Synergistic Action of Anti-Microtubule and Anti-Tropomyosin Agents on Neuroblastoma

We have developed a new therapeutic cancer strategy based on targeting a core component of the cancer cell actin cytoskeleton, tropomyosin Tpm3.1. The first-in-class series of anti-tropomyosin (ATM) compounds prevent the cancer-associated tropomyosin Tpm3.1 from stabilising actin filaments, leading to the collapse of the actin cytoskeleton in cancer cells. We have recently demonstrated that ATM agents strongly synergise with anti-microtubule (anti-MT) chemotherapeutics that are among the most widely used front-line treatment strategies for multiple cancer types. Such synergy has been observed in childhood solid tumours (e.g. neuroblastoma) and adult cancers (e.g. cervical, prostate and lung cancer) both in vitro and in vivo. In this thesis, the mechanism was examined by which the Tpm3.1 inhibitor TR100 synergises with the anti-MT drug vincristine (VCR) in HeLa cells, a model more suitable for imaging analysis. Analysis of the degree of synergy between TR100 and VCR in HeLa cells revealed strong to very strong synergistic combinational effects on inhibiting cell proliferation. Interestingly, TR100 alone exhibited no impact on HeLa cell cycle progression, however it significantly promoted the VCR-induced cell cycle arrest of mitosis, particularly at prometaphase and metaphase. By confocal microscopy imaging, observations were made that this drug combination disrupted spindle assembly and promoted multipolar spindle formation. Given the role of actin in positioning the spindle, a hypothesis was made that Tpm3.1 plays a role in the orientation of the mitotic spindle possibly via interacting with astral MTs or MT-associated proteins at the cell cortex. To test this hypothesis, live cell imaging showed that Tpm3.1 preferentially localises at the cell cortex during mitosis. Further imaging revealed that the drug combination induced astral MT defects extending to the cell cortex, implying that Tpm3.1 is needed for normal mitotic spindle functioning. In summary, these results highlight a novel connection between Tpm3.1-containing actin filaments and microtubules, and provide a clearer understanding of how targeting these structures may lead to improved cancer therapy