# PPARγ Inhibitors as Novel Tubulin-Targeting Agents

Authors: Katherine L. Schaefer
Journal: PPAR Research
 (2008)

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## Abstract

The microtubule-targeting agents (MTAs) are a very successful class of cancer drugs with therapeutic benefits in both hematopoietic and solid tumors.  However, resistance to these drugs is a significant problem.  Current MTAs bind to microtubules, and/or to their constituent tubulin heterodimers, and affect microtubule polymerization and dynamics.  The PPARγ inhibitor T0070907 can reduce tubulin levels in colorectal cancer cell lines and suppress tumor growth in a murine xenograft model.  T0070907 does not alter microtubule polymerization in vitro, and does not appear to work by triggering modulation of tubulin RNA levels subsequent to decreased polymerization.  This observation suggests the possible development of antimicrotubule drugs that work by a novel mechanism, and implies the presence of cancer therapeutic targets that have not yet been exploited.  This review summarizes what is known about PPARγ inhibitors and cancer cell death, with emphasis on the tubulin phenotype and PPAR-dependence, and identifies potential mechanisms of action.


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## Body

## 1. Introduction

The peroxisome proliferator-activated receptors (PPARs) are
ligand-activated nuclear  hormone receptors that act as transcriptional
modulators. They have important roles in
control of metabolism, inflammation, and cell growth and differentiation. There are three PPAR isoforms (γ, β/δ, and α)
with overlapping but distinct tissue expression patterns and cellular functions
[1]. Much evidence suggests that PPARγ
activity can modulate tumor development, implicating PPARγ as an important
therapeutic cancer target [2].PPARγ (NR1C3) is able to both activate and repress transcription, depending on the promoter that is
involved [3]. In the classical pathway, PPARγ binds to promoters containing
PPAR-response elements (PPREs) in combination with its heterodimer partner, the
retinoid X receptor. Activator ligand binding to PPARγ causes a structural shift that increases its ability to
recruit transcriptional coactivators while decreasing its basal ability to bind
to corepressors [4]. PPARγ also exhibits transrepressive functions at promoters lacking a
PPRE [5], by binding in a ligand-dependent
manner to transcription factors, cofactors, or repressor complexes. In these cases, PPARγ binding inhibits
transcription, either by binding/sequestering the transcription factors or by
preventing clearance of repressor complexes. In at least one case of transrepression, the specific PPARγ
conformational shift required is different from that required for cofactor
recruitment at PPREs [6]. It is worth noting here that since PPARγ has basal ligand-independent
repression [5] and activation functions [3], the effects of PPARγ inhibitor
binding and PPARγ knockdown may not be the same.PPARγ can be activated pharmacologically by thiazolidenedione (TZD) compounds, including the antidiabetic drugs
pioglitazone and rosiglitazone. There are multiple studies showing that high doses of TZDs can inhibit tumor growth
in cell lines and mouse models. Clinical trials are currently underway testing TZDs as chemopreventive and therapeutic
agents in human cancers [11]. While TZDs act to stimulate PPARγ activity, they
also have multiple PPARγ-independent effects, and the specific role of PPARγ activation
itself in the therapeutic effects of TZDs is still an active area of research. These topics are reviewed, from the point of
view of cancer therapeutic effects, in several recent reviews [11–18] and elsewhere
in this special issue of PPAR research.Several studies indicate that PPARγ inhibitor compounds are also able to reduce tumor growth in preclinical
models [9, 19–29]. As with the TZDs, the precise role of the loss
of PPARγ activity in cell death is an active research area, and may depend on the
specific cell type. Our recent observation that PPARγ inhibitors can cause rapid dissolution of the
microtubule network in colon cancer cells [26]
suggests that these compounds might act as microtubule-targeting agents (MTAs),
similar to the taxanes or Vinca alkaloids that are in current clinical use. However, unlike MTAs [30], they markedly reduce
concentrations of α and β tubulin proteins long before a commitment to
apoptosis, and do not strongly affect microtubule polymerization in vitro. This review will focus on the strong possibility
that PPARγ inhibitor compounds represent a new class of tubulin-targeting
agents [31].


## 2. Binding Activity of PPARγ Activators and Inhibitors

The PPARγ ligand-binding pocket can accommodate a variety of lipophilic molecules [32]. Many cellular fatty acids activate PPARγ, including oxidized low-density
lipoproteins, unsaturated fatty acids, 15-hydroxyeicosatetraenoic acid, and 9-
and 13-hydroxyoctadecadienoic acids. In addition, the putative  endogenous ligand prostaglandin 15-deoxy delta-(12,14)-prostaglandin J2, as well as the TZD anti-diabetic drugs [32], are able to activate PPARγ. The anti-inflammatory drug 5-aminosalicylic
acid binds to PPARγ at therapeutic doses [33], as do other nonsteroidal
anti-inflammatory drugs [34], although both classes of
medications are lower affinity ligands than the TZDs. Ligand binding introduces PPARγ conformational
shifts that favor recruitment of transcriptional coactivators over corepressors
or that promote specific posttranslational modifications, and it is these changes
that dictate the transcriptional activity of PPARγ. All of these ligands also have multiple
effects that are independent of PPARγ, especially at high doses [13, 32]. In addition, the identity and regulation of true endogenous ligands is
poorly understood at the present time.PPARγ also binds to a number of compounds that are able to inhibit TZD-mediated PPARγ activation
(see [35] for chemical structures). These include halofenate [36] and
its enantiomer metaglidasen [37], SR-202 [38], G3335 and its
derivatives [35, 39], T0070907 [9],
GW9662 [8], and bisphenol-A-diglycidyl-ether (BADGE) [10]. PPARγ inhibitors probably suppress PPARγ activation both by preventing
binding by endogenous or exogenously added ligands, and by inducing specific
conformational shifts that actively promote repression [9]. However, the details of these conformational changes are less well understood
than for the activators. Of the known PPARγ inhibitors, only T0070907, GW9662, and BADGE have been tested for their
effects on cancer cell death; all three can cause cell death in multiple cancer
cell types at high-micromolar concentrations.Interpreting the effects of the cancer-targeting PPARγ inhibitors is difficult, since they can act as activators or inhibitors,
depending on the concentration used. They also bind to multiple members of the PPAR family (and quite possibly to other
molecules) at high doses. At low micromolar doses, T0070907 and GW9662 also bind to and inhibit PPARα and PPARδ (Table 1). In
addition, at low nanomolar doses, GW9662 is a partial activator of PPARα. While the ability of T0070907 to activate PPARα has
not been checked, it is possible that this compound may behave in the same manner. Similarly, there are reports that BADGE can
act as a PPARγ activator at lower doses (10–30 μM) than those needed for
inhibitory effects [28, 40].Table 1
Effects of PPARγ inhibitors on PPARγ, PPARα, and PPARδ activity IC50 (nM) for ability to compete with a PPAR agonist.
BindingDirect binding assayActivation of GAL4 chimeraReferencesPPARγPPARαPPARδPPARγPPARαPPARδGW9662Irreversible4188471[7]53912008630(1)4100[8]T0070907Irreversible185018001 μM completely inhibitsγ with no effect on α or δ(2)[9]BADGEReversible100 000100 μM∼70% inhibits γ with little or no effect on α or δ(2)[10](1)GW9662 is also a partial activator of PPARα with an EC50 of 22 nM [8], leading to the apparently higher concentrations
of GW9662 required to inhibit PPARα than would be predicted by the direct
binding assay.
(2)Dose curves were not performed, but the indicated concentrations
suppressed the GAL4 chimera as indicated.


## 3. Standard Microtubule Targeting Agents Act by Interfering with Microtubule Dynamics

Microtubules are long, tube-shaped polymers, formed by ordered arrays ofα/β tubulin heterodimers (Figure 1), that
make up one of the major components of the cellular cytoskeleton. Precise regulation of microtubule function is
essential for maintenance of cell shape and polarity, migration, regulation of
cell signaling cascades, intracellular transport, and cell division [41]. Microtubule function is governed by a variety
of active changes in microtubule structure collectively termed microtubule dynamics. Microtubules normally alternate between
phases of growth and rapid shrinking (dynamic instability), and also move
tubulin heterodimers from one end of the polymer to the other (treadmilling) [42]. These processes are regulated in the cell by
a host of microtubule-associated proteins with varied functions [43–46]. Both dynamic instability and treadmilling are
required for mitosis, and are almost certainly necessary for the other
functions of microtubules [44].Figure 1
Microtubule formation
depends both on chaperone-mediated production and assembly ofα/β heterodimers
and on microtubule-associated proteins. Production of α and β tubulin
proteins requires assistance from chaperone proteins. The chaperone prefoldin associates with
nascent tubulin polypeptide chains and delivers them to the CCT chaperone. CCT folds them into stable forms, which are
delivered to the tubulin cofactors A and B [47]. CoA and CoB both transfer tubulin monomers to
the CoC/D/E complex, which assembles the monomers into α/β
heterodimers ready for introduction into microtubules. Tubulin reservoirs are held by the
microtubule-associated protein stathmin (green box), which, depending on
phosphorylation, binds to free tubulin and also destabilizes microtubule
polymers. A host of micotubule-associated
proteins (green circles) associate with the microtubule and regulate addition
and removal of heterodimers from both ends of the microtubule; in some cases,
they have been shown to regulate tubulin levels. While MTA therapies like the
taxanes and Vinca alkaloids target
the equilibrium between α/β tubulin heterodimer and the microtubule
polymer, PPARγ inhibitors could be affecting any of the chaperone proteins or
one of the microtubule-associated proteins that is involved in control of
tubulin levels.Given the importance of rapid cell proliferation and migration to tumor development, it is not surprising that the
microtubule-targeting agents (MTAs) are one of the most successful classes of cancer therapeutics, with clinical applications
in hematological cancers and solid tumors of the head/neck, breast, ovaries, testes, lung, gastric tissue,
and prostate [48]. MTAs are a chemically and structurally diverse sets of small molecules that
bind to microtubules, tubulin, or both, and interfere with microtubule function [44]. Despite this diversity, all known MTAs bind
at or near to one of three domains: the taxane domain, the Vinca domain, or the colchicine domain. Historically,
MTAs have been divided into microtubule-stabilizing and microtubule-destabilizing
agents, based on their effects at high doses on polymer mass in vitro polymerization assays. However, while the classification remains in
use, and these effects clearly occur in vivo at high doses, it is becoming generally accepted that MTAs at
clinically relevant concentrations primarily act by disrupting microtubule
dynamics, rather than by affecting bulk polymerization [30, 49]. The microtubule-disrupting effect leads to
cell cycle arrest. In addition, MTAs may also cause apoptosis by mechanisms that ultimately prove to be at least
partially independent of the effect on the mitotic spindle [50, 51].Discovery and development of new microtubule-binding compounds is an area of active research. In
contrast, less effort has been spent on considering whether reducing tubulin
levels directly, and thereby altering microtubule function, could be used to impair
cancer cells. Our recent results, showing that PPARγ inhibitors reduce tubulin levels in HT-29 colon cancer
cells, before a commitment to apoptosis [26], suggest that targeting tubulin itself may be a
viable strategy.


## 4. PPARγ Inhibitors Cause Apoptosis in Multiple Cancer Cell Types and Cause Rapid Loss of Tubulin Proteins in Colon Cancer Cells

Experiments with many different cancer cell lines show that
high doses of PPARγ inhibitors can cause cell death. T0070907 and/or GW9662 exhibited
antiproliferative effects in both hematopoietic cell lines from non-Hodgkin’s
lymphoma and multiple myeloma [27] and
epithelial cancer cell lines, including carcinoma cell lines from renal [27], breast
[21, 22, 27], liver [23, 52], oral
squamous [29],
esophageal [24],  prostate [22], and colon
tissue [22, 26]. IC50 concentrations for inhibition of growth
in the epithelial lines ranged from 3–50 μM for T0070907 and 10–50 μM for
GW9662. While the reasons for this reduction in cell number have not been explored in all cases, T0070907 and
GW9662 clearly caused apoptosis in several epithelial cell lines. BADGE also exhibited cytotoxic effects against
colon cancer cell lines [20, 22, 25, 31] and a T
lymphoma cell line [19, 20], at
doses in the 100–200 μM range.In HT-29 colon cancer cells, treatment with
50 μM T0070907 led to dissolution of the microtubule network within 12 hours [26]. At this timepoint, the effects of T0070907
were reversible. However, after longer exposure,
the cells became committed to caspase-dependent apoptosis. Cells treated with T0070907 also assumed a
rounded shape that occurred prior to commitment to apoptosis, and that was not
affected by caspase inhibitors. Similar
effects and timing were seen with the PPARγ inhibitor GW9662 ([26] and KLS,
unpublished data).Loss of the microtubule network is associated with the microtubule-depolymerizing class of MTAs that includes
vinblastine, vincristine, vinorelbine, and nocodazole [30, 53]. Thus, it was especially striking that, unlike
nocodazole, T0070907 and GW9662 did not affect microtubule polymerization in in vitro assays [26]. Instead, α and β tubulin protein levels in
the cells dropped rapidly after treatment with these compounds, suggesting that
the microtubule network disappeared because tubulin protein levels were below
critical thresholds needed for polymerization.  BADGE also caused tubulin loss, although the
timing of this loss relative to commitment to apoptosis was not
determined. It is not currently known whether PPARγ inhibitors cause loss of tubulin in other cell lines. While many of the other experiments with
inhibitors documented altered cytoskeletal structure [23–25, 29], it was not clear in these papers whether the altered shape was the result or the
cause of apoptosis, and tubulin levels were not measured directly.


## 5. Is Tubulin Loss Required for Cell Death Induced by T0070907 and GW9662?

The effects of T0070907 and GW9662 on tubulin are striking, especially as there have not been any reports of cancer cell-targeting small
molecules that affect tubulin levels without dramatic effects on microtubule polymerization. However, it is not clear
that the ultimate cause of cell death is loss of tubulin. These compounds may independently target a
combination of signaling pathways that ultimately trigger the apoptotic
response as well as modulating tubulin levels. Given the fact that the PPARγ inhibitors also trigger loss of γ and δ
tubulin isoforms (KLS, unpublished data), it will be difficult to do genetic
replacement experiments to address this issue in the absence of other
information about the reasons for tubulin loss. Regardless of whether or not microtubule disruption is the first trigger
for apoptosis, the reduction in tubulin should serve as a barrier that is
impossible for the tumor cells to surmount. This effect could have profound advantages, in that simply modulating
the levels of anti-apoptotic proteins in response to chemotherapy would not be
sufficient to allow tumor escape.Intriguingly, commitment to apoptosis in HT-29 cells occurred
at about the same time that tubulin levels dropped below a threshold level for
normal function observed in yeast. After 12 hours of T0070907 treatment, when
the tumor cells had lost∼50% of their tubulin, the effects of the drug were
still reversible. By the time, 50% of
the cells had committed to apoptosis (∼20 h), the average tubulin level was less
than 10% of control ([26] and data not shown), suggesting that
apoptosis may be triggered by tubulin levels below 50% of normal. These numbers parallel observations in the
yeast Saccharomyes cerevisiae, which was
able to tolerate a 50% reduction in either α or β tubulin, as long as there
were not excess unpaired β tubulin molecules [54, 55], but which began to show defects in
mitosis when levels dropped to ∼20% of normal.


## 6. What Could Be Causing the Loss of Tubulin Induced by T0070907?

The reasons for T0070907-mediated tubulin loss remain to be
elucidated, and may well be the result of multiple coordinated changes taking
place in the context of alterations in PPAR function. This point is of critical interest, as
identification of the mechanism(s) of tubulin loss will serve as an important
step in identifying the therapeutic targets that are exploited by T0070907, and
in design of better ways to target them. In HT-29 cells,α/β tubulin RNA levels were unaffected, suggesting a
post-transcriptional mechanism. Several
aspects of tubulin production could be involved, including degradation,
translation initiation, chaperone-mediated folding/assembly of tubulin
heterodimers (Figure 1), and disruption of microtubule-associated protein
interactions with tubulin.Because the protein half-life of tubulin is believed to be
long (∼50 hours) [56, 57], detectable loss of tubulin within 6
hours suggests a mechanism involving increased decay. Tubulin can be targeted for proteasomal
degradation by the tubulin cofactor-like protein El [58] and probably by other regulatory
factors. However, proteasome inhibitors did not prevent T0070907-induced tubulin loss [26], suggesting that ubiquitin-mediated
proteasomal degradation is not a major factor. Other degradation pathways must be investigated, especially the aggresome
pathway, which can replace proteasomal degradation, resulting in autophagic
clearance and lysosomal degradation [59, 60]. It is also worth noting that the estimate of long tubulin half-lives is
based on measurement in only two cell types, and may not apply to HT-29 cells.T0070907-induced tubulin loss is unlikely to be the result of
acute increases in tubulin monomer protein. Many eukaryotic cells respond to a sudden increase in unpolymerized
tubulin by reducing synthesis of tubulin [61–65] in a process termed autoregulatory control. This mechanism is associated with large
reductions in tubulin mRNA; later work showed that polysomal mRNA in the
process of being translated was specifically susceptible to an increased mRNA
decay [66, 67]. T0070907 induces little difference in tubulin mRNA concentrations as
measured by real-time PCR [26]. In addition, at least in in vitro polymerization assays,
T0070907 did not inhibit polymerization or cause depolymerization, although it
is important to note that depolymerization might occur in the cell as a result
of alterations in microtubule-associated protein function. However, nocodazole, which at high (10 μM)
doses increases the amount of soluble tubulin by inhibiting bulk microtubule
polymerization [53], did not affect the tubulin protein
levels in HT-29 cells. This result strongly suggests that tubulin in these particular cells is not strongly subject to
autoregulatory control. However, it is formally possible that T0070907 might increase the soluble tubulin pool far
more strongly than nocodazole. Direct measurement of the amount of tubulin in the polymerized and free pools after
T0070907 treatment should resolve these questions.There is also the potential for T0070907 to control
translation initiation or other aspects of protein synthesis. The TZD PPARγ activators have been shown to
suppress translation initiation in a PPARγ-independent manner through a
mechanism involving intracellular Ca2+ store depletion and
subsequent inhibition of the eIF2 translation inititation factor [68]. It is possible that T0070907 also affects some aspect(s) of the
translation machinery.The loss of tubulin and cell death phenotypes induced by
T0070907 can be mimicked by knockdown of chaperone proteins involved in folding
and assembly of tubulin, suggesting that PPARγ inhibitors may interfere with
this pathway. Tubulin production and assembly into α/β heterodimers require the presence of multiple
chaperone proteins, including the multisubunit chaperones prefoldin [69] and CCT [70], and tubulin
cofactor proteins A–E [71]  (Figure 1). Additional chaperone modulatory proteins, including PhLP3 [72] and
E-like (El) [58], also modulate the function of the tubulin chaperone system. Knockdown of CCT subunits causes reduced
tubulin levels [73], as
does knockdown of tubulin cofactor A [74]. It is possible that PPARγ inhibitors bind directly to and
inhibit some of the chaperone proteins. Alternatively, they may change the expression or function of any of the
chaperone subunits or cofactors.Knockdown of microtubule-associated proteins (MAPs) can also cause loss of tubulin, and PPARγ inhibitors could interfere
with MAPs or dysregulate MAP expression. MAPs are a functional class of proteins that physically interact with microtubules or microtubule
precursors and regulate microtubule functions. Some MAPs directly control the rate of association or dissociation of
α/β tubulin heterodimers with the ends of the microtubule, as well as the
levels of soluble tubulin in the cell, and thus affect microtubule dynamics
(Figure 1). Others link microtubules to
signaling complexes and other cytoskeletal components [75]. Mutations in stathmin, a multifunctional MAP that both destabilizes
microtubules and sequesters α/β tubulin heterodimers so that they are not part
of the freely polymerizing pool, led to reduced α tubulin levels (β tubulin was
not checked) and fewer microtubules in Drosophila oocytes [76]. In the same system, stathmin overexpression increased tubulin levels. Knockdown of MAP4, generally thought to be a
microtubule-stabilizing MAP, also caused reduced tubulin levels [77]. In both cases, it is possible, but has not been shown directly, that
these effects were subsequent to autoregulatory control.


## 7. Are the Cell Death and Tubulin Effects of T0070907 or GW9662 Dependent upon PPARγ?

It is important to establish whether the effects of T0070907 and GW9662 on cell death can be separated from their ability to target
PPARγ. Although results in two cell lines [23, 29] have shown
that PPARγ knockdown causes cell death or potentiates the effects of the inhibitors, this result
does not occur in all cell lines [26]. In addition, the inhibitor doses required for the cell
death (3–50 μM for T0070907 and 10–50 μM for GW9662) in all cell lines tested are much higher than those needed
to inhibit the transcriptional effect of PPARγ by at least 90%, given an
approximate effective dissociation constant in the low nanomolar range (Table 1). This result suggests that at least
some of the cell death effects are indeed independent of PPARγ. The differences in cell lines may reflect a
true disparity in the role of PPARγ in maintaining cell growth and survival in
different cell types, and suggests that the HT-29 system is ideal for examining
the PPARγ-independent effects of T0070907.The effect of T0070907 and GW9662 on tubulin has been
examined primarily in HT-29 colon cancer cells, although the loss of adhesion, cell
rounding, and cell death occur in multiple colorectal cancer cell lines ([26] and KLS, unpublished data). In HT-29 cells, the effects on tubulin are
not replicated by PPARγ knockdown, or reductions in the closely related PPARδ. However, given the fact that at 50 μM T0070907,
PPARγ, PPARα and PPARδ transcriptional activities are all expected to be at
least partially repressed, based on the predicted dissociation constants (Table 1), it is
possible that multiple PPAR molecules must be inactivated in order to
create the conditions necessary for tubulin loss. In addition, it is entirely possible that a
non-PPAR-dependent event must occur in the context of knockdown of one or more
of the PPAR transcription factors. This idea is somewhat contradicted by the observation that BADGE, which does not
strongly affect PPARα/δ at 100 μM [10], does cause a reduction in the
amount of tubulin [26]. However, in this case, it is possible that the tubulin loss was a
separate phenomenon, secondary to extensive cell death. Further experiments will be needed to address
this issue.


## 8. Advantages and Disadvantages to Using PPARγ Inhibitors as Tubulin-Targeting Therapies

To our knowledge, the PPARγ inhibitors are the first described
instance of a possible small molecule cancer therapeutic that reduces tubulin
levels. This result suggests the exciting possibility that tubulin levels can be modulated therapeutically, in a
tumor-specific manner, to kill cancer cells. While the current microtubule-targeting agents have significant
antitumor activity in many cancer types, they are not effective in all cancers,
and acquired resistance is a problem [78]. In addition, because these drugs differentially targeting rapidly
proliferating cells, they cause leukopenia [78]. For the same reason, they are not expected to target cancer stem cells,
owing to their generally slow proliferation rate [79]. Microtubule-targeting drugs also induce peripheral neuropathy [80] and may interfere with
mental function [81], presumably as a result of their effects
on neuronal microtubule function. T0070907 and/or GW9662, or second-generation compounds, by virtue of
acting by a different mechanism, might ameliorate some of the difficulties of
the standard MTAs.Several questions must be addressed, when considering these
compounds, or others like them, as cancer therapies. To date, preclinical data on the
bioavailability of either T0070907 or GW9662 has not been published, although a
pilot experiment with radiolabeled GW9662 indicates that these compounds are
delivered to tumors [82]. It is also important to consider tumor specificity and whether the
tubulin-targeting effect can be separated from the PPAR inhibitory effect.Inherent or acquired resistance to current microtubule-targeting agents is a serious problem in microtubule-based cancer
therapy. One major source of resistance is the expression of alternate tubulin isoforms with inherently different
microtubule dynamics [83]. These differences antagonize the effects of the drug, and allow the cell
to continue proliferating. It appears that T0070907 causes concurrent loss of multiple tubulin isoforms (KLS,
unpublished data), presumably by some regulatory mechanism common to all isoforms. It is therefore reasonable to suspect that T0070907
would suppress levels of the tumor-specific alternate isoforms as well. The microtubule targeting drugs are also
generally good substrates for drug efflux pumps that prevent accumulation of
therapeutic levels of drug [78]. For this reason, it would be useful to test whether T0070907 and GW9662 are
substrates for the common drug efflux pumps.As tubulin is a constituent of all cells, the effects of
T0070907 or similar compounds on normal cells is a serious consideration. To date, these compounds have only been
tested in one mouse model of cancer. At oral doses that reduced tumor growth (1–5 mg/kg/d), the compounds did not cause weight
loss or malaise in mice [26]. In addition, recent unpublished results from our laboratory showed that 10 mg/kg/d
orally, maintained daily for three weeks, did not cause any alterations in values
from a standard complete blood count with differential. The reasons for this apparent specificity
will need to be examined in more detail, as well as whether tubulin levels are
reduced in normal and tumor tissues in vivo. The fact that radioactively
labeled GW9662 preferentially accumulated in tumor cells as compared to many
normal tissues in mice [82] suggests that part of the
specificity may simply reflect where the compound is accumulating. Another possibility is that these compounds
do act through the tubulin chaperone system. Components of this system are upregulated in some tumor cells [84–86], and tumor cells may require higher
levels of tubulin chaperone function, as they do with the HSP90 chaperone [87, 88]. If this were true, this might explain why tumor cells are preferentially susceptible to the PPARγ inhibitors.A major question is whether suppression of PPARγ (or PPARα/δ) function is required for the tubulin-targeting effects of
T0070907 and/or GW9662. If suppression of PPARγ cannot be separated from the tubulin targeting effects, it will be
necessary to carefully balance the therapeutic effects of PPARγ inhibitors on
tubulin with the possible deleterious effects of PPARγ inhibition on
physiologic processes that affect tumor growth. In addition to the question of whether PPARγ is a tumor suppressor or
has tumor-promoting activity in each cancer cell type, the effects of PPARγ on angiogenesis
[14, 16] and the immune system [32] must also be considered.The role of PPARγ itself in angiogenesis, in contrast
to the effects of TZD PPARγ activators, is still relatively unclear [16]. TZDs can reduce the production of proangiogenic FGF and VEGF
factors, interfere with endothelial cell migration, and inhibit vascular tube
formation, as well as reduce production of inflammatory mediators that
stimulate angiogenesis. However, to date, only CD36 upregulation [89] and decreased
iNOS production [6] are
known to be PPARγ-dependent. It is also noteworthy that standard microtubule-targeting agents in clinical use
disrupt tumor-specific vasculature [90, 91]. It will be interesting to determine whether
the PPARγ inhibitors also suppress tumor angiogenesis, and whether the effects
are linked to PPARγ and/or tubulin.In all probability, the net effect of PPARγ inhibition on the immune
system will depend upon the individual characteristics of the tumor, including the
site of the tumor, the role of PPARγ in tumor-intrinsic biology, and the
presence and type of immune infiltration. A variety of immune
cells infiltrate tumors, including macrophage lineage cells, T lymphocytes, mast
cells, and natural killer (NK) cells [92]. All of these cells have the potential to promote or hinder tumor growth,
depending on the cytokines they secrete and the cell-mediated cytotoxic effects
they are able to promote. Both macrophage and regulatory T cell (Treg) functions are modulated by
PPARγ. Monocytes can differentiate into M1 or M2 macrophages in response to different stimuli [93, 94]. In general, M1 macrophages secrete large quantities of inflammatory
cytokines, have cytotoxic activity toward tumor cells, elicit the adaptive
immune response, and are associated with a better tumor prognosis. In contrast, M2 macrophages secrete
immunosuppressive cytokines, have poor antigen-presenting capacity, and promote
angiogenesis and tissue remodeling; these macrophages are generally associated
with a poorer prognosis [93]. Since PPARγ activation of human
monocytes promotes M2 polarization [95], PPARγ
inhibitors might be expected to favor production of M1 tumor-suppressing
inflammatory macrophages. Treg cells lacking PPARγ are unable to suppress colitis in a regulatory T
cell-dependent model of inflammatory bowel disease [96], arguing that PPARγ is required for
suppressive regulatory function. As Treg activity can impair tumor rejection [97],
PPARγ inhibitors should suppress Treg thereby aiding tumor rejection.


## 9. Conclusions

The microtubule-targeting agents are one of the most
successful classes of cancer therapeutics, but ongoing issues with resistance
make the development of additional strategies for targeting microtubules extremely
desirable. The recent discovery that the small molecule PPARγ inhibitor compounds reduce tubulin protein levels, without
affecting in vitro polymerization rates, suggests the exciting possibility that targeting tubulin levels
directly, rather than microtubule dynamics, might be an additional way to
manipulate microtubule biology to kill cancer cells.  Several questions, including whether
inhibition of PPAR function is required for the tubulin effect, the nature of the
tumor specificity, the ultimate targets of these compounds, and whether better
compounds with similar tubulin targeting effects can be designed, must be
answered before this strategy can be fully realized.



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