The Interplay Between Insulin/IGF-Axis and GRP78 in Colorectal Cancer Development and Response to Fluorouracil

Abstract

Colorectal cancer (CRC) presents significant challenges in cancer therapy due to its tendency to develop drug resistance, often associated with disrupted growth and survival signalling. This PhD thesis focused on the complex role of insulin-like growth factor-binding protein-3 (IGFBP-3) in CRC, specifically in its regulation of IGF-I/II activation of the IGF-1 receptor (IGF-1R) and insulin receptor (IR) and its interaction with the 78 kDa glucose-regulated protein (GRP78), a key regulator of the unfolded protein response. The research established the basal localization of IGFBP-3 and GRP78 in H747 CRC cells using immunofluorescence staining, showing GRP78's primary localization in the nucleus and cytoplasm, with minor surface presence, and IGFBP-3's predominance on the plasma membrane. Physical interactions between GRP78 and IGFBP-3 in H747 and HCT 116 CRC cells were demonstrated using immunoprecipitation, SDS-PAGE electrophoresis, and western blotting. The silencing of GRP78 reduced intracellular IGFBP-3 levels, enhancing the cytotoxicity of both 5-FU and AG1024, underscoring the therapeutic potential of targeting GRP78 in CRC. The study also observed that exogenous IGFBP-3 affects endoplasmic reticulum stress (ERS) and cell proliferation differently in H747 and HCT 116 cells, with IGFBP-3 knockdown reducing ERS and increasing proliferation in HCT 116 cells but inducing ERS without affecting growth in H747 cells. Moreover, 5-FU treatment was found to induce ERS and upregulate IGFBP-3 expression, with added IGFBP-3 enhancing 5-FU cytotoxicity in HCT 116 cells but reducing it in H747 cells, mediated by changes in insulin signalling transduction, including IR protein downregulation and decreased AKT activation. AG1024 treatment in H747 and HCT 116 cells also led to notable apoptotic effects, GRP78 induction, and IGFBP-3 downregulation. Combining IC50 doses of 5-FU with minimal doses of AG1024 resulted in additive or synergistic effects on 5-FU's anti-proliferative and cytotoxic effects, suggesting a novel combination therapy strategy in CRC. In summary, this thesis provides valuable insights into the molecular interplay of IGFBP-3, GRP78, and IR in CRC, advancing the understanding of chemoresistance and highlighting the potential of targeted therapies to improve CRC treatment outcomes.