Science University Research Symposium (SURS)
Publication Date
2025
College
College of Sciences & Mathematics
Department
Biology, Department of
SURS Faculty Advisor
Dr. Erick Spears
Presentation Type
Poster Presentation
Abstract
Immunofluorescence-Based Detection of MYC–EGR1 Pathway Activity in p53-Deficient Human Colorectal Cancer Cells
The Wnt signaling pathway drives proliferation in the intestinal epithelium and maintains those cells in an undifferentiated state. Dysregulation of this pathway results in uncontrolled proliferation of the intestinal epithelium which can lead to colorectal cancer. These effects are driven by the expression of a common Wnt target gene, MYC. Upregulation of MYC expression by the Wnt signaling pathway, either during normal Wnt signaling or dysregulated Wnt signaling, canonically drives intestinal epithelial cells into a proliferative state. Under a specific cellular context, the loss of functional p53, MYC can noncanonically upregulate the expression of a tumor suppressor gene, EGR1, leading to apoptosis instead of proliferation (termed the MYC-EGR1 pathway). This is of particular interest because one of the standard genetic alterations found in late-stage colorectal carcinomas results in the loss of functional p53. This MYC-EGR1 pathway was identified in p53 knockout mouse embryonic fibroblasts but has yet to be observed in human cells. To assess the presence of the MYC-EGR1 pathway in human intestinal epithelial cells, we employed HCT116 variant cell lines with wild-type p53 (p53+/+) or with a knockout of the TP53 locus (p53-/-). These cell lines were transduced with shRNA targeting MYC or EGR1 or with a non-targeting (scrambled) shRNA control. The effect of MYC knockdown on EGR1 expression was used to assess whether MYC was controlling EGR1 expression, and the role of p53 was assessed within the different cell lines. For instance, in MYC shRNA transduced cells (decreased MYC expression), we expect a decrease in EGR1 if the MYC-EGR1 pathway is functional. An immunofluorescence staining technique was used to assess the levels of MYC and EGR1 protein, fluorescence microscopy was used to detect GFP expression; a fluorescence staining technique was used to assess the levels of MYC and EGR1 protein, scence staining technique was used to assess the levels of MYC and EGR1 protein and transduced cells were identified by a GFP marker. Of interest, paraformaldehyde fixation appeared to quench GFP fluorescence. Several fixation techniques were evaluated for GFP quenching before MYC or EGR1 protein levels could be compared between transduced and untransduced cells. Successsful detection of MYC-EGR1 pathway activity in human colorectal cancer cells, particularly those lacking p53 and thus mimicking late-stage tumorigenesis, could lead to this pathway being used as a novel therapeutic target.
Recommended Citation
Schultheiss, Samantha E.; Skojec, Lyndsea; and Spears, Erick Dr., "Immunofluorescence-Based Detection of MYC–EGR1 Pathway Activity in p53-Deficient Human Colorectal Cancer Cells" (2025). Science University Research Symposium (SURS). 255.
https://repository.belmont.edu/surs/255
