Effects of Eribulin on Microtubule Binding and Dynamic Instability Are

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The Effects of Eribulin on Microtubule Binding and Dynamic Instability are Strengthened in the Absence of the #III Tubulin Isotype Leslie Wilson, Manu Lopus, Herbert P Miller, Olga Azarenko, Stephen Riffle, Jennifer Anne Smith, and Mary Ann Jordan Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.5b00745 • Publication Date (Web): 03 Oct 2015 Downloaded from http://pubs.acs.org on October 9, 2015

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The Effects of Eribulin on Microtubule Binding and Dynamic Instability are Strengthened in the Absence of the βIII Tubulin Isotype

Leslie Wilson*, 1, Manu Lopus2, Herbert P. Miller1, Olga Azarenko1, Stephen Riffle1, Jennifer A. Smith1, and Mary Ann Jordan1

1

Neuroscience Research Institute, University of California, Santa Barbara, Santa

Barbara, CA 93106

2

University of Mumbai, Vidyanagari Campus, Mumbai, India 400098

* Leslie Wilson (805) 893-2819 [email protected]

ACS Paragon Plus Environment

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Abbreviations MAPs, microtubule-associated proteins; PEM50 buffer, 50 mM Pipes, 1 mM EGTA, 1 mM MgSO4, 0.1 mM GTP, pH 6.85; PMME buffer, 87 mM Pipes, 36 mM MES, 1 mM EGTA 2 mM MgCl2, 2 mM GTP, pH 6.8.


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ABSTRACT

Eribulin mesylate (Halaven) is a microtubule-targeted anticancer drug used to treat patients with metastatic breast cancer who have previously received a taxane and an anthracycline. It binds at the plus-ends of microtubules and has been shown to suppress plus-end growth selectively. Because the Class III β tubulin isotype is associated with resistance to microtubule targeting drugs, we sought to determine how βIII tubulin might mechanistically influence the effects of eribulin on microtubules. We found that while [3H]-eribulin bound to bovine brain soluble tubulin depleted of βIII tubulin similarly to unfractionated tubulin, it bound to plus ends of microtubules that were depleted of βIII-depleted tubulin with higher maximum stoichiometry (20 ± 3 molecules per microtubule) than unfractionated microtubules (9 ± 2 molecules per microtubule). In addition, eribulin suppressed the dynamic instability behavior of βIIIdepleted microtubules more strongly and in a different way than microtubules containing βIII tubulin. Specifically, with βIII tubulin present in the microtubules, 100 nM eribulin suppressed the growth rate by 32% and marginally reduced the catastrophe frequency (by 17%), but did not modulate the rescue frequency. However, in the absence of βIII tubulin, eribulin not only reduced the growth rate, but also strongly reduced the shortening rate (by 43%) and the catastrophe and the rescue frequencies (by 49% and 32%, respectively). Thus, when present in microtubules, βIII tubulin substantially weakens the effects of eribulin.


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Eribulin mesylate (Halaven, Eisai Inc.) is a structurally simplified synthetic derivative of halichondrin B, a marine polyether macrolide present in several marine sponges including Halichondria okadai 1. It is a microtubule-targeting drug 2-5 used to treat patients with metastatic breast cancer who have previously received a taxane and an anthracycline 6, 7. Eribulin binds both to soluble tubulin and to tubulin in microtubules 3, 5

. At relatively high concentrations it has been shown to inhibit the polymerization of

bovine brain tubulin into microtubules and at its lowest effective concentrations to selectively inhibit steady-state plus end microtubule growth without affecting polymer mass 5. Tubulin, the building block of microtubules, is a heterodimeric protein consisting of one α tubulin and one β tubulin subunit 8. In microtubules, β tubulin is exposed to solvent at the plus ends, while α tubulin is exposed at the minus ends. Several α and β tubulin isotypes are expressed in most cells and organisms 9. Seven different β tubulin isotypes are expressed in mammals, referred to as βI, βII, βIII, βIVa, βIVb, βV, and βIV 9-11

. The amino acid sequences of the isotypes are strongly conserved, differing

predominantly in their C-termini. Class III β tubulin, a product of the TUBB3 gene, here called βIII tubulin, is the most divergent of the major β tubulin isotypes. For example, the βI, βII, and βIV isotypes are approximately 98% homologous to each other, while βIII tubulin is only 92% homologous with the others 9, 12. The βIII tubulin isotype, the best studied of the β tubulin isotypes, is normally expressed in neurons and in testicular Sertoli cells along with the βI, βII and βIV isotypes. Purified bovine brain tubulin, the tubulin used in this work, has been shown to contain ~25% βIII tubulin, along with 58% βII tubulin, 13% βIV tubulin, 3% βI tubulin, and a small but uncharacterized amount of βV tubulin 10. In addition, βIII tubulin expression, amounting to as high as ~6-7% of the


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total β tubulin protein pool, is also found in a number of tumor cells 13, many of which have been shown to be resistant to taxanes 14, 15. Strong evidence indicates that eribulin binds near the vinca binding domain of β tubulin 5 which is exposed at microtubule plus ends. Molecular modeling of eribulin binding to tubulin suggests that its binding is mediated by interactions with the olefin and methyl groups of eribulin 5. Studies on the mechanism of action of eribulin with purified bovine brain tubulin and bovine brain microtubules (containing ~25% βIII tubulin) have shown that eribulin binds with a 1:1 stoichiometry to tubulin dimers when they are in solution 4. Once assembled into microtubules, eribulin does not bind efficiently to tubulin along the length of the microtubule nor does it appear to bind at the minus ends 4. Rather, eribulin binds with relatively high affinity to a maximum of approximately 15 tubulin molecules exposed uniquely at the plus ends. Binding of eribulin at plus ends of microtubules made from unfractionated bovine brain tubulin primarily reduces the rate and extent of microtubule growth without significantly affecting other dynamic parameters 4. Analysis of eribulin binding in relation to its effects on microtubule growth indicates that a single eribulin molecule bound at a microtubule plus end is sufficient to prevent growth. The presence of βIII tubulin in tumor cells is often correlated with increased tumor aggressiveness (reviewed in 15). It is also associated with resistance of tumor cells to a number of microtubule-targeting drugs, including the vinca alkaloids, which bind near the eribulin binding site 5, and paclitaxel. Thus we wanted to determine whether presence of the βIII tubulin isotype affected the action of eribulin. We removed all of the βIII tubulin from unfractionated bovine brain tubulin (here called βIII-depleted


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tubulin) using an immunoaffinity column in which bovine brain tubulin containing βIII tubulin (from here on called unfractionated tubulin) was passed through a column with covalently bound βIII tubulin monoclonal antibodies. We found that [3H]-eribulin bound similarly to unfractionated tubulin and tubulin depleted of βIII tubulin, while [3H]-eribulin bound to βIII tubulin-depleted microtubules with ~2-fold higher stoichiometry than microtubules made from unfractionated tubulin. Most interestingly, eribulin suppressed the dynamic instability behavior of βIII-tubulin-depleted microtubules more strongly and qualitatively differently than with microtubules assembled from βIII-tubulin-containing unfractionated tubulin, indicating that the presence of βIII tubulin in microtubules diminishes the responses of the microtubules to eribulin. These results indicate that the presence of βIII tubulin in microtubules may modify the microtubule lattice in a way that functionally influences dynamic behavior at their plus ends.

MATERIALS AND METHODS Purification of Unfractionated Bovine Brain Tubulin and βIII-Depleted Tubulin. Bovine brain tubulin, used throughout this work, was purified by two cycles of temperature-dependent assembly and disassembly followed by phosphocellulose chromatography as previously described 16. This tubulin, here called unfractionated tubulin, contained 3% βI tubulin, 58% βII tubulin, 25% βIII tubulin, and 13% βIV tubulin 10

. The βIII tubulin isotype was completely removed from the unfractionated tubulin by

immunoaffinity chromatography as follows. βIII tubulin monoclonal IgG antibodies were obtained from TUJ1 hybridomas expressing the anti βIII tubulin antibody (a kind gift from Drs. Anthony Frankfurter and Anthony J. Spano, University of Virginia). Cells were


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grown for 10 to 14 days in Iscove’s modified Dulbecco's medium (Sigma-Aldrich, St. Louis, MO) supplemented with 10% fetal bovine serum (hybridoma quality, Atlanta Biologicals, Atlanta GA). Antibodies were harvested by centrifugation (Sorvall RC5B centrifuge, 10,000 rpm, 20 min, 25 °C), and purified using Pierce Chromatography Protein G cartridges and an FPLC system (Bio Rad, Hercules, CA, BioLogic Workstation), according to the manufacturer's protocol. To remove βIII tubulin, approximately 40 mg of anti-βIII tubulin antibody was coupled to 12 ml of cyanogen bromide (CNBr)-activated Sepharose 4B Fast Flow resin (GE Healthcare, Pittsburgh, PA) in Tris buffer, pH 8.3, containing 50 mM NaCl. After coupling, unoccupied sites on the column were blocked with blocking buffer (Tris buffer, 1 mol/L, pH 8.5). The resin was then poured into a 1.5 x 15 cm column and equilibrated with PEM50 buffer (50 mM Pipes, 1 mM EGTA, 1 mM MgSO4, 0.1 mM GTP, pH 6.85). Unfractionated tubulin (8-12 mg) was added to the top of the column bed and the flow-through was collected in 1 mL fractions. Peak tubulin-containing fractions were pooled and concentrated using Amicon centrifugal concentrators (Millipore, Billerica, MA). The βIII tubulin was completely removed from the unfractionated tubulin as confirmed by Western Blotting using Covance TuJ1 mouse IgG antibodies as shown in Figure 1. Protein concentrations were determined by the method of Bradford 17 using bovine serum albumin as the standard. Binding of Eribulin to Soluble Tubulin in the Absence and Presence of βIII Tubulin. Tubulin (unfractionated or depleted of βIII tubulin, 2 µM) was incubated with different concentrations of [3H]-eribulin (0.5 µM to 80 µM) for 30 min at 30°C (300 µL). Tubulin-bound [3H]-eribulin was separated from free [3H]-eribulin by passing 200 uL of


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each reaction mixture through a 2 mL Zeba Spin desalting column (40,000 molecular weight cut off, Fisher Scientific, Pittsburg, PA). Binding of Eribulin to Steady-State Microtubules Composed of Unfractionated or

βIII-Depleted Tubulin. Thirty µM unfractionated or β-depleted tubulin in PEM50 buffer (total volume 300 µL) was polymerized to steady state in the presence of 3 µM recombinant 4-repeat tau (called 4R tau, a gift from Dr. Nikki LaPointe, UC Santa Barbara) by incubating the tau-tubulin mixture for 30 min at 35 ºC. The tau, present in a 1:10 molar ratio of tau to tubulin, was added to stabilize the microtubules. The microtubules were then sheared six times by passage through a 25 gauge needle. Sheared microtubules were incubated for another 10 min to reestablish steady state. Different concentrations of [3H]-eribulin (between 0.1 µM and14 µM; specific activity 960 mCi/mmol) were incubated with the microtubule suspensions for 2 min (total sample volume, 300 µL) and 225 µL volumes were centrifuged at 200,000 ×g for 2 h at 32 ºC in an SW 50.1 rotor to sediment the microtubules (Beckman Optima L-90K ultracentrifuge, Beckman Coulter, Brea, CA). Short incubation times were necessary to minimize microtubule depolymerization that occurs at the higher eribulin concentrations. By light scattering at 350 nm, no aggregation was detected in the presence or absence of βIII tubulin. Microtubule pellets were washed and dissolved in water (225 µL) by incubating on ice overnight and the protein content determined. The quantity of [3H]-eribulin bound to the microtubules was determined in a Beckman LS 6500 liquid scintillation spectrometer (Beckman Coulter). For obtaining microtubule length distributions, identical samples incubated with unlabeled eribulin were fixed with 0.2% glutaraldehyde, stained with 1.5% uranyl acetate, and imaged at 2500 x magnification


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on a JEOL 1230 transmission electron microscope operating at 80 kV. The lengths of the microtubules were measured using a Zeiss MOPIII digitizer and the mean lengths determined. A minimum of 300 microtubules were measured per condition. The stoichiometries of microtubule-bound [3H]-eribulin were calculated from the mean lengths of the microtubules, the concentration of tubulin in sedimented microtubules, and the specific activity of the eribulin as previously described 4. The apparent equilibrium dissociation constants for the binding of eribulin to the microtubules were obtained from double-reciprocal plots of the binding data. Effects of Eribulin on Microtubule Dynamic Instability In Vitro. The dynamic instability parameters at plus ends of steady-state microtubules were determined by video microscopy as described in detail elsewhere 18. Briefly, purified unfractionated or βIII-depleted tubulin (1.6 mg of tubulin/ml) was assembled onto sea urchin flagellar axoneme seeds at 32 ºC for 30 min to allow the microtubules to achieve polymer mass steady state in the absence or presence of 100 nM or 300 nM eribulin in PMME buffer (87 mM Pipes, 36 mM MES, 1 mM EGTA 2 mM MgCl2, 2 mM GTP, pH 6.8 14. The seed concentration was optimized at 3-6 seeds per microscope field. Plus ends were distinguished from minus ends based upon their relatively longer lengths, the greater numbers of microtubules at these ends, and their more rapid dynamics. The selection of 100 nM eribulin was based upon our previous study which showed that this eribulin concentration selectively suppressed plus end growth of microtubules made from unfractionated tubulin but did not appreciably suppress other parameters 4. In addition, a higher eribulin concentration, 300 nM, was also used in this work to ensure that the lack of eribulin effects on shortening of microtubules made from unfractionated tubulin


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was not due to the use of an eribulin concentration that was too low to act on the shortening rate. Dynamic instability parameters were obtained by video-enhanced differential interference contrast microscopy using an Olympus IX71 inverted microscope with a 100x (na,1.4) oil immersion objective as previously described 4, 18, 19. The dynamic instability parameters analyzed included the rate of growth per growing event, the rate of shortening per shortening event, the transition frequency from the growth or attenuated (paused) state to shortening, called the catastrophe frequency, the switching frequency from shortening to the growth or attenuated state (called rescue), and the overall dynamicity of the microtubules, which was the total visually measurable growth and shortening of the microtubules per unit time. Between 25 and 30 microtubules were analyzed per condition from three independent experiments.

RESULTS Eribulin Binds Similarly to Unfractionated and βIII-Depleted Soluble Tubulin. [3H]eribulin was incubated with βIII-depleted or unfractionated soluble tubulin and unbound [3H]-eribulin was separated from tubulin-bound [3H]-eribulin by passing the mixtures through 2 ml Zeba spin columns (Materials and Methods). [3H]-eribulin bound to unfractionated and βIII-depleted soluble tubulin in a concentration-dependent manner, with maximum stoichiometries of 1.0 ± 0.03 (SEM) mol of eribulin per mol unfractionated tubulin (mean of 5 experiments) and 1.0 ± 0.02 (SEM) (3 experiments) per mol of βIII-depleted tubulin (Figure 2). The affinity (Kd) of [3H]-eribulin for unfractionated tubulin was 1.2 ± 0.12 µM (SEM), while the mean Kd of [3H]-eribulin for βIII-depleted tubulin was 0.85 µM ± 0.02 (SEM). While the mean binding affinities were


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slightly different, the differences were not statistically significant as determined by a Student’s t-test. Because depletion of βIII-tubulin did not significantly change the affinity or stoichiometry of eribulin binding to tubulin, we conclude that [3H]-eribulin binds to soluble βIII-tubulin with a stoichiometry of one mol eribulin per mol of tubulin and an affinity not very different than the remaining β tubulin isotypes. Depletion of βIII Tubulin Enhances Eribulin Binding to Microtubules. We analyzed the binding of [3H]-eribulin to steady-state microtubules assembled from either unfractionated bovine brain tubulin or βIII depleted tubulin. The concentration range used, between 0.1 µM and 14 µM eribulin, was the same as that used previously to analyze eribulin binding to microtubules made from unfractionated tubulin,4 but this time we used a 1:10 molar ratio of recombinant 4R tau to tubulin in the microtubules to stabilize the microtubules rather than a mixture of bovine brain microtubule-associated proteins (MAPs) (Materials and Methods). Similar to previous results with MAPcontaining unfractionated microtubules, small numbers of eribulin molecules were bound per microtubule, which we previously concluded were not bound along the microtubule length but were bound only at plus ends. If eribulin could bind to tubulin along the length of the microtubule, we would expect approximately 1800 molecules of bound eribulin per µm of microtubule length. Because eribulin does not affect growth or shortening dynamics at minus ends, we assume that it binds only at plus ends. Specifically with tau-stabilized microtubules, [3H]-eribulin bound to unfractionated microtubules in a concentration-dependent manner (Figure 3) with a maximum stoichiometry at high eribulin concentrations of 9 ± 2 molecules eribulin per microtubule (closed squares). In contrast, eribulin bound to βIII-tubulin depleted microtubules at


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more sites, with a maximum stoichiometry of 20 ± 3 molecules eribulin per microtubule. The mean Kd for eribulin binding to the plus ends of microtubules made from unfractionated tubulin, estimated from the log concentration dependence of the binding and from a double-reciprocal plot of the binding data (not shown), was ~ 4.3 ± 1.7 µM. We could not determine an accurate binding constant for the binding of eribulin to microtubules made from βIII–depleted tubulin (a double-reciprocal plot of the data was not interpretable as a single affinity class of sites). One possible reason is that the larger number of eribulin binding sites are due to a slight loosening of the lattice at the microtubule plus ends in the absence of βIII tubulin, which may expose additional eribulin binding sites having somewhat different affinities (see Discussion). Depletion of βIII Tubulin Greatly Increases the Suppressive Effects of Eribulin on Microtubule Dynamic Instability. Microtubule ends display relatively slow growth and relatively rapid shortening events, with switching between growth and shortening, a GTP-dependent behavior known as dynamic instability. They also spend an appreciable fraction of time neither growing nor shortening detectably, called an attenuated or paused state 18. Similar to other results with purified microtubules and in living cells, removal of βIII tubulin did not detectably change the dynamic instability parameters of the microtubules at their plus ends (Table 1). However, depletion of βIII tubulin significantly changed the effects of eribulin on dynamic instability. As shown in Table 1, the principal effect of 100 or 300 nM eribulin on dynamic instability at plus ends of unfractionated microtubules was to suppress the growth rate. Specifically, at a concentration of 100 nM, the only statistically significant effect of eribulin was to suppress the growth rate (by 32%). Overall dynamicity was reduced by 37%. Increasing


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the eribulin concentration to 300 nM further increased the effects of eribulin on the growth rate and also significantly reduced the catastrophe frequency (by 30%); no other parameters were affected significantly. Removal of βIII tubulin quantitatively and qualitatively changed the effects of eribulin on plus end dynamic instability. Specifically, 100 nM eribulin suppressed the growth rate by 38%, which was somewhat stronger than with microtubules containing βIII tubulin. However, it strongly reduced the shortening rate by 43%, a value that was unique to βIII-depleted tubulin and statistically highly significant. Also in the absence of βIII tubulin, eribulin caused a 38% reduction in the time microtubules spent growing and a 25% reduction in the time microtubules spent shortening. Eribulin also exerted two additional novel actions after removal of βIII tubulin, significantly reducing both the catastrophe and the rescue frequencies (by 49% and 32%, respectively), leading to an overall suppression of dynamicity by 58%. The results indicate that eribulin suppresses the dynamic instability of βIII tubulin-depleted microtubules very differently than with βIII tubulin-containing microtubules, indicating that the presence of the βIII tubulin isotype in the microtubules diminishes the suppressive effects of eribulin on dynamic instability.

DISCUSSION We studied the interactions of eribulin with purified unfractionated bovine bovine brain tubulin and with microtubules made from this tubulin, containing ~ 58% βII tubulin, 25% βIII tubulin, 13% βIV tubulin and 3% βI tubulin 10 in comparison with tubulin and microtubules depleted of βIII tubulin (here called βIII-depleted microtubules). We estimate that the βIII-depleted tubulin contains ~ 78% βII tubulin, 18% βIV tubulin and


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4% βI tubulin. There also may be a small quantity of βV tubulin present (R.F. Luduena, personal communication). Depletion of βIII tubulin was accomplished by passage of unfractionated tubulin through an immunoaffinity CNBr column linked to a βIII tubulin monoclonal antibody (Materials and Methods). No βIII tubulin was detected in the βIII tubulin-depleted tubulin preparation as determined by western blotting (Figure 1). [3H]-Eribulin Binding to Tubulin and to Microtubules Made from Unfractionated and βIII Tubulin Depleted Microtubules. We previously reported that [3H]-eribulin bound to bovine brain tubulin with maximum stoichiometry of 1.3 ± 0.4 eribulin molecules per tubulin heterodimer, which we interpreted as a single site. The curve was complex. The overall Kd was 46 µM, somewhat weaker than found here (see below), and we also detected an apparent very high affinity region (Kd = 0.4 µM) for a subset of ~25% of the tubulin 4. The column used in that work to separate bound and free eribulin was a 0.5 ml Zeba spin column with 100 µL of sample applied 4. In the present work, we improved the separation by use of a 2 ml Zeba spin column and increasing the sample volume to 200 µL, which separated bound and free eribulin more cleanly than the 0.5 ml column used previously. Here, using [3H]-eribulin, we similarly found that eribulin bound to unfractionated soluble tubulin at a single site (1.0 ± 0.03 (SEM) mol eribulin per mol tubulin), and with a stronger affinity than found previously (Kd) of 1.17 ± 0.12 µM (SEM). We did not detect any very high affinity binding at the low eribulin concentrations as we did previously. We believe that these values more accurately reflect eribulin binding to unfractionated tubulin. We found that [3H]-eribulin also bound to tubulin devoid of detectable βIII tubulin to a single site (1.0 ± 0.02 (SEM) with a slightly stronger but


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similar mean affinity of 0.85 ± 0.02 µM (SEM). The difference in the affinities for the two tubulin preparations was not statistically significant (Student’s t-test). Because the affinities and maximum stoichiometries of eribulin binding to unfractionated tubulin and tubulin depleted of βIII tubulin were similar, we can conclude that when the tubulin is free in solution, eribulin binds to βIII tubulin similarly to its binding to the other tubulin isotypes. In the previous work 4, the microtubules used were MAP-rich, isolated by cycles of assembly and disassembly from bovine brain 16. Thus they contained a mixture of MAPs amounting to approximately 25-30% of the microtubule protein preparation (called MAP-rich microtubules). Eribulin bound to these microtubules with a maximum stoichiometry of 15 molecules of eribulin per microtubule and an affinity (Kd) of ~ 3.5 µM. Thus eribulin bound to a very small number of binding sites per microtubule that we concluded were located at the plus ends 4. In the present work, we simplified the MAP content of the microtubules by stabilizing them with a 1:10 molar ratio of recombinant 4repeat tau to tubulin (Materials and Methods). Thus the microtubules contained a low molar ratio to tubulin of a single stabilizing MAP, tau, rather than a large amount of a mixture of brain MAPs. As shown in Figure 3, [3H]-eribulin bound to the microtubules in a concentration-dependent manner with a Kd of 4.3 ± 1.7 µM (SD) and a maximum stoichiometry of 9 ± 2 (SEM) molecules eribulin per microtubule. Eribulin bound to taustabilized microtubules in similar fashion to MAP-rich microtubules. These results were essentially identical to those found previously, and indicate that the binding of eribulin to its small number of tubulin binding sites at microtubule plus ends is not modified by the MAP content of the microtubules.


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However, eribulin bound to microtubules depleted of βIII tubulin more extensively than to microtubules containing βIII tubulin. Specifically, the maximum number of eribulin binding sites doubled from 9 ± 2 per microtubule in microtubules made from unfractionated microtubules, to 20 ± 3 molecules eribulin per microtubule for microtubules devoid of βIII tubulin. The affinity of eribulin for the microtubules devoid of βIII tubulin was difficult to determine and may represent a mixture of several somewhat different affinities for eribulin binding to its available sites at microtubule plus ends. One possibility is that when βIII tubulin is not present in the microtubule lattice, the binding of eribulin at the plus ends induces a more open lattice structure exposing additional binding sites. When βIII tubulin is present in the lattice, the lattice is stabilized so as to expose fewer eribulin binding sites than in its absence (see model in Figure 4). Absence of βIII Tubulin Altered the Response of Microtubule Dynamics to Eribulin. Microtubule dynamic instability is defined by several parameters at the microtubule ends including the growth rate, the shortening rate, the catastrophe and rescue frequencies, and the overall dynamicity 18, 19. Previously when microtubules were assembled from purified βIII tubulin, their dynamics at plus ends were found to be approximately 2-fold faster than with microtubules assembled from purified βII tubulin 20. In the present work, the dynamics of microtubules containing 25% βIII tubulin and microtubules devoid of βIII tubulin were indistinguishable (Table I). These results indicate that when incorporated into microtubules containing a mixture of other β tubulin isotypes, the presence of the βIII tubulin does not affect the intrinsic dynamic instability properties of microtubules made from the other β tubulin isotypes. Similar results have been found in tumor cells in which the βIII tubulin has been over expressed 21.


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However, eribulin greatly modified the dynamic instability properties of microtubules that were devoid of βIII tubulin. As described previously, the main effect of eribulin on steady state bovine brain microtubules, which contain 25% βIII tubulin, is suppression of growth events 4. In this study as well with βIII tubulin present, eribulin primarily suppressed microtubule growth events and had little if any effect on the rate or extent of shortening or any of the other measured parameters, including the rescue and catastrophe frequencies, even at a very high eribulin concentration (300 nM) (Table 1). However, in the absence of βIII tubulin, 100 nM eribulin not only suppressed the growth rate, but also strongly suppressed the rate and extent of shortening and the catastrophe and rescue frequencies (Table 1). As shown previously, inhibition of growth by eribulin does not affect the off-rate constant for tubulin dissociation at microtubule plus ends, as demonstrated by dilution-induced disassembly of the microtubules 3. In contrast, a drug such as vinblastine, which does inhibit the rate and extent of shortening 22 does reduce the off rate constant for tubulin 3, indicating that such drugs reduce the off rate by strengthening longitudinal or lateral tubulin-tubulin associations in the microtubule lattice at plus ends. These results indicate that when incorporated into the microtubule lattice at plus ends, βIII tubulin prevents such eribulin-induced strengthening but in the absence of βIII tubulin, such strengthening is permitted. We do not understand how the presence of βIII tubulin creates this change. It seems reasonable to think that eribulin binds to all of the β tubulin isotypes present at microtubule plus ends including βIII tubulin because the maximum number of molecules of eribulin that bind to unfractionated microtubules is close to the number of protofilaments exposed at plus ends (Figure 3). Thus the binding of eribulin to plus ends


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when βIII tubulin is present in the microtubule lattice is doing something unique. One possibility is that the binding of eribulin to βIII tubulin prevents a conformational change from occurring that in the absence of βIII tubulin results in an increase in the affinity of tubulin with neighboring tubulin dimers, perhaps longitudinal tubulin associations. Because eribulin suppresses the rate and extent of plus-end shortening in the absence of βIII tubulin, eribulin must be able to induce such changes in the absence of βIII tubulin. How this might occur mechanistically remains a mystery. These results with eribulin indicate that βIII tubulin may be playing a unique role in regulating microtubule dynamics, which occurs in our system only when eribulin binds to the microtubule ends. In the absence of βIII tubulin, the actions of eribulin on plus end dynamics are significantly different than in its presence, becoming similar to the effects of vinblastine which, like eribulin, binds in the vinca binding domain 5, and strongly suppresses the rate and extent of plus end shortening 22. Eribulin is not the only molecule that regulates βIII tubulin-containing microtubules differently than microtubules devoid of βIII tubulin. We have very recently reported that the microtubule-targeting drug ixabepilone, an epothilone B derivative that in contrast with eribulin stabilizes microtubules by binding in the vicinity of the taxane binding site, also displays increased potency in the absence of βIII tubulin 23. Perhaps the role of βIII tubulin when present at the plus ends is to serve as a binding sensor for other small molecules or protein regulators as well, that modulate tubulin-tubulin interactions at plus ends. Is the High Concentration of βIII tubulin in Neuronal Microtubules Responsible for the Low Peripheral Neuropathy Caused by Eribulin? One of the major, dose-limiting


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Biochemistry

side effects of microtubule-targeting cancer chemotherapeutic drugs is peripheral neuropathy. At clinically relevant doses, eribulin causes less peripheral neuropathy than other microtubule targeting drugs 24, 25. For example, in a phase II clinical trial conducted by California Cancer Consortium, of 66 patients treated with eribulin mesylate, only two developed Grade III peripheral neuropathy 26. In another phase II study, of the 103 metastatic breast cancer patients treated with eribulin, only 9.8% (five individuals) developed infrequent Grade III neuropathy and none developed Grade IV neuropathy 24. The incidence of peripheral neuropathy induced clinically with other microtubule targeted drugs such as paclitaxel and ixabepilone appears to be considerably higher than with eribulin. Interestingly, eribulin also causes low neurotoxicity in animals 25. For example, compared to paclitaxel or ixabepilone, eribulin does not cause deleterious effects on nerve conduction parameters such as conduction velocity, caudal amplitude, and digital nerve amplitudes in mouse sciatic nerves 25. Perhaps the high concentration of βIII tubulin in neurons may play an important role in the low neurotoxicity of eribulin. The causes of chemotherapy-induced peripheral neuropathy are poorly understood. Among the possible causes, one may involve impaired trafficking of organelles and other important molecules between nerve cell bodies and nerve endings, a process which is highly dependent upon properly functioning microtubules. As discussed previously, the presence of βIII tubulin in purified microtubules substantially diminishes the effects of eribulin on dynamic instability. Such reduced effects of eribulin on microtubule-dependent processes specifically in neurons could be an important factor in eribulin’s reduced peripheral neuropathy. A substantial fraction of brain tissue


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is comprised of glial cells, which do not contain βIII tubulin (R.F. Luduena, personal communication), so the content of βIII tubulin in neurons is likely to be even higher than the ~25% found in the purified brain tubulin used in this work. If a major aspect of chemotherapy-induced peripheral neuropathy is indeed due to the effects of microtubule-targeting drugs on brain neuronal processes, we suggest the possibility that the high content of βIII tubulin in neurons may be responsible for the diminished deleterious effects of eribulin. Might βIII Tubulin Expression in Some Tumor Cells Diminish the Anticancer Effects of Eribulin? As previously discussed, βIII tubulin is not expressed in most cells and tissues. It is primarily found in neuronal cells, in testicular Sertoli cells, and in some cancer cells. The levels present in cancer cells are far lower than the levels expressed in neurons, amounting to no more than 6-7% of the total β tubulin 13. There has been considerable interest as well as disagreement about the possible role of βIII tubulin in resistance to microtubule targeting drugs (reviewed in ref. 15). For example, expression of βIII tubulin in non-small cell lung cancer cells has been considered responsible for resistance to taxanes 14. In this work, we have found that the high levels (~25%) of βIII tubulin in reconstituted bovine brain microtubules diminishes the effects of eribulin on the dynamics of microtubules. We do not yet know if low levels of βIII tubulin in microtubules can reduce the effects of eribulin the way that high levels of βIII tubulin do. If so, it may be anticipated that expression of βIII tubulin in tumors could cause a weakened drug response and resistance to eribulin in tumor cells expressing it.


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ACKNOWLEDGMENTS We thank Dr. Richard Luduena, University of Texas Health Science Center, San Antonio, TX, for valuable discussions about βIII tubulin isotypes.

AUTHOR INFORMATION Corresponding Author E-mail: [email protected]. Telephone: 805-893-2819 Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding This research was supported by Eisai, Inc, Andover, MA. Notes The authors declare no competing financial Interest


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REFERENCES [1] Hirata, Y., and Uemura, D. (1986) Halichondrins: antitumor polyether macrolides from a marine sponge, Pure Appl. Chem. 58, 701-710. [2] Towle, M. J., Salvato, K. A., Budrow, J., Wels, B. F., Kuznetsov, G., Aalfs, K. K., Welsh, S., Zheng, W. J., Seletsky, B. M., Palme, M. H., Habgood, G. J., Singer, L. A., DiPietro, L. V., Wang, Y., Chen, J. J., Quincy, D. A., Davis, A., Yoshimatsu, K., Kishi, Y., Yu, M. J., and Littlefield, B. A. (2001) In vitro and in vivo anticancer activities of synthetic macrocyclic ketone analogues of halichondrin B, Cancer Res. 61, 1013-1021. [3] Jordan, M. A., Kamath, K., Manna, T., Okouneva, T., Miller, H., Davis, C., Littlefield, B., and L., W. (2005) The primary antimitotic mechanism of action of the synthetic halichondrin E7389 is suppression of microtubule growth., Mol Cancer Ther. 4, 1086-1095. [4] Smith, J. A., Wilson, L., Azarenko, O., Zhu, X. J., Lewis, B. M., Littlefield, B. A., and Jordan, M. A. (2010) Eribulin Binds at Microtubule Ends to a Single Site on Tubulin To Suppress Dynamic Instability, Biochemistry 49, 1331-1337. [5] Bai, R. L., Nguyen, T. L., Burnett, J. C., Atasoylu, O., Munro, M. H. G., Pettit, G. R., Smith, A. B., Gussio, R., and Hamel, E. (2011) Interactions of Halichondrin B and Eribulin with Tubulin, J. Chem. Inf. Model. 51, 1393-1404. [6] Jain, S., and Cigler, T. (2012) Eribulin mesylate in the treatment of metastatic breast cancer, Biologics. 6, 21-29.


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[7] Doherty, M. K., and Morris, P. G. (2015) Eribulin for the treatment of metastatic breast cancer: an update on its safety and efficacy, Int J Womens Health. 7, 4758. [8] Luduena, R. F., Shooter, E. M., and Wilson, L. (1977) Structure of the tubulin dimer, J Biol Chem 252, 7006-7014. [9] Luduena, R. F. (1993) Are tubulin isotypes functionally significant, Mol Biol Cell 4, 445-457. [10] Banerjee, A., Roach, M. C., Wall, K. A., Lopata, M. A., Cleveland, D. W., and Luduena, R. F. (1988) A monoclonal antibody against the type II isotype of betatubulin. Preparation of isotypically altered tubulin, J Biol Chem. 263, 3029-3034. [11] Guo, J., Walss-Bass, C., and Luduena, R. F. (2010) The beta isotypes of tubulin in neuronal differentiation, Cytoskeleton (Hoboken) 67, 431-441. [12] Mariani, M., Karki, R., Spennato, M., Pandya, D., He, S., Andreoli, M., Fiedler, P., and Ferlini, C. (2015) Class III beta-tubulin in normal and cancer tissues, Gene 563, 109-114. [13] Hiser, L., Aggarwal, A., Young, R., Frankfurter, A., Spano, A., Correia, J. J., and Lobert, S. (2006) Comparison of beta-tubulin mRNA and protein levels in 12 human cancer cell lines, Cell. Motil. Cytoskeleton 63, 41-52. [14] Seve, P., Mackey, J., Isaac, S., Tredan, O., Souquet, P. J., Perol, M., Lai, R., Voloch, A., and Dumontet, C. (2005) Class III beta-tubulin expression in tumor cells predicts response and outcome in patients with non-small cell lung cancer receiving paclitaxel, Mol Cancer Ther. 4, 2001-2007.


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[15] Kavallaris, M. (2010) Microtubules and resistance to tubulin-binding agents, Nat. Rev Cancer 10, 194-204. [16] Miller, H. P., and Wilson, L. (2010) Preparation of Microtubule Protein and Purified Tubulin from Bovine Brain by Cycles of Assembly and Disassembly and Phosphocellulose Chromatography, Method Cell Biol 95, 3-15. [17] Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal Biochem 72, 248-254. [18] Yenjerla, M., Lopus, M., and Wilson, L. (2010) Analysis of Dynamic Instability of Steady-State Microtubules In Vitro by Video-Enhanced Differential Interference Contrast Microscopy with an Appendix by Emin Oroudjev, Method Cell Biol 95, 189-206. [19] Lopus, M., Oroudjev, E., Wilson, L., Wilhelm, S., Widdison, W., Chari, R., and Jordan, M. A. (2010) Maytansine and cellular metabolites of antibodymaytansinoid conjugates strongly suppress microtubule dynamics by binding to microtubules, Mol Cancer Ther. 9, 2689-2699. [20] Panda, D., Miller, H. P., Banerjee, A., Luduena, R. F., and Wilson, L. (1994) Microtubule Dynamics in-Vitro Are Regulated by the Tubulin Isotype Composition, Proc Natl Acad Sci U.S.A. 91, 11358-11362. [21] Kamath, K., Wilson, L., Cabral, F., and Jordan, M. A. (2005) BetaIII-tubulin induces paclitaxel resistance in association with reduced effects on microtubule dynamic instability, J. Biol Chem. 280, 12902-12907.


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[22] Toso, R. J., Jordan, M. A., Farrell, K. W., Matsumoto, B., and Wilson, L. (1993) Kinetic stabilization of microtubule dynamic instability in vitro by vinblastine, Biochemistry 32, 1285-1293. [23] Lopus, M., Smiyun, G., Miller, H., Oroudjev, E., Wilson, L., and Jordan, M. A. (2015) Mechanism of action of ixabepilone and its interactions with the betaIIItubulin isotype, Cancer Chemother Pharmacol. DOI 10.1007/s00280-015-2863-z. [24] Vahdat, L. T., Pruitt, B., Fabian, C. J., Rivera, R. R., Smith, D. A., Tan-Chiu, E., Wright, J., Tan, A. R., Dacosta, N. A., Chuang, E., Smith, J., O'Shaughnessy, J., Shuster, D. E., Meneses, N. L., Chandrawansa, K., Fang, F., Cole, P. E., Ashworth, S., and Blum, J. L. (2009) Phase II study of eribulin mesylate, a halichondrin B analog, in patients with metastatic breast cancer previously treated with an anthracycline and a taxane, J Clin Oncol 27, 2954-2961. [25] Wozniak, K. M., Nomoto, K., Lapidus, R. G., Wu, Y., Carozzi, V., Cavaletti, G., Hayakawa, K., Hosokawa, S., Towle, M. J., Littlefield, B. A., and Slusher, B. S. (2011) Comparison of neuropathy-inducing effects of eribulin mesylate, paclitaxel, and ixabepilone in mice, Cancer Res. 71, 3952-3962. [26] Gitlitz, B. J., Tsao-Wei, D. D., Groshen, S., Davies, A., Koczywas, M., Belani, C. P., Argiris, A., Ramalingam, S., Vokes, E. E., Edelman, M., Hoffman, P., Ballas, M. S., Liu, S. V., and Gandara, D. R. (2012) A phase II study of halichondrin B analog eribulin mesylate (E7389) in patients with advanced non-small cell lung cancer previously treated with a taxane: a California cancer consortium trial, J Thorac Oncol. 7, 574-578.


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Table 1. Effects of eribulin on microtubule plus end dynamic instability in the presence and absence of βIII tubulin. 1 2 3Dynamic 4 5instability 6 7parametersa 8 9 10 11Growth rate 12 (µm/min) 13 14Shortening 15 rate 16 (µm/min) 17 18 Percent time 19 growing 20 21 22Percent time 23 shortening 24 25Percent time 26 attenuated 27 28 29Catastrophe 30 frequency 31 (per min) 32 33 34 Rescue 35 frequency 36 (per min) 37 38 39Dynamicity 40 (µm/min) 41 42 43 44 45 46 47 48

Unfractionated microtubules

Control

βIII-depleted microtubules

Eribulin 100 nM

% Change

Eribulin 300 nM

% Change

Control

Eribulin 300 nM

% Change

1.5 ± 0.1**c

-32

1.3 ± 0.1**

-40

2.1 ± 0.1

1.3 ± 0.1**

-38

14.2 ± 1.2

12.2 ± 1.2

-14

13.8 ± 1

-3

13.4 ± 1

7.7 ± 1***

-43

54

55

+2

50

-7

56

35

-38

14

10

-29

8

-43

16

12

-25

32

35

+9

42

+31

28

53

+89

0.30 ± 0.04

0.25 ± 0.04

-17

0.21 ± 0.04*

-28

0.35 ± 0.1

0.2 ± 0.03***

-49

1.3 ± 0.2

1.3 ± 0.3

+2

1.2 ± 0.3

-1

1.3 ± 0.2

0.9 ± 0.2**

-32

2.5

1.6

-37

1.7

-34

2.5

1.1

-58

2.2 ± 0.2b


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a

25-30 microtubules were measured for each condition

b

Data = mean ± SEM

c

*, **, and *** represent significance of differences at

Effects of Eribulin on Microtubule Binding and Dynamic Instability Are

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