Vanderbilt Institute of Chemical Biology

 

 

Discovery at the VICB

 

 

 

 

 

 

New Target for Antimitotic Antitumor Agents

 

By: Carol A. Rouzer, VICB Communications
Published:  April 27, 2016

 

 

Kinesin-12 (Kif15) plays a key role in development of resistance to antimitotic kinesin-5 (Eg5) inhibitors, suggesting Kif15 as a target to block this resistance.

 

Kinesin-5 (Eg5) plays a key role in the correct formation and orientation of the mitotic spindle in mammalian cells. Hence, the discovery of Eg5 inhibitors (K5Is) sparked considerable interest as they were viewed as potential antimitotic cancer chemotherapeutic agents. Translation of K5Is to the clinic has been disappointing, however, due at least in part to the development of drug resistance. Now, Vanderbilt Institute of Chemical Biology member Puck Ohi and his laboratory demonstrate that K5I resistance is highly dependent on the function of another protein, kinesin-12 (Kif15). Their work reveals important new information concerning spindle formation and points to a possible target to overcome K5I resistance in cancer patients [E. G. Sturgill, et al., J. Cell Biol., Published online April 18, DOI: 10.1083/jcb.201507036].

 

The process by which chromosomes are distributed to each incipient daughter cell during mitosis is executed by the spindle, a complex array of microtubules (MTs) that emanates from two centrosomes at opposite poles of the dividing cell. Centrally aligned along the equator of the spindle, the chromosomes are attached to MT fibers at their kinetochores. It is the shortening of these kinetochore MTs that pulls one member of each chromosome pair to the opposite pole, guaranteeing an even distribution of the chromosomes to the daughter cells. Due to the importance of the spindle in this process, cells contain many proteins that function to ensure the correct bipolar orientation of the centrosomes and their associated MTs. In mammalian cells, a key player is Eg5, one of the kinesin class of molecular motors. These proteins use the energy of ATP to move along a track - an MT track in the case of Eg5. Eg5 is a homotetramer formed by two homodimers arranged in opposite orientation to each other, placing two motor heads at each end of the molecule (Figure 1, top). This structure enables Eg5 to bind to two adjacent antiparallel MTs and then move those two MTs in opposite directions relative to one another (Figure 1, bottom), a function that is critical to the process of pushing the centrosomes to the opposite poles of the cell during spindle formation.

 

 



FIGURE 1
. (Top) Diagrammatic representation of the structure of kinesin-5 (Eg5) and kinesin-12 (Kif15). The proteins are multimers of subunits comprising an N-terminal motor domain (light green) joined by a coiled coil (dark green) to a specialized C-terminal domain (blue, kinesin-5 only). Kinesin-5 is a tetramer formed by two dimers aligned in opposition to each other. Kinesin-12 is a dimer. Figure reproduced by permission from Macmillan Publishers, Ltd. from K. J. Verhey & J. W. Hammond, (2001) Nat. Rev. Mol. Cell Biol., 10, 765. Copyright 2009. (Bottom) Representation of an Eg5 tetramer spanning two antiparallel microtubules. The arrangement enables Eg5 to move the two microtubules in opposite directions relative to each other. Figure reproduced under the Creative Commons Attribution License from M. E. Tanenbaum, et al., (2013) eLife, 2:e00943. Copyright 2013, M. E. Tanenbaum, et al.


 

To better understand the mechanisms by which cancer cells acquire K5I resistance, the Ohi lab exposed HeLa cells to the K5I S-trityl-L-cysteine (STLC) over an extended period of time and then isolated the colonies of surviving cells. This process yielded three distinct K5I-resistant clones (KIRCs), designated KIRC-1, -2, and -3. Prior work had shown that the K5I-resistance of KIRC-1 cells depended on overexpression of kinesin-12 (Kif15, Figure 1A); however, this was not found in KIRC-2 or -3. Rather, RNA-seq analysis revealed that these cell lines both expressed a mutant form of Eg5 along with the wild-type protein. The specific mutation, G268V, altered a highly conserved residue in the motor domain of the protein. Similar mutations in other kinesins produce proteins that bind to MTs with very high affinity, leading the Ohi lab investigators to hypothesize that this might be the case with their G268V Eg5 mutant. Their observation that Eg5 was associated with spindle microtubules in KIRC-2 and -3 cells, even in the continual presence of STLC (Figure 2), was consistent with this possibility.

 

FIGURE 2. Fluorescence microscopy reveals the presence of Eg5 associated with spindle MTs in parental HeLa cells, and in KIRC-2 and KIRC-3 cells, but not KIRC-1 cells. Antibody against Eg5 is shown in grayscale (top) and in red (bottom). Antibodies against tubulin and DNA are shown in blue and green, respectively. Figure reproduced under an Attribution-Noncommercial-Share Alike-No Mirror Sites License from E. G. Sturgill, et al., J. Cell Biol., Published online April 18, DOI: 10.1083/jcb.201507036. Copyright 2016 E. G. Sturgill, et al.

 

 

To further test their hypothesis, the investigators compared the ability of the wild-type and G268V mutant Eg5 proteins to promote the movement of MTs in an in vitro gliding assay. As predicted, the wild-type protein functioned normally in this assay, while the mutant protein was incapable of powering MT motion. Further studies demonstrated that the G268V mutant bound more tightly to MTs than the wild-type protein, and that addition of ATP did not lead to MT release by the mutant as it did with the wild-type Eg5. These findings supported the conclusion that G268V is a “rigor” mutant of Eg5, binding to MTs so tightly that ATP-driven motion cannot occur.

 

KIRC-2 and KIRC-3 cells expressed both wild-type and G268V Eg5, leading the investigators to propose that tetramers formed in these cells likely contained at least one copy of the mutant. Tetramers containing one mutant Eg5 protein at each end could theoretically bind opposing MTs, forming a stable cross-link. Consistent with this hypothesis, the investigators showed that, although both the wild-type and G268V mutant Eg5 could bundle MTs under physiological conditions in vitro, only the mutant protein could maintain bundling under conditions of high ionic strength. To further test this possibility in intact cells, the investigators expressed both wild-type and G268V Eg5 proteins labeled with enhanced green fluorescent protein (EGFP) in parental HeLa cells. They found bundled MTs in Eg5-G268V-EGFP-expressing interphase cells, but not in cells expressing Eg5-WT-EGFP. In mitotic KIRC-2 and KIRC-3 cells, mitotic spindles were warped as compared to those of parental HeLa cells (Figure 3, left); however, it was difficult to see MT bundling in the complex spindle array. To solve this problem, the investigators used RNAi to knock down expression of the Nuf2 protein, an important component of the kinetochore. Cells lacking Nuf2 did not form kinetochore-associated MTs, resulting in a failure of the chromosomes to align along the equator of the spindle. However, this simplification of the spindle structure enabled the investigators to observe MT bundling in the remaining non-kinetochore MTs in KIRC-2 and -3 cells (Figure 3, right).

 

 

FIGURE 3. Fluorescence microscopy reveals microtubule bundling in KIRC-2 and KIRC-3 cells. (Left) A representative parental HeLa, KIRC-2, and KIRC-3 cell is shown. Tubulin staining is shown in grayscale (top) and green (bottom). Antibodies directed toward centromeres (CREST) and DNA are shown in red and blue respectively. In these control cells, it is difficult to appreciate the subtle differences in the microtubules. (Right) Experimental conditions are the same as on the left, but the cells were treated with RNAi to knockdown expression of Nuf2, a centromere component. This eliminates centromere-associated MTs, making it easier to see the bundling of the remaining MTs in the KIRC-2 and KIRC-3 cells. Figure reproduced under an Attribution-Noncommercial-Share Alike-No Mirror Sites License from E. G. Sturgill, et al., J. Cell Biol., Published online April 18, DOI: 10.1083/jcb.201507036. Copyright 2016 E. G. Sturgill, et al.

 

 

The results demonstrated that, despite the G268V mutation, or likely because of it, Eg5 remained strongly associated with spindle MTs in KIRC-2 and -3 cells. To determine if Eg5 played a role in spindle formation in these cells, the researchers used RNAi to knockdown Eg5 expression. They found that Eg5 knockdown profoundly increased the mitotic index (MI) and the monopolar index (MPI) in parental HeLa cells as well as in both KIRC-2 and -3 cells. In contrast, the RNAi treatment had no effects on KIRC-1 cells. These findings indicated that Eg5 is required for spindle formation and particularly spindle bipolarity in parental, KIRC-2, and KIRC-3 cells.

 

Prior studies had shown that KIRC-1 cells rely on overexpressed Kif15 rather than Eg5 for spindle assembly, consistent with the failure of Eg5 RNAi treatment to affect their spindle formation. Kif15 is notable for its preferential binding to MT bundles as opposed to individual MTs. The Ohi lab researchers postulated that bundling of MTs by the C268V Eg5 mutant might facilitate the ability of Kif15 to mediate spindle assembly in KIRC-2 and -3 cells, even though they did not overexpress this protein. Consistent with this hypothesis, although Kif15 was readily detected in the spindle microtubules of all of the cell lines studied, it was present in the non-kinetochore MTs of only KIRC-2 and KIRC-3 cells (Figure 4). Furthermore, differential contrast imaging of mitotic KIRC-2 and -3 cells revealed evidence of “reverse jackknifing,” a process by which Kif15 mediates spindle assembly by transitioning to a bipolar state via a monopolar geometry.

 

 

FIGURE 4. Fluorescence microscopy reveals the association of Kif15 with microtubules. Antibodies against Kif15 are shown in grayscale or in red. Antibodies against tubulin and DNA are shown in green and blue, respectively. In the control cells (top two rows) MT-associated Kif15 can be seen in all cell lines. However, in cells treated with RNAi against Nuf2, association of Kif15 is only seen in KIRC-2 and KIRC-3 cells. Figure reproduced under an Attribution-Noncommercial-Share Alike-No Mirror Sites License from E. G. Sturgill, et al., J. Cell Biol., Published online April 18, DOI: 10.1083/jcb.201507036. Copyright 2016 E. G. Sturgill, et al.

 

 

To further explore the importance of Kif15 in spindle formation in KIRC-2 and -3 cells, the researchers used RNAi to knock down expression of this protein. This treatment had little effect on parental HeLa cells but markedly increased both MI and MPI in KIRC-1, -2, and -3 cells. Knockdown of Kif15 expression was also highly toxic to all three K5I-resistant cell lines.

 

These results suggested that Kif15 may play an important role in all forms of K5I resistance. To test this hypothesis, the researchers used the CRISPR-Cas9 gene editing system to create a KIF15 knockout HeLa cell line. They then treated these cells for extended periods with three different K5Is. For all three inhibitors, K5I-resistant colonies emerged in the case of parental HeLa cells, but no KIF15 knockout cells survived any of the treatments. In contrast, restoration of KIF15 expression to the knockout cells enabled them to regain the ability to develop K5I resistance.

 

These results demonstrate that factors that promote bundling of MTs can result in Kif15-mediated spindle assembly (Figure 5). Consequently, Kif15 can fulfill the role of Eg5 under conditions in which it is overexpressed or other factors enable MT bundling. The findings also show that, at least in HeLa cells, Kif15 is absolutely required for development of K5I resistance. Thus, Kif15 becomes a key target in combatting this form of drug resistance in cancer patients. If potent and selective Kif15 inhibitors can be discovered, their use in conjunction with K5Is may prove to be a highly effective combination for the treatment of some forms of cancer.

 

 

FIGURE 5. Proposed model for the cooperation of an Eg5 rigor mutant (orange) and Kif15 (blue) in mitotic spindle formation. Normally, Kif15 only associates with bundled MTs. Overexpression of Kif15 or the presence of an Eg5 rigor mutant can lead to MT bundling (shown by thick green lines). This enables Kif15 to associate with the MTs with sufficient efficiency that it can effect bipolar spindle organization. Figure reproduced under an Attribution-Noncommercial-Share Alike-No Mirror Sites License from E. G. Sturgill, et al., J. Cell Biol., Published online April 18, DOI: 10.1083/jcb.201507036. Copyright 2016 E. G. Sturgill, et al.

 

 

View J. Cell Biol. article: Kinesin-5 inhibitor resistance is driven by kinesin-12

 

 

 

 

TwitterYouTube

 

 

The Vanderbilt Institute of Chemical Biology, 896 Preston Building, Nashville, TN 37232-6304, phone 866.303 VICB (8422), fax 615 936 3884
Vanderbilt University is committed to principles of equal opportunity and affirmative action. Copyright © 2014 by Vanderbilt University Medical Center