Abstract
Focal adhesion kinase (FAK) is a 125 kDa protein recruited as a participant in focal adhesion dynamics and serves as a signaling scaffold for the assembly and subsequent maturation of focal contact. Identification of new FAK binding proteins could reveal potential signaling targets and contribute to further development of therapeutic drugs in the treatment of colon cancer. Here, we applied a functional proteomic strategy to identify proteins that interact with FAK in human colon cancer cell line HCT-116. Proteins were targeted by coimmunoprecipitation with an anti-FAK antibody and resolved on 1D-SDS-PAGE. The gel was excised, reduced, alkylated, and trypsin digested. Tryptic peptides were separated by nano-LC-MS/MS by an LTQ-Orbitrap-Velos spectrometer. We identified 101 proteins in the immunocomplex under epithelial growth factor (EGF) stimulation. Three proteins, zyxin, nesprin-1, and desmoplakin, were discovered and validated using reciprocal immunoprecipitation and Western blot analysis. Then, we sought to study the biological relevance of these proteins by siRNA transfection of HCT-116 cells. According to the results, zyxin might play a central role as an upstream regulator to mediate critical cancer-related signaling pathways. Zyxin and nesprin-1 depletion significantly impaired cell migration and invasion capabilities. Additionally, we performed ELISA assays on serum samples from patients with colon cancer instead of cell models to quantify the protein levels of zyxin and nesprin-1. Our results suggested that zyxin and nesprin-1 are not only promising therapeutic targets but also potential diagnostic biomarkers for colon cancer.
Highlights
Colon cancer is one of the most commonly diagnosed cancers and the leading cause of cancer death, with an estimated 1,096,601 cases and 551,269 deaths in 2018 according to Global Cancer Statistics[1,2]
We have identified among Focal adhesion kinase (FAK) interactors; zyxin, a zinc-binding phosphoprotein located along the actin cytoskeleton which is known to involve in cell motility[34]; nesprin-1, a protein shuttles between actin filaments and the nucleus is crucial for nuclear positioning and anchorage[35]; and desmoplakin, a desmosomal protein which maintains structural integrity at cell-cell interfaces[36]
Our Western blot results showed that FAK protein levels were higher in colon cancer cell line HCT-116 than in gastric cancer cell line MKN-1, breast cancer cell line MD MAB237 and lung cancer cell line A549 (Fig. 1a)
Summary
Colon cancer is one of the most commonly diagnosed cancers and the leading cause of cancer death, with an estimated 1,096,601 cases and 551,269 deaths in 2018 according to Global Cancer Statistics[1,2]. Chemotherapy has served as the backbone of cancer treatment, its cytotoxicity destroys cancer cells as well as surrounding healthy tissues, resulting in severe side effects, including hair loss, nausea, infections, and immune system destruction. VS-4718 inhibits FAK, blocks fibronectin-stimulated FAK autophosphorylation of Tyr[397], and may prevent the integrin-mediated activation of numerous downstream signal transduction cascades, including ERK, JNK/MAPK and PI3K/AKT. This treatment results in a decline in cancer stem cells (CSCs) and suppresses tumor cell migration, proliferation, and survival[22]. Identification of the interactome of FAK is necessary to understand the regulation of this oncogene and target new binding partners as potential therapeutics for colon cancer
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