Anti-Cancer Agents in Medicinal Chemistry, 2011, 11, 719-728
719
Targeting III-Tubulin in Glioblastoma Multiforme: From Cell Biology and Histopathology to Cancer Therapeutics Christos D. Katsetos1,*, Pavel Dráber 2 and Maria Kavallaris3 1
Departments of Pediatrics, Pathology & Laboratory Medicine, and Neurology, Drexel University College of Medicine, St. Christopher's Hospital for Children and Hahnemann University Hospital, Philadelphia, Pennsylvania, USA; 2Laboratory of the Biology of Cytoskeleton, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague, Czech Republic; 3Children's Cancer Institute Australia, Lowy Cancer Research Centre, University of New South Wales, Randwick, New South Wales, Australia Abstract: Glioblastoma multiforme (GBM) is the most common, aggressive, and chemorefractory brain tumor in human adults. Notwithstanding significant discoveries in the elucidation of pathways of molecular signaling and genetics of GBM during the past 20 years there has been no breakthrough in the pharmacological treatment of this high-grade malignancy. We, and others, have previously demonstrated increased expression of III-tubulin in GBM asserting a link between aberrant expression of this -tubulin isotype and a disruption of microtubule dynamics associated either with malignant tumor development de novo, or with progression and malignant transformation of a low-grade glioma into GBM. This article reviews III-tubulin as a promising target in the experimental treatment of GBM and examines the potential use of epothilones, a new family of anticancer agents shown to be active in III-tubulin-expressing tumor cells, as well as the “double hit” therapeutic concept of tumor cell sensitization to tubulin binding agents (TBAs) by III-tubulin silencing. The latest progress regarding the function and potential role of III-tubulin in aggressive tumor behavior, cancer stem cells, tumor cell hypoxia, and resistance to taxane-related compounds, is also critically appraised.
Keywords: Tubulin, glioblastoma multiforme, TBA, tubulin binding agent(s), epothilones. INTRODUCTION Gliomas are the most prevalent group of central nervous system (CNS) neoplasms accounting for more than 70% of all brain tumors [1]. Diffuse gliomas are particularly challenging forms of brain cancer owing to their highly invasive and infiltrative nature. Glioblastoma multiforme (GBM) is the most common and aggressive, as well as the deadliest, histologic type of glioma in adults [1]. To date, one of the principal strategies used in the treatment of malignant tumors has been to disrupt the integrity of microtubules in order to prevent or curtail mitotic division [2-4]. Moreover, targeting microtubules represents a strategy aimed at exerting an inhibitory effect on tumor cell invasiveness, given that microtubules are involved in tumor cell motility, the formation of long dynamic protrusions [5] and the elongation of invadopodia [6]. Unfortunately, the clinical usefulness and potential benefits of several timehonored tubulin-binding agents (TBAs) are hampered by the emergence of drug-resistant tumor cells, which represents a challenging problem responsible for treatment failures [7]. To this example, the efficacy of certain TBAs, such as the taxanes, that stabilize microtubules in tumor cells is hindered by the development of resistance, which is accounted for, in large part, by alterations of the microtubule cytoskeleton including the aberrant overexpression of IIItubulin (also refered to as class III -tubulin; gene name TUBB3)[4, 8-10]. Moreover, the inability of many TBAs to cross the bloodbrain barrier [11] coupled with the develoment of drug-induced neurotoxicity [12] have historically dampened enthusiasm about their use in neuro-oncology. However, major advances in the pathobiology of microtubules in cancer during the past decade have reignited a keen interest in potential microtubule/tubulin targets in cancer therapeutics. In this light, it is critically important to enhance our knowledge and understanding of microtubule dynamics and the disruption of the microtubule cytoskeleton in cancer cells, which may lead to the development of more effective approaches to treatment. *Address correspondence to this author at the Section of Neurology, St. Christopher's Hospital for Children, 3601 A Street, Philadelphia, PA 19134, USA; Tel: (215) 427-5335; Fax: (215) 427-4284; E-mail:
[email protected] 1871-5206/11 $58.00+.00
Microtubules are dynamic polymers that undergo rapid bouts of assembly and disassembly during interphase, and then reorganize into a bipolar spindle during mitosis [13]. A number of different proteins impact the dynamic properties of the microtubules, such as stathmin, which promotes microtubule disassembly, and the classic microtubule-associated proteins, which promote their assembly and stabilization [14]. Molecular motor proteins are also needed to organize microtubules into higher-level structures such as the mitotic spindle, and motors such as kinesin-5 have recently been proposed as potential targets for anti-cancer drugs [15, 16]. Another category of microtubule-related proteins, are the so-called microtubulesevering proteins, which are enzymes (ATPases) capable of severing long microtubules [13] and whose expression may be perturbed in brain cancer [17]. In previous studies, we have shown significant alterations in the microtubule cytoskeleton of neoplastic cells in GBMs, including increased expression of III-tubulin, -tubulin, and the microtubule severing ATPase spastin, which are collectively associated with a trend toward high-grade malignancy [9, 18-24]. The present review lends credence to a new experimental treatment strategy for GBM within the conceptual framework of microtubule dynamics and tubulin targets. In this regard, it focuses specifically on altered isotype composition of tubulin in gliomas with emphasis on III-tubulin as a target of cellular therapeutics in GBM [9]. To this end, we will critically examine the rationale behind the use of epothilones and related compounds as potential new agents that have shown efficacy in taxane-resistant tumors in the light of latest developments regarding the role of III-tubulin in clinically aggressive tumor behavior and resistance to TBAs, particularly taxane-based compounds [4, 8, 10]. MICROTUBULES AND TUBULIN Microtubules are highly dynamic cylindrical cytoskeletal polymers that are indispensable for many cellular functions in eukaryotes such as maintenance of cell shape, division, migration and ordered vesicle transport powered by motor proteins. The basic building components of microtubules are heterodimers of globular and -tubulin subunits. - and -tubulins are highly conserved, and consist of isotypes encoded by different genes [25]. Tubulins are arranged in a head-to-tail fashion to form 13 protofilaments that © 2011 Bentham Science Publishers
720 Anti-Cancer Agents in Medicinal Chemistry, 2011, Vol. 11, No. 8
constitute cylindrical microtubules with outer diameter around 25 nm. Microtubules are thus, inherently polar, and contain two structurally distinct ends: a slow-growing minus end, exposing -tubulin subunits, and a fast-growing plus end, exposing -tubulin subunits [9]. Typically in mammalian cells, microtubule minus ends are stably anchored in microtubule-organizing centers (MTOC), whereas the plus ends are highly dynamic and switch between phases of growth and shrinkage [26, 27]. In vitro microtubules can self-assemble at high concentrations of tubulin heterodimers in the presence of GTP, but such microtubules are randomly organized. Associated with MTOCs is -tubulin, minor member from the tubulin superfamily that is essential for nucleation of microtubules [24]. -TUBULIN ISOTYPES - and -Tubulin subunits are encoded by multiple genes, which are –in large part-- phylogenetically conserved [25, 28]. Mammalian -tubulin exists in the form of (at least) seven distinct isotypes, designated as I, II, III, IVa, IVb, V and VI, each corresponding to a distinct gene product, which is synthesized without alternative splicing [25, 28]. Differences amongst the tubulin isotypes rest principally within the C-terminal tail domain (Box 1), a region of the protein that is positioned on the exterior of microtubules and which corresponds to putative binding sites for microtubule-associated proteins (MAPs) [30]. It is thought that each one of the tubulin isotypes may contribute to differences in microtubule dynamics and binding of anti-mitotic drugs [31]. The tubulin isotypes in the brain undergo extensive posttranslational modifications [32], including phosphorylation [33], polyglutamylation [33], polyglycylation [34] and glycosylation [35]. Diverse posttranslational modification are proposed to form a biochemical “tubulin code” that can be read by factors that interact with microtubules. Specific microtubule regions can be distinguished biochemically and functionally by the presence of post-translational modifications on tubulins [36, 37]. III-Tubulin has certain distinguishing attributes which set it apart it from the other -tubulin isotypes and which might also explain its unique function(s) [28, 38] (Box 2). The expression and cell type distribution of III-tubulin has been widely and thoroughly investigated in normal and neoplastic cells [9, 20, 21, 42]. In the developing and mature cells and tissues, III-tubulin is predominantly (and most highly) expressed in the nervous system, both central and peripheral (CNS and PNS) where it is neuronal Box 1.
Katsetos et al.
associated [42-45]. Recapitulating its predominantly neuronal expression, immunolocalization of III-tubulin has been detected in a population of P19 murine embryonal carcinoma cells induced to undergo neuronal differentiation and neuritogenesis by exposure to retinoic acid [46] (Fig. 1A and 1B). However, in a developmental context, the expression of III-tubulin is not neuron-specific insofar as transient expression of this -tubulin isotype is also detected in human fetal astrocytes in culture (Fig. 1C) and in neuroepithelial precursor cells in the subventricular zones of the cerebral hemispheres from mid-gestational human fetuses, which consist of presumptive glial-restricted precursor cells and/or bipotential neural stem cells [47]. OVEREXPRESSION OF III-TUBULIN IN CANCER Early descriptive cellular/histopathological studies carried out in our laboratories during the 1990s have shown that the expression of different -tubulin isotypes is perturbed in tumor cells in general and brain cancer cells in particular [reviewed in 9, 20, 21, 42]. This gave the impetus for a number of benchmark clinical/translational and pharmacological/functional studies on solid tumors during the past 5 years, which added significantly to our understanding as to how aberrant tumor overexpression of the III-tubulin isotype contributes to tumorigenesis, disease progression and chemoresistance [reviewed in 8-10, 48, 49]. Thus far, clinical trial-based analysis of III-tubulin expression has been carried out, for the most part, in pulmonary and gynecological malignancies. A relationship between expression of III-tubulin and clinical outcomes has been established in non-small cell lung cancer (NSCLC) and carcinomas of the breast and ovary [8, 48, 49]. Moreover, in NSCLC and ovarian carcinomas III-tubulin expression is associated with resistance to taxanes and is an independent predictor of survival [4, 8, 10, 48, 49]. Upregulation of III-tubulin is associated with tumor progression and chemoresistance in lung cancer [50]. Although III-tubulin is strongly associated with drugrefractory and aggressive NSCLC serving both as a prognostic and a predictive factor in certain tumor types, its significance as a tumor biomarker is neither universal nor linear in clinical oncology. From a therapeutic perspective, overexpression or aberrant expression of III-tubulin can affect the response of tumor cells to TBAs [4, 8, 10]. The underlying mechanism(s) accounting for this property are currently unclear but recent studies have unraveled potential leads [4, 8, 10].
Overview of Human -Tubulin Isotypes
Isotype*
Gene Name
Protein ID (NCBI)
Length (a.a.)
C-terminal Sequence (Beyond Residue 430)
I
TUBB
NP_821133
444
EEEEDFGEEAEEEA
II
TUBB2A
NP_001060
445
DEQGEFEEEEGEDEA
II
TUBB2B
NP_821080
445
DEQGEFEEEEGEDEA
III
TUBB3
NP_006077
450
EEEGEMYEDDEEESEAQGPK
IVa
TUBB4
NP_006078
444
EEGEFEEEAEEEVA
IVb
TUBB2C
NP_006079
445
EEEGEFEEEAEEEVA
V
TUBB6
NP_115914
446
NDGEEAFEDEEEEIDG
VI
TUBB1
NP_110400
451
VLEEDEEVTEEAEMEPEDKGH
*The nomenclature of -tubulins is based on a recent reviews [28, 29].
Box 2.
Distinctive Features of III-Tubulin
•
Unlike the I, II and IV isotypes, III-tubulin lacks the widely conserved and oxidation-sensitive residue cys239, which is replaced by ser239 [28, 38, 39].
•
The remarkable conservation of III-tubulin across vertebrate species denotes that cys124 and ser239 may have functional roles [39].
•
Unlike the II- and IV-tubulin isotypes, III-tubulin is phosphorylated at a serine in the C-terminus [38, 40].
•
Unlike other -tubulin isotypes, in the III isotype, the presence of threonine residue Thr(429) strongly favors microtubule assembly [41].
•
Despite its restricted and highly selective (predominantly neuronal) cellular distribution in normal organs and tissues, the III isotype is widely, albeit differentially, expressed in a broad range of human tumors of neuronal and non-neuronal origin [9, 20, 21, 42].
Targeting III-Tubulin in Glioblastoma Multiforme
Anti-Cancer Agents in Medicinal Chemistry, 2011, Vol. 11, No. 8
721
human glioblastoma cell line, III-tubulin is co-expressed, albeit differentially compartmentalized, with -tubulin, a protein associated with microtubule nucleation, with which it forms complexes suggesting a possible functional interaction [23, 24]. Conversely, in medulloblastomas, these two proteins are differentially and inversely expressed, with III-tubulin being consistently expressed in tumor cells undergoing neuronal differentiation and formation of neurite-like cell processes [52]. Some of GBM’s histopathologic features provide leads as to the possible pathobiologic correlates accounting for enhanced IIItubulin expression. These include poorly differentiated tumor cells, resembling glial precursor cells and/or bipotential neural stem cells, commonly encountered adjacent to foci of tumor ischemic necrosis [9]. III-TUBULIN AND CANCER STEM CELLS IN GBM In recent years there has been increased interest regarding the role of cancer stem cells (CSC) in GBMs [reviewed in 9]. Along these lines, there is persuasive evidence to suggest that gliomas may be derived from intermediate gliotypic neural stem cells [57]. Glial fibrillary acidic protein (GFAP)+ non-transformed neural progenitors in culture share phenotypic characteristics with poorly differentiated GBM-derived progenitor-like tumor cells (CD44+/ microtubule associated protein-2+/ GFAP+/vimentin+/IIItubulin+/fibronectin+) [58]. As such, GFAP+/nestin+/III-tubulin + cells in GBMs are akin to normal human fetal astrocytes in vitro [47, 59] (Fig. 1C and 1D) and of glial restricted precursor cells or bipotential neural stem cells of the human telencephalic subventricular zones [47]. It follows then that the immunolocalization of III-tubulin in GBM may set apart a subpopulation of tumor cells with cancer stem cell characteristics [57]. Fig. (1). Differential Distribution of III-Tubulin. (A, B) Localization of III-tubulin in a population of P19 murine embryonal carcinoma cells stimulated to undergo neuronal differentiation and neurite formation by aggregation and exposure to retinoic acid as described [46]. Immunofluorescence staining with monoclonal anti-III-tubulin antibody TU-20 [47] (A, red) and polyclonal antibody to -tubulin dimer (B, green). Nuclei are shown in blue. (C, D) Immunofluorescence staining of normal human astrocytes explanted from the cerebral hemispheres of a mid-gestational male fetus [47] (C) and cells of human glioblastoma line T98G (D) with antibody TU20. Bars, 20 m. Photography E. Dráberová (Institute of Molecular Genetics AS CR, Prague).
III-TUBULIN IN BRAIN TUMORS: DIFFERENTIAL EXPRESSION IN GLIAL VS. NEURONAL TUMORS In the CNS, the -tubulin isotype is differentially expressed in embryonal neuronal/neuroblastic neoplasms as compared to neoplasms of glial origin (gliomas). In embryonal brain tumors, such as cerebellar medulloblastomas, cerebral neuroblastomas/suprtentorial primitive neuroectodermal tumors (PNETs), and in the overtly neuronally differentiated central neurocytomas, III-tubulin is expressed in a constitutive (lineage/differentiation-dependent) fashion, that is, in tumor cells undergoing neuritogenesis and ganglionic maturation concomitant with decreased cell proliferation [20, 21, 51, 52]. Inasmuch as III-tubulin is not normally expressed in nascent, mature glia, its expression in glial tumors is construed as being both ectopic and aberrant [9]. III-Tubulin is present, to a variable extent, in all glioma types and grades but its expression and distribution is significantly increased according to an ascending scale of histological malignancy effectively becoming maximal in GBM [9, 18-21, 53]. In an immunohistochemical study on 378 brain tumors using 37 antibodies and tissue microarray (TMA) technology, III-tubulin was one of six marker proteins to show significant differences between high-grade and low-grade gliomas [54]. III-Tubulin is highly expressed in GBMs [18, 23, 55] and in glioblastoma cell lines [23, 56] (Fig. 1D). In GBMs the T98G
INCREASED III-TUBULIN EXPRESSION IN GBM IN THE CONTEXT OF HYPOXIA AND ANGIOGENESIS Genetic changes associated with tumor progression in highgrade gliomas involve signaling pathways that promote growth and control the cell cycle ultimately causing focal hypoxic/ischemic necrosis, and angiogenesis within the tumor bed [60]. Hypoxic cells in GBM are considered to be resistant to ionizing irradiation and chemotherapy. Tumor necrosis and abundant angiogenesis (microvascular proliferation) are two pathognomonic histological features in GBMs. Geographic areas of ischemic necrosis (“palisading necrosis”) are attributed to the presence of major vasoformative changes and vascular structural abnormalities in the tumor bed, including vasoocclusive phenomena and thrombosis, which, collectively, create a hypoxic environment, mediated, in part, by the hypoxia-inducible factor (HIF-1) [61, 62]. The increased expression of III-tubulin in tumor cells adjacent to areas of “palisading necrosis” suggests a possible link between III-tubulin expression and tumor hypoxia-induced oxidative stress [9]. Intriguingly, the abnormal blood vessels themselves in foci of neoplastic vascular proliferation are, for the most part, devoid of III-tubulin expression [18]. In support of oxidative stress of tumor cells in gliomas, evidence of significant protein nitration has been unraveled, notably in GBMs. Nitric oxide upregulation in malignant gliomas may relate to neoplastic transformation, tumor neovascularization, induction of apoptosis, or free radical damage [63]. Proteomic analysis has identifed -tubulin as one of the targets of protein nitration and to that end, peptide mass fingerprinting has revealed that tubulin is nitrated at Tyr224 in surgically excsised GBM specimens whereas no such modification was found in specimens from low-grade gliomas or from non-tumoral cerebral control tissues [64]. Paclitaxel resistance in GBM is found to be associated with expression of pro-survival members of the Bcl-2 family through
722 Anti-Cancer Agents in Medicinal Chemistry, 2011, Vol. 11, No. 8
Katsetos et al.
generated by different DNA-targeting anti-cancer agents, such as cisplatin, doxorubicin, and etoposide [66]. Moreover, the notion that III-tubulin may constitute a survival factor contributing to chemoresistance is derived from the involvement of this protein in cellular adaptation to oxidative stress and glucose deprivation [35]. Lastly, in the platinum- and paclitaxel-sensitive human ovarian cancer cell line A2780, hypoxia is a strong inducer of III-tubulin expression, which is mediated by HIF-1a through methylation of the 3' enhancer of III-tubulin [67].
hypoxia induced phosphorylation of Bad, effectively acting as a cytoprotective response by the hypoxic cells to paclitaxel-induced apoptosis [65]. Given the prominent neovascularization and hypoxic/ischemic phenomena in GBMs, we have hypothesized that the increased expression of III-tubulin in these neoplasms, especially in the vicinity of necrosis may partially represent an adaptive response of tumor cells to oxidative stress and free radical production [9]. Ludueña and colleagues have previously pointed out that the few normal tissues that express III-tubulin, principally the CNS, also are rich in free radicals and reactive oxygen species and have entertained the idea that one of the functions of III-tubulin is to protect microtubules from the adverse effects of free radicals [28]. These authors posited that lack of cys239 may allow III-tubulin to assemble in the presence of free radicals may explain, in part, the expression and upregulation of III-tubulin in neoplasms, regardless of cell lineage, which are accompanied by high production of free radicals. Knocking down III-tubulin expression augments the susceptibility of cancer cells to antioxidants, that is, independent of and in addition to TBAs. Thus, III-tubulin may subserve the role of a survival factor protecting tumor cells from cell death signals Box 3.
TUBULIN BINDING AGENTS Natural product small molecule inhibitors that bind to the tubulin–microtubule system represent an important group of anticancer agent, also referred to as tubulin-binding agents (TBAs) [68]. They are broadly grouped into microtubule-destabilizing drugs (such as the vinca alkaloids, vincristine, vinblastine and vinorelbine) and microtubule-stabilizing drugs (such as taxanes and epothilones) (Box 3 and Fig. 2). TBAs are also refered to as antimitotic agents as they arrest cells in mitosis and induce cell death [10]. The elucidation of the binding sites of the clinically significant TBAs to tubulin has been accomplished through delineation of the crystal
Tubulin-Binding Agents*
•
At high concentrations, TBAs are broadly classified as microtubule-stabilizing or microtubule-destabilizing agents according to their effects on microtubule polymers [2]. However, these compounds can also exert their lethal effects on cancer cells at drug concentrations that disrupt microtubule dynamics without markedly affecting microtubule polymer levels [2, 69].
•
Vinca alkaloids are microtubule-destabilizing drugs and the first TBAs introduced in the 1970s for the treatment of cancer, transforming from then on leukemia therapy. Nowadays, they are being used in conjunction with other drugs for the treatment of a broad range of malignant tumors [2].
•
Taxanes are microtubule-stabilizing drugs, the first TBAs in their class identified, which are nowadays routinely used in the treatment of solid tumors, principally lung, breast and ovarian cancers [70].
•
Epothilones are the newest clinically approved anti-mitotic agents that target microtubules exhibiting activity in a subset of paclitaxel-refractory tumors [71]. Currently, they are used for the treatment of drug-refractory advanced breast cancer and are under evaluation for the treatment of a range of other solid tumors including GBM [72, 73].
*Modified after Kavallaris [10] by permission (Copyright Nature Publishing Group) Abbreviations: GBM, glioblastoma multiforme; TBA, tubulin-binding agent O
O
O
O
S
O OH
N
O
NH
H
O
OH O
OH
O O
OH
OH
O
H O
O
O
O
O
OO
O
S
H N
HO
N
OH
lxabepilone
Epothilone B
Paclitaxel
C2H3
N
COOH H
H3CO
HN O O
H N O
H O
Vincristine sulfate
HO
O
O S OH
N
H O
HO
OH
N
OH O O
H
HO C H C2H3 H O
COOH
O C CH3 C OCH3 H3C HO O N
CH3
Vinorelbine tartrate
H2SO4 N
OCH 3
CH3
N
O
O
2
N
N H HCO C 3 O
H C OH
NH H 3CO
H N H HO O CH 3
CH 3
O H O OCH3
Vinblastine sulfate
Fig. (2). Chemical structures of Tubulin-Binding Agents (TBAs). This figure depicts the diverse and complex nature of representative natural product small molecule inhibitors belonging to the Taxane, epothilone and Vinca groups.
Targeting III-Tubulin in Glioblastoma Multiforme
Anti-Cancer Agents in Medicinal Chemistry, 2011, Vol. 11, No. 8
structure of the -tubulin heterodimer [74, 75]. TBAs suppress spindle microtubule dynamics, causing a delay or block at the metaphase–anaphase transition during mitosis. Activation of the spindle assembly checkpoint is triggered by disruption of the mitotic spindle, which gives rise to a protracted mitotic arrest potentially culminating in tumor cell death [76]. The exact mechanism of cell death owing to mitotic arrest remains controversial [77]. For the most part, cancer cells are more susceptible to TBA-induced cell death as compared with normal/diploid cells; however, the relationship of the duration of mitotic arrest and the extent of cell death is highly variable among non-transformed versous cancerous cells [78]. In this regard, wide variation of responses to anti-mitotic TBAs, including the archetypical taxane, paclitaxel, has been reported both among different cell lines but also in subpopopulations of cells within the same cell line [79]. Adding to the mechanistic complexity and diversity of TBA-induced cell death, occasionally, the cellular fate appears not to be influenced by the duration of mitotic arrest but by two competing networks: the activation of cell death pathways and another network that regulates degradation of cyclin B1 and exit from mitosis [10, 79]. The mitotic checkpoint delays anaphase and exit from mitosis until all kinetochores are attached to microtubules in order to maintain genomic integrity and lessen the incidence of aneuploidy [10]. Yet, when there are one or more kinetochores that remain unattached, the cell cycle checkpoint cannot be satisfied and, owing to a protracted delay (~20 hours), many cells may undergo “mitotic slippage” (that is, escape from mitosis) and form tetraploid cells [10]. Mitotic slippage occurs because the cyclin B–cyclindependent kinase 1 (CDK1) complex is degraded in the presence of BOX 4.
723
an active cell cycle checkpoint [80]. In the presence of paclitaxel, depending on the drug concentration used in vitro, cells can either die at the G1 phase of the cell cycle [78], arrest in mitosis and die, or, alternatively, escape by virtue of “mitotic slippage” [15, 77]. The elucidation of TBA-mediated cell death remains currently under investigation requiring cutting edge experiments with state-of-theart cell imaging approaches such as intravital microscopy [81]. III-TUBULIN AND RESISTANCE OF CANCER CELLS TO TAXANES: POTENTIAL IMPLICATIONS IN GBM Microtubule-stabilizing drugs, specifically taxanes and epothilones, block mitosis and cell proliferation by targeting the microtubule cytoskeleton [4, 10, 49]. These agents bind to the tubulin subunit of /-tubulin, giving rise to mitotic arrest and apoptosis. The taxanes are extensively used agents employed in chemotherapeutic regimens for the management of diverse tumor types. Paclitaxel binds to -tubulin, causing microtubule polymerization that blocks mitosis by virtue of kinetic stabilization of spindle microtubules [82]. However, the clinical efficacy of TBAs in general and of taxanes in particular is hindered considerably by the development of primary or acquired drug resistance [4, 10]. To date, at least four, possibly five, mechanisms of chemoresistance to TBAs have come to light (Box 4). A schematic diagram pertaining to potential mechanisms of TBA resistance is also presented on Fig. (3). Whilst all mechanisms of drug resistance may be potentially relevant in this connection, there is little or questionable proof in support of multi-drug resistance (MDR)-1 gene overexpression, -tubulin point mutation and/or polymorphisms in solid tumors [10, 49, 86].
Potential Mechanisms of Taxane Resistance to Tubulin-Binding Agents
•
Overexpression of the MDR-1 gene [83].
•
Point mutations of -tubulin at the paclitaxel-binding site [84].
•
Polymorphisms in tubulin isotypes [10].
•
Selective overexpression of class I, III, and IVa -tubulin isotypes [4, 8, 10, 48, 49, 85]
•
Deficient apoptotic signaling downstream of the microtubule-associated insults to which tumor cells are exposed [4].
Tubulin Binding Agents (TBAs) Multidrugg transporters efflux drug Cell membrane
Suppress microtubule dynamics
• Altered MAP expression • Aberrant βIII-tubulin expression
Mitotic arrest Anti-apoptotic factors Apoptosis Fig. (3). Tubulin-Binding Agent Resistance. Drug resistance is complex and multifactorial. Resistance to tubulin-binding agent (TBA) ranges from reduced drug accumulation due to multidrug transporters and drug efflux, alterations in the drug target, e.g., changes in the expression of microtubule associated proteins (MAP) and aberrant expression of III-tubulin, to defects in apoptotic pathways.
724 Anti-Cancer Agents in Medicinal Chemistry, 2011, Vol. 11, No. 8
Ludueña was the first to show that the presence of III-tubulin inhibits paclitaxel-induced -tubulin polymerization [87], whilst Kavallaris was the first to discover that paclitaxel-resistant ovarian cancer cells overexpress classes I, III, and IVa -tubulin isotypes [85]. Several ensuing studies have demonstrated that increased expression of III-tubulin in diverse epithelial malignancies constitutes a most important mechanism of TBA chemoresistance [8, 8890]. Overexpression of III-tubulin has been implicated in clinical resistance encountered in NSCLC, ovarian and breast tumors subjected to combined treatment with tubulin-binding and DNAdamaging agents [10]. Upregulation of III-tubulin expression has emerged as a common mechanism by which taxane resistance develops in patients with advanced epithelial cancers [48, 49, 90]. Potentially, III-tubulin status carries a key role in the therapy of chemorefractory NSCLC [66] and other solid tumors. In TC1 and OVCAR-3 drug-resistant ovarian cancer cell lines III-tubulin exists in two posttranslationally modified forms, a higher molecular weight glycosylated and phosphorylated form associated with the microtubule cytoskeleton, and a lower molecular weight form, which is distinctively associated with mitochondria [35]. Both of these post-translational modifications were found to be associated with drug-resistant tumor phenotypes [35]. The isotype difference in paclitaxel binding may rest with a kinetic effect stemming from the isotype difference at residue serine 275, which is replaced by alanine in the III and VI isotypes [91]. Because III- and V-tubulin may be expressed in a complementary manner in ovarian carcinoma cells, the elucidation of the combined expression of these two proteins may be clinically useful in terms of tumor responses to TBAs [92]. In all, while III-tubulin is probably not an exclusive determinant of drug resistance to TBAs, direct targeting of this protein may enhance the efficacy of TBAs by finally overcoming one of the key factors accounting for the development of chemoresistance [8, 35, 92-94]. Treatment of the human glioblastoma cell line T98G with taxol led to the formation of III-tubulin labeled microtubule bundles while prominent micropunctate and diffuse -tubulin staining in tumor cells remained unaffected [23]. However, the effect of taxanes on III–tubulin enriched microtubules in GBM cells requires further investigation. EPOTHILONES: A PROMISING VISTA FOR GBM TREATMENT Epothilones represent a new class of TBAs with promising preclinical and clinical activity [10]. Like paclitaxel, epothilones belong to the family of microtubule stabilizing agents that disrupt microtubule dynamics by binding to -tubulin and stabilize microtubules, and by inducing mitotic arrest and apoptosis [10]. Their cellular target is -tubulin but the factors influencing intrinsic sensitivity to epothilones are not well understood. Both epothilones and taxanes share overlapping, albeit not identical, binding sites on tubulin [75]. Clinical trials using epothilone B (Patupilone; Novartis) or a novel analogue of epothilone B (Ixabepilone; Bristol-Myers Squibb, BMS247550) (see Fig. 2), have demonstrated antineoplastic activity of these agents alone or in combination with other agents in patients with metastatic breast cancer that is resistant to taxanes and anthracyclines [95-97]. Neurotoxicity, primarily in the context of peripheral neuropathy, remains a concern but in spite of the structural similarity between ixabepilone and patupilone, the latter apparently displays less neurotoxicity [98]. Epothilones have also shown promising clinical activity in a Phase II trial in patients with NSCLC [99], whilst they have been recently approved for use in chemoresistant metastatic breast cancer [49]. A clear benefit of epothilones rests on their ability to bypass the resistance mechanisms associated with drug efflux pumps and paclitaxel resistance associated with -tubulin mutations in cell lines [49, 100] (see Fig. 3). Patupilone was shown to be active in III-tubulin-expressing ovarian tumour cell lines, and molecular modelling of target bind-
Katsetos et al.
ing suggests that it binds III-tubulin more effectively than paclitaxel [94, 100]. Dumontet and colleagues have recently suggested that the significant antitumor activity of ixabepilone in taxane-resistant tumors, may be due to the drug’s preferential suppression of the dynamic instability of /III-microtubules in cells expressing high levels of III-tubulin [49]. That said, the use of ixabepilone in the context of brain tumors may be hampered by the drug’s inability to cross the blood-brain barrier [101]. In the clinical setting of GBM, patupilone may be preferred over ixabepilone, because the former (patupilone) can cross also the blood-brain barrier as its activity is fully P-gp independent [100, 102]. The clinical resistance of GBM to chemotherapeutic agents can be attributed to drug efflux pumps, such as P-glycoprotein, which contributes to reduce drug efficacy [103]. Epothilone B is not a substrate for P-glycoprotein on GBM cells [103]. A clinically attainable concentration of epothilone B has been found to be capable of inducing a significant and sustained cytotoxic response in p53 mutant (but not in p53 wild type) glioblastoma cells suggesting that epothilone B is a potential alternative to traditional TBAs such as vincristine and paclitaxel used to treat clinical glioblastomas with p53 mutations [103]. In a transgenic mouse GBM model, blocking EGFR with EGFR inhibitor AEE788 in combination with sublethal concentrations of the epothilone microtubule stabilizer patupilone induced apoptosis and reduced cell proliferation in GBM cells as compared to EGFR inhibitor alone, which was ineffective [104]. Sagopilone (ZK-EPO), a novel epothilone, under development by Bayer Schering Pharma AG, is a fully synthetic epothilone B analog that is representative of a new class of microtubulestabilizing agents that have a similar mechanism of action to taxanes, but have a decreased propensity for drug resistance [72]. In preclinical studies, sagopilone inhibited cell growth in a wide range of human cancer cell lines whilst it was not recognized by multidrug-resistant (MDR) cellular efflux mechanisms, and maintained its activity in MDR tumor models [72]. Initial reports have shown that sagopilone was clinically active in advanced ovarian cancer, advanced melanoma and metastatic chemotherapy-naïve castration-resistant prostate cancer [72]. Along these lines, sagopilone (ZK-EPO) was found to be more efficacious as compared to other anticancer agents (ie paclitaxel or temozolomide), in orthotopic rodent models of human GBM (U373MG and U87MG glioblastoma lines) and metastatic brain tumors (MDA-MB-435 melanoma, or patient-derived NSCLC) [73]. Sagopilone crosses the blood-brain barrier in both rat and mouse models, leading to therapeutically relevant concentrations in the brain with a long half-life [73]. Interestingly, sagopilone exhibited significant antitumor activity in both U373MG and U87MG models of human GBM cell lines as compared to paclitaxel, which showed only a limited effect in the U373MG GBM model [73]. Moreover, sagopilone significantly inhibited the growth of tumors from CNS metastasis models implanted in the brains of nude mice, in contrast to paclitaxel or temozolomide [73]. In a subsequent study, sagopilone (ZK-EPO) was found to have an acceptable safety profile and clinically relevant activity in patients with pretreated, recurrent malignant gliomas [105]. Peripheral neuropathy [72, 105] and neutropenia [72] are the most common treatment-related adverse effects. Sagopilone is currently being investigated in a broad phase II clinical trial program, including patients with GBM, NSCLC, breast cancer, and melanoma [72, 73]. In a recent study conducted in the laboratory of one of us (M.K.), the functional significance of specific -tubulin isotypes in intrinsic sensitivity to epothilone B was investigated using siRNA gene knockdown against II-, III- or IV-tubulins in two well established NSCLC cell lines, NCI-H460 and Calu-6. Drug-treated clonogenic assays showed that sensitivity to epothilone B was significantly increased after III-tubulin knockdown in contrast to
Targeting III-Tubulin in Glioblastoma Multiforme
knockdown of II-tubulin where no drug sensitization effect was observed. Cell cycle analysis of III-tubulin knockdown cells showed a higher percentage of cell death with epothilone B concentrations as low as 0.5 nM in contrast to IVb-tubulin knockdown cells, which displayed a decrease in epothilone B-induced G2-M cell cycle accumulation compared to control siRNA cells. Importantly, III-tubulin knockdowns displayed a significant dosedependent increase in the percentage of apoptotic cells upon treatment with epothilone B, as determined by caspase 3/7 activity and annexin-V staining. By comparison, higher concentrations of epothilone B were required to induce apoptosis in the IV-tubulin knockdowns compared to control siRNA [personal communication with M.K. / Re: Epothilones and tubulin isotypes]. An apparent contradiction in the available data on III-tubulin is that on the one hand III-tubulin modeling has indicated that epothilones may bind more efficiently to III-tubulin containing microtubules (as compared to other -tubulin isotypes) and yet knockdown of III-tubulin also appears to make cells more sensitive to epothilone B. Two possible explanations are entertained in this regard. First, the original crystal structure of -tubulin bound to epothilone was the result of epothilone A (as opposed to epothilone B) binding [75]. Slight differences in the structure between epothilones A and B may account for some differences in predicted drug-target interactions. Second, there is also increasing evidence that III-tubulin is a survival factor which may be exerting its effects independent of the microtubule structure [50, 66, 100]. This is also supported by the observation that knockdown of III-tubulin does not significantly alter microtubule dynamics [106]. OTHER POTENTIALLY PROMISING MISCELLANEOUS TUBULIN-BINDING AGENTS Aside from the epothilones, taccalonolides, a distinct class of microtubule-stabilizing agents [107] and tasidotin, an analogue of dolastatin 15 [108], can also overcome taxane resistance. In this regard, secotaxoids, which are thought to bind well with high affininty to III-tubulin and retain activity in paclitaxel-resistant preclinical models, appear to be particularly promising although they require further evaluation [93]. Several other promising agents have been reported in preclinical models including eleutherobin, laulimalide, hemiasterlins, peloruside A22, taccalonolide, coumarins, and cyclostreptin [reviewed in 4]. Severalof these novel agents are not substrates of efflux pumps, such as P-gp or other ABC proteins [4]. MOVING FORWARD: “DOUBLE HIT” INTERVENTION USING COMBINED MOLECULAR-PHARMACOLOGICAL APPROACHES Inasmuch as tumor aggressiveness and in some cases sensitivity to TBA chemotherapy is influenced by the degree of expression of III-tubulin, the development of agents targeting this isotype would be of particular interest in patients with high-risk disease owing to the high expression of this isotype. It is becoming increasingly clear that potential treatment of GBM is bound to rest on combined molecular-pharmacological interventions, which are likely to be tailored according to the unique features displayed by individual tumors. Such a strategy was first undertaken by inhibition of III-tubulin by antisense oligonucleotides and by RNA silencinginduced sensitization of tumor cells to various antineoplastic agents [66, 88]. In this regard, lessons of cellular therapeutics may be derived from recent cutting-edge functional studies on lung cancer. According to these studies, III-tubulin mediates not only drug sensitivity but also the incidence and progression of lung cancer [50]. It follows then, that III-tubulin constitutes a cellular survival factor which, when suppressed by silencing approaches, sensitizes cells to chemotherapy with traditional TBAs, such as Vinca alkaloids (vincristine), taxanes (paclitaxel), and DNA damaging agents, via enhanced apoptosis induction and decreased tumorigenesis [50].
Anti-Cancer Agents in Medicinal Chemistry, 2011, Vol. 11, No. 8
725
Doubtless, the concept of tumor cell hypersensitization to TBAs by way of RNA silencing of III-tubulin expression is exemplified in NSCLC [106]. In the absence of drug therapy, III-tubulin knockdown may cause no significant change in microtubule dynamic instability whereas, on the other hand, III-tubulin knockdown may significantly enhance the effectiveness of TBAs through two distinct mechanisms: (a) suppression of microtubule dynamics at low drug concentrations and (b) a mitosis-independent mechanism of cell death at higher drug concentrations [106]. From the practical standpoint, another potentially attractive approach of delivery involves vectorization of microtubule-binding agents to tumor cells using monoclonal antibodies [4]. Considering the widespread expression of III-tubulin in GBM [9, 18-24], taken together, the aforementioned findings reported in solid tumors outside the CNS, raise enthusiasm and provide a sound rationale and a tangible focus for preclinical studies both on GBM cell lines in vitro and on III-tubulin-overexpressing or knockdown human GBM xenografts, to be followed by clinical trials exploring suitable epothilone B compounds, especially patupilone, in the treatment of GBM. DISCLOSURE Part of information included in this chapter has been previously published in the Journal of Cellular Physiology Volume 221, Issue 3, pages 505–513, December 2009” (see reference 9 in the literature cited below: Katsetos, C.D.; Dráberová, E.; Legido, A.; Dumontet, C.; Dráber, P. Tubulin targets in the pathobiology and therapy of glioblastoma multiforme. I. Class III -tubulin. J Cell Physiol. 2009, 221, 505-513.) ACKNOWLEDGEMENTS This work was suported in part by grant #203 from the St. Christopher’s Foundation for Children (CDK), Grant P302/10/1701 from Grant Agency of the Czech Republic (PD), and by Institutional Research Support (AVOZ 50520514) (PD). Children’s Cancer Institute Australia for Medical Research is affiliated with the University of New South Wales and the Sydney Children’s Hospital. MK is supported by grants from the New South Wales Cancer Council (MK) and by a NHMRC Senior Fellowship (MK). ABBREVIATIONS III-tubulin = Class III -tubulin GBM = Glioblastoma multiforme MDR = Multi-drug resistance NSCLC = Non-small-cell lung cancer TBA = Tubulin binding agent(s) REFERENCES [1]
[2]
[3] [4]
[5]
[6]
[7]
Ohgaki, H.; Kleihues, P. Population-based studies on incidence, survival rates, and genetic alterations in astrocytic and oligodendroglial gliomas. J Neuropathol Exp Neurol. 2005, 64, 479-489. Jordan, M.A.; Wilson, L. Microtubules and actin filaments: dynamic targets for cancer chemotherapy. Curr Opin Cell Biol. 1998, 10, 123-130. Pasquier, E.; Kavallaris, M. Microtubules: a dynamic target in cancer therapy. IUBMB Life. 2008, 60, 165-170. Dumontet, C; Jordan, M.A. Microtubule-binding agents: a dynamic field of cancer therapeutics. Nat Rev Drug Discov. 2010, 9, 790-803. Whipple, R.A.; Cheung, A.M.; Martin, S.S. Detyrosinated microtubule protrusions in suspended mammary epithelial cells promote reattachment. Exp Cell Res. 2007, 313, 1326-1336. Schoumacher, M.; Goldman, R.D.; Louvard, D.; Vignjevic, D.M. Actin, microtubules, and vimentin intermediate filaments cooperate for elongation of invadopodia. J Cell Biol. 2010, 189, 541-556. Liaw, T.Y.; Chang, M.H.; Kavallaris, M. The cytoskeleton as a therapeutic target in childhood acute leukemia: obstacles and opportunities. Curr Drug Targets. 2007, 8, 739-749.
726 Anti-Cancer Agents in Medicinal Chemistry, 2011, Vol. 11, No. 8 [8]
[9]
[10] [11]
[12]
[13] [14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
[26]
Ferlini, C.; Raspaglio, G.; Cicchillitti, L.; Mozzetti, S.; Prislei, S.; Bartollino, S.; Scambia, G. Looking at drug resistance mechanisms for microtubule interacting drugs: does TUBB3 work? Curr Cancer Drug Targets. 2007, 7, 704-712. Katsetos, C.D.; Dráberová, E.; Legido, A.; Dumontet, C.; Dráber, P. Tubulin targets in the pathobiology and therapy of glioblastoma multiforme. I. Class III -tubulin. J Cell Physiol. 2009, 221, 505-513. Kavallaris, M. Microtubules and resistance to tubulin-binding agents. Nat Rev Cancer. 2010, 10, 194-204. Régina, A.; Demeule, M.; Ché, C.; Lavallée, I.; Poirier, J.; Gabathuler, R.; et al. Antitumour activity of ANG1005, a conjugate between paclitaxel and the new brain delivery vector Angiopep-2. Br J Pharmacol. 2008, 155, 185-197 Mielke, S.; Sparreboom, A.; Mross, K. Peripheral neuropathy: a persisting challenge in paclitaxel-based regimes. Eur J Cancer. 2006, 42, 24-30. Baas, P.W.; Karabay, A.; Qiang, L. Microtubules cut and run. Trends Cell Biol. 2005, 15, 518-524. Richter-Landsberg, C. The cytoskeleton in oligodendrocytes. Microtubule dynamics in health and disease. J Mol Neurosci. 2008, 35, 55-63. Shi, J.; Orth, J.D.; Mitchison, T. Cell type variation in responses to antimitotic drugs that target microtubules and kinesin-5. Cancer Res. 2008, 68, 3269-3276. Hornick, J.E.; Karanjeet, K.; Collins, E.S.; Hinchcliffe, E.H. Kinesins to the core: The role of microtubule-based motor proteins in building the mitotic spindle midzone. Semin Cell Dev Biol. 2010, 21, 290-299 Dráberová, E.; Vinopal, S.; Morfini, G.; Liu, P.S.; Sládková, V.; Sulimenko, T.; Burns, M.R.; Solowska, J.; Kulandeival, K.; de Chadarevian, J.-P.; Legido, A.; Mörk, S.J.; Janácek, J.; Baas, P.W.; Dráber, P.; Katsetos, C.D. Microtubule severing ATPase spastin in glioblastoma multiforme: Increased expression in human glioblastoma cell lines and roles in cell motility and proliferation. J. Neuropathol. Exp. Neurol., 2011, 70, 811-826. Katsetos, C.D.; Del Valle, L; Geddes, J.F.; Assimakopoulou, M.; Legido, A; Boyd, J.C.; Balin, B.; Parikh, N.A.; Maraziotis, T.; de Chadarevian, J.P.; Varakis, J.N.; Matsas, R.; Spano, A.; Frankfurter, A.; Herman, M.M.; Khalili, K. Aberrant localization of the neuronal class III -tubulin in astrocytomas. Arch Pathol Lab Med. 2001, 125, 613-624. Katsetos, C.D.; Del Valle, L.; Geddes, J.F.; Aldape, K.; Boyd, J.C.; Legido, A.; Khalili, K.; Perentes, E.; Mörk, S.J. Localization of the neuronal class III -tubulin in oligodendrogliomas: comparison with Ki-67 proliferative index and 1p/19q status. J Neuropathol Exp Neurol. 2002, 61, 307-320. Katsetos, C.D.; Herman, M.M.; Mörk, S.J. Class III -tubulin in human development and cancer. Cell Motil Cytoskeleton. 2003, 55, 77-96. Katsetos, C.D.; Legido, A.; Perentes, E.; Mörk, S.J. Class III -tubulin isotype: a key cytoskeletal protein at the crossroads of developmental neurobiology and tumor neuropathology. J Child Neurol. 2003, 18, 851-866. Katsetos, C.D.; Reddy, G.; Dráberová, E.; mejkalová, B.; Del Valle, L.; Ashraf, Q.; Tadevosyan, A.; Yelin, K.; Maraziotis, T.; Mishra, O.P.; Mörk, S.; Legido, A.; Nissanov, J.; Baas, P.W.; de Chadarévian, J.-P.; Dráber, P. Altered cellular distribution and subcellular sorting of -tubulin in astrocytic gliomas and human glioblastoma cell lines. J Neuropathol Exp Neurol. 2006, 65, 465-477. Katsetos, C.D.; Dráberová, E.; mejkalová, B.; Reddy, G.; Bertrand, L.; de Chadarévian, J.-P.; Legido, A.; Nissanov, J.; Baas, P.W.; Dráber, P. Class III -tubulin and -tubulin are co-expressed and form complexes in human glioblastoma cells. Neurochem Res. 2007, 32, 1387-1398. Katsetos, C.D.; Dráberová, E.; Legido, A.; Dráber, P. Tubulin targets in the pathobiology and therapy of glioblastoma multiforme. II. -Tubulin. J Cell Physiol. 2009, 221, 514-520. Ludueña, R.F. The multiple forms of tubulin: different gene products and covalent modifications. Int Rev Cytol. 1998, 178, 207-275. Mitchison, T.; Kirschner, M. Dynamic instability of microtubule growth. Nature. 1984, 312, 237-242.
Katsetos et al. [27] [28]
[29]
[30] [31] [32]
[33]
[34]
[35]
[36] [37] [38]
[39]
[40] [41]
[42]
[43]
[44]
[45]
[46]
[47]
Doxsey, S. Re-evaluating centrosome function. Nat Rev Mol Cell Biol. 2001, 2, 688-698. Ludueña, R.F.; Banerjee, A. The isotypes of tubulin: distribution and functional significance. In: Cancer Drug Discovery and Development: The Role of Microtubules in Cell Biology, Neurobiology, and Oncology, Fojo, T., Ed., Humana Press, Totowa, NJ; 2008, pp. 123-175, Verdier-Pinard, P.; Pasquier, E.; Xiao, H.; Burd, B.; Villard, C.; Lafitte, D.; Miller, L.M.; Angeletti, R.H.; Horwitz, S.B.; Braguer, D. Tubulin proteomics: towards breaking the code. Anal Biochem. 2009, 384,197-206. Downing, K.H.; Nogales, E. Tubulin and microtubule structure. Curr Opin Cell Biol. 1998, 10, 16-22. Jordan, M.A.; Kamath, K. How do microtubule-targeted drugs work? An overview. Curr Cancer Drug Targets. 2007, 7, 730-742. Linhartová, I.; Dráber, P.; Dráberová, E.; Viklick , V. Immunological discrimination of -tubulin isoforms in developing mouse brain. Post-translational modification of non-class-III -tubulins. Biochem J. 1992, 288, 919-924. Alexander, J.E.; Hunt, D.F.; Shabanowitz, J.; Michel, H.; Berlin, S.C.; MacDonald, T.L.; Sundberg, R.J.; Rebhun, L.I.; Frankfurter, A. Characterization of posttranslational modifications in neuron specific class III -tubulin by mass spectrometry. Proc Natl Acad Sci USA. 1991, 88, 4685-4689. Mary, J.; Redeker, V.; Le Caer, J.P.; Promé, J.C.; Rossier, J. Class I and IVa -tubulin isotypes expressed in adult mouse brain are glutamylated. FEBS Lett. 1994, 353, 89-94. Cicchillitti, L.; Penci, R.; Di Michele, M.; Filippetti, F.; Rotilio, D.; Donati, M.B.; Scambia, G.; Ferlini, C. Proteomic characterization of cytoskeletal and mitochondrial class III -tubulin. Mol Cancer Ther. 2008, 7, 2070-2079. Westermann, S.; Weber, K. Post-translational modifications regulate microtubule function. Nat Rev Mol Cell Biol. 2003, 4, 938-947. Verhey, K.J.; Gaertig, J. The tubulin code. Cell Cycle. 2007, 6, 2152-2160. Ludueña, R.F.; Banerjee, A. The post-translational modifications of tubulin. In: Cancer Drug Discovery and Development: The Role of Microtubules in Cell Biology, Neurobiology, and Oncology (Fojo T, ed.), 2008, pp. 105-121, Humana Press, Totowa, NJ. Joe, P.A.; Banerjee, A.; Ludueña, R.F. The roles of cys124 and ser239 in the functional properties of human betaIII tubulin. Cell Motil Cytoskeleton. 2008, 65, 476-486. Khan, I.A.; Ludueña, R.F. Phosphorylation of III-tubulin. Biochemistry. 1996, 35, 3704-3711. Joe, P.A.; Banerjee, A.; Ludueña, R.F. Roles of -tubulin residues Ala428 and Thr429 in microtubule formation in vivo. J Biol Chem. 2009, 284, 4283-4291. Dráberová, E.; Luká , Z.; Ivanyi, D.; Viklick , V.; Dráber, P. Expression of class III -tubulin in normal and neoplastic human tissues. Histochem Cell Biol. 1998, 109, 231-239. Katsetos, C.D.; Herman, M.M.; Frankfurter, A.; Uffer, S.; Perentes, E.; Rubinstein, L.J. Neuron-associated class III -tubulin isotype, microtubule-associated protein 2, and synaptophysin in human retinoblastomas in situ. Further immunohistochemical observations on the Flexner-Wintersteiner rosettes. Lab Invest. 1991, 64, 45-54. Katsetos, C.D.; Frankfurter, A.; Christakos, S.; Mancall, E.L.; Vlachos, I.; Urich, H. Differential localization of class III -tubulin isotype and calbindin-D28k defines distinct neuronal types in the developing human cerebellar cortex. J Neuropathol Exp Neurol. 1993, 52, 655-666. Katsetos, C.D.; Karkavelas, G.; Herman, M.M.; Vinores, S.A.; Provencio, J.; Spano, A.J.; Frankfurter, A. Class III -tubulin isotype (III) in the adrenal medulla: I. Localization in the developing human adrenal medulla. Anat Rec. 1998, 250, 335-343. Kukharskyy, V.; Sulimenko, V.; Mac rek, L.; Sulimenko, T.; Dráberová, E.; Dráber, P. Complexes of -tubulin with nonreceptor protein tyrosine kinases Src and Fyn in differentiating P19 embryonal carcinoma cells. Exp Cell Res. 2004, 298, 218-228. Dráberová, E.; Del Valle, L.; Gordon, J.; Marková, V.; mejkalová, B.; Bertrand, L.; de Chadarévian, J.-P.; Agamanolis, D.P.; Legido, A.; Khalili, K.; Dráber, P.; Katsetos, C.D. Class III -tubulin is constitutively coexpressed with glial fibrillary acidic protein and nestin in midgestational human fetal astrocytes: implications for phenotypic identity. J Neuropathol Exp Neurol. 2008, 67, 341-354.
Targeting III-Tubulin in Glioblastoma Multiforme [48]
[49]
[50]
[51]
[52]
[53]
[54]
[55]
[56]
[57]
[58]
[59]
[60] [61]
[62]
[63]
[64]
[65]
Sève, P.; Dumontet, C. Is class III -tubulin a predictive factor in patients receiving tubulin-binding agents? Lancet Oncol. 2008, 9, 168-175. Dumontet, C.; Jordan, M.A.; Lee, F.F. Ixabepilone: targeting IIItubulin expression in taxane-resistant malignancies. Mol Cancer Ther. 2009, 8, 17-25. McCarroll, J.A.; Gan, P.P.; Liu, M.; Kavallaris, M. III-Tubulin is a multifunctional protein involved in drug sensitivity and tumorigenesis in non-small cell lung cancer. Cancer Res. 2010, 70, 4995-5003. Katsetos, C.D.; Del Valle, L.; Legido, A.; de Chadarevian, J.-P.; Perentes, E.; Mörk, S.J. On the neuronal/neuroblastic nature of medulloblastomas: a tribute to Pio del Rio Hortega and Moises Polak. Acta Neuropathol (Berl). 2003, 105, 1-13. Caracciolo, V.; D'Agostino, L.; Dráberová, E.; Sládková, V.; Crozier-Fitzgerald, C.; Agamanolis, D.P.; de Chadarévian, J.-P.; Legido, A.; Giordano, A.; Dráber, P.; Katsetos, C.D. Differential expression and cellular distribution of -tubulin and III-tubulin in medulloblastomas and human medulloblastoma cell lines. J Cell Physiol. 2010, 223, 519-529. Mao, Y.; Zhou, L.; Zhu, W.; Wang, X.; Yang, G.; Xie, L.; Mao, X.; Jin, K. Proliferative status of tumor stem cells may be correlated with malignancy grade of human astrocytomas. Front Biosci. 2007, 12, 2252-2259. Ikota, H.; Kinjo, S.; Yokoo, H.; Nakazato, Y. Systematic immunohistochemical profiling of 378 brain tumors with 37 antibodies using tissue microarray technology. Acta Neuropathol (Berl). 2006, 111, 475-482. Martinez-Diaz, H.; Kleinschmidt-DeMasters, B.K.; Powell, S.Z.; Yachnis, A.T. Giant cell glioblastoma and pleomorphic xanthoastrocytoma show different immunohistochemical profiles for neuronal antigens and p53 but share reactivity for class III -tubulin. Arch Pathol Lab Med. 2003, 127, 1187-1191. Lopes, M.B.; Frankfurter, A.; Zientek, G.M.; Herman, M.M. The presence of neuron-associated microtubule proteins in the human U-251 MG cell line. A comparative immunoblot and immunohistochemical study. Mol Chem Neuropathol. 1992, 17, 273-287. Walton, N.M.; Snyder, G.E.; Park, D.; Kobeissy, F.; Scheffler, B.; Steindler, D.A. Gliotypic neural stem cells transiently adopt tumorigenic properties during normal differentiation. Stem Cells. 2009, 27, 280-289. Rieske, P.; Golanska, E.; Zakrzewska, M.; Piaskowski, S.; HulasBigoszewska, K.; Wolaczyk, M.; Szybka, M.; WitusikPerkowska, M.; Jaskolski, D.J.; Zakrzewski, K.; Biernat, W.; Krynska, B.; Liberski, P.P. Arrested neural and advanced mesenchymal differentiation of glioblastoma cells-comparative study with neural progenitors. BMC Cancer. 2009, 9, 54. Rieske, P.; Azizi, S.A.; Augelli, B.; Gaughan, J.; Krynska, B. A population of human brain parenchymal cells express markers of glial, neuronal and early neural cells and differentiate into cells of neuronal and glial lineages. Eur J Neurosci. 2007, 25, 31-33. Louis, D.N. Molecular pathology of malignant gliomas. Annu Rev Pathol. 2006, 1, 97-117. Fischer, I.; Gagner, J.P.; Law, M.; Newcomb, E.W.; Zagzag D. Angiogenesis in gliomas: biology and molecular pathophysiology. Brain Pathol. 2005, 15, 297-310. Rong, Y.; Durden, D.L.; Van Meir, E.G.; Brat, D.J. 'Pseudopalisading' necrosis in glioblastoma: a familiar morphologic feature that links vascular pathology, hypoxia, and angiogenesis. J Neuropathol Exp Neurol. 2006, 65, 529-539. Lam-Himlin, D.; Espey, M.G.; Perry, G.; Smith, M.A.; Castellani, R.J. Malignant glioma progression and nitric oxide. Neurochem Int. 2006, 49, 764-768. Fiore, G.; Di Cristo, C.; Monti, G.; Amoresano, A.; Columbano, L.; Pucci, P.; Cioffi, F.A.; Di Cosmo, A.; Palumbo, A.; d'Ischia, M. Tubulin nitration in human gliomas. Neurosci Lett. 2006, 394, 57-62. Merighi, S.; Benini, A.; Mirandola, P.; Gessi, S.; Varani, K.; Leung, E.; Maclennan, S.; Baraldi, P.G.; Borea, P.A. Hypoxia inhibits paclitaxel-induced apoptosis through adenosine-mediated phosphorylation of bad in glioblastoma cells. Mol Pharmacol. 2007, 72, 162-172.
Anti-Cancer Agents in Medicinal Chemistry, 2011, Vol. 11, No. 8 [66]
[67]
[68]
[69] [70] [71] [72]
[73]
[74]
[75]
[76]
[77] [78]
[79]
[80]
[81]
[82]
[83]
[84]
[85]
[86]
[87]
[88]
727
Gan, P.P.; Pasquier, E.; Kavallaris, M. Class III -tubulin mediates sensitivity to chemotherapeutic drugs in non small cell lung cancer. Cancer Res. 2007, 67, 9356-9363. Raspaglio, G.; Filippetti, F.; Prislei, S.; Penci, R.; De Maria, I.; Cicchillitti, L.; Mozzetti, S.; Scambia, G.; Ferlini, C. Hypoxia induces class III -tubulin gene expression by HIF-1alpha binding to its 3' flanking region. Gene. 2008, 409, 100-108. Perez, E.A. Microtubule inhibitors: differentiating tubulininhibiting agents based on mechanisms of action, clinical activity, and resistance. Mol. Cancer Ther. 2009, 8, 2086-2095. Cabral, F. Factors determining cellular mechanisms of resistance to antimitotic drugs. Drug Resist Updat. 2001, 4, 3-8. Schiff, P.B.; Horwitz, S.B. Taxol stabilizes microtubules in mouse fibroblast cells. Proc Natl Acad Sci USA. 1980, 77, 1561-1565. Hunt JT. Discovery of ixabepilone. Mol Cancer Ther. 2009, 8, 275-281. Galmarini, C.M. Sagopilone, a microtubule stabilizer for the potential treatment of cancer. Curr Opin Investig Drugs. 2009, 10, 1359-1371. Hoffmann, J.; Fichtner, I.; Lemm, M.; Lienau, P.; Hess-Stumpp, H.; Rotgeri, A.; Hofmann, B.; Klar, U. Sagopilone crosses the blood-brain barrier in vivo to inhibit brain tumor growth and metastases. Neuro Oncol. 2009, 11,158-166. Downing, K.H. Structural basis for the interaction of tubulin with proteins and drugs that affect microtubule dynamics. Annu. Rev. Cell Dev. Biol. 2000, 16, 89-111. Nettles, J.H.; Li, H.; Cornett, B.; Krahn, J.M.; Snyder, J.P.; Downing, K.H. The binding mode of epothilone A on , -tubulin by electron crystallography. Science. 2004, 305, 866-869. Rieder, C.L.; Maiato, H. Stuck in division or passing through: what happens when cells cannot satisfy the spindle assembly checkpoint. Dev Cell. 2004, 7, 637-651. Gascoigne, K.E.; Taylor, S.S. How do anti-mitotic drugs kill cancer cells? J. Cell Sci. 2009, 122, 2579-2585. Brito, D.A.; Rieder, C.L. The ability to survive mitosis in the presence of microtubule poisons differs significantly between human nontransformed (RPE1) and cancer (U2OS, HeLa) cells. Cell Motil. Cytoskeleton. 2009, 66, 437-447. Gascoigne, K.E.; Taylor, S.S. Cancer cells display profound intraand interline variation following prolonged exposure to antimitotic drugs. Cancer Cell. 2008, 14, 111-122. Brito, D.A.; Rieder, C.L. Mitotic checkpoint slippage in humans occurs via cyclin B destruction in the presence of an active checkpoint. Curr. Biol. 2006, 16, 1194-1200. Vakoc, B.J.; Lanning, R.M.; Tyrrell, J.A.; Padera, T.P.; Bartlett, L.A.; Stylianopoulos, T.; Munn, L.L.; Tearney, G.J.; Fukumura, D.; Jain, R.K.; Bouma, B.E. Three-dimensional microscopy of the tumor microenvironment in vivo using optical frequency domain imaging. Nature Med. 2009, 15, 1219-1223. Jordan, M.A.; Toso, R.J.; Thrower, D.; Wilson, L. Mechanism of mitotic block and inhibition of cell proliferation by taxol at low concentrations. Proc Natl Acad Sci USA. 1993, 90, 9552-9556. Horwitz, S.B.; Lothstein, L.; Manfredi, J.J.; Mellado, W.; Parness, J.; Roy, S.N.; Schiff, P.B.; Sorbara, L.; Zeheb, R. Taxol: mechanisms of action and resistance. Ann N Y Acad Sci. 1986, 466, 733-744. Giannakakou, P.; Sackett, D.L.; Kang, Y.K.; Zhan, Z.; Buters, J.T.; Fojo, T.; Poruchynsky, M.S. Paclitaxel-resistant human ovarian cancer cells have mutant beta-tubulins that exhibit impaired paclitaxel-driven polymerization. J Biol Chem. 1997, 272, 17118-17125. Kavallaris, M.; Kuo, D.Y.; Burkhart, C.A.; Regl, D.L.; Norris, M.D.; Haber, M.; Horwitz, S.B. Taxol-resistant epithelial ovarian tumors are associated with altered expression of specific betatubulin isotypes. J Clin Invest. 1997, 100, 1282-1293. Berrieman, H.K.; Lind, M.J.; Cawkwell, L. Do -tubulin mutations have a role in resistance to chemotherapy? Lancet Oncol. 2004, 5, 158-164. Lu, Q.; Ludueña, R.F. Removal of III isotype enhances taxol induced microtubule assembly. Cell Struct Funct. 1993, 18, 173182. Kavallaris, M.; Burkhart, C.A.; Horwitz, S.B. Antisense oligonucleotides to class III -tubulin sensitize drug-resistant cells to Taxol. Br J Cancer. 1999, 80, 1020-1025.
728 Anti-Cancer Agents in Medicinal Chemistry, 2011, Vol. 11, No. 8 [89]
[90]
[91]
[92]
[93]
[94]
[95]
[96]
[97]
Kamath, K.; Wilson, L.; Cabral, F.; Jordan, M.A. III-Tubulin induces paclitaxel resistance in association with reduced effects on microtubule dynamic instability. J Biol Chem. 2005, 280, 12902-12907. Mozzetti, S.; Ferlini, C.; Concolino, P.; Filippetti, F.; Raspaglio, G.; Prislei, S.; Gallo, D.; Martinelli, E.; Ranelletti, F.O.; Ferrandina, G., Scambia, G. Class III -tubulin overexpression is a prominent mechanism of paclitaxel resistance in ovarian cancer patients. Clin Cancer Res. 2005, 11, 298-305. Freedman, H.; Huzil, J.T.; Luchko, T.; Ludueña, R.F.; Tuszynski, J.A. Identification and characterization of an intermediate taxol binding site within microtubule nanopores and a mechanism for tubulin isotype binding selectivity. J Chem Inf Model. 2009, 49, 424-436. Verdier-Pinard, P.; Shahabi, S.; Wang, F.; Burd, B.; Xiao, H.; Goldberg, G.L.; Orr, G.A.; Horwitz, S.B. Detection of human Vtubulin expression in epithelial cancer cell lines by tubulin proteomics. Biochemistry. 2005, 44, 15858-15870. Ferlini, C.; Raspaglio, G.; Mozzetti, S.; Cicchillitti, L.; Filippetti, F.; Gallo, D.; Fattorusso, C.; Campiani, G.; Scambia, G. The secotaxane IDN5390 is able to target class III -tubulin and to overcome paclitaxel resistance. Cancer Res. 2005, 65, 2397-2405. Magnani, M.; Ortuso, F.; Soro, S.; Alcaro, S.; Tramontano, A.; Botta, M. The I/ III-tubulin isoforms and their complexes with antimitotic agents. Docking and molecular dynamics studies. FEBS J. 2006, 273, 3301-3310. Thomas, E.; Tabernero, J.; Fornier, M.; Conté, P.; Fumoleau, P.; Lluch, A.; Vahdat, L.T.; Bunnell, C.A.; Burris, H.A.; Viens, P.; Baselga, J.; Rivera, E.; Guarneri, V.; Poulart, V.; Klimovsky, J.; Lebwohl, D.; Martin, M. Phase II clinical trial of ixabepilone (BMS247550), an epothilone B analog, in patients with taxane-resistant metastatic breast cancer. J. Clin. Oncol. 2007, 25, 3399-3406. Thomas, E.S.; Gomez, H.L.; Li, R.K.; Chung, H.C.; Fein, L.E.; Chan, V.F.; Jassem, J.; Pivot, X.B.; Klimovsky J.V.; de Mendoza, F.H.; Xu, B.; Campone, M.; Lerzo, G.L.; Peck, R.A.; Mukhopadhyay, P.; Vahdat, L.T.; Roché, H.H. Ixabepilone plus capecitabine for metastatic breast cancer progressing after anthracycline and taxane treatment. J. Clin. Oncol. 2007, 25, 5210-5217. Ten Bokkel Huinink, W.W.; Sufliarsky, J.; Smit, W.M.; Spanik, S.; Wagnerova, M.; Hirte, H.W.; Kaye, S.; Johri, A.R.; Oza, A.M. Safety and efficacy of patupilone in patients with advanced ovarian, primary fallopian, or primary peritoneal cancer: a Phase I, open-label, dose-escalation study. J. Clin. Oncol. 2009, 27, 3097-3103.
Received: November 30, 2010
Revised: January 17, 2011
Accepted: January 17, 2011
Katsetos et al. [98] [99]
[100]
[101]
[102]
[103] [104]
[105]
[106]
[107]
[108]
Sabbatini, P.; Spriggs, D.R. Epothilones: better or more of the same? J. Clin. Oncol. 2009, 27, 3079-3081. Vansteenkiste, J.; Lara, P.N. Jr; Le Chevalier, T.; Breton, J.L.; Bonomi, P.; Sandler, A.B.; Socinski, M.A.; Delbaldo, C.; McHenry, B.; Lebwohl, D.; Peck, R.; Edelman, M.J. Phase II clinical trial of the epothilone B analog, ixabepilone, in patients with non small-cell lung cancer whose tumors have failed firstline platinum-based chemotherapy. J. Clin. Oncol. 2007, 25, 3448-3455. Mozzetti, S.; Iantomasi, R.; De Maria, I.; Prislei, S.; Mariani, M.; Camperchioli, A.; Bartollino, S.; Gallo, D.; Scambia, G.; Ferlini, C. Molecular mechanisms of patupilone resistance. Cancer Res. 2008, 68, 10197-1204. Lee, F.Y.; Borzilleri, R.; Fairchild, C.R.; Kamath, A.; Smykla, R.; Kramer, R.; Vite, G. Preclinical discovery of ixabepilone, a highly active antineoplastic agent. Cancer Chemother Pharmacol. 2008, 63, 157-166. O'Reilly, T.; Wartmann, M.; Brueggen, J.; Allegrini, P.R.; Floersheimer, A.; Maira, M.; McSheehy, P.M. Pharmacokinetic profile of the microtubule stabilizer patupilone in tumor-bearing rodents and comparison of anti-cancer activity with other MTS in vitro and in vivo. Cancer Chemother Pharmacol. 2008, 62, 1045-1054. Quick, Q.A. Epothilone B induces glioblastoma cell death via survivin down-regulation. Exp Oncol. 2008, 30, 195-201. Failly, M.; Korur, S.; Egler, V.; Boulay, J.L.; Lino, M.M.; Imber, R.; Merlo, A. Combination of sublethal concentrations of epidermal growth factor receptor inhibitor and microtubule stabilizer induces apoptosis of glioblastoma cells. Mol Cancer Ther. 2007, 6, 773-781. Silvani, A.; Gaviani, P.; Fiumani, A.; Scaioli, V.; Lamperti, E.; Eoli, M.; Botturi, A.; Salmaggi, A. Systemic sagopilone (ZKEPO) treatment of patients with recurrent malignant gliomas. J Neurooncol. 2009, 95, 61-64. Gan, P.P.; McCarroll, J.A.; Po'uha, S.T.; Kamath, K.; Jordan, M.A.; Kavallaris, M. Microtubule dynamics, mitotic arrest, and apoptosis: drug-induced differential effects of III-tubulin. Mol Cancer Ther. 2010, 9, 1339-1348. Risinger, A.L.; Jackson, E.M.; Polin, L.A.; Helms, G.L.; LeBoeuf, D.A.; Joe, P.A.; Hopper-Borge, E.; Ludueña, R.F.; Kruh, G.D.; Mooberry, S.L. The taccalonolides: microtubule stabilizers that circumvent clinically relevant taxane resistance mechanisms. Cancer Res. 2008, 68, 8881-8888. Bai, R.; Edler, M.C.; Bonate, P.L.; Copeland, T.D.; Pettit, G.R.; Ludueña, R.F.; Hamel, E. Intracellular activation and deactivation of tasidotin, an analog of dolastatin 15: correlation with cytotoxicity. Mol Pharmacol. 2009, 75, 218-226.
