Investigating the Relationship Between Cystic Fibrosis Transmembrane Conductance Regulator and Colorectal Cancer Under Endoplasmic Reticulum Stress
2018-04
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Investigating the Relationship Between Cystic Fibrosis Transmembrane Conductance Regulator and Colorectal Cancer Under Endoplasmic Reticulum Stress
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2018-04
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Our group has identified Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) as a tumor suppressor for colorectal cancer (CRC) (Than et al., 2016). However, the mechanism by which CFTR protects against CRC is unknown. Determining this mechanism may lead to new therapies for CRC. Gene Set Enrichment Analysis (GSEA) pathway analysis showed that CFTR-deficient normal and tumor tissues from mice and humans were enriched in genes involved in the unfolded protein response (UPR) to endoplasmic reticulum (ER) stress. Altered genes, environmental stress and increased protein synthesis induce UPR activation in cancer cells (Kaufman & Wang, 2014; Ma & Hendershot, 2004; Dejeans, Barroso, Fernandez-Zapico, Samali, Chevet, 2015). The UPR has two potentially opposing effects on cancer development: on the one hand, some cancer cells require a higher level of UPR to support their growth and survival (Corazzari, Gagliardi, Fimia & Piacentini, 2017); on the other hand, increased UPR leads to loss of stemness which could inhibit cancer development (Heijmans et al., 2013). To identify the correlation between UPR and cancer-supporting effects of CFTR-deficiency, a pair of CFTR knockout (KO) and wildtype (WT) Caco2 cell lines created by CRISPR-Cas9 engineering were utilized to test the working model that UPR activation might be enhanced in CFTR KO cells, and this activation might support CRC cell survival under ER stress. To test the working model, the Caco2 cell lines were treated with chemical inducers to trigger ER stress. To evaluate the UPR activities, we used Thapsigargin (TG) to induce ER stress in CFTR KO and WT cell lines and compared iii mRNA expression changes of key genes in the two cell lines by performing RT- qPCR. The heat shock protein A member 5 (HSPA5) gene encodes the protein that initiates all three UPR sub-pathways. Although the expression of HSPA5 increased after TG treatment in both CFTR WT and CFTR KO cell lines, the TG treatment had more of an effect on HSPA5 expression in CFTR KO cells, suggesting a trend that CFTR deficiency might enhance the effect of ER stress on the expression of UPR gatekeeper HSPA5, the common regulator for initiating three UPR sub-pathways. The ATF4 expression levels in both CFTR WT and CFTR KO cell lines increased to the same extent, which reflects that the loss of CFTR might not promote the activation of PERK signaling. The decreased total XBP1 mRNA expression levels in CFTR KO cells suggests that CFTR deficiency might not activate the specific IRE1α-XBP1 signaling pathway. The TXNIP, which is part of an apoptotic pathway, is down- regulated in CFTR KO cells, which might provide protection from apoptosis. These studies suggest that UPR activation might be enhanced in CFTR KO cells, but the specific pathway might not be the one detected. Additionally, the down regulation of TXNIP might offer protection for CRC cells from apoptosis. Although many cancer cells require an enhanced UPR (Corazzari, Gagliardi, Fimia & Piacentini, 2017), excessive, prolonged activation triggers a switch to an apoptotic pathway (Wang & Kaufman, 2016; Niederreiter, L. et al., 2013). To examine this possibility, TG and Tunicamycin (TM) were used to induce ER stress in cells, and cell viability in CFTR KO and WT cell lines over time was compared by conducting MTT assay. Although the results showed decreased cell viability after treatment in both iv CFTR WT and CFTR KO cell lines, the ratios of treated over untreated viability at each point after TG and TM treatment was higher in CFTR KO cells, indicating that the loss of CFTR might cause TG-induced ER stress to have less of an effect on cell viability. These data suggest that the loss of CFTR increases protection from ER stress, and that this protection supports the survival of CRC cells. Based on the fact that CFTR deficiency can activate the NF-κB signaling pathway (Crites et al., 2015), and that UPR can activate the NF-κB (Grootjans, Kaser, Kaufman & Blumberg, 2016), the NF-κB activity in CFTR KO cells in response to UPR was also examined. RT-qPCR analysis was performed to compare the gene expression changes relating to NF-κB signaling pathway in CFTR WT and CFTR KO Caco2 cell lines treated with TG to trigger UPR activation. Although the results showed enhanced expression of IL8 and TNF in both cell lines, the TG treatment had less of an effect on the expression of these genes in CFTR KO cells. This suggests that CFTR deficiency might cause TG to produce less of an effect on the expression of NF-κB signaling target genes IL8 and TNF. These data suggest that the loss of CFTR might cause TG-induced UPR to have less of an effect on NF-κB activation. The enhanced basal expression levels of the NF-κB target gene IL8 in CFTR KO cells might suggest an increased activation of NF-κB signaling in CFTR KO cells and this activation might support the growth of cancer cells. In conclusion, in Caco2 CRC cells, CFTR deficiency may alter the response to UPR in several pathways, which may promote cancer cell survival. Additional experiments v must be conducted to determine which pathways among those that are altered are vital for cancer development.
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University of Minnesota M.S. thesis.April 2018. Major: Integrated Biosciences. Advisors: Patricia Scott, Robert Cormier. 1 computer file (PDF); vii, 64 pages.
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Zhang, Zishan. (2018). Investigating the Relationship Between Cystic Fibrosis Transmembrane Conductance Regulator and Colorectal Cancer Under Endoplasmic Reticulum Stress. Retrieved from the University Digital Conservancy, https://hdl.handle.net/11299/198350.
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