B. D. Honick1, A. K. Dilly1, S. Hong1, Y. J. Lee1,2, H. J. Zeh1, D. L. Bartlett1, H. A. Choudry1 2University Of Pittsburg,Department Of Pharmacology & Chemical Biology,Pittsburgh, PA, USA 1University Of Pittsburg,Department Of Surgery,Pittsburgh, PA, USA
Introduction: Cancer cells up-regulate endoplasmic reticulum (ER) stress response related molecular signaling pathways, known as unfolded protein response (UPR), in order to accommodate high protein turnover associated with rapidly proliferating cells. We hypothesized that mucinous colon cancers would have higher basal ER stress owing to high mucin 2 (MUC2) protein production, and that ER stress aggravation in these tumors would overwhelm cytoprotective capacity of UPR and induce apoptosis.
Methods: In vitro studies were conducted using explant tissue from mucinous and non-mucinous colon cancers, normal colon, LS174T cells (MUC2-secreting human colon cancer cells), and stably transfected LS174T cells expressing dominant negative TCF4 (differentiated goblet-like cells controlled by Tet-on system that express higher MUC2 levels than wild-type LS174T cells). ER stress aggravators included ER calcium pump inhibitor celecoxib and fatty acid synthase inhibitor orlistat.
Results: Higher levels of ER stress response proteins, including GRP78/BiP (immunoglobulin heavy chain-binding protein), ATF4 (activating transcription factor 4), CHOP (C/EBP homologous protein) assessed by real-time PCR and immunofluorescence, were detected in mucinous colon cancer explant tissue and MUC2 over-expressing LS174T-dnTCF4 cells as compared to non-mucinous cancer explant tissue, normal colon epithelium and wild-type LS174T cells. These data suggest a correlation between MUC2 protein production and basal ER stress levels. Treatment of LS174T cells with ER stress inducers celecoxib or orlistat alone decreased cell viability (assessed by MTS assay and phase-contrast microscopy) and induced apoptosis (measured by flow cytometry with annexin-V and propidium iodide staining) and ER stress response proteins (BiP, ATF4, CHOP evaluated by western blot) in a dose-dependent fashion, with doses > 75 µM (celecoxib) and > 200 µM (orlistat) required to achieve > 50% cell death. Combination treatment of LS174T cells with celecoxib (50 µM) and orlistat (100 µM) demonstrated synergistic loss of cell viability (> 50% cell death by MTS assay) and induction of apoptosis (flow cytometry) and ER stress response proteins (western blot). Western blot demonstrated synergistic induction of PUMA (p53 upregulated modulator of apoptosis), cleaved caspases 9 and 3, and cleaved PARP (poly ADP-ribose polymerase) as the downstream mechanism for apoptosis following combination therapy in LS174T cells.
Conclusion: Mucinous cancers have higher basal ER stress than non-mucinous cancers, perhaps due to the higher protein turnover associated with abundant MUC2 production. These tumors may be especially vulnerable to drugs that overwhelm the ER stress response through disrupting protein homeostasis. Ongoing studies will determine whether ER stress aggravation by this combination therapy triggers a switch from autophagy to apoptosis.