Apoptotic and Necroptotic Mediators are Differentially Expressed in Mucinous and Non-Mucinous Colorectal Cancer

Abstract

Background: Mucinous colorectal cancer (CRC) represents 10% of all CRC and is associated with chemotherapy resistance. This study aimed to determine expression of apoptosis and necroptosis mediators in mucinous CRC.

Methods: RNA gene expression data were extracted from TCGA. Protein levels in 14 mucinous and 39 non-mucinous tumors were measured by multiplexed immunofluorescence. Levels of apoptosis and necroptosis signalling proteins were analysed in SW1463 (mucinous rectal), SW837 (non-mucinous rectal), LS174T (mucinous colon) and HCT116 (non-mucinous colon) cell lines by western blot. Cell death was investigated by flow cytometry measurement of propidium iodide stained cells.

Results: High cleaved-Caspase 3 expression was noted in resected mucinous tumors. Western blot identified alterations in apoptosis proteins in mucinous CRC, most prominently downregulation of Bcl-xL protein levels (p=0.029) which was also observed at the mRNA level in patients by analysis of TCGA gene expression data (p<0.001). Treatment with 5-FU did not significantly elevate cell death in mucinous cells, while non-mucinous cells showed robust cell death responses. However, 5-FU-induced phosphorylation of MLKL in mucinous cancer cells, suggestive of a switch to necroptotic cell death signaling.

Conclusion: Apoptotic and necroptotic mediators are differentially expressed in mucinous and non-mucinous colorectal cancers and represent targets for investigation of cell death mechanisms in the mucinous subtype.

Keywords: apoptosis; chemotherapy; colorectal; mucinous; necroptosis.

Figure 1

Caspase protein levels in mucinous versus non-mucinous colorectal cancer. (A) Mucinous rectal cancer tumor core stained with pro-Caspase 3 (red), cleaved-Caspase 3 (green), DAPI (blue=nucleus); (B) Non-mucinous rectal cancer tumor core stained with pro-Caspase 3 (red), cleaved-Caspase 3 (green), DAPI (blue=nucleus); (C) Quantification of pro-Caspase 3 protein level in 14 mucinous and 39 non-mucinous rectal cancers, *=p<0.05. p value for t-test comparing mean protein level between mucinous and non-mucinous cancers; (D) Quantification of cleaved-Caspase 3 protein level in 14 mucinous and 39 non-mucinous rectal cancers, *p<0.05. p value for t-test comparing mean protein level between mucinous and non-mucinous cancers.

Figure 2

BCL-2 family protein levels in mucinous versus non-mucinous colon and rectal cancer cell lines. (A) Representative blots of BCL-2 family proteins at baseline; (B) BCL-2 family protein expression normalized to loading control. *p<0.05. p value for t test comparing mucinous versus non mucinous cell lines in colon and rectal cancer. N=3 experimental repeats.

Figure 3

Pro-Caspase protein and Necroptosis protein levels in mucinous versus non-mucinous colon and rectal cancer cell lines. (A) Representative blots of pro-Caspase proteins at baseline; (B) Pro-Caspase protein expression normalized to loading control; (C) Representative blots of necroptosis proteins at baseline; (D) Necroptosis protein expression normalized to loading control. *p<0.05. p value for t test comparing mucinous versus non mucinous cell lines in colon and rectal cancer. N=3 experimental repeats.

Figure 4

Apoptosis and necroptosis protein levels in response to 5-FU treatment in mucinous versus non-mucinous colon and rectal cancer cell lines. (A) Representative blots of apoptosis and necroptosis proteins in response to 5-FU; (B) Protein expression normalized to loading control. *p<0.05. p value for t test comparing mucinous versus non mucinous cell lines in colon and rectal cancer. N=3 experimental repeats.

Figure 5

Cell death assay to determine cell death rate by measurement of PI positive cells. Cells treated with DMSO control versus 25μM 5-FU. * indicates p<0.05 for comparison between control and treated cells within cell line.