Notch Signaling Mediates Drug Resistance in Colorectal Cancer Cells via Effects on DNA Repair Proteins

Abstract

Colorectal cancer (CRC) is the second leading cause of cancer deaths in the United States and the third most commonly diagnosed cancer in men and women. Despite tremendous progress in diagnosis, prevention, and treatment efforts, tumor recurrence and chemoresistance remain as considerable challenges. Treatment options are limited if surgery and chemotherapy are unsuccessful. Several studies have implicated the Notch signaling pathway in conferring drug resistance to tumor cells, in addition to increased CRC aggressiveness and potential for metastatic spread. Our group previously showed that Notch-1 signaling is highly associated with promoting cancer stem cell properties in CRC cells. Furthermore, we have also confirmed that human colon tissue samples isolated from CRC patients indicate higher expression of DNA repair proteins associated with Base Excision Repair (BER). Herein, we hypothesized that Notch signaling confers drug resistance to tumor cells via signaling effects on critical proteins associated with DNA repair. Methods: The experiments conducted in this study utilized the colon cancer cell line HCT-116. These cells were transduced with an IRES-GFP retrovirus expressing human intracytoplasmic domain of Notch-1 (ICN1). Another group of HCT-116 cells was transduced with a small hairpin mRNA construct (sh59) that effectively knocked out Notch-1 action. Cell lines were exposed to varying concentrations of cytarabine and cisplatin, potent DNA damaging agents, and observed for chemosensitivity. Western blot analysis was performed using standard methodology. Results: Small hairpin mRNA (sh59) transduction into the colon tumor cell line HCT-116 resulted in a significantly decreased expression of critical BER DNA repair enzymes, Poly (ADP-Ribose) Polymerase 1 (PARP1) and 8-Oxoguanine glycosylase (OGG1). These changes were accompanied by significantly higher chemosensitivity of these cells to cytarabine and cisplatin treatment as opposed to cells expressing constitutively active Notch-1 signaling. Furthermore, cells with intact Notch signaling expressed upto two-fold increase in expression of APE1 following treatment with cytarabine compared to cells with no Notch-1 expression. Conclusions: These data indicate a key role for Notch signaling in conferring drug resistance to CRC cells via effects on critical proteins of DNA repair. This finding highlights the potential use of Notch-1 inhibitors in combination with PARP1 inhibitors to effectively target highly drug resistant CRC cells.