Pharmacological inhibition of Bmi1 by PTC-209 impaired tumor growth in head neck squamous cell carcinoma
Wang et al. Cancer Cell Int
Pharmacological inhibition of Bmi1 by PTC-209 impaired tumor growth in head neck squamous cell carcinoma
Qiong Wang 0
Zhongwu Li 0
Yaping Wu 0 2
Rong Huang 0 2
Yumin Zhu 2
Wei Zhang 1
Yanling Wang 1
Jie Cheng 0 1
0 Jiangsu Key Laboratory of Oral Disease, Nanjing Medical University , 136 Hanzhong Road, Nanjing 210029, Jiangsu , China
1 Department of Oral Pathology, School of Stomatology, Nanjing Medical University , 136 Hanzhong Road, Nanjing 210029, Jiangsu , China
2 Department of Oral and Maxillofacial Surgery, Affiliated Stomatological Hospital, Nanjing Medical University , 136 Hanzhong Road, Nanjing 210029, Jiangsu , China
Background: Bmi1 (B lymphoma Mo-MLV insertion region 1 homolog) contributes to human tumorigenesis via epigenetic transcriptional silencing and represents a novel therapeutic target with great potentials. Here we sought to determine the therapeutic efficiency of PTC-209, a potent and selective Bmi1 inhibitor, in head neck squamous cell carcinoma (HNSCC) cells and a HNSCC xenograft model. Methods: The mutation pattern, mRNA level of Bmi1 in HNSCC and its associations with clinicopathological parameters were determined through comprehensive data mining and interrogation using publicly available databases GENT, cBioPortal, Oncomine and TCGA. The PTC-209, a selective and potent Bmi1 inhibitor, was exploited and its effect on Bmi1 expression was measured in two HNSCC cell lines Cal27 and FaDu. The phenotypical changes of HNSCC cells were observed upon PTC-209 treatment in vitro. Moreover, the therapeutic effects of PTC-209 for HNSCC were determined in a xenograft animal model. Results: Through comprehensive data mining and interrogation, we found that Bmi1 mRNA was frequently overexpressed in a subset of HNSCC samples. Our data revealed that PTC-209 robustly reduced the expression of Bmi1 in Cal27 and FaDu cells presumably by post-transcriptional repression and ubiquitin-proteasomal degradation. PTC-209 treatment resulted in impaired cell proliferation, G1-phase cell cycle arrest, compromised migration and invasiveness, and increased cell apoptosis and chemosensitivity to 5-FU and cisplatin in vitro. Moreover, PTC-209 exposure reduced colony formation, tumorsphere formation and the percentage of ALDH1+ subpopulation in both Cal27 and FaDu cells. Importantly, in vivo PTC-209 administration significantly reduced tumor growth in a HNSCC xenograft model probably by Bmi1 inhibition and impaired cell proliferation. Conclusions: Our findings indicate that pharmacological inhibition of Bmi1 is a novel therapeutic strategy for HNSCC patients, especially with those with aberrant Bmi1 overexpression.
Head neck squamous cell carcinoma; Polycomb; Bmi1; PTC-209
Head neck squamous cell carcinoma (HNSCC) is the
sixth most common cancers and one of the leading
cancer-related death worldwide. The major etiological
risks for this malignancy include smoking abuse, alcohol
consumption, betel quid chew and human
papillomavirus (HPV) infection [
]. Despite tremendous
advancement in multimodal therapies against HNSCC over the
past decades, however, the long-term survival rate for
these devastating diseases, especially for patients with
advanced diseases, has not been markedly improved [
Locoregional relapse, cervical lymph node metastasis
and therapeutic resistance are recognized as the
prevalent factors affecting patient prognosis. Although
intensive efforts have been made to unravel the genetic and
environmental factors driving HNSCC tumorigenesis,
however, mechanistic understanding about HNSCC
tumorigenesis still remains far from complete [
Therefore, identification and verification of new biomarkers
and therapeutic targets for HNSCC are paramount and
urgent to improve treatment outcome .
Polycomb group proteins are key chromatin modulators
governing cell fate decision, stem cell self-renewal and
differentiation, and tumorigenesis largely via
transcriptional repression of a plethora of downstream targets [
B lymphoma Mo-MLV insertion region 1 homolog (Bmi1)
is a core member of the Polycomb Repressive Complex 1
(PRC1) required for monoubiquitination of histone 2A,
usually functions as an epigenetic silencer of target genes
such as Ink4a-arf locus [
]. Accumulating evidence has
been indicated that Bmi1 is critically involved in diverse
fundamental cellular processes such as cell proliferation,
apoptosis, senescence, epithelial–mesenchymal transition
(EMT) and stem cell maintenance [
overexpression of Bmi1 has been found in multiple human
malignancies such as lung, liver, breast and head neck
cancer. Its overexpression often correlates with advanced
stages, aggressive clinicopathological behaviors,
therapeutic resistance and unfavorable prognosis in these
]. Enforced overexpression of Bmi1 facilitated
malignant transformation, cancer cell proliferation, EMT
and metastatic spreading, whereas its depletion inhibited
cell proliferation, migration and invasion and induced
cell apoptosis and senescence both in vitro and in vivo
]. Noticeably, Bmi1 plays critical and
indispensable roles in governing self-renewal capacity of normal
and malignant cancer stem cells (CSCs) which the latter
has been increasingly recognized to largely responsible for
cancer initiation, therapeutic resistance, disease relapse
and metastatic dissemination [
]. Several target
genes including p16 and E-cadherin have been identified
to be responsible for these essential oncogenic roles of
Bmi1 in diverse cancer contexts including HNSCC [
These abovementioned findings demonstrate that Bmi1
is not only an oncogenic driver during tumorigenesis,
but also represents an cancer biomarker and
therapeutic target with translational significance. Indeed, genetic
silencing and pharmacological inhibition of Bmi1 induced
apoptosis and senescence, enhanced chemosensitivity,
and diminished invasive and metastatic potentials, thus
ultimately compromised cancer progression [
9, 19, 20
Particularly, targeting Bmi1 with a selective
small-molecule inhibitor (PTC-209) resulted in loss of colorectal
CSCs and caused long-term, irreversible impairment of
tumor progression [
12, 21, 22
]. Thus, these findings
support the notion that chemical targeting Bmi1 might be an
attractive and plausible way to eradicate cancers as a novel
therapeutic strategy .
Several lines of evidence have revealed that Bmi1
associates with malignant transformation of precancerous
lesions, EMT and CSCs maintenance of HNSCC [
]. Elevated Bmi1 usually correlated with aggressive
features and unfavorable patients’ survival [
11, 13, 20, 25
Our previous study has found that Bmi1 was aberrantly
overexpressed in oral tongue cancer and it was potently
inhibited by HDACi (inhibitors of histone deacetylases)
chemicals, which in turn resulted in impaired tumor
growth. However, these chemicals are not specific and
might induced unwanted downstream effects beyond
Bmi1 inhibition . In the present study, we sought to
determine the therapeutic efficiency of PTC-209, a novel
and selective inhibitor of Bmi1, against HNSCC using
both in vitro cell culture and in vivo xenograft animal
Materials and methods
HNSCC cell lines and chemical reagents
Two HNSCC cell lines Cal27 and FaDu were purchased
from American Type Culture Collection (ATCC) and
authenticated by short tandem repeat (STR) profiling and
tested for mycoplasma. Cells were grown in DMEM/F12
(Invitrogen) supplemented with 10% FBS (Gibco) and
penicillin–streptomycin (1%), and maintained at 37 °C in
a 5% CO2-humidified incubator. PTC-209 was purchased
from Selleck (catalog No. S7372) and dissolved in DMSO.
Two common anticancer agents 5-fluorouracil (5-FU)
and cisplatin were purchased from Sigma-Aldrich,
dissolved as stocking solutions and diluted with culture
medium upon additions to cell plates. HNSCC cells were
treated with diverse concentrations of PTC-209 alone or
in combination with 5-FU or cisplatin for indicated times
and then harvested for further analyses.
RNA extraction and real‑time RT‑PCR
Total RNA was extracted from cells using Trizol
reagent (Invitrogen) and then reversely transcribed into
first strand cDNA using PrimeScript™ RT reagent kit
(Takara). Then the cDNA was subjected to real-time PCR
reaction using SYBR Premix Ex Taq™ kit (Takara)
following the manufacturer’s instructions. The gene-specific
primers for human Bmi1, Ezh2, p16 and GAPDH were
purchased commercially (Invitrogen). Relative mRNA
expression of each gene as compared to internal control
GAPDH was quantified using comparative CT method.
Western blot and immunoprecipitation assay
Cells in culture flasks or plates were lysed in ice-cold
buffer containing protease inhibitor cocktail (Roche).
Equal amounts of protein samples were loaded and
separated by 8–12% SDS-PAGE and transferred to PVDF
membranes (Millipore) followed by 5% non-fat milk or
3% BSA blocking. These blots were incubated at 4 °C
overnight with primary antibodies against Bmi1, p16,
cleaved-PARP, Sox2, Nanog, Oct4 (Cell signaling, 1:1000
dilution), E-cadherin, N-cadherin and Vimentin (BD
Biosciences, 1:2000 dilution), Lin28B (Abcam, 1:1000
dilution), ubiquitinated histone 2A and total histone 2A
(Millipore, 1:1000 dilution), and GAPDH (Santa Cruz,
1:1000 dilution) followed by incubations with the
corresponding secondary antibodies. The relative levels of
each protein were quantified with Quantity One software
(Bio-Rad). The protein–protein interaction was
determined by protein immunoprecipitation using
Pierce™CoImmunoprecipitation Kit (ThermoFisher) and performed
according to the manufacturer’s protocol. The
antibodies for IP and following western blot were anti-Bmi1
(Cell signaling, #6964) and anti-ubiquitin (Cell signaling,
Cell proliferation and viability were determined by
absorbance using MTT assay. Approximately 1000–3000
cells per well were seeded in the 96-well plates. At the
indicated time-points, 5 mg/ml MTT (Sigma) was added
to the plates and incubated at 37 °C for another 4 h.
Absorbance at 490 nm was measured with an automatic
enzyme-linked immunosorbent assay reader (BioTek
Flow‑cytometric analysis and ALDEFLUOR assay
For cell apoptosis assay, cells were stained with Annexin
V: PI Apoptosis Detection Kit (BD Bioscience). The
ALDEFLUOR assay was used to monitor the
percentage of ALDH1+ cells in HNSCC treated with or without
PTC-209 with a FACSCalibur flow cytometer. A specific
ALDH1 inhibitor, diethylaminobenzaldehyde, was used
as a negative control.
In vitro cell migration and wound healing assay
Cell invasion assay was performed using chambers
(8-μm pore size, Corning) in 24-well plates. Forty-eight
hours after PTC-209 treatment, cells were detached and
resuspended, then seeded into the upper chambers with
medium containing 1% FBS. Complete medium
containing 10% FBS in the lower chambers served as
chemoattractant. The non-invading cells were gently removed
with a cotton swab and those migratory cells located on
the lower side were stained with crystal violet. The
number of migrated cells were counted and averaged in
randomly selected 10 fields under microscope. For wound
healing assay, cells were grown into confluent monolayers
and scratched using a sterile 200 µl pipette. Cell
migration was observed at various time-points later by
microscopy. Images of 10 scratches per cells were captured
during the experiment and compared with Image J
Colony formation and tumorsphere formation assays
For colony formation assay, 1000 single cells pretreated
with PTC-209 or vehicle were seeded into 6-well plates
or dishes and allowed to grow for 10–14 days. The cells
were then fixed and stained with crystal violet. The
colonies were further visualized under an invert microscope
and photographed. Cell aggregations with more than 50
cells were defined as colonies and counted.
For tumorsphere assay, single cells pretreated with
PTC-209 or vehicle were seeded at a density of 1000/well
in ultra-low attachment plates and cultured in
serumfree DMEM/F12 supplemented with 1% B27 and N2
supplements as well as human recombinant epidermal
growth factor (EGF, 20 ng/ml, R&D systems) and basic
fibroblast growth factor (bFGF, 10 ng/ml, R&D systems).
The fresh media were changed every other day until the
tumorsphere formed. For passaging, the tumorspheres
were collected by centrifugation and dissociated by 0.5%
trypsin to obtain single cells which were further cultured
for secondary tumorsphere formation. The tumorsphere
with a diameter over 100 μm were counted and recorded
2 weeks after plating.
PTC‑209 treatment in HNSCC xenograft model
All the animal protocols (2016-168) were in accordance
with institutional animal welfare guidelines of Nanjing
Medical University. Both 2 × 106 Cal27 and FaDu cells
in 100 μl PBS were injected subcutaneously into nude
mice (male, aged 8 weeks). Three weeks later, these mice
bearing tumors (approximate 100 mm3 in volume) were
randomly divided into two groups (6 mice per group)
which were scheduled to receive the following
treatments: 30 mg/kg PTC-209 every day by subcutaneous
injection or vehicle (PBS) only in control animals for
consecutive 15 days. The tumor diameters were measured
by calipers every 3 days. Tumor volume was calculated
by the formula volume (mm3) = [length (mm) × width
(mm)2] × 0.5. Tumor weight was also measured upon
tumor samples were harvested.
Immunohistochemical staining for Bmi1 and Ki-67
in these samples was performed and scored similarly as
our previous reports [
]. The immunoreactivity in
each slide was assessed independently by two senior oral
pathologists without knowledge about the clinical and
pathological information. Negative controls (without
primary antibody incubation) were included in each staining
run. Immunoreactivity was semi-quantitatively evaluated
on the basis of staining intensity and distribution using
the immunoreactive score. Immunoflurescent staining of
CD44 (ab51037, Abcam) in samples were also conducted
as we previously documented. The images were further
visualized under fluorescence microscope.
Data mining and analysis of Bmi1 in HNSCC via publicly available database
The original data concerning mutational landscape and
expression of Bmi1 mRNA in HNSCC were retrieved
from three publicly available databases including GENT
cBioPortal (http://www.cbioportal.org/) [
] and TCGA (https://
cancergenome.nih.gov/). The expression levels of Bmi1
mRNA (log2-transformed) in HNSCC and normal
counterparts were retrieved and statistically compared. The
associations between expression status of Bmi1 (high or
low using median value as cutoff ) and clinicopathological
parameters as well as overall survival were determined by
Chi square test or Kaplan–Meir analysis (Log-rank test),
All quantitative data in the present study was shown as
mean ± SD of two or three independent experiments and
compared with Student’s t test or ANOVA with
Bonferroni post hoc test unless otherwise specified. p values
less than 0.05 (two-sided) were considered statistically
significant. All statistical analyses were performed using
Graphpad Prism 6 or SPSS 18.0 software.
Bmi1 mRNA is overexpressed in a subset of HNSCC samples
Our previous studies and others have indicated that
Bmi1 is aberrantly overexpressed in oral tongue cancer
and HNSCC samples with both diagnostic and
prognostic values [
9, 15, 20, 25
]. To extend these findings
and reinforce this notion, we utilized the publicly
available databases including GENT, cBioPortal, Oncomine
and TCGA to interrogate the mutational landscape and
expression pattern of Bmi1 in HNSCC. As shown in
Fig. 1a, transcriptional profiling data of human cancer
in GENT revealed that the mRNA level of Bmi1 varied
remarkably among diverse human cancers and its
abundance was frequently higher in cancers than those
normal counterparts including HNSCC. Data mining from
Oncomine database indicated marked overexpression of
Bmi1 mRNA in HNSCC samples from Toruner’s  and
] patient cohorts, but comparable in HNSCC
samples from Cromer’s [
], Kuriakose’s [
] as well
as Peng’s [
] cohorts (Fig. 1b, c and data not shown).
Unexpectedly, interrogation of TCGA HNSCC dataset
revealed that the abundance of Bmi1 mRNA in HNSCC
samples (502 cases) was comparable to normal
epithelial (44 cases). No significant associations between Bmi1
expression (median value as cutoff between low and high
expression) and aggressive clinicopathological
parameters and patients survival were identified (Additional
file 1: Figure S1, Additional file 2: Table S1). Further
bioinformatics analyses in cBioPortal database revealed that
total frequencies of Bmi1 genetic amplification, mutation
and deletion in HNSCC samples were less than 3%,
suggesting that gene structural variations might not be
primarily responsible for its expression pattern in HNSCC.
Taken together, these analyses from bioinformatics data
mining and interrogation suggest that Bmi1 mRNA is
aberrantly overexpressed in a fraction of HNSCC and
might serve as putative oncogene during HNSCC
initiation and progression.
PTC‑209 reduces Bmi1 expression probably by inhibiting its transcription and inducing its protein degradation in HNSCC cells
Accumulating evidence has revealed that Bmi1
represents a promising therapeutic target with considerable
translational potentials [
]. The pioneering studies have
identified PTC-209 as an novel chemical inhibitor of
Bmi1 through chemical library compound screen and
demonstrated its potency and specificity against Bmi1
in vitro and in vivo [
]. We wondered whether
PTC209 had the similar inhibitory effect on Bmi1 and induced
therapeutic effects in HNSCC. To address this issue, we
initially selected two HNSCC cell lines Cal27 and FaDu
with relatively high endogenous Bmi1 and incubated
them with diverse concentrations of PTC-209 and then
monitored the expression changes of Bmi1. Following the
exposure of PTC-209 for 48 h, the protein abundance of
Bmi1 in both cells was significantly reduced as compared
to vehicle-treated cells (Fig. 2a, left panel). Moreover,
when cells were exposed during a 72 h time course, the
Bmi1 protein levels were greatly diminished in a
timedependent manner (Fig. 2a, right panel). To understand
the mechanisms underlying Bmi1 reduction upon
PTC209 exposure, the changes of Bmi1 mRNA abundance
were measured by real-time RT-PCR. As displayed in
Fig. 2b, PTC-209 treatment (10 μM, 48 h) resulted in
significant downregulation of Bmi1 transcripts and
derepression of its well-established downstream target p16.
In addition, global ubiquitinated histone 2A (uH2A), the
hallmark of Bmi1-mediated repressive chromatin
structures and transcriptional silencing, was significantly
downregulated upon PTC-209 treatment (10 μM, 48 h,
Additional file 3: Figure S2). However, the expression
of other polycomb members EZH2, SUZ12 and EED
was not affected (data not shown), thus partially
supporting the highly selective nature of PTC-209 against
Bmi1. Moreover, considering the relatively instability of
Bmi1 protein [
], we next sought to explore whether
PTC-209 affected the turnover of Bmi1 protein in
HNSCC cells. To this regard, we monitored the changes
of Bmi1 protein when cells were treated with PTC-209
alone or in combination with the proteinase inhibitor
MG132. Intriguingly, as shown in Fig. 2c, Bmi1
reduction induced by PTC-209 exposure (10 μM) was partially
attenuated by MG132, thus suggesting that PTC-209
might induce Bmi1 downregulation partially by
triggering its protein degradation. Furthermore, as shown
in Fig. 2d, our data revealed that the ubiquitination of
Bmi1 protein was markedly increased following PTC-209
exposure in vitro. Collectively, these data clearly indicate
that Bmi1 can be pharmacologically inhibited by
PTC209 in HNSCC and this effect is presumably induced by
post-transcriptional repression and protein degradation.
PTC‑209 inhibited cell proliferation, migration and invasion, and induced cell cycle arrest and apoptosis in HNSCC cells
Having demonstrated the inhibitory effects of
PTC209 on Bmi1 expression in HNSCC, we next sought to
determine the phenotypic changes upon PTC-209 in
detail. As shown in Fig. 3a, when cells were treated with
diverse concentrations of PTC-209, the cell proliferation
was markedly suppressed as measured in MTT assay. In
addition, when cells were treated with PTC-209 alone
or together with 5-FU or cisplatin, the common
therapeutic agents against HNSCC, our data revealed more
obviously anti-proliferative effects of combinational
treatments (PTC-209+5-FU, PTC-209+cisplatin)
compared to single agent treatment (Fig. 3b), suggesting the
potential synergic effects of PTC-209 and these agents.
Moreover, cell cycle analysis revealed G1 arrest upon
PTC-209 treatment in Cal27 and FaDu cells (Fig. 3c
and data not shown). The proportions of cell
undergoing apoptosis after PTC-209 exposure were significantly
increased (Cal27 3.16% vs 7.81%; FaDu 2.84 vs 9.03%;
p < 0.01) as evidenced by Annexin V-PI double staining
and increased apoptosis marker cleaved-PARP (Fig. 3d,
e). Given the proposed roles of Bmi1 during EMT and
(See figure on next page.)
Fig. 3 PTC-209 inhibited cell proliferation and induced cell cycle arrest, cell apoptosis and enhanced chemosensitivity in HNSCC cells. a Cell
proliferation was remarkably suppressed in Cal27 and FaDu cells upon treatment with PTC-209 (10 μM) as measured by MTT assay. b Cell viability was
more significantly impaired in cells treated with PTC-209 (10 μM) pus 5-FU (2.5 μg/ml) or cisplatin (2.5 μg/ml) than those treated with single agent. c
Increased percentages of cells in G1 stage was observed in Cal27 treated with PTC-209 (10 μM) for 48 h. d Increased percentages of cell
undergoing apoptosis (both early and late apoptosis) were detected in cells treated with PTC-209 (10 μM) as compared to control (vehicle-treated cells) as
assayed by Annexin V-PI double staining. e The abundances of apoptosis marker cleave-PARP were concomitantly increased after PTC-209 exposure
(10 μM) for 48 h. Representative images are shown. Data showed here are mean ± SD from three independent experiments, *p < 0.05, **p < 0.01,
Student’s t test and ANOVA analyses
invasion in HNSCC [
9, 13, 20
], we next proceeded to
determine whether PTC-209 affected the migratory and
invasive potential of HNSCC cells. As anticipated, our
data from wound healing and transwell assays showed
that cell migration and invasion of both Cal27 and FaDu
cells were significantly inhibited after PTC-209
exposure (10 μM, 48 h) as compared to vehicle-treated cells
(Fig. 4a, b). In support of these findings, the
invasionassociated EMT markers E-cadherin was upregulated
while N-cadherin and Vimentin was concomitantly
downregulated following PTC-209 addition (Fig. 4c).
PTC‑209 inhibited colony and tumorsphere formation and decreased the proportion of ALDH1+ subpopulation in HNSCC cells
Considering the critical roles of Bmi1 for CSCs
13, 16, 22
], we next sought to investigate the
effects of PTC-209 on the CSCs-associated traits in
HNSCC. Our data from colony formation assay revealed
that PTC-209 pretreatment significantly reduced the
numbers and size of colonies derived from both Cal27
and FaDu cells (Fig. 5a). Moreover, we utilized the
tumorsphere formation assay, a surrogate test for CSCs
stemness, to evaluate the effects of PTC-209 on the
selfrenewal properties of HNSCC cells. As expected, the
number of tumorsphere formed in vitro was significantly
decreased upon PTC-209 addition. Noticeably,
secondary tumorsphere formation was also remarkably impaired
in PTC-209-treated cells, thus suggesting that the
selfrenewal properties were compromised in HNSCC cells
treated with PTC-209 (Fig. 5b). In parallel, the ALDH1+
subpopulation which was functionally enriched with
putative CSCs [
] was also measured following PTC-209
treatment. As shown in Fig. 5c, d, the ratios of ALDH1+
cells were significantly reduced from 2.20 to 0.42% in
Cal27 and from 2.85 to 1.32% in FaDu, respectively.
Complemented with these findings, PTC-209 treatment also
resulted in remarkable downregulation of several CSCs
markers and modulators such as CD44, CD133, Sox2,
Nanog et al. (Fig. 5e).
Pharmacological inhibition of Bmi1 by PTC‑209 impaired
HNSCC overgrowth in vivo
Having demonstrated the anticancer effects of
PTC209 in vitro, we then wanted to determine its
therapeutic efficiency in vivo to further reinforce the
translational potential of PTC-209 as a novel
chemical against HNSCC. The HNSCC xenograft model was
developed by subcutaneous transplantation of HNSCC
cells in nude mice. After subcutaneous inoculation of
Cal27 and FaDu cell for consecutive 3 weeks, tumor
masses were established with approximate 100 mm3. The
mice bearing tumors were randomly divided into groups
for PTC-209 or vehicle treatment by subcutaneous
injection. Tumor volume in recipient animals were monitored
every 3 days by manual gauge with a caliber. During the
whole experiment, all animals survived and had normal
weight gains and undisturbed activities (data not shown),
thus suggesting these treatments were well-tolerated in
experimental animals. As shown in Fig. 6a, b, PTC-209
administration significantly retarded and impaired
tumor overgrowth as compared with vehicle as
measured by tumor volume and final weight of tumor masses,
although tumor regression was not achieved under our
experimental conditions. The samples derived from
animals in each group were proceeded to routine H&E
staining and immunohistochemical staining. As displayed in
Fig. 6c, the number of Bmi1-positive cancer cells in
PTC209-treated samples was significantly less than those in
vehicle-treated samples, therefore suggesting that Bmi1
was inhibited in vivo by PTC-209. Similarly, cancer cell
proliferation was compromised in PTC-209-treated
samples as evidenced by weaker and less Ki-67 staining
(Fig. 6c and data not shown). Moreover,
immunofluorescence staining of the putative HNSCC CSCs marker
] revealed that the number of CD44-positive
cells was much less in PTC-209-treated samples in
relative to controls (Fig. 6d).
The polycomb complex mediates gene repression by
modifying chromatin structure and is critically involved
in various fundamental biological processes including
stem cell homeostasis, cell differentiation and
]. In particular, Bmi1 has been increasingly
recognized as a key bona fide oncogene driving tumorigenesis
and a novel therapeutic target against human malignancy
]. In the present study, our data confirm that Bmi1
can be successfully inhibited by PTC-209 with high
potency and specificity in HNSCC. Pharmacological
targeting of Bmi1 resulted in impaired cell proliferation,
migration and invasion in parallel to increased cellular
apoptosis and reduced CSCs subpopulation. These
findings highlight the Bmi1 is a feasible and viable
therapeutic target against HNSCC with translational potentials.
Mounting evidence has established that Bmi1 is a
key oncogene intricately linked to cell transformation,
EMT and CSCs propagation in diverse cancer contexts
including HNSCC [
6, 9, 18
]. Ectopic Bmi1
overexpression results in senescence bypass and immortalization
of normal keratinocytes and malignant progression of
precancerous lesions into cancers [
24, 39, 40
bioinformatics analyses by data mining and interrogation
reveal that Bmi1 mRNA is aberrantly overexpressed in
a fraction of, although not all, HNSCC samples.
Previous studies including ours and others have indicated that
Bmi1 protein is usually upregulated in HNSCC as
compared to normal counterparts as determined by
immunohistochemical staining in clinical samples [
9, 15, 20,
]. Moreover, its expression status associated with
aggressive clinicopathological features and outcomes,
and served as an prognostic predictor for HNSCC [
15, 20, 24
]. In contrast, Hayry V, etc. reported that Bmi1
protein expression didn’t correlate with any clinical and
histopathological parameters and its negative expression
served as adverse prognostic factor in patients with
primary T1N0M0 tongue cancers . Moreover, our
findings using TCGA–HNSCC data also failed to reveal the
differential expression of Bmi1 between cancer and
normal epithelial and associations between Bmi1 expression
and clinicopathological characteristics or prognosis. The
reasons for this discrepancy remain unknown. We
speculated that such conflicting findings might be partially due
to diverse patient’s inclusion criteria, sample size, genetic
background, immunohistochemical scoring and potential
inconsistence between mRNA and protein abundance
of a specific gene. Collectively, these findings indicate
that aberrant Bmi1 overexpression is found in a subset
of HNSCC and might serve as one of the key molecular
events underlying HNSCC initiation and progression.
The well-established oncogenic roles of Bmi1 have
spurred intensive efforts to uncover its potential as a
therapeutic target for cancer. Previous studies have revealed
that Bmi1 expression and activity were potently inhibited
by HDACi presumably via transcriptional repression in
multiple texts of malignancies [
]. However, these
HDACi compounds are not specific to Bmi1 and might
induce unwanted side-effects as evidenced by our
previous findings that Trichostatin A (TSA) potently inhibited
Bmi1 but paradoxically induced EMT-like changes in
HNSCC cells . Recent pioneering studies have
identified PTC-209 as a potent and specific chemical inhibitor
of Bmi1 and revealed promising therapeutic efficiency of
PTC-209 against cancer [
]. Our data presented here
are consistent with previous findings and further
experimentally confirmed that PTC-209 is a potent inhibitor
of Bmi1 in HNSCC as evidenced by remarkable
downregulation of Bmi1 in vitro and in vivo. Furthermore,
Bmi1 loss induced by PTC-209 resulted in similar
phenotypic changes reminiscence of shRNA-mediated Bmi1
depletion in HNSCC cells, thus supporting Bmi1 as the
target of PTC-209. Noticeably, we found that in addition
to post-transcriptional regulation of Bmi1 expression by
PTC-209 , the proteasomal degradation might be
partially responsible for PTC-209-mediated Bmi1
downregulation in HNSCC as evidenced by the attenuation of
Bmi1 loss by MG132 addition following PTC-209
treatment and increased ubiquitination of Bmi1 after
PTC209 exposure. This is conceivable and in agreement with
previous findings Bmi1 protein is relatively unstable and
has short half-life in cancer cells [
36, 44, 45
]. Of course,
further investigations into mechanistic insights into
PTC-209-induced Bmi1 loss in diverse cancers are still
needed and warranted.
Accumulative evidence has revealed that Bmi1 has
critical roles implicated in normal and malignant stem
cell self-renewal and has been recognized as a
functional marker as well as regulator for CSCs in
malignancies including HNSCC [
13, 17, 22, 46
]. For example,
CSCs properties in human colorectal cancer are highly
dependent on Bmi1, as downregulation of Bmi1 inhibits
their self-renewal and abrogates their tumorigenic
potentials in vitro and in animal models . Not surprisingly,
our studies revealed that Bmi1 depletion mediated by
PTC-209 significantly inhibited tumorsphere formation,
reduced the percentage of CSCs subpopulation with
concomitant downregulation of CSCs markers. In
addition, PTC-209 also enhanced chemosensitivity and
vulnerability of HNSCC cells to 5-FU and cisplatin. These
findings largely phenocopied the Bmi1 knockdown [
thus supporting that PTC-209 might selectively target
the CSCs subpopulation via reducing Bmi1 in HNSCC.
Indeed, when our manuscript was under preparation,
Wang CY and his colleagues have reported that Bmi1 is
a marker for HNSCC CSCs subpopulation through
lineage tracing and also serves as a key driver mediating the
metastasis and chemoresistance of HNSCC. Genetic
ablation or pharmacological inhibition of Bmi1 markedly
impaired 4NQO-induced tongue tumorigenesis, tumor
overgrowth and cervical node metastasis [
given conventional chemotherapy usually enriched
putative CSCs [
] and sometimes induced Bmi1
upregulation in HNSCC , combinational therapy consisting
chemical agents and Bmi1 inhibitors might yield better
outcomes than monotherapy for HNSCC. Collectively,
these data strongly suggest that Bmi1 is a pivotal
regulator for CSCs maintenance and can be therapeutically
manipulated against HNSCC with promising benefits.
Here our findings indicate that pharmacological
depletion of Bmi1 in HNSCC by PTC-209 induces
anti-neoplastic effects both in vitro and in vivo xenograft animal
model. Disruption of Bmi1 by selective and potent
chemical inhibitors might represent a novel therapeutic
strategy against HNSCC, especially for patients with aberrant
Additional file 1: Figure S1. Bmi1 mRNA expression in HNSCC samples
derived from TCGA database. The original data of Bmi1 mRNA in HNSCC
samples and normal epithelial from TCGA patient cohort were download
and log2 transformed, and then statistically compared. #p > 0.05, Mann–
Whitney U test.
Additional file 2: Table S1. Associations between Bmi1 mRNA
expression and selected clinicopathological parameters in HNSCC.
Additional file 3: Figure S2. Global ubiquitinated histone 2A (uH2A), the
hallmark of Bmi1-mediated repressive chromatin structures and
transcriptional silencing, is significantly downregulated upon PTC-209 treatment
(10 μM, 48 h). The representative images of western blot are shown.
HNSCC: head neck squamous cell carcinoma; HPV: human papillomavirus;
PRC2: polycomb repressive complex 2; Bmi1: B lymphoma Mo-MLV insertion
region 1 homolog; PRC1: Polycomb Repressive Complex 1; EMT: epithelial–
mesenchymal transition; CSCs: cancer stem cells; HDACi: inhibitors of histone
deacetylases; EGF: epidermal growth factor; bFGF: basic fibroblast growth
QW, ZL, YW and RH performed the experimental study, data collection and
analysis and manuscript writing. YZ, YW and WZ performed in vivo animal
study and analyzed the IHC data. JC conceived and supervised the whole
project. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Availability of data and materials
All original data are available upon request.
Consent for publication
No parts of this manuscript are being considered for publication elsewhere. All
authors have read and finally approved this manuscript.
Ethics approval and consent to participate
Our study protocol was reviewed and approved by the Research Ethic
Committee of Nanjing Medical University (2016-1056). Written informed consent
was obtained from all subjects in our study.
This work is financially supported, in whole or in part, by National Natural
Science Foundation of China (81572669, 81602378), Natural Science Foundation
of Jiangsu Province (BK20151561, BK20161564, BK20161024), A Project Funded
by the Priority Academic Program Development of Jiangsu Higher Education
Institutions (2014-37) and Jiangsu Medical Talent Project (QNRC2016851).
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
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