We are interested in how drugs that target microtubules exert their therapeutic and toxic actions in man, and in the potential for new medical uses of these drugs. Drugs that stabilize (taxanes, epothilones) and destabilize (vinca alkaloids, eribulin) microtubules are important components of combination chemotherapy for many cancers. Colchicine, a destabilizing drug, has been used to treat gout since ancient times. Recently, microtubule stabilizing epothilones have been proposed for treatment of neurodegenerative disease and spinal cord damage. Figure 1 illustrates how representative anti-microtubule drugs affect microtubule polymerization.
Figure 1. Drugs that stabilize and destabilize microtubules provide useful medicines. Two classic examples are illustrated. Colchicine and paclitaxel are both plant-derived natural products.
In tissue culture, anti-microtubule drugs mainly kill cancer cells by perturbing the mitotic spindle, leading to mitotic arrest and apoptosis at high concentration, and chromosome miss-segregation at low concentration. However, the rate of cell proliferation is much slower in human tumors than in cell culture or mouse tumor models, and it is currently unclear to what extent these drugs exert their therapeutic effect through anti-mitotic actions(Komlodi-Pasztor et al., 2011)(Mitchison, 2012). We hypothesize they also act by triggering changes in cell signaling in quiescent cells, and these are sufficient to kill sensitive cancer cells without the need for passage through mitosis. To test this hypothesis we measured the cell cycle state of cancer cells responding to microtubule-targeting drugs in a mouse tumor model by intravital imaging in collaboration with Ralph Weissleder (Chittajallu et al., 2015). We are currently developing 3D cell culture models where cancer and normal breast epithelial cells leave the cell cycle and become quiescent, so we can test the effect of anti-microtubule drugs in the absence of cell division. We are also profiling the effects of anti-microtubule drugs on cell signaling pathways using proteomics and transcriptomics. Understanding how taxanes act to selectively kill solid tumor cells will lead to methods for matching individual cancer patients to effective treatments (“precision medicine”), and perhaps to better future drugs. Figure 2 is a snapshot from the method we developed to score the cell cycle phenotype of drug-treated cells in a mouse tumor model.
Figure 2. Cell cycle profiles and tumor cell killing following treatment of mouse model tumors with the kinesin-5 inhibitor ispinesib or the anti-microtubule drug eribulin. From (Chittajallu et al., 2015).
Colchicine is an ancient drug that is still widely used to treat gout and certain other inflammatory diseases, including Familial Mediterranean Fever (FMF) and recurrent pericarditis. At a biochemical level it binds to tubulin dimers and inhibits their polymerization into microtubules – indeed tubulin was discovered in part as the binding partner of colchicine by Ed Taylor’s group (Weisenberg et al., 1968). In man, it reduces adhesive activity of neutrophils (Fig 3) and inhibits accumulation at sites of inflammation(Fordham et al., 1981)(Malawista, 1968). How the biochemical action of colchicine leads to it’s therapeutic action is unknown. The textbook hypothesis is that it accumulates in neutrophils and directly inhibits their chemotactic activity. We question this model, because of the lack of anti-mitotic side effects of colchicine at therapeutic doses. We are exploring alternative models, which is taking us deep into in vivo pharmacology. Solving the mechanism of colchicine action might reveal new pathways for regulating inflammation in man, and to new treatments for inflammatory disease.
Figure 3. Colchicine treatment in man inhibits the adhesive activity of neutrophils tested ex vivo. From (Fordham et al., 1981)
Neurodegenerative disease and spinal cord damage
We currently lack treatments for neurodegenerative diseases such as Alzheimer’s and motor neuron disease (ALS). A common feature of these diseases, and to some extent also of normal neuronal aging, is a reduction in microtubule density in axons. Axonal microtubules provide the tracks for axonal transport, and probably also play important roles in signaling pathways. Stabilizing axonal microtubules with drugs might provide symptomatic relief in neurodegenerative disease, and perhaps even inhibit disease progression. Consistent with this hypothesis, treatment of mice genetically engineered to model neurodegenerative diseases with microtubule-stabilizing drugs that enter the CNS caused improvement in neuropathology and behavioral symptoms (Brunden et al., 2014). Stabilizing microtubules was also shown to promote repair of spinal cord injury in animal models (Ruschel et al., 2015)(Fig 4). This, stabilizing microtubules in the brain and spinal cord might have multiple therapeutic benefits. A major challenge for translating this hypothesis to man is the potent anti-mitotic toxicity of current microtubule stabilizing drugs that penetrate the CNS, such as epothilones. We will pursue several strategies to try and overcome this problem, including development of novel chemical scaffolds that stabilize microtubules and exhibit improved ratios of neuro-protective to antimitotic activity in the hope of better treatments for these devastating diseases.
Figure 4. Epothilone treatment induces microtubule polymerization and neurite outgrowth in a cell culture model of spinal cord damage. From (Ruschel et al., 2015)
Brunden, K.R., Trojanowski, J.Q., Smith, A.B., Lee, V.M.-Y., and Ballatore, C. (2014). Microtubule-stabilizing agents as potential therapeutics for neurodegenerative disease. Bioorg. Med. Chem. 22, 5040–5049.
Chittajallu, D.R., Florian, S., Kohler, R.H., Iwamoto, Y., Orth, J.D., Weissleder, R., Danuser, G., and Mitchison, T.J. (2015). In vivo cell-cycle profiling in xenograft tumors by quantitative intravital microscopy. Nat. Methods 12, 577–585.
Fordham, J.N., Kirwan, J., Cason, J., and Currey, H.L. (1981). Prolonged reduction in polymorphonuclear adhesion following oral colchicine. Ann. Rheum. Dis. 40, 605–608.
Komlodi-Pasztor, E., Sackett, D., Wilkerson, J., and Fojo, T. (2011). Mitosis is not a key target of microtubule agents in patient tumors. Nat Rev Clin Oncol 8, 244–250.
Malawista, S.E. (1968). Colchicine: a common mechanism for its anti-inflammatory and anti-mitotic effects. Arthritis Rheum. 11, 191–197.
Mitchison, T.J. (2012). The proliferation rate paradox in antimitotic chemotherapy. Mol. Biol. Cell 23, 1–6.
Ruschel, J., Hellal, F., Flynn, K.C., Dupraz, S., Elliott, D.A., Tedeschi, A., Bates, M., Sliwinski, C., Brook, G., Dobrindt, K., et al. (2015). Axonal regeneration. Systemic administration of epothilone B promotes axon regeneration after spinal cord injury. Science 348, 347–352.
Weisenberg, R.C., Borisy, G.G., and Taylor, E.W. (1968). The colchicine-binding protein of mammalian brain and its relation to microtubules. Biochemistry 7, 4466–4479.