Temozolomide-resistant Glioblastoma Depends on HDAC6
Activity Through Regulation of DNA Mismatch Repair

Abstract. Background/Aim: Histone deacetylase 6 (HDAC6)
is considered as one of the most promising targets in drug
development for cancer therapy. Drug resistance is a major
cause of treatment failure in many cancers including
glioblastoma (GBM), the most lethal malignant tumor. The
role of HDAC6 in GBM resistance and its underlying
mechanisms have not been well elucidated. Herein, we
investigated the function of HDAC6 in modulating GBM
resistance. Materials and Methods: The anticancer effects of
four structurally distinct selective HDAC6 inhibitors were
addressed using western blot, flow cytometry, CCK-8 assay,
and CI in temozolomide (TMZ)-resistant GBM cells. Results:
We showed that HDAC6-selecitve inhibitors block activation
of the EGFR and p53 pathways in TMZ-resistant GBM cells.
Importantly, the inhibition of HDAC6 correlates with
increased levels of MSH2 and MSH6, key DNA mismatch
repair proteins, in TMZ-resistant GBM cells. In addition to the
MSH, HDAC6 inhibitors decrease MGMT expression in TMZ￾resistant GBM cells. Furthermore, HDAC6 inhibitors increase
TMZ sensitivity and efficiently induce apoptosis in TMZ￾resistant GBM cells. Conclusion: Selective inhibition of
HDAC6 may be a promising strategy for the treatment of
TMZ-resistant GBM.
Glioblastoma (GBM), also known as World Health
Organization grade IV glioma or glioblastoma multiforme,
is the most common and lethal type of primary brain cancer
(1, 2). With the introduction of combined chemotherapy and
radiation therapy, the median survival time has increased to
about 15 months (3). Temozolomide (TMZ) was used for the
standard treatment of GBM for over a decade, irrespective
of O6-methylguanine (O6-MG)-DNA methyltransferase
(MGMT) status (4, 5). However, almost all patients treated
with TMZ eventually develop acquired resistance and tumor
recurrence.
Several factors involved in epigenetic and genetic
mechanisms have been associated with TMZ resistance. The
mechanisms of resistance include increased DNA repair
activity, overexpression of murine double minute 2 (MDM2),
epidermal growth factor receptor (EGFR), and galetin-1; and
mutations or alterations of phosphatase, tensin homolog
(PTEN) and p53 (6). The MGMT DNA repair enzyme and
DNA mismatch repair (MMR) system are the major known
mechanisms of TMZ resistance in GBM (7-11). MGMT is a
DNA repair protein, which removes the cytotoxic O6-MG
DNA lesions made by TMZ, and high levels of MGMT
expression are associated with TMZ resistance in GBM cells
(8). The DNA MMR pathway is a system that corrects errors
of nucleotide base mismatches made during DNA replication
(12). Although there are six MutS homologs (MSH), MSH2
plays the major role in mismatch recognition, while other
homologs such as MSH3 and MSH6 enhance the specificity
of such recognition (13). A defective MMR system caused
by mutations in MMR protein complexes leads to failure to
recognize and repair O6-MG adducts generated by TMZ,
thereby making TMZ less effective (6, 14). Thus, strategies
to rescue the effect of the MMR system need to improve the
effect of TMZ and to overcome TMZ resistance.
Since the acetylation status of histone proteins and non￾histone proteins affects gene expression and protein activity
in various biological processes, histone deacetylase (HDAC)
inhibitors have emerged as a potential therapeutic approach
to cancer (15, 16). However, because more than 11 classical
HDACs (class I, II and IV HDACs) exist in human cells, the
use of HDAC inhibitors targeting only a few types of HDACs
6731
*These Authors contributed equally to this study.
Correspondence to: So Hee Kwon, College of Pharmacy, Yonsei
Institute of Pharmaceutical Sciences, Yonsei University, 85
Songdogwahak-ro, Yeonsu-gu, Incheon, 21983, Republic of Korea. Tel:
+82 327494513, Fax: +82 327494105, e-mail: [email protected]
Key Words: Histone deacetylase 6, glioblastoma, temozolomide
resistance, DNA mismatch repair, MutSα.
ANTICANCER RESEARCH 39: 6731-6741 (2019)
doi:10.21873/anticanres.13888
Temozolomide-resistant Glioblastoma Depends on HDAC6
Activity Through Regulation of DNA Mismatch Repair
GO WOON KIM1*, DONG HOON LEE1*, SOO-KEUN YEON1, YU HYUN JEON1,
JUNG YOO1, SANG WOO LEE1 and SO HEE KWON1,2
1College of Pharmacy, Yonsei Institute of Pharmaceutical Sciences, Yonsei University, Incheon, Republic of Korea;
2Department of Integrated OMICS for Biomedical Science, Yonsei University, Seoul, Republic of Korea
for treatment of cancer or experimental studies is frequently
more effective, with fewer side-effects (17). Class IIb
HDAC6 has a unique structure (18). HDAC6 not only can
function as a protein deacetylase, but also interacts with other
regulatory proteins through ubiquitination and acetylation￾related mechanisms (19). HDAC6 is associated with cell
sensitivity to drugs and emerging resistance during
chemotherapy (20, 21). HDAC6 is overexpressed in GMB
cell lines and tissues (21, 22), the overexpression of HDAC6
promotes GBM proliferation, and confers resistance to TMZ
(21). Interestingly, HDAC6 and HDAC10 have a role in DNA
MMR (23, 24). HDAC6 regulates MutSα by deacetylating
and ubiquitinating MSH2 for degradation (24). HDAC6
significantly decreases sensitivity to DNA-damaging agents
and decreases MMR activities by down-regulating MSH2.
Additionally, MutSα inactivation is associated with TMZ
resistance in GBM (9, 11). However, little is known on the
relevance of HDAC6, MGMT, and the MMR system to the
development of TMZ resistance in GBM.
To shed more light on this issue, we investigated the
functions of HDAC6 in GBM resistance using four
structurally distinct HDAC6 selective inhibitors in TMZ￾sensitive and TMZ-resistant GBM cells. Therefore, our
findings provide a scientific rationale for targeting HDAC6
to overcome chemoresistance in TMZ-resistant GBM.
Materials and Methods
Cell culture. Human glioblastoma cell lines U87 (wtp53, no basal
MGMT-TMZ sensitive cell), and T98G (mutp53, high basal MGMT￾TMZ resistant cell) were purchased from the American Type Culture
Collection (Manassas, VA, USA). Cells were cultured in Dulbecco’s
modified Eagle’s medium (Gibco; Thermo Fisher Scientific, Inc.,
Waltham, MA, USA) containing 10% fetal bovine serum (FBS;
HyClone; GE Healthcare, Logan, UT, USA), 100 U/ml penicillin,
and 100 μg/ml streptomycin (Gibco; Thermo Fisher Scientific) in a
humidified atmosphere of 5% CO2 and 95% air at 37˚C.
Reagents. ACY-1215 (ricolinostat), CAY10603, temozolomide, and
tubastatin A were purchased from Selleck Chemicals (Houston, TX,
USA). A452 (purity 99%) is a γ-lactam-based HDAC6 inhibitor
(25) and was kindly provided by Dr. Gyoonhee Han (Yonsei
University, Seoul, Republic of Korea).
Cell viability assay. Cell viability was assessed by measuring the
dye absorbance of a water-soluble tetrazolium salt WST-8 [Cell
Counting Kit (CCK)-8 kit, Dojindo Molecular Technologies,
Kumamoto, Japan] according to the manufacturer’s protocol and
was performed as previously described (26). Cells were seeded in
triplicates at a density of 1.5×103 cells in 200 μl of medium in 96-
well plates. The drugs were added to the cells at the indicated
concentrations 24 h after seeding at 37˚C for 72 h. The cells in each
well were pulsed with 20 μl of WST-8 for the final 3 h of a 72-h
incubation, and absorbance was then measured at 450 nm using a
multimode microplate reader (Tecan Group, Mannedorf,
Switzerland). Absorbance was normalized to that of the negative
control (no DMSO vehicle) at each time interval. To analyze cell
viability, the percentage absorbance was calculated relative to
negative control cultures. The results from three independent
experiments performed in triplicate are presented.
Apoptosis assay. Apoptosis was assessed using Annexin
V/propidium iodide (PI) double staining according to the
manufacturer’s protocol (FITC Annexin V Apoptosis Detection Kit;
BD Biosciences, Franklin Lakes, NJ, USA), as previously described
(26). The cells were then analyzed using a flow cytometer and BD
FACSDiva software version 7 (both from BD Biosciences).
Western blot analysis. Cells grown and treated as indicated, were
collected, lysed, and separated by sodium dodecyl sulfate￾polyacrylamide gel electrophoresis (SDS-PAGE); western blotting
was performed as previously described (27). The blots were semi￾quantified using FusionCapt software version 16.08a (Viber
Lourmat Sté, Collégien, France). The protein expression levels were
semi-quantified relative to GADPH and the levels in the 0.1%
DMSO treated groups were set at 1. GADPH was used as a loading
control. The sources of the primary antibodies are available upon
the request.
Drug combination analysis. For combined drug analysis, a constant
ratio of TMZ and A452 or ACY-1215 was evaluated. Drug dilutions
and combinations were prepared in media immediately prior to use.
Cells (1.5×103/well) in 96-well plates were incubated with the drugs
for 72 h at 37˚C. A CCK-8 assay was performed to determine cell
viability. Drug interactions were determined according to the
combination index (CI) method described by Chou (28); CI>1
implies antagonism, CI=1 is additive and CI<1 implies synergism.
CIs for the combination treatment groups were generated using
CalcuSyn software version 2.11 (Biosoft, Cambridge, UK). The
fraction affected (FA) was calculated from the percentage viability,
as follows: FA=(100 – percentage viability)/100.
Statistical analysis. Statistical analyses were performed with
GraphPad Prism software (version 7.0, Graphpad Software). Data
are presented as the means±standard deviation of three independent
experiments. Statistical differences were determined by one-way or
two-way analysis of variance (ANOVA) with post-hoc analysis
using Bonferroni's multiple comparison test. p<0.05 was considered
to indicate a statistically significant difference.
Results
HDAC6-selective inhibitor activates MMR pathways in
TMZ-resistant GBM cells but not in TMZ-sensitive cells.
Loss of DNA MMR proteins is associated with GBM
recurrence during TMZ treatment (29) and HDAC6 regulates
MSH2 protein stability via de-acetylation and ubiquinination
(24). Therefore, we assessed whether inhibition of HDAC6
could up-regulate MSH2 protein levels in GBM cells. To this
end, we examined the expression level of MSH2 in TMZ￾sensitive U87 and TMZ-resistant T98G GBM cells treated
with four structurally distinct HDAC6-selective inhibitors,
A452, ACY-1215, CAY10603, and tubastatin A. A452 and
ACY-1215 resulted in slightly increased levels of MSH2
ANTICANCER RESEARCH 39: 6731-6741 (2019)
6732
Kim et al: HDAC6 Regulates MMR in TMZ-resistant GBM
6733
Figure 1. HDAC6 inhibitor up-regulates MSH2 and MSH6 proteins in TMZ-resistant GBM cells but not in TMZ-sensitive GBM cells. (A) TMZ-resistant
T98G and (B) TMZ-sensitive U87 GBM cells were treated with 0.1% DMSO (control) or HDAC6 inhibitor at the indicated concentrations (0.1, 1, and
10 μM) for 24 h. Whole-cell lysates were subjected to immunoblotting with the indicated antibodies. Acetylation of H3 (Ace-H3) and α-tubulin (Ace-
α-tub) are markers for HDAC1 and HDAC6 inhibition, respectively. The protein expression levels were semi-quantified relative to GAPDH, and the
levels in the 0.1% DMSO-treated groups were set at 1. Levels of Ace-α-tub and Ace-H3 were semi-quantified relative to α-tub and H3, respectively;
GAPDH, α-tub and histone H3 were used as equal loading controls. *p<0.05, **p<0.01, and ***p<0.001 vs. the DMSO control group.
protein in TMZ-resistant T98G cells (Figure 1A). Previous
studies suggest that MSH6 stabilizes MSH2 by forming
MSH2-MSH6 heterodimers (30). Thus, we examined
whether HDAC6 inhibitors could up-regulate MSH6 protein
levels. In addition to MSH2, four HDAC6 inhibitors
substantially increased MSH6 levels in TMZ-resistant T98G
cells. In contrast, all tested HDAC6 inhibitors decreased the
levels of both MSH6 and, to a lesser extent, MSH2 proteins
in TMZ-sensitive U87 cells (Figure 1B). These results
indicate that selective inhibition of HDAC6 markedly
increases MSH protein levels in TMZ-resistant GBM cells,
but not in TMZ-sensitive GBM cells.
Next, we investigated whether HDAC6 inhibitor affects
the expression of another DNA repair protein, MGMT,
related to DNA MMR and TMZ resistance. As shown in
Figure 1A, A452 and ACY-1215 reduced MGMT levels in
TMZ-resistant T98G cells with high basal MGMT
expression. On the other hand, TMZ-sensitive U87 cells with
no MGMT expression due to gene silencing by promoter
methylation appeared ubiquitinated MGMT, but not
unmodified MGMT. A452, ACY-1215 and CAY10603
slightly increased the levels of ubiquitinated MGMT. No
detectable alterations in MGMT protein level were observed
in TMZ-sensitive U87 cells treated with tubastatin A (Figure
ANTICANCER RESEARCH 39: 6731-6741 (2019)
6734
Figure 2. HDAC6 inhibitors lead to a pronounced inhibition of EGFR in both TMZ-resistant and TMZ-sensitive GBM cells. (A) TMZ-resistant T98G
and (B) TMZ-sensitive U87 GBM cells were treated with 0.1% DMSO (control) or HDAC6 inhibitor at the indicated concentrations for 24 h. Whole￾cell lysates were subjected to immunoblotting with the indicated antibodies. Relative protein expression levels were semi-quantified by densitometric
analysis of the blots. pEGFR was semiquantified relative to EGFR. Levels of Ace-α-tub were semiquantified relative to α-tub; GAPDH and α-tub
were used as equal loading controls. *p<0.05, **p<0.01, and ***p<0.001 vs. the DMSO control group.
1B). Overall, this result indicates that HDAC6-selective
inhibitors modulate MGMT protein levels in TMZ-resistant
GBM cells, but not in TMZ-sensitive GBM cells.
HDAC6-selective inhibitor inactivates EGFR pathway by
destabilizing EGFR in GBM cells. Previous studies have
shown that EGFR plays a pivotal role in TMZ resistance (31)
and HDAC6 inhibitors inactivate the EGFR pathway (21).
To test whether HDAC6 inhibitors regulate EGFR in TMZ￾sensitive and TMZ-resistant GBM cells, we tested the
phosphorylation level of EGFR and total EGFR by western
blotting. A452, ACY-1215 and CAY10603 destabilized
EGFR at higher concentrations in TMZ-sensitive U87 and
TMZ-resistant T98G GBM cells (Figure 2). Moreover,
inhibition of HDAC6 by these three HDAC6-selective
inhibitors decreased EGFR phosphorylation levels in both
GBM cells. This finding suggests that inhibition of HDAC6
controls the survival and chemoresistance of GBM cells via
destabilization of EGFR and inactivation of the EGFR
pathway.
HDAC6-selective inhibitor differentially modulates p53 by
up-regulating wild-type and down-regulating mutant p53 in
GBM cells. Previously, we reported that inhibition of
HDAC6 by A452 differentially modulates p53 by up￾regulating wtp53 and down-regulating mutp53 in cancer cells
(32). In the present study, we assessed whether HDAC6
altered the p53 signaling pathway in TMZ-sensitive and
TMZ-resistant GBM cells. Four tested HDAC6 inhibitors
robustly down-regulated mutp53 protein in TMZ-resistant
T98G cells in a dose-dependent manner (Figure 3A).
Conversely, A452 and CAY10603 slightly up-regulated
Kim et al: HDAC6 Regulates MMR in TMZ-resistant GBM
6735
Figure 3. HDAC6 inhibitor differentially modulates p53 in both TMZ-resistant and TMZ-sensitive GBM cells. (A) TMZ-resistant T98G and (B) TMZ￾sensitive U87 GBM cells were treated with 0.1% DMSO (control) or HDAC6 inhibitor at the indicated concentrations for 24 h. Whole-cell lysates
were subjected to immunoblotting with the indicated antibodies. *p<0.05, **p<0.01, and ***p<0.001 vs. the DMSO control group.
wtp53 protein in TMZ-sensitive U87 cells (Figure 3B).
MDM2, a key negative regulator of p53, down-regulated in
GBM cells, independently of p53 and TMZ resistance status.
HDAC6-selective inhibitor sensitizes GBM cells to TMZ.
Among four tested HDAC6 inhibitors, we selected and used
for further study the two more effective HDAC6 inhibitors,
A452 and ACY-1215, based on MutSα and EGFR
destabilization result. Next, we examined the effects of the two
HDAC6 inhibitors, A452 and ACY-1215, on cell viability in
TMZ-sensitive and TMZ-resistant GBM cells. Cells were
cultured with an HDAC6 inhibitor for up to 72 h, and cell
viability was measured by CCK-8 assays. A452 and ACY-1215
resulted in a dose-dependent decrease in cell viability in both
TMZ-sensitive U87 and TMZ-resistant T98G GBM cells
(Figure 4). Next, we tested whether HDAC6 inhibitor enhanced
the antiproliferative effects of TMZ in TMZ-sensitive and
TMZ-resistant GBM cells. The combination of A452 with
TMZ augmented cell death compared with cultures treated with
TMZ alone in both cells (Figure 4). Similarly, the combination
of TMZ and ACY-1215 showed synergistic cytotoxicity.
Furthermore, the CI values, a quantitative measure of drug
interaction according to the Chou and Talalay method, were
evaluated (28). The CI for A452-TMZ and ACY-1215-TMZ in
both GBM cells was <1, indicating the synergistic effect of the
combination (Figure 4). In particular, A452 demonstrated
significant synergy in the TMZ-resistant T98G GBM cells
compared with TMZ-sensitive U87 GBM cells. The ACY-
1215-TMZ combination exhibited similar synergistic effects on
both GBM cells, albeit weaker synergy than the A452-TMZ
combination. This finding indicates that the HDAC6 inhibitor–
TMZ combination may be more efficient on TMZ-resistant
GBM cells with mutp53 and MGMT expression and that the
HDAC6 inhibitor may overcome TMZ resistance.
HDAC6-selective inhibitor in combination with TMZ
synergistically increase apoptosis in TMZ-resistant GMB
cells. Selective inhibition of HDAC6 increased MMR
proteins in TMZ-resistant T98G cells, but not in TMZ￾sensitive U87 GBM cells. Therefore, we focused on T98G
cells which express MGMT and have mutated p53. To
investigate the mechanism of cell death in GBM cells
cultured with a combination of HDAC6 inhibitor and TMZ,
we evaluated their apoptotic effects. Cells treated with the
combination of TMZ and A452 or ACY-1215 had increased
levels of cleaved poly(ADP ribose) polymerase (PARP) and
of active caspase-3, which are apoptosis markers (Figure
5A). Furthermore, Annexin V/PI staining revealed that cell
apoptosis was significantly increased following combination
treatment of cells with TMZ and A452 or ACY-1215 (Figure
5B). Overall, our results suggest that combination treatment
with an HDAC6-selective inhibitor and TMZ triggers
apoptosis by activating caspase 3.
Discussion
The ultimate hindrance to the clinical success of GBM
treatment is drug resistance causing high recurrence rate of
this cancer. HDACs, the most promising target in drug
development for cancer therapy, are known to be associated
with oncogenesis of GBM in various human GBM cell lines
(33). In this paper, we analyzed the effects of treatment of
HDAC6-selective inhibitors in order to find potential
relationship between HDAC6 and GBM resistance. Thus, we
conducted further research into the detailed mechanism of
HDAC6 inhibition and the potential of combination of
treatment of HDAC6-selective inhibitor with TMZ, a widely
used chemotherapeutic drug.
We have shown the different impact of each HDAC6
inhibitor on GBM proliferation and resistance by differential
modulation of DNA MMR, EGFR, and p53 pathways. EGFR
targeting has been studied as a cancer treatment strategy.
Reducing EGFR protein levels as well as inhibiting EGFR
kinase activity is a pivotal strategy to block the oncogenic
effects of EGFR in cells. HDAC6 deacetylates the molecular
chaperone Hsp90, and the deacetylated active form of Hsp90,
regulates its chaperone activity for its client proteins, including
EGFR (34, 35). Thus, the use of HDAC6 inhibitors is another
strategy to down-regulate EGFR protein levels in cancer cells.
Overexpression of EGFR is also associated with
chemoresistance in GBM (31). In consistent with previous
results, EGFR expression was higher in TMZ-resistant GBM
cells than TMZ-sensitive GBM cells. First, we showed that
HDAC6 inhibitors reduced the phosphorylation levels of EGFR
and total EGFR in both TMZ-sensitive and TMZ-resistant GBM
cells although the altered levels of EGFR were different. This
result suggests that selective inhibition of HDAC6 controls the
survival and chemoresistance of GBM cells by destabilizing
EGFR and inactivating the EGFR pathway.
ANTICANCER RESEARCH 39: 6731-6741 (2019)
6736
→
Figure 4. Combined treatment of HDAC6 inhibitor and TMZ triggers
synergistic cytotoxicity. (A) TMZ-resistant T98G and (B) TMZ-sensitive
U87 GBM cells were treated with 0.1% DMSO (control) or the
indicated compounds alone or in combination for 72 h. Combination
treatments were then performed in cells maintaining a constant ratio
between the doses of TMZ and A452 or ACY-1215 and cell viability was
assessed at 72 h by CCK-8 assay. The combination index (CI) value and
the relative fraction affected (FA) were determined at each dose
combination (actual) and a simulation was run to estimate the CI values
and confidence intervals across the entire FA range (simulation). CI<1,
CI=1 and CI>1 indicate synergistic, additive and antagonistic effects,
respectively. Significance was determined by two-way analysis of
ANOVA with Bonferroni’s post-hoc test to compare groups. *p<0.05,
**p<0.01, and ***p<0.001 vs. the DMSO control group; #p<0.05, #p<0.01, and ###p<0.001 vs. the HDAC6 inhibitor-treated groups;
$p<0.05 and $$$p<0.001 vs. the TMZ-treated group.
Kim et al: HDAC6 Regulates MMR in TMZ-resistant GBM
6737
Second, we demonstrated that four tested HDAC6
inhibitors robustly down-regulated mutp53 protein in TMZ￾resistant T98G cells. In contrast, A452 and CAY10603 up￾regulated wtp53 protein in TMZ-sensitive U87 cells.
Although mutp53 is associated with resistance to TMZ and
most studied, the results have been mixed. Furthermore, p53
status was not always correlated with MGMT status in GBM
cell lines; TMZ-sensitive GBM cells (e.g., U87, A172)
possess wtp53 and p53 gene mutations were found in TMZ￾resistant GBM cells (e.g., LN-18, T98G, U138) as well as
TMZ sensitive-GBM cells (e.g., U251, U373). Thus, these
data indicate that a mutation in the TP53 gene does not seem
to be a primary indicator of resistance to TMZ (36). MDM2,
which is a p53-negative regulator related to resistant GBM
cell lines, decreased tumor size when down-regulated with
the antisense oligonucleotide (37). Interestingly, HDAC6
inhibitors caused decreased levels of MDM2 in both TMZ￾sensitive and TMZ-resistant GBM cells, independently of
ANTICANCER RESEARCH 39: 6731-6741 (2019)
6738
Figure 5. HDAC6 inhibitors in combination with TMZ synergistically increase apoptosis in TMZ-resistant T98G cells. (A) T98G cells were treated
with 0.1% DMSO (control), TMZ, A452, or ACY-1215, or in combination with these compounds for 24 h. Whole-cell lysates were subjected to
immunoblotting with the indicated antibodies. *p<0.05, **p<0.01, and ***p<0.001 vs. the DMSO control group. (B) TMZ-resistant T98G cells
were treated with 0.1% DMSO (control), TMZ, A452, or ACY-1215 or in combination with these compounds for 48 h. Cell death was assessed by
flow cytometry and Annexin V/PI staining (n=3). Significance was determined by two-way analysis of ANOVA with Bonferroni's post-hoc test to
compare groups. *p<0.05, **p<0.01, and ***p<0.001 vs. apoptotic cells in the DMSO control group; ##p<0.01 and ###p<0.001 vs. apoptotic cells
in the HDAC6 inhibitor-treated group; $$p<0.01 and $$$p<0.001 vs. the TMZ-treated group.
their p53 and TMZ resistance status. This finding indicates
that HDAC6-selective inhibitors may cause cell death by
reduction of hyperstable mutp53 as an oncogene.
Third, we found that HDAC6-selective inhibitor activates
MMR pathways in TMZ-resistant cells, but not in TMZ￾sensitive cells. In line with previous findings, HDAC6-
selective inhibitors increased the levels of MSH2 protein in
TMZ-resistant GBM cells. Zhang M et al. have reported that
HDAC6 interacts with MSH2 and MSH6, deacetylating
these MMR proteins. Subsequently, the deacetylated MSH2
is degraded by proteasome, reducing DNA MMR activity
(24). However, they did not provide the evidence that
MSH6 protein stability can be directly regulated by
HDAC6. In our finding, HDAC6-selective inhibitors
significantly increased the protein levels of MSH6 as well
as MSH2 in TMZ-resistant GBM cells. In contrast, the
effect of HDAC6 inhibitor on activation of the MMR
pathway was not observed in TMZ-sensitive GBM cells,
probably because MSH2 and MSH6 are already highly
expressed in TMZ-sensitive cells. It has also been reported
that mutation of MSH6 occurs after TMZ treatment and
knockdown of MSH6 increases TMZ resistance in U251
GBM cells (38). In addition, the MSH6 mutation was
mostly found in recurrent GBM patients, but not in newly￾diagnosed GBM patients (39). Based on these findings,
MSH6 may play a crucial role in TMZ resistance in GBM.
Since our findings show that HDAC6 regulates MSH6
levels, HDAC6 inhibitor may be a potential therapeutic
agent to overcome TMZ resistance.
Thus, further work will be required to determine the
molecular mechanisms whereby HDAC6 regulates MSH6
stability.
Another DNA repair protein, MGMT, which is related to
DNA MMR and TMZ resistance, was reduced by treatment
with A452 and ACY-1215 in TMZ-resistant GBM cells.
These results are hopeful for overcoming TMZ-resistance
and, perhaps, suggest a possible utility of the inhibition of
HDAC6 in combination with traditional chemotherapy for
GBM. Indeed, HDAC6 sensitized GBM cells to the inhibition
of TMZ-induced cell proliferation and the induction of
apoptosis, which occur as a result of the activation of
caspases and DNA MMR and the destabilization of EGFR
and p53 in TMZ-resistant GBM cells. Our findings indicate
that HDAC6-mediated destabilization of MutSα and
stabilization of MGMT may partly explain the oncogenic
function of HDAC6 in GBM resistance. Taken together, our
results suggest that selective inhibition of HDAC6 may be a
promising strategy for the treatment of GBM and overcoming
TMZ resistance.
Conflicts of Interest
The Authors report no conflicts of interest regarding this study.
Authors’ Contributions
GWK and DHL designed and performed experiments, as well as
analyzed the data. SKY, YHJ, YJ, and SWL performed experiments.
S.H.K conceived the general design of the study, participated in the
development of the approaches, wrote the initial draft of the
manuscript, extensively edited the manuscript and supervised the
work.
Acknowledgements
The Authors would like to thank Dr. Gyoonhee Han (Yonsei
University, Seoul, Korea) for providing A452. This research was
supported by the Basic Science Research Program through the
National Research Foundation of Korea (NRF) funded by the
Ministry of Education, Science and Technology (2016R1D1A1A
02937071, 2018R1A6A1A03023718, 2019R1I1A1A01058601, and
2019R1A2C1008619).
References
1 Ng K, Kim R, Kesari S, Carter B and Chen CC: Genomic
profiling of glioblastoma: Convergence of fundamental biologic
tenets and novel insights. J Neurooncol 107(1): 1-12, 2012.
PMID: 22002595. DOI: 10.1007/s11060-011-0714-2
2 Rich JN and Bigner DD: Development of novel targeted
therapies in the treatment of malignant glioma. Nat Rev Drug
Discov 3(5): 430-446, 2004. PMID: 15136790. DOI:
10.1038/nrd1380
3 Stupp R, Pavlidis N, Jelic S and Force EGT: Esmo minimum
clinical recommendations for diagnosis, treatment and follow￾up of malignant glioma. Ann Oncol 16(Suppl 1): i64-65, 2005.
PMID: 15888760. DOI: 10.1093/annonc/mdi834
4 Stupp R, Brada M, van den Bent MJ, Tonn JC, Pentheroudakis
G and Group EGW: High-grade glioma: Esmo clinical practice
guidelines for diagnosis, treatment and follow-up. Ann Oncol
25(Suppl 3): iii93-101, 2014. PMID: 24782454. DOI: 10.1093/
annonc/mdu050
5 Mellai M1, Caldera V, Annovazzi L, Chiò A, Lanotte M, Cassoni
P, Finocchiaro G and Schiffer D: MGMT promoter
hypermethylation in a series of 104 glioblastomas. Cancer
Genomics Proteomics 6(4): 219-227, 2009. PMID: 19656999.
6 Messaoudi K, Clavreul A and Lagarce F: Toward an effective
strategy in glioblastoma treatment. Part i: Resistance
mechanisms and strategies to overcome resistance of
glioblastoma to temozolomide. Drug Discov Today 20(7): 899-
905, 2015. PMID: 25744176. DOI: 10.1016/j.drudis.2015.02.011
7 Cahill DP, Codd PJ, Batchelor TT, Curry WT and Louis DN:
Msh6 inactivation and emergent temozolomide resistance in
human glioblastomas. Clin Neurosurg 55: 165-171, 2008. PMID:
19248684.
8 Kitange GJ, Carlson BL, Schroeder MA, Grogan PT, Lamont
JD, Decker PA, Wu W, James CD and Sarkaria JN: Induction of
mgmt expression is associated with temozolomide resistance in
glioblastoma xenografts. Neuro Oncol 11(3): 281-291, 2009.
PMID: 18952979. DOI: 10.1215/15228517-2008-090
9 McFaline-Figueroa JL, Braun CJ, Stanciu M, Nagel ZD,
Mazzucato P, Sangaraju D, Cerniauskas E, Barford K, Vargas A,
Chen Y, Tretyakova N, Lees JA, Hemann MT, White FM and
Kim et al: HDAC6 Regulates MMR in TMZ-resistant GBM
6739
Samson LD: Minor changes in expression of the mismatch repair
protein msh2 exert a major impact on glioblastoma response to
temozolomide. Cancer Res 75(15): 3127-3138, 2015. PMID:
26025730. DOI: 10.1158/0008-5472.CAN-14-3616
10 Shinsato Y, Furukawa T, Yunoue S, Yonezawa H, Minami K,
Nishizawa Y, Ikeda R, Kawahara K, Yamamoto M, Hirano H,
Tokimura H and Arita K: Reduction of mlh1 and pms2 confers
temozolomide resistance and is associated with recurrence of
glioblastoma. Oncotarget 4(12): 2261-2270, 2013. PMID:
24259277. DOI: 10.18632/oncotarget.1302
11 Stark AM, Doukas A, Hugo HH, Hedderich J, Hattermann K,
Maximilian Mehdorn H and Held-Feindt J: Expression of DNA
mismatch repair proteins mlh1, msh2, and msh6 in recurrent
glioblastoma. Neurol Res 37(2): 95-105, 2015. PMID:
24995467. DOI: 10.1179/1743132814Y.0000000409
12 Fishel R and Wilson T: Muts homologs in mammalian cells.
Curr Opin Genet Dev 7(1): 105-113, 1997. PMID: 9024626.
DOI: 10.1016/s0959-437x(97)80117-7
13 Jiricny J and Nystrom-Lahti M: Mismatch repair defects in
cancer. Curr Opin Genet Dev 10(2): 157-161, 2000. PMID:
10753784. DOI: 10.1016/s0959-437x(00)00066-6
14 Liu L, Markowitz S and Gerson SL: Mismatch repair mutations
override alkyltransferase in conferring resistance to
temozolomide but not to 1,3-bis(2-chloroethyl)nitrosourea.
Cancer Res 56(23): 5375-5379, 1996. PMID: 8968088.
15 Marks P, Rifkind RA, Richon VM, Breslow R, Miller T and
Kelly WK: Histone deacetylases and cancer: Causes and
therapies. Nat Rev Cancer 1(3): 194-202, 2001. PMID:
11902574. DOI: 10.1038/35106079
16 Garmpis N, Damaskos C, Garmpi A, Kalampokas E, Kalampokas
T, Spartalis E, Daskalopoulou A, Valsami S, Kontos M, Nonni A,
Kontzoglou K, Perrea D, Nikiteas N, Dimitroulis D: Histone
deacetylases as new therapeutic targets in triple-negative breast
cancer: Progress and promises. Cancer Genomics Proteomics 14(5):
299-313, 2017. PMID: 28870998. DOI: 10.21873/cgp.20041
17 Gupta PK, Reid RC, Liu L, Lucke AJ, Broomfield SA, Andrews
MR, Sweet MJ and Fairlie DP: Inhibitors selective for hdac6 in
enzymes and cells. Bioorg Med Chem Lett 20(23): 7067-7070,
2010. PMID: 20947351. DOI: 10.1016/j.bmcl.2010.09.100
18 Seigneurin-Berny D, Verdel A, Curtet S, Lemercier C, Garin J,
Rousseaux S and Khochbin S: Identification of components of the
murine histone deacetylase 6 complex: Link between acetylation
and ubiquitination signaling pathways. Mol Cell Biol 21(23): 8035-
8044, 2001. PMID: 11689694. DOI: 10.1128/MCB.21.23.8035-
8044.2001
19 Lee YS, Lim KH, Guo X, Kawaguchi Y, Gao Y, Barrientos T,
Ordentlich P, Wang XF, Counter CM and Yao TP: The
cytoplasmic deacetylase hdac6 is required for efficient
oncogenic tumorigenesis. Cancer Res 68(18): 7561-7569, 2008.
PMID: 18794144. DOI: 10.1158/0008-5472.CAN-08-0188
20 Ai J, Wang Y, Dar JA, Liu J, Liu L, Nelson JB and Wang Z:
Hdac6 regulates androgen receptor hypersensitivity and nuclear
localization via modulating hsp90 acetylation in castration￾resistant prostate cancer. Mol Endocrinol 23(12): 1963-1972,
2009. PMID: 19855091. DOI: 10.1210/me.2009-0188
21 Wang Z, Hu P, Tang F, Lian H, Chen X, Zhang Y, He X, Liu W
and Xie C: Hdac6 promotes cell proliferation and confers
resistance to temozolomide in glioblastoma. Cancer Lett 379(1):
134-142, 2016. PMID: 27267806. DOI: 10.1016/j.canlet.
2016.06.001
22 Aldape K, Zadeh G, Mansouri S, Reifenberger G and von
Deimling A: Glioblastoma: Pathology, molecular mechanisms
and markers. Acta Neuropathol 129(6): 829-848, 2015. PMID:
25943888. DOI: 10.1007/s00401-015-1432-1
23 Radhakrishnan R, Li Y, Xiang S, Yuan F, Yuan Z, Telles E, Fang
J, Coppola D, Shibata D, Lane WS, Zhang Y, Zhang X and Seto
E: Histone deacetylase 10 regulates DNA mismatch repair and
may involve the deacetylation of muts homolog 2. J Biol Chem
290(37): 22795-22804, 2015. PMID: 26221039. DOI:
10.1074/jbc.M114.612945
24 Zhang M, Xiang S, Joo HY, Wang L, Williams KA, Liu W, Hu
C, Tong D, Haakenson J, Wang C, Zhang S, Pavlovicz RE, Jones
A, Schmidt KH, Tang J, Dong H, Shan B, Fang B,
Radhakrishnan R, Glazer PM, Matthias P, Koomen J, Seto E,
Bepler G, Nicosia SV, Chen J, Li C, Gu L, Li GM, Bai W, Wang
H and Zhang X: Hdac6 deacetylates and ubiquitinates msh2 to
maintain proper levels of mutsalpha. Mol Cell 55(1): 31-46,
2014. PMID: 24882211. DOI: 10.1016/j.molcel.2014.04.028
25 Choi E, Lee C, Park JE, Seo JJ, Cho M, Kang JS, Kim HM, Park
SK, Lee K and Han G: Structure and property based design,
synthesis and biological evaluation of gamma-lactam based hdac
inhibitors. Bioorg Med Chem Lett 21(4): 1218-1221, 2011.
PMID: 21256006. DOI: 10.1016/j.bmcl.2010.12.079
26 Lee DH, Won HR, Ryu HW, Han JM and Kwon SH: The hdac6
inhibitor acy1215 enhances the anticancer activity of oxaliplatin
in colorectal cancer cells. Int J Oncol 53(2): 844-854, 2018.
PMID: 29749542. DOI: 10.3892/ijo.2018.4405
27 Kwon S, Zhang Y and Matthias P: The deacetylase hdac6 is a
novel critical component of stress granules involved in the stress
response. Genes Dev 21(24): 3381-3394, 2007. PMID:
18079183. DOI: 10.1101/gad.461107
28 Chou TC: Drug combination studies and their synergy
quantification using the chou-talalay method. Cancer Res 70(2):
440-446, 2010. PMID: 20068163. DOI: 10.1158/0008-5472.CAN-
09-1947
29 Cahill DP, Levine KK, Betensky RA, Codd PJ, Romany CA,
Reavie LB, Batchelor TT, Futreal PA, Stratton MR, Curry WT,
Iafrate AJ and Louis DN: Loss of the mismatch repair protein
msh6 in human glioblastomas is associated with tumor
progression during temozolomide treatment. Clin Cancer Res
13(7): 2038-2045, 2007. PMID: 17404084. DOI: 10.1158/1078-
0432.CCR-06-2149
30 Verhaak RG, Hoadley KA, Purdom E, Wang V, Qi Y, Wilkerson
MD, Miller CR, Ding L, Golub T, Mesirov JP, Alexe G,
Lawrence M, O’Kelly M, Tamayo P, Weir BA, Gabriel S,
Winckler W, Gupta S, Jakkula L, Feiler HS, Hodgson JG, James
CD, Sarkaria JN, Brennan C, Kahn A, Spellman PT, Wilson RK,
Speed TP, Gray JW, Meyerson M, Getz G, Perou CM, Hayes
DN and Cancer Genome Atlas Research N: Integrated genomic
analysis identifies clinically relevant subtypes of glioblastoma
characterized by abnormalities in pdgfra, idh1, egfr, and nf1.
Cancer Cell 17(1): 98-110, 2010. PMID: 20129251. DOI:
10.1016/j.ccr.2009.12.020
31 Munoz JL, Rodriguez-Cruz V, Greco SJ, Ramkissoon SH, Ligon
KL and Rameshwar P: Temozolomide resistance in glioblastoma
cells occurs partly through epidermal growth factor receptor￾mediated induction of connexin 43. Cell Death Dis 5: e1145,
2014. PMID: 24675463. DOI: 10.1038/cddis.2014.111
32 Ryu HW, Shin DH, Lee DH, Choi J, Han G, Lee KY and Kwon
SH: Hdac6 deacetylates p53 at lysines 381/382 and differentially
ANTICANCER RESEARCH 39: 6731-6741 (2019)
6740
coordinates p53-induced apoptosis. Cancer Lett 391: 162-171,
2017. PMID: 28153791. DOI: 10.1016/j.canlet.2017.01.033
33 Svechnikova I, Almqvist PM and Ekstrom TJ: Hdac inhibitors
effectively induce cell type-specific differentiation in human
glioblastoma cell lines of different origin. Int J Oncol 32(4):
821-827, 2008. PMID: 18360709.
34 Kovacs JJ, Murphy PJ, Gaillard S, Zhao X, Wu JT, Nicchitta CV,
Yoshida M, Toft DO, Pratt WB and Yao TP: Hdac6 regulates
hsp90 acetylation and chaperone-dependent activation of
glucocorticoid receptor. Mol Cell 18(5): 601-607, 2005. PMID:
15916966. DOI: 10.1016/j.molcel.2005.04.021
35 Murphy PJ, Morishima Y, Kovacs JJ, Yao TP and Pratt WB:
Regulation of the dynamics of hsp90 action on the
glucocorticoid receptor by acetylation/deacetylation of the
chaperone. J Biol Chem 280(40): 33792-33799, 2005. PMID:
16087666.
36 Lee SY: Temozolomide resistance in glioblastoma multiforme.
Genes Dis 3(3): 198-210, 2016. PMID: 30258889. DOI:
10.1016/j.gendis.2016.04.007
37 Kondo S, Barnett GH, Hara H, Morimura T and Takeuchi J:
Mdm2 protein confers the resistance of a human glioblastoma
cell line to cisplatin-induced apoptosis. Oncogene 10(10): 2001-
2006, 1995. PMID: 7761100.
38 Yip S, Miao J, Cahill DP, Iafrate AJ, Aldape K, Nutt CL and
Louis DN: MSH6 mutations arise in GBM during temozolomide
therapy and mediated temozolomide resistance. Clin Cancer Res
15(14): 4622-4629, 2009. PMID: 19584161. DOI: 10.1158/
1078-0432.CCR-08-3012
39 Hunter C, Smith R, Cahill DP, Stephens P, Stevens C, Teague J,
Greenman C, Edkins S, Bignell G, Davies H, O’Meara S, Parker
A, Avis T, Barthorpe S, Brackenbury L, Buck G, Butler A,
Clements J, Cole J, Dicks E, Forbes S, Gorton M, Gray K,
Halliday K, Harrison R, Hills K, Hinton J, Jenkinson A, Jones
D, Kosmidou V, Laman R, Lugg R, Menzies A, Perry J, Petty
R, Raine K, Richardson D, Shepherd R, Small A, Solomon H,
Tofts C, Varian J, West S, Widaa S, Yates A, Easton DF, Riggins
G, Roy JE, Levine KK, Mueller W, Tubastatin A Batchelor TT, Louis DN,
Stratton MR, Futreal PA and Wooster R: A hypermutation
phenotype and somatic msh6 mutations in recurrent human
malignant gliomas after alkylator chemotherapy. Cancer Res
66(8): 3987-3991, 2006. PMID: 16618716.