Nuclear localization of Coiled Coil Domain-Containing 3 protein in breast cancer cells
A preliminary study from our lab shows that Coiled Coil Domain-Containing 3 (CCDC3) or “Favine” plays a significant role as a tumor suppressor protein in breast cancer cells. This thesis aims to dig deeper into the mechanisms behind the protein’s nuclear localization. Next, it pursues CCDC3’s nuclear localization’s role in cancer cell tumor suppression. This thesis suggests that CCDC3 may be imported into the nucleus via the importin-β pathway, suggesting that CCDC3 may contain a classical nuclear localization sequence. To better understand the mechanism behind this protein’s import, research in our lab mutated human CCDC3 protein via a K233T mutation to try to inhibit nuclear localization. This single nucleotide substitution still presented nuclear localization when overexpressed in cells. Thus, this thesis took more drastic measures. Primers were designed to further mutate the CCDC3 gene via mutagenesis and transfect this mutated gene into Cal51 cells, a breast cancer cell line. It was found by cell immunofluorescence that CCDC3 mutants K233T + K234M and R232G + K233T + K234M are not imported into the Cal51 cell nucleus. These promising results expose specific amino acids required for a functional nuclear localization sequence of CCDC3. After determination of how the protein enters the cell’s nucleus and what sequence is used to signal that, the next mystery was how that would affect the protein’s nuclear function. This research demonstrates the fast growth of breast cancer cells when CCDC3 double or triple mutants were overexpressed. This suggests that inhibiting nuclear import disrupts the tumor suppressive function of CCDC3. These findings will deepen the understanding of the molecular mechanisms underlying the anti-breast cancer function of CCDC3 and provide new information for the future study of this protein.