The overall goal of the experimental work undertaken in this thesis is to better understand the cellular and molecular mechanisms responsible for the onset, progression, and recovery from peripheral neuropathy, a painful and dose-limiting side effect of microtubule targeting agent (MTA) chemotherapies. The frequency of severe peripheral neuropathy varies among the different MTAs, and since the microtubule binding interactions and mechanisms of action also vary, we hypothesized that these distinct mechanisms may underlie the variability in pathophysiology of chemotherapy induced peripheral neuropathy (CIPN).
This work began with a correlative investigation of the morphological and biochemical effects that occur in peripheral nerves upon in-vivo treatment with MTAs in a maximum tolerated dose mouse model. We analyzed sciatic nerves from mice treated with the chemotherapy drugs paclitaxel (frequent severe neuropathy) or eribulin (infrequent severe neuropathy) and found that morphologically, paclitaxel increased the frequency of observed signs of axon degeneration more significantly than did eribulin. Alternatively, eribulin but not paclitaxel induced occasional myelin "halo" structures. Biochemically, paclitaxel, and eribulin both induced α-tubulin expression (~1.9- and ~2.5-fold, respectively) and α-tubulin acetylation, a marker for microtubule stability, (~5- and ~11.7-fold, respectively). Eribulin but not paclitaxel-induced microtuble end-binding protein 1 (EB1) expression ~2.2-fold while paclitaxel but not eribulin mildly suppressed end-binding protein 3 (EB3) expression. Both EB proteins are associated with microtubule growth. Eribulin's combination of relatively mild deleterious morphological effects coupled with more potent biochemical changes promoting microtubule stability and growth in mice correlate with lower frequencies of severe neuropathy in humans. We hypothesized that these eribulin-induced effects created a relatively stable microtubule network that compensated, in part, for the toxic anti-cancer effects of the drug, leading to fewer reported incidences of peripheral neuropathy than for paclitaxel.
We then extended these comparative studies to acquire a more detailed understanding of the resolution from these initial effects over the course of 6 months after the completion of either paclitaxel, eribulin, or ixabepilone dosing. In paclitaxel-treated mice, axon area density was significantly decreased through 3 months of recovery. In contrast, axon area density in eribulin-treated mice recovered fully from initial deficits by the 2 week time point, with ixabepilone showing no change at any time point. Evidence of myelin abnormalities, likely secondary to axonopathy, was prominent at 2 weeks and 3 months and was consistently most frequent in paclitaxel-treated animals. Also, only paclitaxel-treated mice displayed a significant and persistent increase in the number of non-neuronal nuclei at the 2 week, 3 month and 6 month recovery time points, although ixabepilone-treated mice showed a similar trend at 2 weeks. These additional nuclei were positive for known Schwann cell markers S100B and GFAP, indicating that they are likely Schwann cells, the resident glia of the sciatic nerve. Biochemically, we found that two weeks into the recovery phase, α-tubulin acetylation in eribulin-treated mice returned to control levels while it was greatly reduced but still significantly higher than vehicle treated mice in paclitaxel-treated mice. In contrast, axonal levels of both α-tubulin and end-binding protein 1 (EB1) rapidly returned to control values at 14 day from initially induced levels at the end of the MTD treatment in both paclitaxel and eribulin treated mice. In summary, we found that (i) morphologically, sciatic nerve axons recovered more rapidly from eribulin and ixabepilone-induced morphological and biochemical effects than did paclitaxel-treated mice, and (ii) biochemically, drug-induced increases in protein expression levels following paclitaxel and eribulin treatment are relatively transient. Taken together, our data in mice indicate a milder onset and faster recovery with eribulin and ixabepilone treatment than for paclitaxel.
Finally, we made progress developing an in-vitro cell culture model for studying the cellular and molecular mechanisms that regulate the celllular response to microtubule inhibition. We found that all MTAs reduced neurite area of neuronal cells at concentrations that coincided with a possible shift in microtubule mass and a change in cytosolic α-tubulin acetylation. When we compared the IC50 concentrations for neurite area and inhibition of cancer cell proliferation, we found that eribulin was far less potent to neurons than it was to cancer cells, indicating that neurons are more tolerant of eribulin’s action as compared to ixabpilone, paclitaxel, and vincristine.
