Defining the Functional Biology of E3 Ubiquitin Ligase TRIP12 in Mammalian Intestine and Colorectal Cancer

Abstract

The E3 ubiquitin ligase, TRIP12, belongs to the HECT domain family that is found to interact with many proteins, including ones that control the cell cycle. Several studies found its involvement in cell cycle progression due to interactions with USP7 or other mechanisms that are not yet understood. Recently, Dr. Khan's lab showed that TRIP12 acts as a negative regulator of FBW7, another E3 ligase that is highly mutated in colorectal cancers. These FBW7 mutations are thought to contribute to chemotherapy resistance by accumulating its anti-apoptotic substrate, MCL-1. This study aimed to characterize the functional role of TRIP12 deletion in multiple colorectal cancer cell lines. We hypothesized that downregulating TRIP12 would increase sensitivity to chemotherapy by stabilizing FBW7 and would also affect stem cell homeostasis. Briefly, we measured the TRIP12-/- cells' ability to form colonies compared to wild type cells. Additionally, we studied the effect of TRIP12 knockout on the response to conventional chemotherapeutic agents, such as 5-FU. Proliferation and differential phosphorylation patterns between TRIP12-/- and wild type cells were also studied. Our data revealed that TRIP12 deletion reduced the spheroid clonogenicity in HT29 and SW837 cells but does not affect the colony formation in monolayer cultures. Consistently, staining for the proliferation marker, Ki67, indicated a lower proliferation rate in TRIP12-/-. Moreover, proteins that regulate cell cycle progression, specifically p53, is highly phosphorylated in TRIP12-/-. This finding suggests that TRIP12 possibly interacts with different signaling pathways in CRC and interferes with cell cycle regulators' function. Furthermore, the difference in response to conventional colorectal cancer treatment upon TRIP12 knockout was visible compared to wild type cells.

Date of Award	2021
Original language	American English
Awarding Institution	HBKU College of Health & Life Sciences