Colorectal cancer, a highly heterogeneous cancer, continues to be a leading cause of mortality worldwide. While the 5-year survival rates for patients with Stage I and II are high, there has been little or no improvement of survival for patients with metastases. To make matters worse, 20% of the patients already present with metastasis at the time of diagnosis. Therefore, early detection of patients who are at high risk of developing metastases using biomarkers is key to improving patient survival.
Metastasis-associated in colon cancer 1 (MACC1) is one such biomarker that has been directly linked to metastasis development, reduced survival, and worse overall outcomes. In addition to identifying high-risk patients, MACC1 is biologically linked to tumor and metastasis development. Specifically, the MACC1 structure contains diverse domains and several tyrosine sites capable of versatile interactions. Therefore, the aim of the first part of the project was to study the role of tyrosine sites close to the N-terminus of MACC1. Employing computational tyrosine phosphorylation prediction tools, site Tyr379 and SRC kinase as one of the promising kinases responsible for its phosphorylation were identified. Preliminary examination reveals an association between MACC1 and SRC.
Despite extensive evidence describing the functional diversity of MACC1, little is known about the structural features and self-association property of MACC1. To address this gap in knowledge, the goal of the second part of this project was to systematically evaluate the structural properties of MACC1 and the self-association capability of MACC1. Using AlphaFold2, the structures of MACC1 and MACC1 dimer were revealed. Val212, Ileu214, and Cys216 present in the ZU5 domain of MACC1 were found to be critical for dimerization. The knowledge gained from the AI prediction was transferred to set up a bioluminescence resonance energy transfer (BRET) assay to analyze MACC1 dimerization and the effect of mutation on dimerization. In addition to validating the presence of MACC1 dimer in living cells, the BRET assay confirmed reduced MACC1 self-association when the above residues were mutated. Ultimately, the impact of these mutations on MACC1 signaling and metastasis properties was verified using an in vitro metastasis assay.
In summary, these results shed new light on the MACC1 structural characteristics particularly the presence of MACC1 homodimer, and reveal the residues important for dimerization, thus providing a framework for future development of intervention strategies.
