Targeting the actin cytoskeleton in HER2+ metastatic cancer cells using a macrolide toxin

Breast and ovarian cancers are among the leading causes of cancer related deaths in women worldwide. In a subset of these cancers, dysregulation of the human epidermal growth factor receptor 2 (HER2) leads to overexpression of the receptor on the cell surface. Previous studies have found that these HER2+ cancers show high rates of progression to metastatic disease. Metastasis is driven by cytoskeletal rearrangements that produce filamentous actin (F-actin) based structures that penetrate and degrade extracellular matrix to facilitate tumour invasion. Advancements in targeted therapy have made F-actin an attractive target for the development of new cancer therapies. In this thesis, we tested the actin-depolymerizing macrolide toxin, Mycalolide B (MycB), as a potential warhead for a novel antibody drug conjugate (ADC) to target highly metastatic HER2+ breast and ovarian cancers. We found that MycB treatment of HER2+ breast (SKBR3, MDA-MB-453) and ovarian (SKOV3) cancer cells led to loss of viability (IC50 values ≤ 64 nM). Sub-lethal doses of MycB treatment caused potent suppression of leading edge protrusions, migration and invasion potential of HER2+ cancer cells (IC50 ≤ 32 nM). In contrast, other F-actin based processes such as receptor endocytosis were less sensitive to MycB treatment. MycB treatment skewed the size of endocytic vesicles, which may reflect defects in F-actin based vesicle motility or maturation. Given that HER2+ cancers have been effectively targeted by Trastuzumab and Trastuzumab-based ADCs, we tested the effects of a combination of Trastuzumab and MycB on cell migration and invasion. We found that MycB/ Trastuzumab combination treatments inhibited motility of SKOV3 cells to a greater degree than either treatment alone. Altogether, our results provide proof-of-principle that actin toxins such as MycB can be used as a novel class of warheads for ADCs to target and combat highly metastatic cancers.

Structural and functional studies of bacterial protein tyrosine kinases

While protein tyrosine kinases (PTKs) have been extensively characterized in eukaryotes, far less is known about their emerging counterparts in prokaryotes. Studies of close to 20 homologs of bacterial protein tyrosine (BY) kinases have inaugurated a blooming new field of research, all since just the end of the last decade. These kinases are key regulators in the polymerization and exportation of the virulence-determining polysaccharides which shield the bacterial from the non-specific defenses of the host. This research is aimed at furthering our understanding of the BY kinases through the use of X-ray crystallography and various in vitro and in vivo experiments. We reported the first crystal structure of a bacterial PTK, the C-terminal kinase domain of E. coli tyrosine kinase (Etk) at 2.5Å resolution. The fold of the Etk kinase domain differs markedly from that of eukaryotic PTKs. Based on the observed structure and supporting evidences, we proposed a unique activation mechanism for BY kinases in Gram-negative bacteria. The phosphorylation of tyrosine residue Y574 at the active site and the specific interaction of P-Y574 with a previously unidentified key arginine residue, R614, unblock the Etk active site and activate the kinase. Both in vitro kinase activity and in vivo antibiotics resistance studies utilizing structure-guided mutants further support the novel activation mechanism. In addition, the level of phosphorylation of their C-terminal Tyr cluster is known to regulate the translocation of extracellular polysaccharides. Our studies have significantly clarified our understanding of how the phosphorylation status on the C-terminal tyrosine cluster of BY kinases affects the oligomerization state of the protein, which is likely the machinery of polysaccharide export regulation. In summary, this research makes a substantial contribution to the rapidly progressing research of bacterial tyrosine kinases.