Intrinsic and Extrinsic Factors Affecting Microtubule Dynamics in Normal and Cancer Cells

Microtubules (MTs), highly dynamic structures composed of α- and β-tubulin heterodimers, are involved in cell movement and intracellular traffic and are essential for cell division. Within the cell, MTs are not uniform as they can be composed of different tubulin isotypes that are post-translationally modified and interact with different microtubule-associated proteins (MAPs). These diverse intrinsic factors influence the dynamics of MTs. Extrinsic factors such as microtubule-targeting agents (MTAs) can also affect MT dynamics. MTAs can be divided into two main categories: microtubule-stabilizing agents (MSAs) and microtubule-destabilizing agents (MDAs). Thus, the MT skeleton is an important target for anticancer therapy. This review discusses factors that determine the microtubule dynamics in normal and cancer cells and describes microtubule–MTA interactions, highlighting the importance of tubulin isoform diversity and post-translational modifications in MTA responses and the consequences of such a phenomenon, including drug resistance development.

Among the investigated β-tubulin isotypes (βII, βIII, and βIV), βIII is the most divergent, with substitutions in globular protein body including: (i) helix H3 (serine in positions 124 and 126 is substituted by cysteine and asparagine, respectively), (ii) loops H1-S2 and H2-S3 (structures involved in the formation of H3 surface), (iii) structures located proximal to the ML surface (H6-H7 loop threonine 217 and S7-H9 loop serine 275 substituted to alanine) [68], and (iv) T7 loop (minus end surface) near colchicine-binding site (cysteine 239 substituted by serine). Moreover, the C-terminal tail of βIII is very divergent. It contains positively charged lysine (Table 1) and a phosphorylatable serine [69,70]. These variations are potentially important for the overall tubulin structure; in fact, an atomic model of βIII-containing MTs shows slight but significant displacement of the H1-S2 loop and part of the ML surface-forming structures with respect to βII-containing MTs [68]. However, how βIII substitution relates to its specific properties is mainly unknown.
For more than two decades, it has been known that MTs assembled from βIII tubulin are more dynamic than MTs containing βII or βIV [9]. More recent data indicate that the main difference is caused by the significantly increased catastrophe rate (depolymerization) of βIII-containing MTs [27,71,72], while the growth rate seems to be similar [27,71] or only slightly lower [72]. The dynamic features of MTs assembled with specific isotypes were retained when the C-terminal tails of βII and βIII were interchanged, indicating that the dynamic properties of these two tubulins are "encoded" within the main globular protein body [71]. Remarkably, not only dynamics, but also resistance to depolymerizing factors and structural features vary between βIIand βIII-containing MTs, with the former showing lower resistance to depolymerizing agents and more protofilaments in the MT wall (14) than the latter (βIII contains 13 protofilaments and shows more resistance to depolymerizing factors) [68]. Interestingly, when two populations of tubulin heterodimers are mixed with different stoichiometry, assembled MTs show intermediate dynamics [68,71,72].
On the other hand, at low tubulin concentration, the MTs nucleate much more slowly if the tubulin heterodimers contain βIII-tubulin compared to βII or βIV. Interestingly, this difference can be abolished by the proteolytic removal of C-terminal tail [8]. This result indicates that although it is not crucial for dynamic tubulin properties, the tubulin C-terminus can influence other features that could be important for MT cytoskeleton formation within the cell.
To summarize, growing evidence indicates that the intrinsic properties of tubulin isotypes, the expression of specific isotypes, and their ratio within the cell are significant factors influencing MT dynamics.

Post-Translational Modifications of Microtubules
Tubulin modification sites, modifying enzymes, and functions of post-translational modifications (PTMs), including the impact on MT dynamics, have recently been broadly reviewed [12,33,73,74]. Thus, here, we will only briefly summarize how PTMs affect MT dynamics.
Both αand β-tubulin undergo a number of post-translational modifications that change the properties of the free tubulin heterodimers and microtubules. Tubulin PTMs can modulate MT dynamics directly or indirectly by influencing the interactions between MTs and microtubule-interacting proteins (which can stabilize, destabilize, or cut microtubules).
While for the vast majority of modifying enzymes, a tubulin heterodimer already incorporated into the microtubule lattice is a preferred substrate, in some cases, free tubulin heterodimers are effectively modified. The latter can affect the binding of tubulin heterodimers to the microtubule plus end and, thus, affect microtubule growth and stability. For instance, acetylation of lysine 252 of β-tubulin by San acetyltransferase slows down the rate of tubulin incorporation into the microtubule and consequently reduces the rate of MT assembly [75]. Phosphorylation of serine 172 of β-tubulin by minikinase/DYRK1a (neurons) or cyclin-dependent kinase Cdk1 (mammalian mitotic cells) inhibits the incorporation of tubulin heterodimers [76,77], while heterodimers containing αIc-tubulin phosphorylated at serine 165 assemble more effectively than unmodified ones [78,79]. Serine and threonine residues of αand β-tubulin can also be modified by O-Glc-NAcylation [80,81] and, in vitro, such modified tubulins are not incorporated into microtubules [81].
The presence of differentially modified MTs within the cell is crucial in the assembly, disassembly, and rearrangement of the microtubular cytoskeleton [82]. Newly polymerized dynamic microtubules are highly tyrosinated (besided αIVa, tyrosine is encoded in the C-terminus of α-tubulins, see Table 1). With time, the tyrosine is removed by the vasohibin family, VASH1 and VASH2 tubulin detyrosinases, generating so-called detyrosinated tubulin. Detyrosinated tubulin is found in spindle and is essential for correct chromosome congression [83]. Glutamic acid residue, which is the most frequent penultimate residue in α-tubulin, can also be removed by cytosolic carboxypeptidases, irreversibly forming so-called ∆2-tubulin [84][85][86][87]. After depolymerization, free detyrosinated α-tubulin can be re-tyrosinated by tubulin tyrosine ligase (TTL) [88,89].
Polyamination of αand β-tubulin by a transglutaminase causes the formation of hyperstable, cold-resistant microtubules. This modification is important for neuronal development and axon maturation [99]. The positions of the main polyamination sites near the GTP pocket (glutamine 15 in β-tubulin) and α-tubulin minus end suggest that tubulin polyamination could affect GTP binding or hydrolysis and microtubule lattice stabilization [99].
Glycylation and glutamylation of αand β-tubulin can occur as mono-or polymodification, and glycyl or glutamyl residues are ligated to the glutamic acid residues within the C-terminal tail [73,92]. These tubulin modifications are catalyzed by enzymes related to TTL, called tubulin tyrosine ligase-like (TTLL) [73]. The reverse reaction (deglutamylation) is carried out by cytoplasmic carboxypeptidases (CCPs). To date, the identity of tubulin deglycylase remains unknown [92].

Microtubule-Associated Proteins and Microtubule Dynamics
Microtubule-associated proteins (MAPs) are another intrinsic factor affecting microtubule dynamics (reviewed in [100][101][102]). Generally, MAPs function as MT stabilizers or destabilizers; however, stabilization/destabilization of MTs can be achieved by affecting one of several processes, including MT nucleation [1] and stabilization/destabilization of the MT ends [2] or of the MT lattice [102,103]. For this reason, MAPs are divided into several functional categories: (i) microtubule nucleators, (ii) MT end-binding proteins, (iii) lattice-binding proteins also known as structural MAPs, (iv) enzymes severing or depolymerizing microtubules, and (v) motor MAPs (kinesin, dynein) that generate forces and use microtubules as tracks for intracellular transport [102]. Intriguingly, some MAPs can participate in several MT dynamics-related processes. For example, XMAP215 can be classified as both an MT nucleator and a plus end-binding protein [1,2] while kinesin-13 family proteins are motor proteins that bind to microtubule plus ends and have MT-depolymerizing properties [104,105]. Because MAPs form a large class of proteins and a number of high-quality reviews on this topic are already available, we will only provide a short overview of these proteins, highlighting the relationship between MAPs and MT dynamics.
Proteins that bind to MT ends are specific to either the plus (so-called +TIPs [107]) or minus end (so-called −TIPs [108]). Plus TIPs belong to approximately 20 different families of proteins [107]. EB (end-binding) proteins form a core of +TIP network and the majority of studies suggest that they stabilize or protect the MT plus end [107]. EB proteins interact with both the MT plus end and other +TIP proteins that can be either stabilizers (as CLIP-170 (cytoplasmic linker protein 170)/CLIP1 and CLASP proteins), destabilizers (as kinesin-13 family proteins), or polymerases (as XMAP215 protein). Within the cell, the interplay between these proteins results in MT growth or shrinkage (reviewed in [2,23,107]).
Stathmin-1, also known as Op18 (oncoprotein 18), binds to the MT plus end but is not included in the +TIP protein class. It was first discovered as an oncoprotein highly expressed in some types of leukemia, breast, and ovarian cancers [109]. Stathmin-1 causes a decrease of the MT polymer mass by two mechanisms: (i) by binding two tubulin dimers in a curved conformation and inhibition of their incorporation into microtubule and (ii) by interfering with the lateral bonding between tubulin subunits, leading to destabilization of the microtubule tip and MT shrinkage [110,111].
A recently described class of −TIP, includes CAMSAP proteins and the KANSL complex [108]. In mammals, CAMSAP proteins protect the MT minus end against the depolymerizing activity of kinesin-13. Additionally, CAMSAP2 and 3 proteins decrease the rate of tubulin incorporation at the minus end, decreasing its dynamics [112]. The functions of KANSL complex are still unknown [108].
The lattice-binding MAPs include classical MAPs, MAP1, MAP2, MAP4, MAP6, MAP7, and Tau, which promote polymerization, stabilization, and bundling of microtubules (reviewed in [102]). They also regulate the association of MTs with other cytoskeletal fibers, organelles, and membranes, and influence the ratio of transport along MTs and MT severing by physically blocking the access of motors and severing enzymes. Additionally, structural MAPs can regulate the number of MT protofilaments [102]. Interestingly, it seems that MAP6 has unique properties and functions as it is a microtubule luminal protein and protects MTs against drug and cold-dependent destabilization [113]. With the exception of MAP4, which is a ubiquitous protein, and the expression of MAP7 in epithelial cells, the expression of structural MAPs is mainly restricted to the brain [114].
Microtubule organization is also regulated through the microtubule-severing proteins katanin, spastin, and fidgetin, whose activity can lead to MT shortening or even depolymerization, but also to the formation of numerous microtubule seeds that serve as MT nucleation templates and free tubulin dimers that can be incorporated into new microtubules. Therefore, severing activity can have both a negative and positive effect on MT dynamics and microtubule polymer mass (reviewed in [103]).
It should be noted that, within a cell, the organization and dynamics of MTs is a result of the interplay between tubulin isotypes, their posttranslational modifications, and microtubule-associated proteins. For example, tyrosination increases the affinity of MT to the stabilizing protein, CLIP-170/CLIP-1 but, also to depolymerizing proteins from the kinesin-13 family [115][116][117]. Similarly, tubulin polyglutamylation affects the interactions of MT with several MAPs, including Tau, MAP1, and MAP2, but also with MT-severing proteins [118][119][120].

Microtubule-Targeting Agents
The surface of the globular part of tubulins contains several pockets that can be intercalation sites for MTAs. These compounds, while embedded in the tubulin structure, can alter the microtubule dynamics. This feature of MTAs is used in cancer therapy, as it was shown that treatment of cancer cells with MTAs led to the mitotic arrest and consequent cell death [18,121]. Many MTA compounds are produced by plants, fungi, and invertebrates as a natural defense against antagonists, competitors, or parasites (for a review, see [122]).
Currently, six MTA-binding sites, named after the main compounds with affinity to them, have beem described (Figure 2). Four pockets are located on β-tubulin: taxane, laulimalide/peloruside, vinca, and maytansine sites. The colchicine site is islocated near the intradimer interface between the αand β-tubulin subunits, while the pironetin site is a binding pocket located on the α-tubulin surface (for a review, see [18]). Taxane and laulimalide/peloruside sites bind compounds that stabilize microtubules (MSAs), while the other four pockets accommodate factors that destabilize MTs (MDAs).

Taxane Site
Paclitaxel, a tetracyclic diterpenoid originally isolated from Taxus brevifolia in the 1960s [123], was approved for the treatment of ovarian cancer in 1992 by the US Food and Drug Administration (FDA) as Taxol ® . Now, paclitaxel is produced by a semi-synthetic route by modifying 10deacetylbaccatins III derived from the European yew Taxus baccata [124].

Taxane Site
Paclitaxel, a tetracyclic diterpenoid originally isolated from Taxus brevifolia in the 1960s [123], was approved for the treatment of ovarian cancer in 1992 by the US Food and Drug Administration (FDA) as Taxol ® . Now, paclitaxel is produced by a semi-synthetic route by modifying 10-deacetylbaccatins III derived from the European yew Taxus baccata [124].
Paclitaxel and its derivates are used in diverse cancer therapies and are characterized by high neurotoxicity, myelosuppression, poor water solubility, and the occurrence of multidrug resistance (MDR) in treated tumors (see below). This led to the search for and discovery of other compounds that enhance microtubule stabilization, including epothilones A, B, and D; discodermolide (DDM) and the DDM-paclitaxel hybrid KS-1-199-32; dictyostatin; taccalonolide A and J; and zampanolide ( Figure 3) (for reviews, see [18,125,126] Paclitaxel and its derivates are used in diverse cancer therapies and are characterized by high neurotoxicity, myelosuppression, poor water solubility, and the occurrence of multidrug resistance (MDR) in treated tumors (see below). This led to the search for and discovery of other compounds that enhance microtubule stabilization, including epothilones A, B, and D; discodermolide (DDM) and the DDM-paclitaxel hybrid KS-1-199-32; dictyostatin; taccalonolide A and J; and zampanolide ( Figure 3) (for reviews, see [18,125,126]). Epothilones A and B are macrolide drugs (natural products that consist of a large macrocyclic lactone ring) produced by the myxobacterium Sorangium cellulosum [127]. Unlike paclitaxel, they are highly soluble in water and are not a substrate for P-glycoprotein, which actively transports drugs out of the cell. A semi-synthetic derivate of epothilone B, ixabepilone, was approved for cancer treatment [128,129].
Zampanolide, a sponge-derived macrolide, and taccalonolide A and J, polycyclic steroids isolated from plants of the genus Tacca, were more recently discovered and are still under investigation, but are promising antitumor drug candidates [130]. They both have the unique ability to bind covalently to taxane-site residues asparagine 228/histidine 229 and aspartic acid 226 [130,131].
The taxane ( Figure 2) site is located near the ML surface, on the "inside" side of the tubulin (which in MT, faces the lumen), and is formed by hydrophobic residues of H7, S7, and loops H6-H7, S7-H9 (M loop), and S9-S10 ( Figure 2) [19,20,138]. All compounds ( Figure 3) that bind to the taxane site form hydrophobic and polar contacts with pocket amino acids and strengthen the lateral contacts between heterodimers of adjacent protofilaments, leading to MT stabilization ( Figure 2). The mechanism of microtubule stabilization is compound-specific [25,131,138,139]. Several compounds that contain side chains, such as epothilone A and zampanolide, engage with the M loop, stabilizing it into a short helix [138]. Significantly, a similar helical conformation of the M loop was observed in Epothilones A and B are macrolide drugs (natural products that consist of a large macrocyclic lactone ring) produced by the myxobacterium Sorangium cellulosum [127]. Unlike paclitaxel, they are highly soluble in water and are not a substrate for P-glycoprotein, which actively transports drugs out of the cell. A semi-synthetic derivate of epothilone B, ixabepilone, was approved for cancer treatment [128,129].
Zampanolide, a sponge-derived macrolide, and taccalonolide A and J, polycyclic steroids isolated from plants of the genus Tacca, were more recently discovered and are still under investigation, but are promising antitumor drug candidates [130]. They both have the unique ability to bind covalently to taxane-site residues asparagine 228/histidine 229 and aspartic acid 226 [130,131].
The taxane ( Figure 2) site is located near the ML surface, on the "inside" side of the tubulin (which in MT, faces the lumen), and is formed by hydrophobic residues of H7, S7, and loops H6-H7, S7-H9 (M loop), and S9-S10 ( Figure 2) [19,20,138]. All compounds ( Figure 3) that bind to the taxane site form hydrophobic and polar contacts with pocket amino acids and strengthen the lateral contacts between heterodimers of adjacent protofilaments, leading to MT stabilization ( Figure 2). The mechanism of microtubule stabilization is compound-specific [25,131,138,139]. Several compounds that contain side chains, such as epothilone A and zampanolide, engage with the M loop, stabilizing it into a short helix [138]. Significantly, a similar helical conformation of the M loop was observed in native polymerized MTs [25,138], indicating its importance in the formation of stable MT. Other compounds (e.g., taccalonolide A and J) cause displacement of the M-loop into a more open conformation, facilitating lateral interactions between adjacent protofilaments [131]. By contrast, paclitaxel was suggested to stabilize MTs via an allosteric mechanism by preventing dimer compaction after GTP hydrolysis [25,138]. A similar indirect effect was also proposed as an additional mechanism of MT stabilization for taccalonolide AJ and zampanolide [130,131].
native polymerized MTs [25,138], indicating its importance in the formation of stable MT. Other compounds (e.g., taccalonolide A and J) cause displacement of the M-loop into a more open conformation, facilitating lateral interactions between adjacent protofilaments [131]. By contrast, paclitaxel was suggested to stabilize MTs via an allosteric mechanism by preventing dimer compaction after GTP hydrolysis [25,138]. A similar indirect effect was also proposed as an additional mechanism of MT stabilization for taccalonolide AJ and zampanolide [130,131].
After binding laulimalide or peloruside to the β-tubulin pocket, MTs are stabilized by two main mechanisms which strengthen the lateral contacts between neighboring protofilaments. First, the βtubulin M loop shifts to an "open" conformation (without forming the regular secondary structure). Second, the position of both agents within the pocket allows their interaction with the H3 surface of the adjacent heterodimer, which leads to bridging of neighboring protofilaments ( Figure 2) [143]. In the case of peloruside A, an especially strong effect is observed in the seam, where the lateral contacts are weaker. It was also proposed that both compounds fix structures located near the M loop and, thus, have an additional allosteric effect on MT stabilization [143].

Vinca Site
The naturally occurring vinca alkaloids (vincristine and vinblastine) were discovered in periwinkle (Catharantus roseus G. Don.) in the late 1950s ( Figure 5). These are first-generation vinca alkaloids that have achieved significant clinical progress [147,148]. The therapeutic success of vinca alkaloids in the treatment of hematological cancers (mainly childhood leukemia) [149] led to the development of diverse semi-synthetic analogs ( Figure 5) [150], including vindesine, vinorelbine, and vinflunine, the latter used for the treatment of solid tumors, particularly metastatic breast cancer [151]. However, similar to taxanes, vinca alkaloids have severe side effects (peripheral neuropathies and reversible myelosuppression) [152,153]. The laulimalide/peloruside site is positioned at the opposite side of the ML surface with respect to the taxane site, i.e., the "outside" surface of the MT wall (Figure 2), and is formed by hydrophobic and polar residues of H9 (including a short loop that divides H9 into H9 and H9'), H10, and loop H10-S9 of β-tubulin ( Figure 2) [138,143].
After binding laulimalide or peloruside to the β-tubulin pocket, MTs are stabilized by two main mechanisms which strengthen the lateral contacts between neighboring protofilaments. First, the β-tubulin M loop shifts to an "open" conformation (without forming the regular secondary structure). Second, the position of both agents within the pocket allows their interaction with the H3 surface of the adjacent heterodimer, which leads to bridging of neighboring protofilaments ( Figure 2) [143]. In the case of peloruside A, an especially strong effect is observed in the seam, where the lateral contacts are weaker. It was also proposed that both compounds fix structures located near the M loop and, thus, have an additional allosteric effect on MT stabilization [143].

Vinca Site
The naturally occurring vinca alkaloids (vincristine and vinblastine) were discovered in periwinkle (Catharantus roseus G. Don.) in the late 1950s ( Figure 5). These are first-generation vinca alkaloids that have achieved significant clinical progress [147,148]. The therapeutic success of vinca alkaloids in the treatment of hematological cancers (mainly childhood leukemia) [149] led to the development of diverse semi-synthetic analogs ( Figure 5) [150], including vindesine, vinorelbine, and vinflunine, the latter used for the treatment of solid tumors, particularly metastatic breast cancer [151]. However, similar to taxanes, vinca alkaloids have severe side effects (peripheral neuropathies and reversible myelosuppression) [152,153]. Besides vinca alkaloids, several other groups of compounds were also shown to target the vinca site, including peptides, depsipeptides, and macrolides, and some have been used in clinical trials (reviewed in [18,125,154,155]). Currently, vincristine and vinblastine are used for the treatment of breast cancer, lymphomas, and sarcomas [150], vinorelbine for breast and lung cancer, sarcomas, and glioma [150,156], vindesine for lung cancer [150], vinflunine for urothelial cancer [157,158], vintafolide (vinflunine and folate) for lung, ovarian, and endometrial cancer [159], eribulin for liposarcomas, bladder cancer, and metastatic breast cancer [160,161], and dolastatin 10 for solid tumors [162].
The vinca site is located at the plus end surface of β-tubulin and is formed by residues of H6 and loops T5 and H6-H7; however, several agents also bind to H7 and β-tubulin-bound nucleotide sites [163][164][165]. The vinca-site ligands also form connections with α-tubulin of the subsequent dimer, interacting with its minus end surface structures, including H10, S9, and T7 loop [163].
The binding of ligands to the vinca site alter the surface of the β-tubulin plus end, forming a socalled wedge (Figure 2) [163], thus interfering with the incorporation of new heterodimers at the MT plus end. As a result, the plus end heterodimers remain in curved conformation, which inhibits formation of the MT wall and leads to destabilization [163]. It was also shown that vinca-site ligands can cause the formation of ring-like tubulin oligomers, decreasing the level of free tubulin available for polymerization ( Figure 2) [163,166]. Additionally, several vinca-site compounds were shown to have an allosteric effect on the inhibition of lateral contacts between dimers by stabilizing the M loop in the interaction-incompetent conformation [165].
The vinca site is located at the plus end surface of β-tubulin and is formed by residues of H6 and loops T5 and H6-H7; however, several agents also bind to H7 and β-tubulin-bound nucleotide sites [163][164][165]. The vinca-site ligands also form connections with α-tubulin of the subsequent dimer, interacting with its minus end surface structures, including H10, S9, and T7 loop [163].
The binding of ligands to the vinca site alter the surface of the β-tubulin plus end, forming a so-called wedge ( Figure 2) [163], thus interfering with the incorporation of new heterodimers at the MT plus end. As a result, the plus end heterodimers remain in curved conformation, which inhibits formation of the MT wall and leads to destabilization [163]. It was also shown that vinca-site ligands can cause the formation of ring-like tubulin oligomers, decreasing the level of free tubulin available for polymerization ( Figure 2) [163,166]. Additionally, several vinca-site compounds were shown to have an allosteric effect on the inhibition of lateral contacts between dimers by stabilizing the M loop in the interaction-incompetent conformation [165].
It is worth noting that PM060184 is currently under clinical evaluation [168], while ado-trastuzumab emtansine was recently approved for adjuvant treatment of patients with HER2-positive early breast cancer [169] and was shown to prolong patient survival with a manageable safety profile [170].
The maytansine site is located in close vicinity to NBP and the vinca site, but is formed by other structures, including H3', H11, H11', and loops H11-H11', T3, and T5 ( Figure 2) [171]. It is worth noting that PM060184 is currently under clinical evaluation [168], while adotrastuzumab emtansine was recently approved for adjuvant treatment of patients with HER2positive early breast cancer [169] and was shown to prolong patient survival with a manageable safety profile [170].
The maytansine site is located in close vicinity to NBP and the vinca site, but is formed by other structures, including H3', H11, H11', and loops H11-H11', T3, and T5 ( Figure 2) [171]. The experimental evidence indicates that the inhibitory effect of maytansine-site ligands is a direct consequence of the occupation of the β-tubulin pocket. In growing microtubules, the maytansine-binding pocket of the MT plus end β-tubulin accommodates the minus end structures of the α-tubulin of a newly added heterodimer, including S8, H8, and loop H10-S9 [171]. Incorporation of maytansine-site ligands prevent this interaction, impeding MT elongation ( Figure 2).

Colchicine Site
Colchicine (Figure 7) was isolated from autumn crocus Colchicum autumnale. This alkaloid contains three rings, of which rings A and C bind to β-tubulin, while aromatic ring B binds to αtubulin [172]. Colchicine-tubulin binding is a slow, strongly temperature-dependent, and practically irreversible process [173]. Although colchicine has been used clinically in the treatment of nonneoplastic diseases (gout, familial Mediterranean fever), neither colchicine nor other related compounds were successful as chemotherapeutic agents owing to their severe toxicity to normal tissues at doses required for antitumor effects [174,175]. The experimental evidence indicates that the inhibitory effect of maytansine-site ligands is a direct consequence of the occupation of the β-tubulin pocket. In growing microtubules, the maytansine-binding pocket of the MT plus end β-tubulin accommodates the minus end structures of the α-tubulin of a newly added heterodimer, including S8, H8, and loop H10-S9 [171]. Incorporation of maytansine-site ligands prevent this interaction, impeding MT elongation ( Figure 2).

Colchicine Site
Colchicine (Figure 7) was isolated from autumn crocus Colchicum autumnale. This alkaloid contains three rings, of which rings A and C bind to β-tubulin, while aromatic ring B binds to α-tubulin [172]. Colchicine-tubulin binding is a slow, strongly temperature-dependent, and practically irreversible process [173]. Although colchicine has been used clinically in the treatment of nonneoplastic diseases (gout, familial Mediterranean fever), neither colchicine nor other related compounds were successful as chemotherapeutic agents owing to their severe toxicity to normal tissues at doses required for antitumor effects [174,175]. Over the last few decades, compounds of low toxicity which target the colchicine site have been reported (Figure 7), including derivatives of stilbenoid-combretastatins (combretastatin A-1 phosphate/OXi4503, combretastatin A-1, combretastatin A-4, fosbretabulin, ombrabulin), chalcones (MDL 27048), compounds with furonaphthodioxole skeleton (podophyllotoxin), derivatives of indole (indibulin), and natural metabolite of estradiol (2-methoxyestradiol). While no colchicine-site MTAs are currently approved for cancer treatment, several are in phase I/III clinical trials [174] (for a review, see [175]).
Recently, a compound initially designed as MTH1 (Mut T homolog 1) inhibitor, TH588, was shown to dock into the colchicine-binding pocket [176]. By reducing microtubule plus end dynamics, this cyclopropyl analog affects tubulin polymerization, resulting in disruption of mitotic spindles, prolongation of mitosis and, eventually, apoptosis [176][177][178]. Preclinical studies show promising results for the use of TH588 as an anticancer drug [179,180].
The colchicine-binding site is located near the plus end surface of β-tubulin in the center of the tubulin heterodimer at the interface between α-and β-tubulin. It is a big pocket formed by the hydrophobic and polar residues of H7, H8, S7, S8, and loop H7-H8 (T7 loop) that can be divided into three zones: central zone 2 and two accessory zones, zone 1 facing α-tubulin and zone 3 buried deeper within the β-tubulin pocket [181,182].
Binding of colchicine-site ligands to heterodimer causes its stabilization in the curved conformation ( Figure 2) [181]. As mentioned, during MT polymerization, tubulin dimers at the MT tip undergo a transition from curved to straight conformation, which requires a shift of several βtubulin structures (S8-S9 and H8) closer to each other. As a result, the colchicine pocket is contracted [24,181]. While the colchicine pocket is occupied by a ligand, such conformational changes cannot occur, making colchicine ligand-bound heterodimer incompetent for polymerization [24,181].

Pironetin Site
Pironetin (Figure 8), a polyketide, is a natural product that was first extracted from fermentation broths of Streptomyces sp. [183,184]. It is worth noting that pironetin is, to date, the only known compound that exclusively targets the α-tubulin subunit and covalently binds to Cys316 of α-tubulin [185,186]. The molecule and its derivates are currently under investigation and display promising anticancer properties (reviewed in [187]). Over the last few decades, compounds of low toxicity which target the colchicine site have been reported (Figure 7), including derivatives of stilbenoid-combretastatins (combretastatin A-1 phosphate/OXi4503, combretastatin A-1, combretastatin A-4, fosbretabulin, ombrabulin), chalcones (MDL 27048), compounds with furonaphthodioxole skeleton (podophyllotoxin), derivatives of indole (indibulin), and natural metabolite of estradiol (2-methoxyestradiol). While no colchicine-site MTAs are currently approved for cancer treatment, several are in phase I/III clinical trials [174] (for a review, see [175]).
Recently, a compound initially designed as MTH1 (Mut T homolog 1) inhibitor, TH588, was shown to dock into the colchicine-binding pocket [176]. By reducing microtubule plus end dynamics, this cyclopropyl analog affects tubulin polymerization, resulting in disruption of mitotic spindles, prolongation of mitosis and, eventually, apoptosis [176][177][178]. Preclinical studies show promising results for the use of TH588 as an anticancer drug [179,180].
The colchicine-binding site is located near the plus end surface of β-tubulin in the center of the tubulin heterodimer at the interface between αand β-tubulin. It is a big pocket formed by the hydrophobic and polar residues of H7, H8, S7, S8, and loop H7-H8 (T7 loop) that can be divided into three zones: central zone 2 and two accessory zones, zone 1 facing α-tubulin and zone 3 buried deeper within the β-tubulin pocket [181,182].
Binding of colchicine-site ligands to heterodimer causes its stabilization in the curved conformation ( Figure 2) [181]. As mentioned, during MT polymerization, tubulin dimers at the MT tip undergo a transition from curved to straight conformation, which requires a shift of several β-tubulin structures (S8-S9 and H8) closer to each other. As a result, the colchicine pocket is contracted [24,181]. While the colchicine pocket is occupied by a ligand, such conformational changes cannot occur, making colchicine ligand-bound heterodimer incompetent for polymerization [24,181].

Pironetin Site
Pironetin (Figure 8), a polyketide, is a natural product that was first extracted from fermentation broths of Streptomyces sp. [183,184]. It is worth noting that pironetin is, to date, the only known compound that exclusively targets the α-tubulin subunit and covalently binds to Cys316 of α-tubulin [185,186]. The molecule and its derivates are currently under investigation and display promising anticancer properties (reviewed in [187]). The pironetin-binding site is the only known pocket on α-tubulin targeted by MTAs. Its main part is formed by residues of S8, S10, and H7, but residues of S4, S5, and S6 also participate in the ligand accommodation [185]. The binding of pironetin leads to conformational changes of α-tubulin within the minus end, including disordering of loop H7-H8 (T7 loop) and part of H8 [185]. Since these structures are required for the formation of longitudinal interactions within protofilaments, it was proposed that pironetin prevents MT polymerization by the formation of assembly-incompetent pironetin-bound tubulin dimers ( Figure 2) [185].

Factors Affecting Microtubule Dynamics in Cancer Cells
Carcinogenesis is a multistep process involving, among other actions, a remodeling of the cytoskeleton. The transformation from highly polarized epithelial cells to multipolar spindle-like metastatic cells that are able to detach from the extracellular matrix and migrate requires extensive reorganization of the cytoskeleton, including microtubules, during a process called epithelialmesenchymal transition (EMT) (for a review, see [188]). The abnormalities of mitotic spindle and consequent aberrant cell cycle progression and division lead to genomic instability (for a review, see [189]). Thus, it is not surprising that numerous alterations in tubulins, including mutations and variations in isotype expression level, post-translational modifications, and MAP composition, were identified in cancer cells.

Tubulin Isotypes in Cancer and Anticancer Drug Resistance
Altered expression of tubulin isotypes is considered to be a hallmark in a range of cancers. Analysis of clinical specimens has shown that in many cancers, a high expression of several β-tubulin isotypes correlates with aggressive clinical behavior, chemotherapy drug resistance, and poor patient outcome [121]. Strikingly, little is known about the level of tubulin isoforms in primary nontreated cancers, while numerous studies indicate their variations after chemotherapy, especially after taxanebased treatment [121]. In fact, some data show that in cancer cell lines, paclitaxel can itself induce the expression of specific tubulin isoforms [190].
An increase in βI expression was observed in several cancers, including breast, colon, and kidney cancer, while its level was decreased in prostate cancer (Table 2) [43,[191][192][193]. In the case of ovarian cancer, the data are inconsistent as both increased and invariant levels of βI expression were reported [43,191,194,195]. High βI expression is associated with the acquisition of chemoresistance to MTAs and poor prognosis in ovarian serous carcinoma and lung adenocarcinoma, but not in lung squamous cell carcinoma [191,193,196]. Interestingly, recent data show that experimentally lowering βI level by siRNA or mir-195 microRNA sensitizes adenocarcinoma cell lines to paclitaxel and eribuline [193], indicating a direct correlation between βI level and MTA resistance, at least in non-small-cell lung adenocarcinomas.
βIIa and b differ by only two amino acids and, thus, the expression of these two isotypes can be distinguished at the RNA level but not at the protein level. Using PCR, it was shown that βIIa is increased in NSCLC, prostate, and ovarian cancers, and decreased in kidney, colon, and breast cancers [43], while βIIb is increased in ovarian cancer and decreased in kidney, colon, and breast cancers [43]. Increased βIIb isoform was also recently associated with the metastatic stage of melanoma, indicating its role in EMT transition [197]. βII was also examined at the protein level in The pironetin-binding site is the only known pocket on α-tubulin targeted by MTAs. Its main part is formed by residues of S8, S10, and H7, but residues of S4, S5, and S6 also participate in the ligand accommodation [185]. The binding of pironetin leads to conformational changes of α-tubulin within the minus end, including disordering of loop H7-H8 (T7 loop) and part of H8 [185]. Since these structures are required for the formation of longitudinal interactions within protofilaments, it was proposed that pironetin prevents MT polymerization by the formation of assembly-incompetent pironetin-bound tubulin dimers ( Figure 2) [185].

Factors Affecting Microtubule Dynamics in Cancer Cells
Carcinogenesis is a multistep process involving, among other actions, a remodeling of the cytoskeleton. The transformation from highly polarized epithelial cells to multipolar spindle-like metastatic cells that are able to detach from the extracellular matrix and migrate requires extensive reorganization of the cytoskeleton, including microtubules, during a process called epithelial-mesenchymal transition (EMT) (for a review, see [188]). The abnormalities of mitotic spindle and consequent aberrant cell cycle progression and division lead to genomic instability (for a review, see [189]). Thus, it is not surprising that numerous alterations in tubulins, including mutations and variations in isotype expression level, post-translational modifications, and MAP composition, were identified in cancer cells.

Tubulin Isotypes in Cancer and Anticancer Drug Resistance
Altered expression of tubulin isotypes is considered to be a hallmark in a range of cancers. Analysis of clinical specimens has shown that in many cancers, a high expression of several β-tubulin isotypes correlates with aggressive clinical behavior, chemotherapy drug resistance, and poor patient outcome [121]. Strikingly, little is known about the level of tubulin isoforms in primary nontreated cancers, while numerous studies indicate their variations after chemotherapy, especially after taxane-based treatment [121]. In fact, some data show that in cancer cell lines, paclitaxel can itself induce the expression of specific tubulin isoforms [190].
An increase in βI expression was observed in several cancers, including breast, colon, and kidney cancer, while its level was decreased in prostate cancer (Table 2) [43,[191][192][193]. In the case of ovarian cancer, the data are inconsistent as both increased and invariant levels of βI expression were reported [43,191,194,195]. High βI expression is associated with the acquisition of chemoresistance to MTAs and poor prognosis in ovarian serous carcinoma and lung adenocarcinoma, but not in lung squamous cell carcinoma [191,193,196]. Interestingly, recent data show that experimentally lowering βI level by siRNA or mir-195 microRNA sensitizes adenocarcinoma cell lines to paclitaxel and eribuline [193], indicating a direct correlation between βI level and MTA resistance, at least in non-small-cell lung adenocarcinomas.
βIIa and b differ by only two amino acids and, thus, the expression of these two isotypes can be distinguished at the RNA level but not at the protein level. Using PCR, it was shown that βIIa is increased in NSCLC, prostate, and ovarian cancers, and decreased in kidney, colon, and breast cancers [43], while βIIb is increased in ovarian cancer and decreased in kidney, colon, and breast cancers [43]. Increased βIIb isoform was also recently associated with the metastatic stage of melanoma, indicating its role in EMT transition [197]. βII was also examined at the protein level in several cancer types, including head and neck carcinomas (LASCCHN), ovarian carcinoma, colorectal cancer, and breast cancer cell lines [194,196,198].  [191] breast not determined docetaxel [200] NSCLC adenocarcinomas poor paclitaxel and eribulin [193] βII breast not determined docetaxel [196,201] [204] bladder, cisplatin resistant poor after paclitaxel chemotherapy n/a [205] gastric poor n/a [206] gastric metastatic poor after taxane chemotherapy n/a [207] uterine serous carcinoma poor paclitaxel, sensitivity to epothilone [208] lung carcinoma cell line n/a epothilone [ Similar to βI, in lung adenocarcinoma, breast cancer, and breast cancer cell lines, an increased level of βII was associated with resistance to MTA [191,196,201], while in LASCCHN, it was associated with poor survival after chemotherapy [198]. By contrast, in taxane-treated ovarian carcinomas, poor outcome is associated with a low level of βII [194]. Interestingly, silencing of βII by siRNA in NSCLC adenocarcinoma and large-cell carcinoma cell lines increases cell sensitivity to vinca alkaloids but not to paclitaxel treatment [220].
In a number of cancers (mainly of epithelial origin) βII was observed within the nucleus of both cancer cells and nontransformed cells in tissues adjacent to the cancer [221]. Nuclear localization of βII was recently associated with poor outcomes in colorectal cancer patients [222].
It was surprising when the neural tubulin isoform βIII, which increases MT dynamics (see above), was discovered to be expressed in tumors with different origins. An analysis of the significant number of different types of tumors revealed that the contribution of βIII to the total tubulin pool depended on the cancer type [223]. For example, in nearly 70-80% of the examined cases of small-cell lung cancer, mesothelioma, NSCLC, adenocarcinoma and large-cell cancer, neuroendocrine pancreatic cancer, malignant melanoma, and gallbladder carcinoma, βIII was expressed at high levels [223]. By contrast, 70-95% of cases of breast cancer, colon adenoma, stomach cancer, basalioma, Warthin's tumor, and hepatocellular carcinoma were βIII-negative [223].
The role of the βIII isoform in tumorigenesis was confirmed in a pancreatic cell line model. Silencing of βIII expression by shRNA or mir-200c microRNA reduced cancer cell growth and tumorigenic potential both in vitro and in vivo in orthotopic and xenographic pancreatic cancer mouse models [190,230].
A high level of βIII is generally believed to be a bad prognostic marker for MTA resistance and survival. However, it was recently shown that a paclitaxel-resistant NSCLC adenocarcinoma cell line with increased βIII expression was sensitive to vinblastine and its analogs to the same extent as "parental" cells with low taxane resistance and lower βIII expression [235]. This indicates that βIII-induced taxane resistance may not influence resistance to other MTAs. In contrast to these observations, overexpression of βIII-tubulin in ovarian clear cell adenocarcinoma is a predictor of a good response to taxane-based chemotherapy, and cases with higher βIII-tubulin expression are associated with a significantly more favorable prognosis than those with lower βIII-tubulin expression [214]. A similar observation was made for early stages (I/II) of breast cancer [212].
Altered expression of the βIV isotype was also reported in numerous cancers, including ovarian, lung, prostate, breast, and kidney cancers and breast and lung cancer cell lines [43,191,194,196,220]; the data, however, are frequently inconsistent. For example, some data indicate decreased βIV in lung and breast cancer [43], while studies on cancer cell lines indicate increased βIV expression, especially in taxane-resistant cell lines [191,196].
Similar to βII, βIVb overexpression is associated with resistance to vinca alkaloids rather than taxanes. In fact, siRNA knockdown of IVb β-tubulin expression in NSCLC and pancreatic ductal carcinoma cell lines increases the response to vinca alkaloids [220,236]. Interestingly, downregulation of βIVb was recently observed in an EMT-induced colon cancer cell line and transformation of epithelium-like to spindle-like cell morphology in these cells was reversed by βIVb overexpression [237].
The level of βV expression was tested on RNA and protein levels. The level of βV RNA was shown to be reduced in most tumors (colon, ovary, prostate, breast, lung) except for kidney [43], while βV protein level was shown to be elevated in lung, breast, and ovarian cancers and decreased in prostate cancer [238,239]. In NSCLC, a low level of βV-tubulin was associated with poor prognosis after paclitaxel-based chemotherapy [219].
Based on the available data, it appears that different β-tubulin isotypes play specific roles in the cancer cell response to extrinsic factors influencing MT dynamics. For example, in breast cancer patients with both βI and βIII upregulation, response to taxane-based therapy was poor; in the group with a low level of both, the majority of patients responded well to the therapy, while in groups where one β-tubulin isotype high and another low, the response was intermediate [200]. Similar, in NSCLC patients with a low level of βV and high level of βIII expression, the outcome was much worse than in patients with high βV and low βIII, while patients with either a high or low level of both isotypes had an intermediate outcome [219].
Thus, the outcome of levels and ratios of particular β tubulin isotypes in terms of the progression of carcinogenesis appears to be specific to tumor type.
MTA resistance in cancer could also be related to mutations of β-tubulin. However, analyses of the clinical samples revealed that mutations in β-tubulin are either not present or very rare. Thus, it seems unlikely that mutations in β-tubulin could play an important role in drug resistance (reviewed in [240]).
Studies conducted on cell lines showed that mutations of predominating β-tubulin isotypes within the taxane-, colchicine-, and vinca-binding sites can be associated with altered MT dynamics and/or resistance to MTA (reviewed in [240][241][242]). Most β-tubulin mutations located within close proximity to the taxane-binding site did not change the affinity of tubulin to taxanes or epothilones, but probably destabilized MTs in the absence of any drugs [242]. Only mutation at F270V, T274I, and R282N residues were reported to have a direct effect on drug-binding affinity [242]. A similar effect was observed when point mutations were located in the M-loop (T274I, R282Q) or in helix H9, which is essential for interdimer interactions (Q292E) [242].
A recent study on a large number of samples of breast cancer tumors identified several mutations in βI, βIIa, and βIVb tubulins in which a gene-encoded residue was replaced by the amino acid present in the corresponding position in βIII [243]. It was proposed that such mutations could influence the clinical outcome in a similar manner as overexpression of βIII-tubulin [243].

Microtubule PTMs and Cancer
Changes in the level of tubulin modifications were linked to tumorigenesis (Table 3) [29,244]. Downregulation of TTL and increased α-tubulin detyrosination were reported during the epithelial-mesenchymal transition (EMT) that occurs during tumor invasion [245] in prostate cancer cells [246], in aggressive subtypes of breast cancer cells [247], and in primary neuroblastomas with poor prognosis [248]. The recent discovery of the vasohibin (VASH)/small vasohibin-binding protein (SVBP) complex, reported as a detyrosinating enzyme, tubulin carboxypeptidase (TCP) [249,250], provides new links between this tubulin modification and already known associations between vasohibin dysfunction and cancer [251,252].
A high ∆2 α-tubulin level in non-small-cell lung cancer (NSCLC) cells was associated with shorter overall patient survival and resistance to vinorelbine [211]. On the contrary, ∆2 α-tubulin was undetectable in prostate cancer cell lines (LNCaP and PC3), but was present in control cells [246].
Tubulin acetylation is associated with several types of cancer. An increased level of acetylation was reported in head and neck squamous cell carcinoma, for which it can be used as a prognostic marker [253]. An elevated level of tubulin acetylation in breast cancer cell line (MCF-7) is associated with the development of colchicine-resistance [254]. Additionally, a higher level of acethylated tubulin in primary breast tumors is linked to the basal-like subtype of breast cancer, in which it promotes adhesion and invasion of breast cancer cells, increasing the risk of disease progression and death [255]. Overexpression of ATAT1 in cultured nonmetastatic lines of breast cancer cells promoted the formation of microtubule-based membrane protrusions, structures characteristic of metastasis [255]. The level of microtubule acetylation was also shown to affect epithelial-mesenchymal transition and cell polarity [256].
Recent studies provide evidence that phosphorylation of serine 21 of HDAC6 by G protein-coupled receptor kinase 5 (GRK5) promotes deacetylase activity in ovarian (HeLa) and breast (MDA MB 231) cancer cell lines. An increased level of acetylated α-tubulin sensitizes these cells to the anti-apoptotic activity of paclitaxel [257]. The high expression of HDAC6 was also linked to poor prognosis of oral squamous cell carcinoma (OSCC) [258], oncogenic transformation [259], and EMT [260]. Phosphorylation of α-tubulin (Ser 165) dephosphorylated (S165D) α-tubulin breast cancer cell lines hyperproliferation and increased metastatic potential [267] Phosphorylation of α-tubulin at Ser 165 residue by protein kinase C, in turn, stimulates microtubule dynamics in human breast cancer cells [78,79,267]. It seems that phosphorylation of α-tubulin at Ser 165 can act as a switch that controls the expression of EMT markers in nontransformed human breast cells and the rate of proliferation of breast tumors [79,267].
A few studies suggested that changes in the levels of tubulin glutamylation [246] and glycylation [266] are observed during tumorigenesis. Some unusual post-translational modifications have been detected in lung and hepatic cancers. The removal of the final two residues of the β IVb-tubulin C-terminal tail was identified in more advanced stages of liver cancer and metastasis to lung in a rat model of hepatic carcinoma [263].
Some studies suggested that the silencing of the Stathmin-1-encoding gene can inhibit cancer cell migration and metastatic potential [281]. The data concerning correlation of the level of Stathmin-1 expression and resistance of cancer to chemotherapy are contradictory. Several studies show that Stathmin-1 overexpression increases the sensitivity of breast and lung cancer cells to taxanes and/or vinca alkaloids [282,283]. However, in epithelial carcinomas, nasopharyngeal carcinomas, breast cancer, and esophageal squamous cell cancer, the increased taxane sensitivity was correlated with Stathmin-1 silencing [284][285][286][287][288]. Interestingly, not only the protein level but also its phosphorylation state was correlated with cancerogenesis and drug resistance [289].
Increased levels of EB1 and CLIP-170/CLIP1, two +TIP proteins, enhances paclitaxel sensitivity in breast cancer cell lines and the response to taxane-containing therapy in patients [290,291]. On the other hand, a decrease of CLIP-170/CLIP1 expression correlates with patients survival in the case of glioma [292].
Structural MAPs have also been related with carcinogenesis. The expression of neuronal MAPs, Tau, MAP2, and MAP4 was detected in non-neuronal cancer tissues. For example, Tau overexpression observed in breast and ovarian cancer cells was correlated with a poor outcome [293][294][295], while downregulation of Tau in breast and ovarian cancer cell lines increased sensitivity to paclitaxel [293,295]. Because Tau and taxanes bind to the same tubulin surface, it was proposed that Tau may compete with paclitaxel for binding to β-tubulin, causing taxane ineffectiveness [293,295]. On the other hand, in mice, docetaxel-sensitive pancreatic neoplasms show a higher level of Tau and MAP2 with respect to those that are docetaxel-resistant [296,297].
MAP2 was proposed as a diagnostic marker in pulmonary neuroendocrine carcinomas, some non-small-cell lung carcinomas [298], Merkel cell carcinomas [299], and oral squamous cell carcinoma [300], but not in metastatic melanomas (while abundant in primary melanomas) [301]. Overexpression of MAP2 in melanoma cell lines leads to microtubule stabilization, associated with G2-M phase cell cycle arrest, growth inhibition, and cancer cell apoptosis, both in vitro and in a nude mouse model [301,302]. Decrease of MAP2c accompanied with a decrease in βIII-tubulin expression was also observed in vinca-resistant neuroblastoma cell lines [303].
Recently, also, MAP1B was shown to be expressed and a marker of a poor outcome in urothelial cancer [270]. Silencing of MAP1B in urothelial cancer cell lines decreased the cell migration and invasiveness [270].
An elevated level of MAP4 and resistance to vinca alkaloids have been observed in childhood acute lymphoblastic leukemia (ALL) cells [304], while leukemia cell lines resistant to the epothilone and hypersensitive to microtubule-destabilizing agents increased the levels of both MAP4 and βIII tubulin [305]. In esophageal squamous cell carcinoma, an increased level of MAP4 was shown to be a poor outcome marker, and its intratumor silencing inhibited cell growth in nude mice [306]. Very similar observations were also made in lung adenocarcinoma [307].
An increased level of MAP7 is a marker of poor prognosis in leukemia and cervical cancers [269,308,309]. Moreover, it was shown that MAP7 promotes migration and invasiveness of cervical cancer cell lines by inducing EMT transition [309].

Conclusions and Perspectives
Microtubule dynamics play a key role in the proper execution of cell division. Thus, it is not surprising that a large number of MTAs have found application as clinical drugs against different types of cancers. Unfortunately, MTAs are also toxic to healthy tissues. Therefore, reducing the toxicity of anticancer MTAs and understanding the causes of cancer cell resistance are extremely important. The main direction of research worldwide includes: (i) a comprehensive understanding of the tubulin code in cancer cells and the selective manipulation of tubulin isotype expression [121], (ii) an improvement of the potency of drugs and increased tumor specificity [18], (iii) combination therapy, with nanoparticles and anticancer drugs working synergistically to delay the onset of drug resistance [310], and (iv) the use of antibody-drug conjugates (ADCs) as a potent class of anticancer therapeutics that confer selective and sustained cytotoxic drug delivery to tumor cells.
Author Contributions: All authors searched and organized data concerning selected chapter. H.F. and E.J. prepared initial version of the manuscript, F.B. and E.J. also prepared figures. All authors worked on the final version of the manuscript. All authors have read and agreed to the published version of the manuscript.