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miR-23a promotes invasion of glioblastoma via HOXD10-regulated glial-mesenchymal transition


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Glioblastoma is the most aggressive and invasive brain tumor and has a poor prognosis; elucidating the underlying molecular mechanisms is essential to select molecular targeted therapies. Here, we investigated the effect of microRNAs on the marked invasiveness of glioblastoma. U373 glioblastoma cells were infected with 140 different microRNAs from an OncomiR library, and the effects of the invasion-related microRNAs and targeted molecules were investigated after repeated Matrigel invasion assays. Screening of the OncomiR library identified miR-23a as a key regulator of glioblastoma invasion. In six glioblastoma cell lines, a positive correlation was detected between the expression levels of miR-23a and invasiveness. A luciferase reporter assay demonstrated that homeobox D10 (HOXD10) was a miR-23a-target molecule, which was verified by high scores from both the PicTar and miRanda algorithms. Forced expression of miR-23a induced expression of invasion-related molecules, including uPAR, RhoA, and RhoC, and altered expression of glial-mesenchymal transition markers such as Snail, Slug, MMP2, MMP9, MMP14, and E-cadherin; however, these changes in expression levels were reversed by HOXD10 overexpression. Thus, miR-23a significantly promoted invasion of glioblastoma cells with polarized formation of focal adhesions, while exogenous HOXD10 overexpression reversed these phenomena. Here, we identify miR-23a-regulated HOXD10 as a pivotal regulator of invasion in glioblastoma, providing a novel mechanism for the aggressive invasiveness of this tumor and providing insight into potential therapeutic targets.
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miR-23a promotes invasion of glioblastoma via HOXD10-
regulated glial-mesenchymal transition
Kazuhiro Yachi
, Masumi Tsuda
, Shinji Kohsaka
, Lei Wang
, Yoshitaka Oda
, Satoshi Tanikawa
, Yusuke Ohba
Shinya Tanaka
Glioblastoma is the most aggressive and invasive brain tumor and has a poor prognosis; elucidating the underlying molecular
mechanisms is essential to select molecular targeted therapies. Here, we investigated the effect of microRNAs on the marked
invasiveness of glioblastoma. U373 glioblastoma cells were infected with 140 different microRNAs from an OncomiR library, and the
effects of the invasion-related microRNAs and targeted molecules were investigated after repeated Matrigel invasion assays.
Screening of the OncomiR library identied miR-23a as a key regulator of glioblastoma invasion. In six glioblastoma cell lines, a
positive correlation was detected between the expression levels of miR-23a and invasiveness. A luciferase reporter assay
demonstrated that homeobox D10 (HOXD10) was a miR-23a-target molecule, which was veried by high scores from both the
PicTar and miRanda algorithms. Forced expression of miR-23a induced expression of invasion-related molecules, including uPAR,
RhoA, and RhoC, and altered expression of glial-mesenchymal transition markers such as Snail,Slug,MMP2,MMP9,MMP14, and E-
cadherin; however, these changes in expression levels were reversed by HOXD10 overexpression. Thus, miR-23a signicantly
promoted invasion of glioblastoma cells with polarized formation of focal adhesions, while exogenous HOXD10 overexpression
reversed these phenomena. Here, we identify miR-23a-regulated HOXD10 as a pivotal regulator of invasion in glioblastoma,
providing a novel mechanism for the aggressive invasiveness of this tumor and providing insight into potential therapeutic targets.
Signal Transduction and Targeted Therapy (2018) 3:33 ;
Glioblastoma (GBM) is the most invasive and aggressive primary
brain tumor and has a poor prognosis, showing a 5-year survival
rate of 7%. Conventional therapy for GBM involves surgical
resection followed by fractionated radiotherapy and concomitant
adjuvant chemotherapy with alkylating drugs such as temozolo-
mide (TMZ).
However, the effects of treatment are limited due to
the complexity of GBM, involving tumor heterogeneity, rapid
invasion, clonal populations maintaining glioma stem cells (GSCs),
and a high frequency of recurrence. Genome-wide analyses such
as the Cancer Genome Atlas (TCGA) along with other efforts, have
identied gene mutations, amplication, modication, and
rearrangement as the principal genetic causes of GBM.
To date,
more than 140 gene mutations have been reported in GBM, most
IDH1,RB1,LZTR1, and PTPN11, while TMZ-dependent hypermuta-
tions are highly expressed in recurrent tumors.
various molecular targeted agents have been attempted to be
used either as a single agent or in combination therapy, few have
been reported to be effective in phase II trials thus far. Notably,
many gene mutations in primary tumors are distinct from those in
recurrent tumors. In addition, mutations in genes at diagnosis,
such as those in EGFR,PDGFRA, and TP53, can switch to different
mutations in the same gene at relapse,
suggesting that
complicated spatiotemporal clonal evolution is a primary mechan-
ism of treatment failure. Therefore, new approaches are urgently
needed to understand the unique biology of GBM and design
optimized therapies.
One unique characteristic of GBM cells is aggressive inltration
and invasion into the surrounding normal tissues along the
vascular tracks, preventing complete resection of all malignant
cells and limiting the effect of localized radiotherapy. CD44
ligation with hyaluronic acid (HA) has been shown to trigger PI3K/
Rho GTPase signaling, leading to GBM invasion via regulation of
actin polymerization and formation of focal adhesions.
lating evidence has indicated that cancer stem cells (CSCs),
epithelialmesenchymal transition (EMT) modulated by PI3K/AKT/
mTOR signaling,
proneural-mesenchymal shifts via NF-κB and
JAK-STAT pathways,
angiogenesis-invasion shifts, tumor-derived
and miRNAs play pivotal roles in GBM migration and
invasion. We have previously identied Snail as the master
regulator of the irradiation-induced glial-mesenchymal transition
(GMT), resulting in promoted migration and invasion.
Thus, a
better understanding of the invasive biology of GBM cells is
needed to develop innovative therapies to suppress GBM
Received: 11 June 2018 Revised: 2 September 2018 Accepted: 5 November 2018
Department of Cancer Pathology, Faculty of Medicine, Hokkaido University, Sapporo, Japan;
Global Station for Soft Matter, Global Institution for Collaborative Research and
Education, Hokkaido University, Sapporo, Japan;
Institute for Chemical Reaction Design and Discovery (WPI-ICReDD), Hokkaido University, Sapporo, Japan and
Department of
Cell Physiology, Faculty of Medicine, Hokkaido University, Sapporo, Japan
Correspondence: Shinya Tanaka (
Present address: Division of Cellular Signaling, National Cancer Center Research Institute, Tokyo, Japan
These authors contributed equally: Kazuhiro Yachi, Masumi Tsuda
Signal Transduction and Targeted Therapy
©The Author(s) 2019
MicroRNAs (miRNAs) are small, non coding RNAs ranging from
18 to 24 nucleotides in length that negatively regulate gene
expression at the post transcriptional level, primarily through base
pairing to the 3UTR of target mRNA.
Because miRNAs modulate
fundamental cell functions such as proliferation, migration,
metabolism, and apoptosis,
dysregulation of miRNA expression
causes diverse diseases, including cancers.
miRNAs can
function as tumor suppressor genes or oncogenes and as
potential specic cancer biomarkers.
Accumulating studies
have demonstrated the roles of miRNAs in cancer stem cell self-
sensitivity to tyrosine kinase inhibitors,
and cancer
therapy targeted to the tumor microenvironment.
miRNAs have been reported to contribute to the promotion of
tumor invasion and metastasis in various cancers, including miR-
10b, miR-373, and miR-520c for breast cancer;
miR-17 and miR-
19 for colon cancer;
and miR-216a for pancreatic cancer.
Recently, the signicant role of miRNAs in the pathogenesis of
GBM has been increasingly elucidated. In GBM, overexpression of
miR-221, miR-10b, miR-130a, miR-125b, miR-9-2, and miR-21 has
been reported.
Among these miRNAs, miR-10b, which regulates
homeobox D10 (HOXD10), and miR-21, which targets RECK, are
important in facilitating glioblastoma invasion.
miR-23a has been reported to regulate several physiological
phenomena by targeting MURF1,MAFbx, and GLS, leading to
promotion of cardiac hypertrophy, inhibition of muscular atrophy,
and suppression of glutamine metabolism, respectively.
addition, dysregulated expression of miR-23a has been reported
in various types of human cancers, including upregulation in
hepatocellular carcinoma, glioblastoma, bladder cancer, and
pancreatic cancer and downregulation in acute promyelocytic
leukemia and oral squamous carcinoma.
When overexpressed in
cancers, miR-23a directly regulates some target genes such as
NOXA,MTSS1, and PTEN, consequently preventing apoptosis,
inducing the EMT, and promoting tumorigenesis, respectively.
Recently, miR-23a was shown to be encapsulated in exosomes
derived from patients with colorectal cancer,
raising the
possibility of its use as a diagnostic and predictive marker.
However, the pathobiological role of miR-23a in GBM has
remained obscure.
In this study, we identied miR-23a as an oncogene that confers
aggressive invasion of GBM cells by directly inhibiting HOXD10
expression. In miR-23a-overexpressing GBM cells, HOXD10 protein
levels were dramatically decreased, and mRNA levels of invasion-
and GMT-related molecules were markedly altered with polarized
formation of focal adhesions, resulting in profound tumor
invasion. These ndings suggest that miR-23a and HOXD10 are
potentially powerful therapeutic targets for GBM treatment.
Cell culture
The human GBM cell lines LN308, LN443, and U373 were kindly
provided by Dr. Erwin G. Van Meir (Emory University School of
Medicine, Atlanta, Georgia). The KMG4 cell line was kindly
provided by Dr. Kazuo Tabuchi (Saga University, Saga, Japan).
U87 cells (ATCC#HTB-14) and U251 cells (ATCC#CRL2219) were
purchased from the American Type Culture Collection (ATCC). All
cell lines, including embryonic kidney 293FT cells, were cultured in
Dulbeccos modied Eagles medium (DMEM) (Wako, Osaka,
Japan) supplemented with 10% fetal bovine serum (FBS; Invitro-
gen, Carlsbad, CA, USA) and maintained in a humidied atmo-
sphere of 5% CO
at 37 °C.
Establishment of miR-23a and HOXD10-overexpressing cells
miR-23a-overexpressing cells were established using the BLOCK-iT
HiPerform Lentiviral Pol II miR RNAi Expression System with EmGFP
(Invitrogen). 293FT cells were transfected with pLenti6.4/Promoter/
MSGW/miR-23a and pLenti6.4/Promoter/MSGW/HOXD10 using
Fugene HD transfection reagent (Promega, Madison, WI, USA).
After 48 h of incubation, the supernatant was treated with U373
and LN443 glioblastoma cells, and miR-23a and HOXD10-
overexpressing cells were selected in DMEM containing blasticidin.
Anti-miR23a oligonucleotide transfection in KMG4 cells
Anti-miR-23a or comparable scramble oligonucleotides were
transfected into KMG4 cells using HiPerfect reagent (Qiagen,
Valencia, CA). The sequences utilized were as follows: anti-miR-
TATACGCCCA-3. After 48 h, the cells were utilized for real-time
PCR of Snail,MMP2,MMP9, and MMP12 and for Matrigel invasion
assays, as described below.
Identication of microRNA that promotes glioblastoma invasion
The OncoMir Precursor Virus Library (System Bioscience, Mountain
View, CA, USA) was infected into U373 cells, and the Matrigel
invasion assay (BD Biosciences, MA, USA) was performed in
triplicate as described below. RNA was isolated from cells with
elevated invasion ability, and semi quantitative RT-PCR using the
OncoMir Precursor Library primers (System Bioscience) and
sequencing were performed to identify the infected oncomiRs.
Matrigel invasion assay
A Matrigel invasion assay was performed as described previously
using a BioCoat Matrigel invasion chamber (24-well chambers)
with 8-µm pores (BD Biosciences, MA). U373 and LN443 cells with
or without enforced miR-23a and HOXD10 were seeded at a
density of 5 × 10
cells into the upper chamber with serum-free
medium. Medium containing 10% FBS was added to the lower
chamber as a chemo attractant. After incubation for 8 or 24 h, the
cells were xed with 3% paraformaldehyde (PFA) for 10 min and
stained with 0.2% crystal violet solution. Non invading cells on the
upper surface of each lter were removed by scrubbing. The
invaded cells were counted in microscopic elds at ×200
magnication. To minimize bias, cells in at least ve randomly
selected elds per well were counted. The experiments were
performed in triplicate independently, and the mean and standard
deviation (SD) of the invading cells were analyzed.
Prediction of miR-23a-targeting molecules
To predict miR-23a-targeting molecules, PicTar (http://pictar.mdc- and miRanda ( algorithms were
Luciferase reporter assay to target the HOXD10-3UTR
The HOXD10-3UTR was amplied from BJ/t cells, converted to
cDNA, and sequenced. The HOXD10-3UTR was cloned into the
region downstream of the Fireyluciferase gene in a pGL3-
promoter luciferase reporter vector (Promega), designated pGL3-
SV40-HOXD10. The luciferase reporter vector was co transfected
with a miR-23a-overexpression vector (pLenti-6.4/miR-23a) or
control vector (pLenti-6.4/nega) into U373 and LN443 cells using
Fugene HD transfection reagent (Promega). The Renilla luciferase
plasmid pCX4-Bleo-RL-Luc (Promega) was utilized as a control for
transfection efciency. After 48 h, a dual-luciferase reporter assay
(Promega) was performed as described previously.
RNA extraction and gene expression analysis
Total RNA from U373 and LN443 cells with or without enforced
miR-23a and HOXD10 expression was extracted using an RNeasy
Mini kit (Qiagen), and cDNA was synthesized using Superscript
VILO (Invitrogen). For semi-quantitative RT-PCR, GoTaq Green
Master Mix was utilized, and PCR was performed at 2333 cycles of
denaturation for 30 s at 94 °C, annealing for 30 s at 55 °C, and
extension for 30 s at 72 °C. qRT-PCR was performed using
a StepOne Real-Time PCR System (Applied Biosystems, Foster
City, CA) as described previously.
The primer sequences utilized
miR-23a promotes invasion of glioblastoma via HOXD10-regulated. . .
Yachi et al.
Signal Transduction and Targeted Therapy (2018) 3:33
were as follows: miR-23a: forward 5-TGCTGGGCCGGCTGGG
Slug: forward 5-TGGTTGCTTCAAGGACACAT-3, reverse 5-GTTGC
GG-3, reverse 5-GCCACCCGAGTGTAACCATA-3;MMP14: forward
CCTTCCCAGACTTTG-3; and glyceraldehyde 3-phosphate dehydro-
genase (GAPDH): forward 5-AGCCACATCGCTCAGACAC-3, reverse
The relative expression levels of total RNA in experimental and
control samples were normalized to the GAPDH mRNA levels.
Immunoblotting and antibodies
Immunoblot analyses were carried out as described previously.
Briey, cells were lysed with RIPA buffer containing 1 mM
phenylmethylsulfonyl uoride (Sigma), 1 mM sodium orthova-
nadate (Na
) and a complete protease inhibitor cocktail
(Roche) for 10 min on ice. The membrane was treated
with primary antibodies (Abs) at 4 °C overnight, followed by
incubation with secondary antibodies for 2 h. The primary
antibodies were purchased as follows: HoxD10 (E-20) was from
Santa Cruz Biotechnology (Santa Cruz, CA), phospho-ERK1/2 was
from Cell Signaling Technology (Beverly, MA), and α-tubulin was
from Sigma Aldrich. The signals were developed using
ECL reagents (GE Healthcare, Little Chalfont, UK) and were
visualized using an ImageQuant LAS4000 mini system (Fujilm,
Tokyo, Japan).
Immunouorescence for focal adhesions
\U373 and LN443 cells with or without forced miR-23a
and HOXD10 expression were cultured on glass-based dishes
(IWAKI, Tokyo, Japan) coated with type I-collagen and xed in 3%
PFA in PBS for 15 min. The cells were permeabilized with 0.1%
Triton X-100 for 4 min and blocked with 1% BSA for 20 min. To
detect focal adhesions, the cells were treated with anti-paxillin Ab
(BD Transduction Laboratories, USA) overnight at 4°C, followed by
incubation with AlexaFluor488-conjugated anti-mouse IgG (Invi-
trogen) for 1 h at room temperature (RT). F-actin was stained with
AlexaFluor594-conjugated phalloidin (Invitrogen) for 30 min at 37°
C. Fluorescent images were obtained using a confocal laser
scanning microscope (Olympus, Tokyo, Japan).
Fig. 1 MiR-23a promotes invasion of GBM cells. aThe Matrigel invasion assays were performed using six human GBM cell lines: LN443, U373,
LN308, U87, U251, and KMG4. *P< 0.05, **P< 0.005, and ***P< 0.0005 vs. LN443. bSchematic diagram identifying microRNAs to confer
marked invasion of GBM cells. U373 cells were infected with the OncoMir Precursor Virus Library, and the Matrigel invasion assay was repeated
three times to enrich the cells that acquired elevated invasion ability. Total RNA was isolated from the cells and subjected to semi quantitative
RT-PCR using OncoMir Precursor Library primers, followed by sequencing. cEndogenous expression levels of miR-23a in the six GBM cell lines
were examined by semi quantitative RT-PCR. GAPDH was utilized as an internal control. dIn the six GBM cell lines shown in c, the correlations
between the invasion ability and expression levels of miR-23a were analyzed. R
=0.95741. eThe scores for HOXD10,Sprouty2, and Marcks from
the PicTar and miRanda algorithms are shown
miR-23a promotes invasion of glioblastoma via HOXD10-regulated. . .
Yachi et al.
Signal Transduction and Targeted Therapy (2018) 3:33
Proliferation assay
U373 and LN443 cells with or without forced miR-23a and
HOXD10 expression were seeded into 35-mm dishes at a density
of 2 × 10
cells per dish. The medium was changed every 48 h, and
the numbers of cells were counted using a cell counter 5 days
after cell inoculation.
Survival analysis of glioma patients
The relationship between HOXD10 expression and survival in
glioma patients was analyzed using the public database
PrognoScan (
Statistical analysis
All data were represented as the means and SD of experiments
performed in triplicate and subjected to one-way analysis of
variance, followed by comparison with Studentst-tests. Pvalues
less than 0.05 were considered statistically signicant, as
described in the Figure legends.
Expression levels of miR-23a are correlated with GBM invasion
To evaluate the invasion potential of human GBM, we performed
Matrigel invasion assays using six human GBM cell lines. The cells
were divided into two groups according to their invasion
capabilities: low invasion (LN443, U373, and LN308 cells) and
high invasion (U87, U251, and KMG4 cells) (Fig. 1a). To identify
microRNAs that confer aggressive invasion in GBM cells, an
OncoMir Precursor Virus Library including 140 cancer-related
oncomiRs was infected into U373 cells with low intrinsic inva-
sion capabilities. The Matrigel invasion assay was repeated three
times to select for the cells that acquired high invasion by
oncomiRs (Fig. 1b). Total RNA was isolated, and semi quantitative
RT-PCR, followed by sequencing identied miR-23a from the cells
that ultimately acquired high invasion (Fig. 1b). miR-23a was
expressed in all human GBM cell lines tested with various degrees
(Fig. 1c), and the expression levels were signicantly correlated
with invasion capability (Fig. 1d, R
=0.95741), suggesting the
pivotal role of miR-23a in GBM invasion. MiR-181b was also
identied in the same context in LN443 cells; however, extrinsic
overexpression of miR-181b did not promote invasion in LN443
cells (data not shown).
The PicTar algorithm nominated 472 target genes of miR-23a
(data not shown). Among them, three genes, HOXD10 (11/472),
Sprouty2 (73/472), and Marcks (332/472), were implicated in tumor
invasion. The distinct algorithm miRanda also identied these genes
as miR-23a target genes with low values of mirSVR values (Fig. 1e).
The prediction scores from the PicTar and miRanda algorithms for
miR-23a targeting HOXD10 were 7.52 and 0.4376, respectively,
nominating HOXD10 as a reliable target of miR-23a (Fig. 1e).
Fig. 2 MiR-23a directly targets the HOXD10-3UTR in GBM cells. aU373 and LN443 cells were infected with miR-23a-producing lentivirus or
control lentivirus, and the expression levels of miR-23a and HOXD10 mRNA were examined by semi quantitative RT-PCR. GAPDH was utilized as
an internal control. bThe expression levels of HOXD10 protein were examined by immunoblotting in U373 and LN443 cells with or without
forced expression of miR-23a. α-Tubulin was used as a loading control. cDiagram of the luciferase reporter vector fused to the 3UTR of
HOXD10 utilized in the luciferase assay. The sequences of miR-23a and the targeted HOXD10-3UTR are shown. dDual luciferase assay. HOXD10-
3UTR luciferase activity were measured in miR-23a-overexpressing U373 and LN443 cells. *P< 0.01 and **P< 0.001 vs. without miR-23a.
eThe expression levels of sprouty2 mRNA in miR-23a-overexpressing U373 and LN443 cells were examined by semi quantitative RT-PCR. fThe
phosphorylation levels of ERK were investigated by immunoblotting in the indicated cells. gThe cell proliferation of U373 and LN443 cells
with or without forced miR-23a was investigated and graphed as the means ± SD. * P<0.05 vs. control cells
miR-23a promotes invasion of glioblastoma via HOXD10-regulated. . .
Yachi et al.
Signal Transduction and Targeted Therapy (2018) 3:33
MiR-23a directly targets HOXD10 in GBM cells via translational
To determine the miR-23a target genes involved in GBM invasion,
we established stably miR-23a-overexpressing U373 and LN443
cells by infecting them with miR-23a-producing lentivirus.
Although the expression levels of HOXD10 mRNA were invariant
irrespective of miR-23a overexpression (Fig. 2a), the protein levels
were signicantly decreased by forced miR-23a expression in both
cell lines (Fig. 2b), indicating miR-23a-dependent post transcrip-
tional degradation of HOXD10. To analyze whether miR-23a
directly targets the HOXD10-3UTR in GBM cells, we developed a
luciferase reporter vector fused to the 3UTR of HOXD10 (Fig. 2c).
In cells stably overexpressing miR-23a, the luciferase activity of
HOXD10-3UTR was reduced compared with that in control cells
(Fig. 2d), conrming HOXD10 as a direct target of miR-23a. Lower
expression of HOXD10 was associated with a shorter survival rate
in glioma patients by KaplanMeier analysis using the PrognoScan
database (data not shown).
Sprouty2 has been reported to suppress invasion of GBM cells
by inhibiting Ras GTPase,
raising the possibility that miR-23a
might activate the Ras/ERK signaling pathway via translational
inhibition of Sprouty2, facilitating GBM invasion. However, forced
expression of miR-23a had no effect on the expression of sprouty2
mRNA or the phosphorylation levels of ERK1/2 (Fig. 2e, f). In
addition, miR-23a overexpression exhibited a distinct effect on the
proliferation of U373 and LN443 cells (Fig. 2f), suggesting a
relatively low possibility of Sprouty2 as a universal target of miR-
MiR-23a regulates the expression levels of invasion- and glial-
mesenchymal transition (GMT)-related genes via HOXD10
HOXD10 is a member of the Homeobox (Hox) superfamily and has
been shown to suppress invasion of GBM by inhibiting the
expression levels of urokinase-type plasminogen activator receptor
(uPAR),matrix metalloproteinase (MMP) 14, and RhoC.
we next examined the effect of miR-23a on the expression of
these genes.
MiR-23a overexpression markedly increased uPAR and RhoC
expression in both U373 and LN443 cells and MMP14 and RhoA
expression in LN443 cells (Fig. 3a, b). We found no miR-23a-
dependent elevation in MMP14 expression in U373 cells, probably
due to substantial endogenous expression levels (Fig. 3b). Notably,
Fig. 3 MiR-23a regulates expression of invasion- and glial-mesenchymal transition (GMT)-related genes via HOXD10. aThe expression levels
of uPAR, RhoA, and RhoC mRNAs were examined in control and miR-23a-overexpressing U373 and LN443 cells by semi quantitative RT-PCR.
GAPDH was utilized as an internal control. b,cIn U373 and LN443 cells with or without miR-23a overexpression, the mRNA expression levels of
the indicated GMT-related genes were investigated by semi quantitative RT-PCR (b) and real-time RT-PCR (c). *P< 0.05 and **P< 0.005 vs.
without miR-23a. d,eThe expression levels of miR-23a and the indicated molecules were examined by semi quantitative RT-PCR (d) and
immunoblotting (e) in LN443 cells with or without miR-23a and HOXD10 overexpression
miR-23a promotes invasion of glioblastoma via HOXD10-regulated. . .
Yachi et al.
Signal Transduction and Targeted Therapy (2018) 3:33
forced expression of miR-23a triggered marked alterations in the
expression levels of glial-mesenchymal transition (GMT)-related
genes, as previously reported,
with increased expression of Snail,
Slug,MMP2,MMP9, and MMP14 and decreased expression of E-
Cadherin, especially in LN443 cells (Fig. 3b, c); these genes are
perceived as so-called epithelial-mesenchymal transition (EMT)-
related genes in other cancers. These alterations were completely
reversed by HOXD10 overexpression (Fig. 3d, e), demonstrating
the signicant contribution of the miR-23a-HOXD10 axis in these
gene expression levels.
MiR-23a produces mesenchymal morphology with polarized focal
Because miR-23a signicantly alters invasion- and GMT-related
gene expression, we further investigated morphological changes
with or without miR-23a overexpression (Fig. 4a). Forced expres-
sion of miR-23a induced elongation of spindle morphology in
U373 and LN443 cells, and HOXD10 overexpression reversed these
alterations, returning the morphology to that of control cells
(Fig. 4b). Immunouorescence analysis revealed that the number
of focal adhesions represented by paxillin was decreased upon
miR-23a overexpression, with shortened actin laments (Fig. 4c). In
addition, extrinsic overexpression of HOXD10 recovered the
paxillin count to 80% of that in control cells (Fig. 4c). For a more
detailed analysis regarding the assembly of focal adhesions, focal
adhesion polarity was investigated with or without miR-23a
overexpression. The cell was divided into three regions by angles
of 120°, and the Aregion was congured as the movement
direction based on cell morphology and the arrangement of actin
laments. In U373 and LN443 cells control cells, paxillin was
Fig. 4 MiR-23a produces mesenchymal changes in cell morphology and affects the polarity of focal adhesions. aThe expression levels of miR-
23a, HOXD10 mRNA, and HOXD10 protein were examined by semi quantitative RT-PCR (upper three panels) and immunoblotting (lower two
panels) in U373 and LN443 cells. GAPDH and α-tubulin were utilized as internal controls for semi quantitative RT-PCR and immunoblotting,
respectively. bPhotomicrographs of U373 and LN443 cells with or without miR-23a or HOXD10-overexpression are displayed. The scale bars
indicate 100 µm. cImmunouorescence of focal adhesions. (Left panels) U373 and LN443 cells with or without forced miR-23a or HOXD10
expression were subjected to immunouorescence analysis for paxillin (green) and actin (red). (Right panels) The paxillin counts in the
indicated cells are shown. dCells stained with anti-paxillin Ab were divided into three regions by angles of 120°, and the Aregion was set as
the movement direction based on cell morphology and the structures of actin laments. In U373 and LN443 cells with or without forced miR-
23a or HOXD10 expression, the paxillin counts were determined and are shown
miR-23a promotes invasion of glioblastoma via HOXD10-regulated. . .
Yachi et al.
Signal Transduction and Targeted Therapy (2018) 3:33
localized equivalently in all regions (Fig. 4d). However, miR-23a
overexpression produced polarity in paxillin distribution, causing
signicant reductions in the rear regions of cells, namely, the B
and C regions (Fig. 4d). Notably, overexpression of HOXD10
abolished the polarity of the focal adhesions (Fig. 4d).
miR-23a promotes GBM tumor invasion via reduced HOXD10
To assess the effectiveness of miR-23a in GBM cell migration and
invasion, we performed a wound-healing assay and a Matrigel
invasion assay using U373 and LN443 cells with or without
extrinsic miR-23a expression. In the wound-healing assay, the
effect of miR-23a on cell motility alone differed across cell types
(Fig. 5a, b). However, miR-23a overexpression strikingly promoted
invasion in both U373 and LN443 cells to levels 4.0-fold and
5.0-fold higher than those in control cells, respectively (Fig. 5c, d).
These increases were reversed by extrinsic expression of HOXD10
(Fig. 5c, d). U87 cells with intrinsic high expression of miR-23a
natively possessed higher invasion potential (Fig. 1a, c). Enhanced
expression of HOXD10 triggered dramatic alterations in morphol-
ogies, changing cells from having a distinct piled-up spindle shape
to having a at shape with an enlarged cytoplasmic compartment
and resulting in a substantial decline in invasiveness (Fig. 5c). Anti-
miR-23a oligonucleotide treatment to KMG4 cells, which had the
highest miR-23a expression and invasiveness, signicantly
decreased the expression levels of Snail,MMP2,MMP9, and
MMP14 (Fig. 6a), resulting in marked suppression of invasion
(Fig. 6b).
GBM is an extremely aggressive tumor with a 5-year survival rate
of 7% due to high invasion into surrounding normal brain tissue;
elucidation of the underlying molecular mechanisms is therefore
essential to develop effective therapies and improve prognoses. In
this study, we addressed a part of this long-standing issue using
the OncoMir library infection system and found that miR-23a
directly targets the HOXD10-3UTR and promotes tumor cell
invasion by elevating the expression levels of invasion- and glial-
mesenchymal transition (GMT)-related genes and inducing
polarity of focal adhesions in GBM (Fig. 6c).
In this analysis, two GBM cell lines, LN443 and U373, with
intrinsically low invasion potential were infected with the OncoMir
library. After repeating the Matrigel invasion assay, we identied
miR-23a as the microRNA responsible for the conferring on U373
cells aggressive invasion capabilities (Fig. 1b). In addition, miR-
181b was also identied in the same context in LN443 cells.
However, extrinsic overexpression of miR-181b did not promote
invasion in LN443 cells (data not shown), in accordance with a
Fig. 5 MiR-23a promotes tumor invasion of glioblastoma via reduced HOXD10. aWound-healing assays were performed with miR-23a-
overexpressing U373 and LN443 cells and their respective control cells. Representative photomicrographs at 0 and 24 h are shown. bThe
distances moved are displayed as the mean ± SD. N.S. indicates not statistically signicant. cMatrigel invasion assays were performed with
both U373 and LN443 cells with or without forced miR-23a or HOXD10 expression. Micrographs of invading cells stained with crystal violet are
displayed. dIn the Matrigel invasion assays, the invaded cells under the lter were counted in three randomly selected regions, and graphed
as the mean ± SD. e(Left) Micrographs of U87 cells with or without forced HOXD10 expression are displayed. (Right) In the Matrigel invasion
assays, invaded cells were counted, and the data are presented as the mean ± SD. * P< 0.01 vs. control cells
miR-23a promotes invasion of glioblastoma via HOXD10-regulated. . .
Yachi et al.
Signal Transduction and Targeted Therapy (2018) 3:33
previous report showing miR-181b-suppressed invasion in GBM.
Therefore, we focused on the role of miR-23a. cAMP response
element-binding protein 1 (CREB1) directly binds to the promoter
of miR-23a to promote its expression, while STAT3 indirectly
induces miR-23a expression.
Given that both CREB1 and STAT3
are up regulated in glioma,
transcription factor-dependent
upregulation of miR-23a might contribute to the aggressive
invasion of GBM.
Hox superfamily genes, including HOXD10, encode transcrip-
tional factors regulating cell differentiation and morphogenesis
during development.
Dysregulation of the Hox gene disrupts
various signaling pathways related to tumorigenesis and metas-
A positive correlation exists between reductions in HOXD10
mRNA and increased malignancy of breast cancer and endome-
trial adenocarcinoma.
Forced expression of HOXD10 mRNA
strikingly suppresses tumor motility and invasion in breast
suggesting a potent inhibitory role of HOXD10 in tumor
invasion. In GBM, miR-10b has been previously reported to inhibit
invasiveness by targeting HOXD10 and regulating the transcription
of MMP14 and uPAR.
Previously, miR-23a has been reported to
regulate expression of HOXD10 in glioma cells.
In this study, we
also identied miR-23a as a novel direct regulator of HOXD10 via
translational but not transcriptional regulation (Fig. 2a, b). Forced
expression of miR-23a increased the expression levels of uPAR,
RhoC,Snail, and Slug but decreased those of E-cadherin in a
HOXD10-dependent manner (Fig. 3d, e). uPAR,MMP14,RhoC, and
Fig. 6 Mechanisms of miR-23a-regulated promotion of GBM invasion through targeting of HOXD10. a,bKMG4 cells were transfected with
anti-miR-23a and scramble DNA as a control, and the expression levels of Snail,MMP2,MMP9, and MMP14 (a) and invasion ability (b) were
investigated. *P< 0.05, **P< 0.005, and ***P< 0.0005 vs. the indicated samples. cmiR-23a directly targets the HOXD10-3-UTR, triggering
dramatic alterations in the expression of genes associated with invasion (uPAR,MMP14,RhoA, and RhoC) and glial-mesenchymal transition
(GMT) events (Snail,Slug,MMP2,MMP9, and E-cadherin), and inducing polarity of focal adhesions, ultimately resulting in cooperatively
aggressive invasion of GBM
miR-23a promotes invasion of glioblastoma via HOXD10-regulated. . .
Yachi et al.
Signal Transduction and Targeted Therapy (2018) 3:33
RhoA have been reported to be targets of miR-23a.
MMP14 seems to regulate the expression levels of MMP2 and
MMP9 in inammatory breast cancer.
Notably, our ndings
suggest that the miR-23a-HOXD10 axis is a novel regulator of the
expression of Snail,Slug, and E-cadherin, which are GMT-regulated
genes in GBM. Taken together, the results suggest that miR-23a-
triggered consecutive gene expression might evoke aggressive
invasion of GBM cells with extensive vascularization through
degradation of extracellular matrices.
MiR-23a overexpression induced polarity in the distribution of
focal adhesions as shown by paxillin, which was completely
reversed by additional forced expression of HOXD10 (Fig. 4c, d).
RhoA has been shown to reduce interactions between focal
adhesion proteins by partly inhibiting the phosphorylation of
paxillin, causing reassembly of the actin cytoskeleton, which leads
to invasion and migration of melanoma and breast cancer.
addition, RhoC induces colocalization of focal adhesion compo-
nents such as paxillin, paxillin kinase linker (PKL), and FAK,
resulting in promoted invasion in prostate cancer cells.
Based on
this evidence, spatiotemporal coordination of RhoA and RhoC
might produce miR-23a-induced polarity of focal adhesions, one
of pivotal events promoting the high invasiveness of GBM.
Although kinase-targeted therapy seems to be a promising
therapeutic approach in GBM, such therapies have been
ineffective in the clinical setting due to the complexity of GBM
with regards to spatiotemporal clonal evolution. Therefore, a
kinase-irrelevant strategy using anti-miRNAs might be an innova-
tive and effective approach to target numerous genes. A distinct
effect of miR-23a on cell growth was observed in LN443 and U373
cells (Fig. 2g), likely because miR-23a regulates different targets;
the PicTar algorithm predicted 472 genes as direct targets of miR-
23a. Because the effect of miR-23a on cell growth remains
controversial and is dependent on the cellular context, special
attention should be paid to assessing the subset of miR-23a-
targeted genes that determines the pathophysiological properties
of GBM cells.
Our studies demonstrated that glioblastoma cells acquire
prominent invasive potential via a miR-23a-HOXD10-GMT-related
pathway. Forced expression of miR-23a promotes invasion by
directly targeting the HOXD10-3UTR. Upregulation of the HOXD10
protein by miR-23a depletion might be an effective approach to
suppress invasion of human GBM.
This work was supported, in part, by Grants-in-Aid from the Ministry of Education,
Culture, Sports, Science, and Technology; Japanese Society for the Promotion of
Science; and Ministry of Health, Labor, and Welfare of Japan as well as a grant from
the Japanese Science and Technology Agency. In addition, this research was
supported by Global Station for Soft Matter, a project of Global Institution for
Collaborative Research and Education at Hokkaido University. Institute for Chemical
Reaction Design and Discovery (ICReDD) was established by World Premier
International Research Initiative (WPI), MEXT, Japan.
Competing interests: The authors declare no competing interests.
Publishers note: Springer Nature remains neutral with regard to jurisdictional claims
in published maps and institutional afliations.
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miR-23a promotes invasion of glioblastoma via HOXD10-regulated. . .
Yachi et al.
Signal Transduction and Targeted Therapy (2018) 3:33
... [13][14][15] Even though the biological function of circPTK2 had been identified in diverse cancers, the specific role of circPTK2 in GBM is unknown. We observed a close correlation between circPTK2 and miR-23a, a critical player in GBM, 16 in our preliminary microarray dataset (data not shown). We, therefore, analyzed the interactions between circPTK2 and miR-23a in GBM. ...
... MiR-23a also plays different roles in different cancers. 16,17 For instance, miR-23a is under-expressed in osteosarcoma and targets RUNX2 and CXCL12 to suppress cancer cell invasion and migration. 17 In contrast, miR-23a is upregulated in GBM and increases cancer cell invasion through HOXD10-regulated glialmesenchymal transition. ...
... 17 In contrast, miR-23a is upregulated in GBM and increases cancer cell invasion through HOXD10-regulated glialmesenchymal transition. 16 Consistently, we also observed the upregulation of mature miR-23a in GBM and its enhancing effects on cancer cell invasion and migration. ...
Full-text available
Background: CircRNA circPTK2 plays opposite roles in different cancers, while its role in glioblastoma is unknown. The aim of this study was to explore the involvement of circPTK2 in glioblastoma. Methods: Expression of circPTK2, mature miR-23a, and premature miR-23a in paired cancer and non-cancer tissues from glioblastoma patients (n = 60) was analyzed by RT-qPCR. Pearson's correlation coefficient was used to analyze the correlations between gene expressions. The effects of circPTK2 overexpression on miR-23a maturation were analyzed by transfecting circPTK2 expression vector into glioblastoma cells, followed by determining the expression of mature miR-23a and premature miR-23a by RT-qPCR. Transwell assays were carried out to explore the role of circPTK2 and miR-23a in regulating glioblastoma cell invasion and migration. Results: We found that circPTK2 was downregulated in GBM and was inversely correlated with mature miR-23a, but not premature miR-23a. GBM cells transfected with circPTK2 expression vector showed significantly downregulated mature miR-23a, but not premature miR-23a. Transwell assay analysis showed that circPTK2 overexpression decreased cell invasion and migration, while miR-23a increased cell invasion and migration. Moreover, miR-23a overexpression reversed the inhibitory effects of circPTK2 overexpression on cell behaviors. Conclusion: CircPTK2 might suppress cancer cell invasion and migration by inhibiting the maturation of miR-23a.
... For example, while HOX genes from across the whole cluster are involved in regulation of wnt/β-catenin signalling, 22,27,49,51,83,[126][127][128][129][130][131][132][133][134][135][136][137][138][139][140] for other HOX target pathways regulation is by a far more restricted group of genes. The later include the matrix metalloproteinases (MMPs), one of the most common HOX targets, which are only regulated by HOX genes from paralogous groups 6 to 10, 20,37,43,51,70,71,110,111,113,115,117,118,[141][142][143][144][145][146] while integrins are regulated by paralog group genes 3 to 5, 140,[147][148][149][150][151][152] and targets related to the NF-κB/p65 signalling pathway by groups 9to13. 63,67,72,92,94,139 The most commonly reported targets of HOX proteins are genes involved in the EMT (Figure 4). ...
... 83 and HOXB13, are notable for having oncogenic functions reported in around 50% of publications, and tumour suppressor functions in the other 50%. Only one HOX gene, HOXD10, seems to act almost exclusively as a tumour suppressor, albeit with a notable exception in head and neck squamous cell carcinoma (HNSCC).[109][110][111][112][113][114][115][116][117][118][119][120][121] How can apparently conflicting functions of genes such as HOXA5 and HOXB13 be explained? ...
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The HOX genes are a highly conserved group of transcription factors that have key roles in early development, but which are also highly expressed in most cancers. Many studies have found strong associative relationships between the expression of individual HOX genes in tumours and clinical parameters including survival. For the majority of HOX genes, high tumour expression levels seem to be associated with a worse outcome for patients, and in some cases this has been shown to result from the activation of pro-oncogenic genes and pathways. However, there are also many studies that indicate a tumour suppressor role for some HOX genes, sometimes with conclusions that contradict earlier work. In this review, we have attempted to clarify the role of HOX genes in cancer by focusing on their downstream targets as identified in studies that provide experimental evidence for their activation or repression. On this basis, the majority of HOX genes would appear to have a pro-oncogenic function, with the notable exception of HOXD10, which acts exclusively as a tumour suppressor. HOX proteins regulate a wide range of target genes involved in metastasis, cell death, proliferation, and angiogenesis, and activate key cell signalling pathways. Furthermore, for some functionally related targets, this regulation is achieved by a relatively small subgroup of HOX genes.
... Homeobox D10 (HOXD10) is a member of the homeobox gene family that serves as a key transcription factor in the development of various types of cancer (9)(10)(11)(12). The overexpression of HOXD10 can reverse miR-23a-mediated invasion in glioblastoma (13). Moreover, HOXD10 was found to inhibit cell invasion and induce apoptosis via the RhoC-AKT signaling pathway (14,15). ...
Full-text available
Emerging evidence suggests that hypermethylation of HOXD10 plays an important role in human cancers. However, the biological and clinical impacts of HOXD10 overmethylation and its downstream targets in colorectal cancer remain unknown. We evaluated the methylation level of HOXD10 in paired cancer and normal tissues ( n = 42) by using pyrosequencing, followed by validation of the methylation status of HOXD10 from The Cancer Genome Atlas (TCGA) datasets with 302 cancer tissues and 38 normal tissues. The biological function of HOXD10 was characterized in cell lines. We further evaluated the effects of HOXD10 and its targets on chemoresistance in our established resistant cell lines and clinical cohort ( n = 66). HOXD10 was found frequently methylated in colorectal cancer, and its hypermethylation correlates with its low expression level, advanced disease, and lymph node metastasis. Functionally, HOXD10 acts as a tumor suppressor gene, in which HOXD10 -expressing cells showed suppressed cell proliferation, colony formation ability, and migration and invasion capacity. Mechanistically, DNMT1, DNMT3B, and MeCP2 were recruited in the HOXD10 promoter, and demethylation by 5-Aza-2′-deoxycytidine (5-Aza-CdR) treatment or MeCP2 knockdown can sufficiently induce HOXD10 expression. HOXD10 regulates the expressions of miR-7 and IGFBP3 in a promoter-dependent manner. Restoration of the expression of HOXD10 in 5-fluorouracil (5-FU)-resistant cells significantly upregulates the expressions of miR-7 and IGFBP3 and enhances chemosensitivity to 5-FU. In conclusion, we provide novel evidence that HOXD10 is frequently methylated, silenced, and contributes to the development of colorectal cancers. Restoration of HOXD10 activates the expressions of miR-7 and IGFBP3 and results in an inhibited phenotype biologically, suggesting its potential therapeutic relevance in colorectal cancer (CRC).
... Targeted delivery of antisense-miR-21 and antisense-miR-10b coloaded in uPAR-targeted polymer nanoparticles (NPs)-treated mice show a substantial reduction in tumor growth [109]. As reported, miR-378a-5p and miR-23a promote tumor cell metastasis by upregulating the expression of uPAR [110,111]. However, miR-324-5p, miR-193b and miR-143 can inhibit the expression of uPA and uPAR, thus inhibiting the migration and invasion of cancer cells [112][113][114]. ...
Tumorigenesis is closely related to the loss of control of many genes. Urokinase-type plasminogen activator receptor (uPAR), a glycolipid-anchored protein on the cell surface, is controlled by many factors in tumorigenesis and is expressed in many tumor tissues. In this review, we summarize the regulatory effects of the uPAR signaling pathway on processes and factors related to tumor progression, such as tumor cell proliferation, adhesion, metastasis, glycolysis, tumor microenvironment and angiogenesis. Overall, the evidence accumulated to date suggests that uPAR induction by tumor progression may be one of the most important factors affecting therapeutic efficacy. An improved understanding of the interactions between uPAR and its coreceptors in cancer will provide critical biomolecular information that may help to better predict the disease course and response to therapy.
... Moreover, HOXD10 is demonstrated to repress hepatocellular carcinoma progression by restraining ERK signaling.31 Additionally, some previous studies have reported that HOXD10 is the downstream target of several miRNAs.[33][34][35] For instance, miR-10b facilitates the migration and invasion of gastric cancer cells by targeting HOXD1036 ; another study reports that miR-501 participates in hemangioma pathogenesis by targeting HOXD10. ...
Full-text available
Circular RNAs (circRNAs) are a class of noncoding RNAs that are widely expressed in cancer tissues and play a pro‐ or anticancer role in modulating cancer progression. This work is aimed to probe the biological role of circ_0000317 in colorectal cancer (CRC) and its underlying mechanism. Circ_0000317 was selected from the circRNA microarray datasets (GSE121895). Quantitative real‐time polymerase chain reaction was utilized to examine circ_0000317, microRNA (miR)‐520g, and homeobox D10 (HOXD10) mRNA expression in CRC. Cell Counting Kit‐8 and Transwell experiments were conducted to examine the effects of circ_0000317 on proliferation, migration, and invasion of CRC cells. Bioinformatic analysis and dual‐luciferase reporter gene experiments were implemented to predict and validate the targeting relationship between circ_0000317 and miR‐520g, miR‐520g, and HOXD10. Western blot was employed to examine HOXD10 expression at protein level in CRC cells. Circ_0000317 and HOXD10 mRNA expression were unveiled to be down‐modulated and miR‐520g expression was up‐modulated in CRC. Functionally, circ_0000317 overexpression repressed CRC cell proliferation, migration, and invasion. Mechanistically, miR‐520g was a direct target of circ_0000317 and miR‐520g specifically modulated HOXD10 expression. Furthermore, miR‐520g mimics partially counteracted the suppressing effect of circ_0000317 on malignant phenotype of CRC cells. Circ_0000317 represses CRC progression by targeting miR‐520g and modulating HOXD10 expression. Hence, circ_0000317 may be a promising diagnostic biomarker and a therapeutic target for CRC.
... HOXD10, a member of the Abd-B homeobox family, encodes a protein with a homeobox DNA-binding domain, which serves as an important transcription factor targeting downstream proteins [15,16]. It is abnormally expressed in several cancers, such as prostate cancer, esophageal squamous cell carcinoma, endometrial cancer, colon cancer glioblastoma and ovarian cancer [15,[17][18][19][20][21]. HOXD10 has also been verified as the primary effector that negatively regulates metastasis in cancers, and it was demonstrated to have an inhibitory influence on tumor cell proliferation [22]. ...
Full-text available
Background Several studies have shown the crucial role of miR-501 in regulating cellular pathology in various cancers. However, the function and expression of miR-501 in endometrial cancer (EC) remain obscure. Methods The expression of miR-501 was determined using quantitative real-time PCR. MTT assay, colony formation assay and cell cycle analysis were used to evaluate the proliferation ability. Migration and invasion were assessed using transwell assay. Tumor formation in nude mice was used to observe the effects of miR-501 on cell proliferation and migration in vivo. Luciferase assay, quantitative real-time PCR and western blot were applied to determine that HOXD10 was the target gene of miR-501. Results In this study, we observed significantly up-regulated expression of miR-501 in endometrial cancer, which correlated with higher pelvic lymph node metastasis and shorter overall survival in high-grade endometrial cancer. High expression of miR-501 was also found in the copy-number-high group than other groups. Moreover, in vitro and in vivo assay showed that overexpression of miR-501 can promote proliferation and metastasis. Mechanistically, we found that miR-501 promotes tumor progression by directly targeting HOXD10. Further study also indicated that miR-501 overexpression can activate the AKT/mTOR pathway. Conclusions MiR-501, which functions as an oncomir in endometrial cancer, might be a potential therapeutic target in high grade endometrial cancer.
Identification of prognostic or predictive molecular markers in glioblastoma resection specimens may lead to strategies for therapy stratification and personalized treatment planning. Here, we analyzed in primary glioblastoma stem cell (pGSC) cultures the mRNA abundances of 7 stem cell (MSI1, Notch1, nestin, Sox2, Oct4, FABP7, ALDH1A3), and 3 radioresistance or invasion markers (CXCR4, IKCa , BKCa ). From these abundances, an mRNA signature was deduced which describes the mesenchymal-to-proneural expression profile of an individual GSC culture. To assess its functional significance, we associated the GSC mRNA signature with the clonogenic survival after irradiation with 4 Gy and the fibrin matrix invasion of the GSC cells. In addition, we compared the molecular pGSC mRNA signature with the tumor recurrence pattern and the overall survival of the glioblastoma patients from whom the pGSC cultures were derived. As a result, the molecular pGSC mRNA signature correlated positively with the pGSC radioresistance and matrix invasion capability in vitro. Moreover, patients with a mesenchymal (> median) mRNA signature in their pGSC cultures exhibited predominantly a multifocal tumor recurrence and a significantly (univariate log rank test) shorter overall survival than patients with proneural (≤ median mRNA signature) pGSCs. The tumors of the latter recurred predominately unifocally. We conclude that our pGSC cultures induce/select those cell subpopulations of the heterogeneous brain tumor that determine disease progression and therapy outcome. In addition, we further postulate a clinically relevant prognostic/predictive value for the 10 mRNAs-based mesenchymal-to-proneural signature of the GSC subpopulations in glioblastoma.
Full-text available
The homeobox (HOX) genes encoding an evolutionarily highly conserved family of homeodomain-containing transcriptional factors are essential for embryogenesis and tumorigenesis. HOX genes are involved in cell identity determination during early embryonic development and postnatal processes. The deregulation of HOX genes is closely associated with numerous human malignancies, highlighting the indispensable involvement in mortal cancer development. Since most HOX genes behave as oncogenes or tumor suppressors in human cancer, a better comprehension of their upstream regulators and downstream targets contributes to elucidating the function of HOX genes in cancer development. In addition, targeting HOX genes may imply therapeutic potential. Recently, novel therapies such as monoclonal antibodies targeting tyrosine receptor kinases, small molecular chemical inhibitors, and small interfering RNA strategies, are difficult to implement for targeting transcriptional factors on account of the dual function and pleiotropic nature of HOX genes-related molecular networks. This paper summarizes the current state of knowledge on the roles of HOX genes in human cancer and emphasizes the emerging importance of HOX genes as potential therapeutic targets to overcome the limitations of present cancer therapy.
Cancer is also determined by the alterations of oncogenes and tumor suppressor genes. These gene expressions can be regulated by microRNAs (miRNA). At this point, researchers focus on addressing two main questions: “How are oncogenes and/or tumor suppressor genes regulated by miRNAs?” and “Which other mechanisms in cancer cells are regulated by miRNAs?” In this work we focus on gathering the publications answering these questions. The expression of miRNAs is affected by amplification, deletion or mutation. These processes are controlled by oncogenes and tumor suppressor genes, which regulate different mechanisms of cancer initiation and progression including cell proliferation, cell growth, apoptosis, DNA repair, invasion, angiogenesis, metastasis, drug resistance, metabolic regulation, and immune response regulation in cancer cells. In addition, profiling of miRNA is an important step in developing a new therapeutic approach for cancer.
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Neuroinflammation and oxidative stress cooperate to compromise the function of the central nervous system (CNS). Colloidal platinum nanoparticles (Pt NPs) are ideal candidates for reducing the deleterious effects of neuroinflammation since they act as free radical scavengers. Here we evaluated the effects of Pt NPs on several markers of lipopolysaccharide (LPS)-induced inflammation in cultured BV-2 microglial cells. BV-2 cells were treated with increased dilutions (1–100 ppm) of Colloidal Pt and/or LPS (1–10 µg/mL) at different exposure times. Three different protocols of exposure were used combining Pt NPs and LPS: (a) conditioning-protective effect (pre–post-treat), (b) therapeutic effect (co-treat) and (c) conditioning-therapeutic effect (pre-co-treat). After exposure to LPS for 24 h, cells were used for assessment of cell viability, reactive oxygen species (ROS) generation, lactate dehydrogenase (LDH) activity, apoptosis and caspase-3 levels, cell proliferation, mitochondrial membrane potential, inducible nitric oxide (iNOS) activity, pro-inflammatory cytokine (IL-1β, TNF-α and IL-6) levels, and phagocytic activity. Low concentrations (below or equal to 10 ppm) of Colloidal Pt prevented or ameliorated the LPS-induced increase in ROS formation, loss of mitochondrial membrane potential, induction of apoptosis, increase in LDH release, increase in pro-inflammatory cytokines and iNOS, inhibition of phagocytosis linked to microglial persistence in the M1 phase phenotype, loss of cell adhesion, differentiation and/or proliferation, as well as loss of cell viability. These protective effects were evident when cells were preconditioned with Pt NPs prior to LPS treatment. Collectively, the findings demonstrate that at low concentrations, Pt NPs can regulate the function and phenotype of BV-2 cells, activating protective mechanisms to maintain the microglial homeostasis and reduce inflammatory events triggered by the inflammatory insults induced by LPS. These preventive/protective effects on the LPS pro-inflammatory model are linked to the antioxidant properties and phagocytic activity of these NPs.
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Tumor-associated macrophages (TAMs) originate as circulating monocytes, and are recruited to gliomas, where they facilitate tumor growth and migration. Understanding the interaction between TAM and cancer cells may identify therapeutic targets for glioblastoma multiforme (GBM). Vascular cell adhesion molecule-1 (VCAM-1) is a cytokine-induced adhesion molecule expressed on the surface of cancer cells, which is involved in interactions with immune cells. Analysis of the glioma patient database and tissue immunohistochemistry showed that VCAM-1 expression correlated with the clinico-pathological grade of gliomas. Here, we found that VCAM-1 expression correlated positively with monocyte adhesion to GBM, and knockdown of VCAM-1 abolished the enhancement of monocyte adhesion. Importantly, upregulation of VCAM-1 is dependent on epidermal-growth-factor-receptor (EGFR) expression, and inhibition of EGFR effectively reduced VCAM-1 expression and monocyte adhesion activity. Moreover, GBM possessing higher EGFR levels (U251 cells) had higher VCAM-1 levels compared to GBMs with lower levels of EGFR (GL261 cells). Using two- and three-dimensional cultures, we found that monocyte adhesion to GBM occurs via integrin α4β1, which promotes tumor growth and invasion activity. Increased proliferation and tumor necrosis factor-α and IFN-γ levels were also observed in the adherent monocytes. Using a genetic modification approach, we demonstrated that VCAM-1 expression and monocyte adhesion were regulated by the miR-181 family, and lower levels of miR-181b correlated with high-grade glioma patients. Our results also demonstrated that miR-181b/protein phosphatase 2A-modulated SP-1 de-phosphorylation, which mediated the EGFR-dependent VCAM-1 expression and monocyte adhesion to GBM. We also found that the EGFR-dependent VCAM-1 expression is mediated by the p38/STAT3 signaling pathway. Our study suggested that VCAM-1 is a critical modulator of EGFR-dependent interaction of monocytes with GBM, which raises the possibility of developing effective and improved therapies for GBM.Oncogene advance online publication, 1 May 2017; doi:10.1038/onc.2017.129.
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MicroRNAs play fundamental roles in mammalian development, differentiation and cellular homeostasis by regulating essential processes such as proliferation, migration, metabolism, migration and cell death. These small non-coding RNAs are also responsible in RNA silencing, and in many developmental and pathological processes. Not surprisingly, miR-23a misexpression contributes to numerous diseases including cancer where certain miRNA genes have been classified as either oncogenes or tumor suppressor genes. Since a single microRNA is capable of targeting a large number of mRNA sequences, de-regulated miRNA expression has the ability to alter various transcripts and activate a wide range of cancer-related pathways. This review article documents reduced levels of mature miR-23a in various tumors, primarily due to epigenetic silencing or alterations in biogenesis pathways. Moreover, inhibition of miR-23a in stressed cells represent a general mechanism for inducing apoptosis and these microRNAs are showed to be regulated by molecular chaperon HSP70. Microarray expression analysis of miRNA overexpression or depletion is now used in the characterization of cancer development pathways and as a biomarker for early cancer detection.
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MicroRNAs (miRNAs) participate in most aspects of cellular differentiation and homeostasis, and consequently have roles in many pathologies, including cancer. These small non-coding RNAs exert their effects in the context of complex regulatory networks, often made all the more extensive by the inclusion of transcription factors as their direct targets. In recent years, the increased availability of gene expression data and the development of methodologies that profile miRNA targets en masse have fuelled our understanding of miRNA functions, and of the sources and consequences of miRNA dysregulation. Advances in experimental and computational approaches are revealing not just cancer pathways controlled by single miRNAs but also intermeshed regulatory networks controlled by multiple miRNAs, which often engage in reciprocal feedback interactions with the targets that they regulate.
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In treating bladder cancer, determining the molecular mechanisms of tumor invasion, metastasis, and drug resistance are urgent to improving long-term patient survival. One of the metabolic enzymes, aldo-keto reductase 1C1 (AKR1C1), plays an essential role in cancer invasion/metastasis and chemoresistance. In orthotopic xenograft models of a human bladder cancer cell line, UM-UC-3, metastatic sublines were established from tumors in the liver, lung, and bone. These cells possessed elevated levels of EMT-associated markers, such as Snail, Slug, or CD44, and exhibited enhanced invasion. By microarray analysis, AKR1C1 was found to be up-regulated in metastatic lesions, which was verified in metastatic human bladder cancer specimens. Decreased invasion caused by AKR1C1 knockdown suggests a novel role of AKR1C1 in cancer invasion, which is probably due to the regulation of Rac1, Src, or Akt. An inflammatory cytokine, interleukin-1β, was found to increase AKR1C1 in bladder cancer cell lines. One particular non-steroidal anti-inflammatory drug, flufenamic acid, antagonized AKR1C1 and decreased the cisplatin-resistance and invasion potential of metastatic sublines. These data uncover the crucial role of AKR1C1 in regulating both metastasis and drug resistance; as a result, AKR1C1 should be a potent molecular target in invasive bladder cancer treatment.
Precision medicine in cancer proposes that genomic characterization of tumors can inform personalized targeted therapies. However, this proposition is complicated by spatial and temporal heterogeneity. Here we study genomic and expression profiles across 127 multisector or longitudinal specimens from 52 individuals with glioblastoma (GBM). Using bulk and single-cell data, we find that samples from the same tumor mass share genomic and expression signatures, whereas geographically separated, multifocal tumors and/or long-term recurrent tumors are seeded from different clones. Chemical screening of patient-derived glioma cells (PDCs) shows that therapeutic response is associated with genetic similarity, and multifocal tumors that are enriched with PIK3CA mutations have a heterogeneous drug-response pattern. We show that targeting truncal events is more efficacious than targeting private events in reducing the tumor burden. In summary, this work demonstrates that evolutionary inference from integrated genomic analysis in multisector biopsies can inform targeted therapeutic interventions for patients with GBM.
Exosome-encapsulated microRNAs are being suggested as a new class novel biomarker and diagnostic and predictive markers in colorectal cancer. These particles are released from many cell types into the extracellular space upon fusion of multivesicular bodies (MVB) with the plasma membrane. They contain a wide variety of information, including proteins, lipids, RNAs, non-transcribed RNAs, microRNAs, which can be circulated in various body fluids (e.g., blood, salvia, ascites, urine). Exosomes can be taken up by neighboring or distant cells and thereby modulate the functional of recipient cells and play a key role in disease progression or facilitate metastasis in cancers. The aim of current review is to give an overview about origin and trafficking of exosomes between cells, techniques to isolate exosomal microRNAs as well as the potential applications of exosome-encapsulated microRNAs as diagnostic markers in clinical settings in colorectal cancer. There is growing body of evidence showing the prognostic and diagnostic value of some exosomal microRNAs in colon cancer (e.g., miR-150, miR-21, miR-192, let-7a, miR-223, and miR-23a). These findings provide a new insight on novel application of these markers as being novel non-invasive biomarkers for early detection and risk assessment of patients with colorectal cancer, although further investigations in larger population are required to explore the clinical utility of exosomal microRNAs in colorectal cancer patients.
Although whole-genome sequencing has uncovered a large number of mutations that drive tumorigenesis, functional ratification for most mutations remains sparse. Here, we present an approach to test functional relevance of tumor mutations employing CRISPR/Cas9. Combining comprehensive sgRNA design and an efficient reporter assay to nominate efficient and selective sgRNAs, we establish a pipeline to dissect roles of cancer mutations with potential applicability to personalized medicine and future therapeutic use.
The significant parallels between cell plasticity during embryonic development and carcinoma progression have helped us understand the importance of the epithelial-mesenchymal transition (EMT) in human disease. Our expanding knowledge of EMT has led to a clarification of the EMT program as a set of multiple and dynamic transitional states between the epithelial and mesenchymal phenotypes, as opposed to a process involving a single binary decision. EMT and its intermediate states have recently been identified as crucial drivers of organ fibrosis and tumor progression, although there is some need for caution when interpreting its contribution to metastatic colonization. Here, we discuss the current state-of-the-art and latest findings regarding the concept of cellular plasticity and heterogeneity in EMT. We raise some of the questions pending and identify the challenges faced in this fast-moving field.
Glioblastoma (GBM) is the most common and aggressive primary brain tumor. To better understand how GBM evolves, we analyzed longitudinal genomic and transcriptomic data from 114 patients. The analysis shows a highly branched evolutionary pattern in which 63% of patients experience expression-based subtype changes. The branching pattern, together with estimates of evolutionary rate, suggests that relapse-associated clones typically existed years before diagnosis. Fifteen percent of tumors present hypermutation at relapse in highly expressed genes, with a clear mutational signature. We find that 11% of recurrence tumors harbor mutations in LTBP4, which encodes a protein binding to TGF-β. Silencing LTBP4 in GBM cells leads to suppression of TGF-β activity and decreased cell proliferation. In recurrent GBM with wild-type IDH1, high LTBP4 expression is associated with worse prognosis, highlighting the TGF-β pathway as a potential therapeutic target in GBM.