M.Sc Thesis


M.Sc StudentJalfon Joelle
SubjectSub-Cellular Foci Dynamics Associated with the Anti-Cancer
Drug SBI-756
DepartmentDepartment of Biomedical Engineering
Supervisor PROF. Amit Meller


Abstract

The eIF4F complex, a heterotrimeric protein complex, is responsible for protein translation initiation (TI) in all eukaryotic cells. TI is the first step of protein synthesis, in which a polypeptide chain is assembled based on the encoded information on an mRNA molecule, before it becomes a functional protein. The eIF4F complex is comprised of three protein subunits- eIF4G, large scaffolding protein; eIF4E, cap-binding protein that recruits the mRNA molecule; and eIF4A, helicase that unwinds mRNA secondary structures and recruits the ribosome. eIF4F complex has an important role of facilitating the cap-dependent translation process by bringing together proteins necessary for translation. It is mostly crucial in the translation of longer, more complex mRNA molecules such as tumor suppressors and oncogenes, implying its importance in the process of cell division, cell cycle control, programmed cell death and cancerous processes.

Cancer cells have the ability to divide and proliferate quickly and repeatedly, avoiding cell cycle control check points and apoptosis (cellular suicide). Therefore, they critically rely on rapid protein translation, making TI a strategical hot spot for interference with cancer proliferation. The TI complex assembly is the rate limiting step of many molecular pathways making it one of the most regulated processes in the cell. For that reason, many anti-cancer drugs take advantage of the central role of TI and try to target it in order to slow down cellular activities hence suppressing cell division or forcing them to surrender to the apoptotic process. Most drugs used today target a cascade upstream of the assembly of the complex, thereby, inhibiting its functionality. Unfortunately, cancer cells often manage to overcome chemotherapy treatment and become resistant to it by bypassing the targeted cascade and upregulating a parallel pathway to compensate for the inhibition. Thus, targeting the eIF4F complex assembly directly remains a challenge that could prove to be a potential anti-cancer treatment.

Using direct microscopy analysis of cancer cell lines, we studied the cellular response to a novel small molecule inhibitor, SBI-756, designed to target the eIF4G. SBI-756 was proven to slow down protein translation rate in cells and turn already-resistant cancer cells back to being chemo-sensitive. It was also shown to disrupt the eIF4E-eIF4G interaction. In this work, we employed multi-color confocal microscopy of thousands of cells analyzed by a custom image processing algorithm. Results reveal that SBI-756 induces formation of processing bodies, increasing their number 2.5-fold and confirming the assumption that it disrupts the eIF4E-eIF4G interaction directly. Furthermore, we show that while SBI-756 treatment does not prevent the formation of stress granules entirely- their number increases by 13% after heat (stress) and drug combined treatment; it rather inhibits the recruitment of eIF4E into them, 4-fold decrease in co-localizations with eIF4G after the same treatment, despite eIF4E’s importance in their formation.

This study of cell behavior, comparing protein localization in normal conditions versus drug treatment in combination with stress environment, reveals that the drug changes the homeostasis and suggests an explanation to the altered cytoplasmic localization changes of eIF4F complex partners.