Dataset Information

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.

SUBMITTER: O’Connell E

PROVIDER: S-EPMC9334008 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BACKGROUND:Colorectal cancer (CRC) is still a leading cause of death worldwide. Recent studies have pointed to an important role of microRNAs in carcinogenesis. Several microRNAs are described as aberrantly expressed in CRC tissues and in the serum of patients. However, functional outcomes of microRNA aberrant expression still need to be explored at the cellular level. Here, we aimed to investigate the effects of microRNAs aberrantly expressed in CRC samples in the proliferation and cell death of a CRC cell line. METHODS:We transfected 31 microRNA mimics into HCT116 cells. Total number of live propidium iodide negative (PI-) and dead (PI+) cells were measured 4?days post-transfection by using a high content screening (HCS) approach. HCS was further used to evaluate apoptosis (via Annexin V and PI staining), and to discern between intrinsic and extrinsic apoptotic pathways, by detecting cleaved Caspase 9 and 8, respectively. To reveal mRNA targets and potentially involved mechanisms, we performed microarray gene expression and functional pathway enrichment analysis. Quantitative PCR and western blot were used to validate potential mRNA targets. RESULTS:Twenty microRNAs altered the proliferation of HCT116 cells in comparison to control. miR-22-3p, miR-24-3p, and miR-101-3p significantly repressed cell proliferation and induced cell death. Interestingly, all anti-proliferative microRNAs in our study had been previously described as poorly expressed in the CRC samples. Predicted miR-101-3p targets that were also downregulated by in our microarray were enriched for genes associated with Wnt and cancer pathways, including MCL-1, a member of the BCL-2 family, involved in apoptosis. Interestingly, miR-101-3p preferentially downregulated the long anti-apoptotic MCL-1?L isoform, and reduced cell survival specifically by activating the intrinsic apoptosis pathway. Moreover, miR-101-3p also downregulated IL6ST, STAT3A/B, and MYC mRNA levels, genes associated with stemness properties of CRC cells. CONCLUSIONS:microRNAs upregulated in CRC tend to induce proliferation in vitro, whereas microRNAs poorly expressed in CRC halt proliferation and induce cell death. We provide novel evidence linking preferential inhibition of the anti-apoptotic MCL-1?L isoform by miR-101-3p and consequent activation of the intrinsic apoptotic pathway as potential mechanisms for its antitumoral activity, likely due to the inhibition of the IL-6/JAK/STAT signaling pathway.