Functional Studies of CtBP in Breast Cancer Using the Lentiviral pINDUCER System

Abstract

As a transcriptional corepressor, CtBP plays a significant role in tumorigenesis and tumor progression through epigenetic regulation. Interacting with transcription factors and chromatin modifying enzymes, CtBP mediates repression of tumor suppressor genes that maintain genome stability, repress cellular proliferation and inhibit EMT. This contributes to the evolution of more aggressive forms of multiple epithelial malignancies, including breast cancer. CtBP is also considered a metabolic sensor due to its ability to bind to NADH, the high energy intermediate generated during carbohydrate metabolism. However, despite its potential role in tumor progression and its link to metabolic imbalance, a detailed mechanism explaining how CtBP promotes aggressive breast cancer behavior is unclear. Thus, we are employing an inducible gene expression approach that utilizes the lentiviral pINDUCER system to inducibly manipulate the levels of CtBPs using Doxycycline. The fidelity of this system allows us to characterize CtBP‐regulated pathways including EMT, invasion, metastasis, genome instability, and uncontrolled self‐renewal. To explore the role of CtBP in increasing cancer malignancy, we are implementing a broad array of biological functional assays including wound healing migration assays, Boyden chamber invasion assays, and 3D gland formation assays. We are also conducting clonogenic assays, comet assays, gamma‐H2AX foci assays to further understand how CtBP drives genome instability and activates self‐renewal pathways. Eventually, we will develop orthotopic xenograft mouse models to study breast cancers in vivo. Together, the functional assays manipulated by the pINDUCER system will shed light on the role of CtBP in breast tumorigenesis and progression.