Colorectal carcinoma cells (LS174T and SW1463) were grown in 2D and 3D-spheroid culture. BCL3 and Bim were suppressed using RNA interference. BCL3 was stably over-expressed in LS174T cells using a pcDNA plasmid. Protein expression was measured with western blotting. Cells were irradiated in plates using a Caesium 137 irradiator and cell viability was measured using a crystal violet cell viability assay.

Results

Cell viability was reduced in BCL3 knockout cells undergoing irradiation compared to control (LS174T 53% reduction at 2.5Gy p=0.028; SW1463 ; furthermore, in 3D-spheroid cultures stable over-expression of BCL3 inhibits g-irradiation induced cell death, suggesting that BCL3 promotes resistance to radiation-induced cell death. Suppression of BCL3 expression in colorectal cancer cells induced apoptosis (LS174T 2-fold, p=0.004; SW1463 2.5-fold, p=0.011), confirming previous work (3); moreover, BCL3 suppression upregulated pro-apoptotic Bim protein expression. Importantly Bim knockdown abrogated the effect of BCL3 suppression (LS174T, -0.38 fold reduction p=0.026; SW1463, -0.38 fold reduction p=0.0193 respectively of BCL3 induced apoptosis) highlighting that the observed apoptosis was Bim dependent.

Conclusion

Evasion of apoptosis is an important mechanism in therapy resistance. Here we show that BCL3 promotes resistance to radiation in colorectal cancer by inhibiting apoptosis through repressing Bim. Targeting BCL3 therapeutically may enhance clinical response to neoadjuvant radiation in patients with LARC.