Antitumor activity of miR-34a in peritoneal mesothelioma relies on c-MET and AXL inhibition: persistent activation of ERK and AKT signaling as a possible cytoprotective mechanism
El Bezawy et al. Journal of Hematology & Oncology
Antitumor activity of miR-34a in peritoneal mesothelioma relies on c-MET and AXL inhibition: persistent activation of ERK and AKT signaling as a possible cytoprotective mechanism
Rihan El Bezawy 0
Michelandrea De Cesare 0
Marzia Pennati 0
Paolo Gandellini 0
Valentina Zuco 0
Nadia Zaffaroni 0
0 Molecular Pharmacology Unit, Department of Experimental Oncology and Molecular Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori , 20133 Milan , Italy
Background: The value of microRNAs (miRNAs) as novel targets for cancer therapy is now widely recognized. However, no information is currently available on the expression/functional role of miRNAs in diffuse malignant peritoneal mesothelioma (DMPM), a rapidly lethal disease, poorly responsive to conventional treatments, for which the development of new therapeutic strategies is urgently needed. Here, we evaluated the expression and biological effects of miR-34a-one of the most widely deregulated miRNAs in cancer and for which a lipid-formulated mimic is already clinically available-in a large cohort of DMPM clinical samples and a unique collection of in house-developed preclinical models, with the aim to assess the potential of a miR-34a-based approach for disease treatment. Methods: miR-34a expression was determined by qRT-PCR in 45 DMPM and 7 normal peritoneum specimens as well as in 5 DMPM cell lines. Following transfection with miR-34a mimic, the effects on DMPM cell phenotype, in terms of proliferative potential, apoptotic rate, invasion ability, and cell cycle distribution, were assessed. In addition, three subcutaneous and orthotopic DMPM xenograft models were used to examine the effect of miR-34a on tumorigenicity. The expression of miRNA targets and the activation status of relevant pathways were investigated by western blot. Results: miR-34a was found to be down-regulated in DMPM clinical specimens and cell lines compared to normal peritoneal samples. miR-34a reconstitution in DMPM cells significantly inhibited proliferation and tumorigenicity, induced an apoptotic response, and declined invasion ability, mainly through the down-regulation of c-MET and AXL and the interference with the activation of downstream signaling. Interestingly, a persistent activation of ERK1/2 and AKT in miR-34a-reconstituted cells was found to counteract the antiproliferative and proapoptotic effects of miRNA, yet not affecting its anti-invasive activity. (Continued on next page) © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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Conclusions: Our preclinical data showing impressive inhibitory effects induced by miR-34a on DMPM cell proliferation,
invasion, and growth in immunodeficient mice strongly suggest the potential clinical utility of a miR-34a-replacement
therapy for the treatment of such a still incurable disease. On the other hand, we provide the first evidence of a potential
cytoprotective/resistance mechanism that may arise towards miRNA-based therapies through the persistent activation of
RTK downstream signaling.
Diffuse malignant peritoneal mesothelioma (DMPM) is
an uncommon though locally aggressive tumor that
develops from mesothelial cells lining the peritoneal
cavity . DMPM prognosis is dismal and standard
therapy, including palliative surgery, systemic/intraperitoneal
chemotherapy, and abdominal irradiation, showed to be
ineffective, with a median survival of about 1 year .
Currently, the most effective treatment is a loco-regional
approach combining aggressive cytoreductive surgery
(CRS) with hyperthermic intraperitoneal chemotherapy
(HIPEC), which significantly extended survival in selected
series of patients . However, for recurrent patients and
for those who are not eligible to CRS+HIPEC, the
prognosis remains severe due to the lack of alternative treatment
options . Considering the rarity of the disease and the
unavailability of experimental models, the biology of
DMPM is still largely unknown. It is anticipated that
advances in the knowledge of the mechanisms responsible
for the biological aggressiveness and the relative
chemoresistance of DMPM will allow the identification of relevant
targets for the development of novel therapeutic strategies.
MicroRNAs (miRNAs) are single-stranded endogenous
evolutionary conserved, non-coding RNA molecules acting
as post-transcriptional regulators of gene expression .
Deregulated miRNA expression and/or function have been
observed in a variety of human solid and hematological
tumors and have been causatively linked to the
pathogenesis of cancer . Depending on their expression levels,
cellular context, and target functions, miRNAs can act as
oncogenes or tumor suppressors  and may represent
novel targets or tools for cancer therapy .
MiR-34a is one of the most widely studied miRNAs in
cancer. Its expression has been found to be decreased in a
variety of human tumors  due to DNA copy number
loss or epigenetic silencing through aberrant CpG
methylation [6, 7]. Results of studies where the expression of
miR-34a was manipulated in human tumor experimental
models clearly showed that the miRNA acts as a tumor
suppressor by regulating highly relevant processes such as
proliferation, cell cycle, apoptosis, invasion, and metastasis
. Reinforced expression of miR-34a has been found to
positively modulate drug response in cancer cells . In
addition, a liposomal nanoparticle-formulated synthetic
miR-34 (MRX34) recently entered a phase I clinical study
for patients with different tumor types .
No information concerning the expression and/or the
functional role of miRNAs in DMPM is currently available
in the literature. Based on the knowledge that several
receptor tyrosine kinases (RTKs) are validated targets of miR-34a
 and our previous results indicating that activation of
downstream RTK signaling, in terms of phosphorylation/
overexpression of extracellular signal regulated kinase ½
(ERK1/2), AKT, and mTOR, is present in a considerable
fraction of DMPM clinical specimens , we proposed to
investigate the possible relevance of miR-34a in the disease,
with the final aim to develop novel therapeutic strategies.
Here, we report that miR-34a is down-regulated in DMPM
clinical specimens and demonstrate that miR-34a
replacement in a unique collection of in-house-developed human
DMPM experimental models [11–13] inhibits cell
proliferation and invasion and impairs tumor growth formation in
SCID mice, mainly as a consequence of c-MET and AXL
inhibition. These findings identified miR-34a-AXL and
-cMET axes as promising therapeutic targets for DMPM.
Moreover, we provide evidence of persistent activation of
ERK1/2 and AKT as a possible cytoprotective mechanism to
RTK inhibition by miR-34a.
Forty-five DMPM specimens classified as epitheliod (40),
sarcomatoid (1), and biphasic (4) from patients treated with
CRS+HIPEC at the Fondazione IRCCS Istituto Nazionale
dei Tumori, Milan (INT) from October 1997 to February
2013, and 7 normal peritoneum specimens from patients
who underwent surgery for non-oncologic disease were
available for miR-34a expression analysis.
This study was approved by the Institutional Review
Board and Ethical Committee and each patient provided
written informed consent to donate to INT the leftover
tissue after diagnostic and clinical procedures.
Cell lines and culture conditions
The human mycoplasma-free DMPM cell lines MesoII,
STO, MP115, MP4, and MP8 were established in our
laboratory [11–13]. All cells were cultured in DMEM F-12
medium (Lonza, Milano s.r.l., Treviglio, Italy) supplemented
with 10% fetal bovine serum in a 37 °C humidified 5% CO2
incubator. Cell lines were authenticated by single-tandem
repeat analysis by the AmpFISTR Identifiler PCR
amplification kit (Applied Biosystems, Foster City, CA, USA).
Mimic pre-miR-34a precursor (miR-34a) and mimic
negative control (Neg) were purchased as Pre-miR™
miRNA precursor molecules (Thermo Fisher Scientific,
Monza, Italy). Knockdown of AXL and c-MET was
performed using specific siRNAs (siAXL and siMET;
ONTARGET plus SMART pool) and, as a control, a siRNA
with a nonsense/scrambled sequence (siNeg,
ONTARGET plus non-Targeting Pool) (Dharmacon, CO,
USA) was used. Cells were transfected for 24 h with
20 pM miR-34a or Neg, or 100 nM siAXL, siMET, or
siNeg, using Lipofectamine® RNAiMAX Transfection
Reagent (Thermo Fisher Scientific) with Opti-MEM I
(Gibco, NY, USA) according to the manufacturer’s
RNA extraction, cDNA synthesis, and qRT-PCR
Quantification of miR-34a expression levels was assessed
by qRT-PCR. Total RNA was isolated using the miRNeasy
Mini Kit (QIAGEN, Hilden, Germany) and 1 μg of RNA
was reverse transcribed by miScript II RT Kit (QIAGEN).
Mature miRNA expression was assayed by miScript Primer
Assays specific for miR-34a (MS00003318) and normalized
on SNORD48 (MS00007511) (QIAGEN). Quantitative
RTPCR was conducted using miScript SYBR Green PCR Kit
(QIAGEN). The reaction was carried out in a 96-well PCR
plate at 95 °C for 15 min followed by 40 cycles of 94 °C for
15 s, 55 °C for 30 s, and 70 °C for 30 s and a dissociation
step to distinguish specific from non-specific amplification
products. Each sample was analyzed in triplicate.
Amplifications were run on the 7900HT Fast
RealTime PCR System (Applied Biosystem). Data were
analyzed by SDS 2.2.2 software (Applied Biosystems) and
reported as -ΔCt, that is the difference between the Ct of
the target gene and the Ct of the housekeeping gene
(where Ct is the threshold cycle), or as relative quantity
(RQ) or -ΔΔCt with respect to a calibrator sample (i.e.,
negative control transfected cells) according to the 2
Cell growth assay
To assess the effect of miR-34a restoration on cell
proliferation, DMPM cells were transfected with Neg or
miR34a as described above. At different intervals from
transfection, cells were trypsinized and counted in a particle
counter (Beckman Coulter, Cassina de’ Pecchi, Italy).
Results were expressed as percent variation in the number
of miR-34a-transfected cells compared with
Cell lysates were fractionated by SDS-PAGE, transferred
to nitrocellulose membranes, and probed with specific
antibodies, as described in . Cells were lysed and
western blot was performed using the following primary
antibodies: anti-c-MET, −CDK6, −uPA, −pospho-FAK
(Tyr 576/577) (Santa Cruz Biotechnology, CA, USA);
anti-AXL, −phospho-AKT (Ser473), −phospho-p44/42
MAPK (ERK1/2) (Thr202/Tyr204), −p44/42 MAPK
(ERK1/2), −FAK, −cleaved CPP32 (Cell signaling,
Beverly, USA); anti-AKT (BD Biosciences, San Jose, CA,
USA); and anti-actin and -vinculin (Sigma Chemical
Company, St. Louis, MO, USA).
Secondary antibodies used were conjugated to
horseradish peroxidase (GE Healthcare, Little Chalfont, UK).
Immunostained bands were detected by
chemoluminescence method (ECL, GE Healthcare). In many
experiments, membranes were stripped and reblotted with a
second antibody. Moreover, membranes were cropped to
allow simultaneous incubation of different primary
antibodies on the same samples. For the preparation of
figures, we cropped the original western blot to generate
the appropriate figure panels with the relevant lanes.
This cropped image was then subjected to uniform
image enhancement of contrast and brightness.
Molecular weights were determined using the Precision Plus
Protein™ Standard (Bio-Rad, Segrate, Italy), which yields
a colorimetric image only and has been removed from
the chemoluminescent blot image.
The AKT-1/2 inhibitor trifluoroacetate salt hydrate
(A6730, Sigma Chemical Company) and the MEK
inhibitor CI-1040 (PD184352, Selleck Chemicals, Houston,
TX, USA) were dissolved and diluted in DMSO. Final
concentration of DMSO in cell cultures never exceeded
0.5%. The antiproliferative activity was evaluated by cell
counting at different times after exposure of
miR34areconstituted MesoII cells to drug concentrations able to
inhibit cell proliferation by 20% (IC20).
Apoptosis detection and cell cycle analysis
At different time points after transfection with miRNAs
mimic or siRNAs, floating and adherent cells were
harvested and processed for apoptosis evaluation by
TUNEL assay according to manufacturer’s instructions
(Roche, Mannheim, Germany) and for cell cycle .
For cell cycle, cells were fixed in 70% ethanol 96 h after
transfection, stained in phosphate-buffered saline (PBS)
containing 10 μg/ml propidium iodide (PI; Sigma
Chemical Company), and RNase A (66 U/ml; Sigma Chemical
Company) for 18 h and analyzed by FACScan flow
cytometer (Becton Dickinson, Mountain View, CA, USA).
Senescence-associated β-galactosidase staining
Cells were transfected with miR-34a or Neg for 24 h.
Samples were washed in PBS 72 h after transfection and
processed for senescence-associated β-galactosidase (SA-β-Gal)
staining. Cells were fixed for 5 min (room temperature) in
2% formaldehyde/0.2% glutaraldehyde, washed and
incubated overnight at 37 °C (no CO2) with fresh solution as
previously described . At least 300 cells were examined,
and the results were expressed as percentage of SA-β-Gal
positive cells over the whole population.
Transwell invasion assay
Invasion assay was performed 72 h after transfection using
a 24-well Boyden chamber with 8-mm pore size filter in
the inset chambers (Costar, Corning Inc., NY, USA). The
Transwell membranes were previously coated with
3.47 μg Matrigel/well (BD Biosciences) and dried for
30 min. Cells were suspended in 300 μL serum-free
medium and seeded into the insert chambers. After 24 h
of incubation at 37 °C in 5% CO2, cells that migrated into
the bottom chamber containing 1 ml of serum-free
medium were fixed in 95% ethanol, stained with a solution
of 0.4% sulforhodamine B in 0.1% acetic acid, counted
under an inverted microscope, and then photographed.
Antibody arrays and ELISA
Cells were seeded at 2 × 104 cells/dish in complete medium
and transfected with Neg or miR-34a for 24 h before serum
starvation for 72 h. Conditioned media were then harvested
and clarified by centrifugation at 13,000 rpm for 15 min.
Cells were trypsinized, counted, and lysed for assaying
protein content. Supernatant aliquots were used to assess
angiogenesis-related protein content by Antibody Arrays
(R&D System, SPACE Import Export, Milan, Italy)
according to manufacturer’s instructions. The ELISA kit for
Maspin (human Maspin “Super X” ELISA Kit, Antigenix
America, Huntington Station, NY, USA) was used according
to the manufacturer’s instructions for quantitative analysis.
In vivo experiments
All experimental protocols were approved by the Ethics
Committee for Animal Experimentation of INT.
Experiments were performed using 8-week-old female SCID
mice (Charles River, Calco, Italy). Each group contained
five to six mice. Cells were transfected with miR-34a or
Neg for 24 h, as described above, and then inoculated
subcutaneously or intraperitoneally after the analysis of
the transfection efficiency by qRT-PCR.
Subcutaneous tumor models
STO, MesoII, and MP8 cells were injected subcutaneously
into the right flank (1–1.2 × 107 cells/mouse). Inoculated
animals were inspected daily to establish the time of tumor
onset. Tumor growth was measured every 2 to 3 days using
a Vernier caliper (Table 1). The subcutaneous tumor
volume was calculated as follows: TV (mm3) = d2 × D/2
where d and D are the shortest and the longest diameter,
respectively. Volume inhibition percentage (TVI%) in
tumors derived from miR-34a- over Neg-transfected cells
was calculated as follows: TVI% = 100 − (mean miR-34a
TV/mean Neg TV × 100).
Proteins were obtained as described previously 
from frozen s.c. tumors derived from two additional mice
sacrified at different time points. Briefly, samples were
pulverized by Mikro-Dismembrator II (B. Brown Biotech
International, Melsungen, Germany) and suspended in
lysis buffer supplemented with protease and phosphatase
inhibitors. Proteins were processed as described .
Intraperitoneal (orthotopic) tumor models
STO and MP8 cells were injected into the peritoneal
cavity (107 and 2.5 × 107 cells/mouse, respectively).
Animals were monitored and weighed daily and sacrificed at
different times from cell injection (Table 2). A careful
necropsy was performed to evaluate the take rate and
spread of mesothelioma cells in the abdominal cavity.
Solid masses were gently detached from organs and
abdominal walls, removed, and weighed for calculating the
percentage of tumor weight inhibition (TWI %) in mice
inoculated with miR-34a- over Neg-transfected cells.
If not otherwise specified, in vitro data are presented as
mean values ± SD from at least three independent
experiments. Statistical analysis of the data was performed by
two-tailed Student’s t test. For in vivo data, two-tailed
Student’s t and Fisher’s exact test were used to compare
tumor volumes/weights and tumor takes, respectively.
Patient survival analysis was performed using Cox
proportional regression model . p values <0.05 were
considered statistically significant.
miR-34a is down-regulated in DMPM clinical samples and
We first evaluated miR-34a expression by qRT-PCR in 45
DMPM and 7 normal peritoneum specimens as well as in
5 unique cell lines established in our laboratory from
clinical samples of epithelioid (STO, MP4, MesoII, MP8) and
biphasic (MP115) DMPM. Results indicated that miR-34a
abundance is significantly reduced in DMPM compared to
normal tissues (Fig. 1). Consistently, miR-34a expression
was found down-regulated in all DMPM cell lines, thus
indicating an oncosuppressive function of the miRNA also
in this disease.
No significant difference in miR-34a expression was
observed as a function of demographic and
clinicopathologic characteristics, including gender, histologic
Table 1 Effect of miR-34a reconstitution on DMPM cell tumorigenicity following s.c. injection in SCID mice
Model miRNA Tumor onsetb TV (mm3)
aNumber of mice presenting s.c. tumors out of number of cell-injected mice
bMedian day of tumor appearance
cTumor volume inhibition % in miRNA34a- over Neg-transfected cell-injected mice
dBy Student’s t test over Neg-transfected cell-injected mice
subtype, and peritoneal cancer index  (data not
shown). In addition, at 5 years of follow-up, miR-34a
expression did not significantly affect the probability of
disease-free survival of DMPM patients (high expressing
versus low expressing—categorized on the basis of the
median miR-34a expression value—36 versus 20%; hazard
ratio, 1.85; 95% confidence interval, 0.86–4.01; p = 0.11).
Overall, such findings suggest a role for miR-34a as a
possible therapeutic target rather than a
prognostic/predictive biomarker in DMPM.
miR-34a reconstitution variably affects DMPM cell growth
To functionally assess the possible role of miR-34a as a
novel therapeutic target in DMPM, we transiently
transfected cells with miR-34a synthetic mimic and miRNA
negative control. As assessed by qRT-PCR, marked
increase in miRNA abundance was consistently observed in
all cell lines at 24 h from transfection (Additional file 1:
Figure S1) and was still maintained, although to a lesser
extent, at 168 h (Additional file 1: Figure S1 and data not
shown). miR-34a reconstitution significantly inhibited the
proliferation of four out of five DMPM cell lines in a
time-dependent manner, though with a different kinetics
(Fig. 2a). Specifically, a more rapid cell growth decline was
observed in STO and MP4 cells (~80% inhibition at 96 and
168 h, respectively), whereas, in MesoII and MP8 cells, the
same level of inhibition was recorded at later time points
(192 and 216 h, respectively) (Fig. 2a). Conversely, ectopic
expression of miR-34a only induced a weak inhibition
(~30%) of MP115 cell growth, which was almost constant
until the end of the experiment (Fig. 2a).
The variable antiproliferative effects consequent to
miR34a reconstitution in the different DMPM cell lines was
paralleled by a different kinetics of apoptosis induction, as
detected by TUNEL assay (Fig. 2b). Specifically, a
significant enhancement in the percentage of apoptotic cells was
already appreciable at 96 h upon transfection of STO and
MP4 cells, whereas the apoptotic response was induced at
a later time point (168 h) in MP8 and MesoII cells
(Fig. 2b). No induction of apoptotic cell death was
observed in MP115 cells until 216 h after miR-34a
reconstitution (Fig. 2b).
To investigate whether validated targets of miR-34a
were modulated by its synthetic mimic in DMPM cells,
we assessed protein expression levels of c-MET, AXL,
and CDK6 considering their established role in the
control of cell proliferation and apoptosis in different
tumor types [8, 19–21]. A marked down-modulation of
the three proteins was consistently observed in all
DMPM cell lines (Fig. 2c), regardless of the effects
induced by miR-34a reconstitution on cell growth and
ERK1/2 and AKT activation as a possible cytoprotective
mechanism following miR-34a reconstitution
Based on the evidence that the activation of downstream
RTK signaling pathways, including PI3K/AKT and RAF/
aNumber of mice presenting i.p. tumors out of number of cell-injected mice
bBy Fisher’s exact test over Neg cell-injected mice
cTumor weight inhibition % in miRNA34a- over Neg-transfected cell-injected mice
dBy Student’s t test over Neg-transfected cell-injected mice
Table 2 Effect of miR-34a reconstitution on DMPM cell tumorigenicity following i.p. injection in SCID mice
80, 90, 70, 120, 80
Fig. 1 Expression levels of miR-34a. qRT-PCR analysis of miR-34a expression using total RNA from fresh normal peritoneum tissues (n = 7), DMPM
clinical samples (n = 45), and DMPM cell lines (STO, MP4, MesoII, MP8, MP115). Data were presented as 2−ΔCt (miR-34a-SNORD48) values (**p < 0.01;
***p < 0.001; ****p < 0.0001 by Student’s t test)
MEK/MAPK cascades, seems to be crucial in both
malignant pleural  and peritoneal  mesothelioma, we
evaluated the effect of miR-34a reconstitution on the
phosphorylation status of AKT and ERK1/2 in DMPM cell lines.
A reduced abundance of phospho-ERK1/2 and
phosphoAKT was appreciable in STO cells at 72 and 96 h after
transfection with miR-34a mimic (Fig. 2c). A reduced
expression of phospho-ERK1/2 was also observed in
miR34a-reconstituted MP4 and MP8 cells at the latter time
point (Fig. 2c). Consistent with the delayed antiproliferative
and proapoptotic response following miR-34a
reconstitution, a decline in the expression levels of phospho-ERK1/2
and phospho-AKT was observed in MesoII cells at only
168 h (Fig. 2c, d), whereas no decrease in the abundance of
the two phopho-proteins was found in the less sensitive
MP115 cell line at either time point (Fig. 2c, d).
To assess whether activation of AKT or MAPK/ERK1/2
signaling pathways, which is a well-known mechanism of
resistance to RTK inhibitors [23–26], could also represent
a cytoprotective mechanism to the oncosuppessive effects
of miR-34a, we exposed miRNA-reconstituted MesoII
cells to subtoxic concentrations of small-molecule AKT
(A6730) and MEK1 (CI-1040) inhibitors (Fig. 3a).
Interestingly, inactivating ERK1/2 or impeding the reactivation of
AKT only slightly affected MesoII response to miR-34a,
whereas the concomitant blockade of the two pathways,
made the sensitivity profile of MesoII cells superimposable
on that of the inherently sensitive STO cell line (Fig. 3b).
Such a growth inhibitory effect was paralleled by an earlier
onset of apoptosis, as detected by caspase-3 cleavage
(CPP32) at 96 h (Fig. 3a).
To better characterize the cytostatic—rather than
cytotoxic—effect observed in MP115 cells following miR-34a
reconstitution, we assessed cell cycle distribution by flow
cytometry. A cell accumulation in the G1-phase, which was
paralleled by an enhanced fraction of senescence-associated
β-galactosidase-positive cells, was observed (Fig. 4a, b).
Such a senescence-like phenotype, which was not
appreciable in the other DMPM cell models (Fig. 4b and data
not shown), could be related to the marked phospho-AKT
accumulation observed in miR-34a-reconstituted MP115
cells, according to previous evidence indicating that
constitutively active AKT induces senescence in human
endothelial cells and human fibroblasts [27, 28].
miR-34a oncosuppressive activities mainly rely on c-MET
and AXL inhibition
To corroborate the hypothesis that miR-34a
oncosuppressive functions mainly rely on the down-regulation of
cMET and AXL, we performed siRNA-based phenocopy
experiments (Additional file 2: Figure S2). When
transfected into STO cells, which do not inherently express
AXL, siMET was able to recapitulate the effects induced
by miR-34a reconstitution, in terms of cell growth
inhibition (Fig. 5a), apoptosis induction (Fig. 5b), impairment
of invasive capability (Fig. 5c), and inactivation of both
ERK1/2 and AKT pathways (Additional file 2: Figure S2).
In MesoII cells, siMET did not appreciably affect cell
proliferation, apoptosis, or invasion (Fig. 5a–c).
Conversely, siAXL reduced MesoII proliferation, although a
cell growth inhibition comparable to that induced by
miR34a reconstitution was only observed following combined
silencing of c-MET and AXL (Fig. 5a). Interestingly, siAXL
alone phenocopied the effects of miR-34a, in terms of
apoptosis and invasion (Fig. 5b, c), suggesting a main role
of this RTK in mediating the oncosuppressive effects of
miR-34a in MesoII cells. Moreover, AXL silencing did not
inhibit AKT and ERK1/2 signaling pathways similarly to
miR-34a reconstitution (Additional file 2: Figure S2).
The decreased EGFR abundance observed in
miR-34areconstituted STO and MesoII cells (Additional file 3:
Figure S3a), together with preliminary evidence indicating
that the RTK is expressed/activated in DMPM clinical
specimens , prompted us to investigate a possible role
for EGFR down-regulation in sustaining the
miR-34ainduced cell phenotype. However, siEGFR failed to affect
cell growth and invasion capability in both cell lines
(Additional file 3: Figure S3b).
Fig. 2 Effects of miR-34a reconstitution on DMPM cell proliferation, apoptosis, and RTK signaling pathways. a Antiproliferative effects of miR-34a. DMPM cell
growth was assessed by cell counting. Data are expressed as percentage of the proliferation of miR-34a- versus miRNA negative control (Neg)-transfected
cells. Means ± SD values of three independent experiments are reported. b Proapoptotic effects of miR-34a. Quantitative analysis of TUNEL-positive DMPM
cells transfected with transfection reagent (Ctrl), Neg, or miR-34a was carried out by flow cytometry at different time points after transfection. Mean ± SD
values of three independent experiments are reported (**p < 0.01; ***p < 0.001 by Student’s t test). c, d Effects of miR-34a on validated miRNA targets and
RTK downstream signaling cascades as assessed by western blot analysis at 72, 96 (c) and 168 (d) h after cell transfection with Ctrl, Neg, or miR-34a. Actin
was used to confirm equal protein loading. A representative experiment of three was reported. Cropped images of selected proteins are shown
miR-34a reconstitution inhibits DMPM cell invasion and
impairs the secretion of angiogenesis-related factors
Enforced expression of miR-34a significantly inhibited
invasion of all DMPM cell models, as detected in a
matrigelbased assay at 72 h after transfection (Fig. 6a), likely as a
consequence of c-MET or AXL down-regulation. c-MET
and AXL signaling pathways are indeed known to affect cell
motility and invasion primarily through the activation of
mitogen-activated protein kinase (MAPK) [29–32].
However, since a decreased invasive potential was observed also
in MesoII and MP115 cells in spite of MAPK iperactivation
(Fig. 2c), the status of focal adhesion kinase (FAK), known
to mediate cell migration and anchorage-independent
growth downstream of RTKs [29–33], was assessed.
Interestingly, miR-34a ectopic expression consistently reduced
FAK posphorylation at Y576/577 in STO, MesoII, and
MP115 cell models, suggesting that miR-34a-induced
inhibition of cell invasion can occur regardless of MAPK and
AKT activation (Fig. 6b).
Since AXL and c-MET inhibition by drugs and
monoclonal antibodies was shown to induce antiangiogenic
effects in tumors [34, 35], we investigated whether miR-34a
reconstitution was able to affect the production/release of
angiogenesis-related proteins by DMPM cells. We found
Fig. 3 Inhibition of AKT and ERK1/2 signaling pathways increases the antiproliferative and proapoptotic effects of miR-34a. MesoII cells were
transfected with Ctrl, Neg, or miR-34a for 24 h and successively exposed to vehicle (DMSO, unt) or low concentrations (corresponding to IC20 values) of
A6730 (AKT inhibitor, 5 μM) and/or CI-1040 (MEK inhibitor, 3 μM). a Expression and phosphorylation status of AKT and ERK1/2 and amount of cleaved
CPP32 at 72 h after drug treatment were assessed by western blot. Actin was used to confirm equal protein loading. A representative experiment of
three was reported. The panel shows cropped blots. b The effects of AKT and MEK inhibitors on the growth of miR-34a-transfected cells were assessed
by cell counting. Data are expressed as percentage of the proliferation of miR-34a- versus Neg-transfected cells. The cell growth inhibition curve of
STO cells after enforced expression of miR-34a was reported for comparison. Means ± SD values of three independent experiments are shown
that miR-34a reconstitution in MesoII cells impaired the
secretion of angiogenesis-related molecules (Fig. 7).
Indeed, antibody array results obtained in conditioned
medium of miR-34a-reconstituted cells (at 72 h following
transfection) showed a reduced secretion of the
urokinase-type plasminogen activator (uPA), which plays
a major role in promoting angiogenesis , together with
and increased release of maspin, a member of the serine
protease inhibitor (serpin) superfamily, which exerts
antiangiogenic effects through the inhibition of both the
growth and migration of endothelial cells [37, 38] (Fig. 7a).
Western blot and ELISA experiments carried out on the
same conditioned media confirmed a reduced expression
miR-34a reconstitution inhibits tumor formation in SCID mice
To investigate whether miR-34a reconstitution affected
DMPM formation in vivo, we subcutaneously (STO,
MesoII, MP8) and intraperitoneally (STO, MP8) inoculated
miR-34a mimic- or miRNA negative control-transfected
cells into SCID mice. Results indicate that miR-34a
consistently impaired the growth of all s.c. xenograft models, with
maximum tumor volume inhibitions ranging from 57 to
98% (Fig. 8 and Table 1). In addition, an appreciably delayed
tumor onset was observed for MesoII and MP8 cell models
Fig. 4 miR-34a induces a senescence-like phenotype in MP115 cells. a At 96 h following transfection of STO and MP115 cells with Ctrl, Neg, or
miR-34a, nuclei were stained with propidium iodide and analyzed for DNA content by FACScan. Data (mean ± SD of three independent experiments)
represent the percentage of cells in the different cell cycle phases. b Left panel: representative micrographs of SA-β-gal staining of STO and MP115 cells
transfected with Neg or miR-34a. Blue precipitation in the cytoplasm was observed in the senescent cells. Original magnification, × 40. One representative
experiment of three was shown. Right panel: histogram bars represent the mean percentage of SA-β-gal positive cells ± SD of at least three independent
experiments (**p < 0.01 by Student’s t test)
(Table 1). Western blot carried out in tumors collected
from additional mice sacrificed at different time points after
DMPM cell inoculum indicated a decreased expression of
c-MET in all xenograft models and AXL in MesoII and
MP8 models (Fig. 8b), in accordance with in vitro results.
As regards i.p. xenograft models, at necropsy, all control
mice showed a large tumor mass at the site of cell injection
mainly invading the peritoneum wall and widespread small
nodules in the peritoneum and attached to the diaphragm,
liver, and bowel. Conversely, only one mouse out of the five
mice receiving miR-34a-reconstituted STO cells developed
small tumor nodules in the abdominal cavity (Table 2).
miR-34a ectopic expression did not influence the take of
MP8 cells but markedly reduced their growth, as indicated
by a significantly reduced tumor weight (Table 2).
No information is currently available on the expression and
functional role of miRNAs in DMPM. Here, we
demonstrated that miR-34a is down-regulated in a large series of
DMPM clinical samples and in a unique panel of cell lines,
established from DMPM patients in our laboratories,
compared to normal peritoneum specimens. We also illustrated
that miR-34a exerts oncosuppressive functions in our
tumor models, consistent to what previously observed in a
variety of human tumor types [5, 39–41]. Indeed, miR-34a
reconstitution impaired proliferation and induced an
apoptotic response in DMPM cell lines, although at a
variable extent and with different kinetics, mainly through the
down-regulation of c-MET and AXL and the interference
with the activation of downstream signaling. Interestingly,
results also indicated that a transient or persistent activation
of ERK1/2 and AKT can delay or prevent the cytotoxic and
proapoptotic effects of miR-34a reconstitution, as observed
in MesoII and MP115 cells, respectively. Noteworthy,
DMPM cell feedback to AXL and c-MET down-regulation
induced by miR-34a reconstitution is to directly activate
ERK1/2 and AKT survival signaling cascades rather than
up-regulate the expression levels of the receptors, thus
ensuring a more prompt counter-response. Such findings
provide the first evidence that tumor cells can exploit a
wellknown mechanism of resistance to RTK inhibitors—i.e., the
activation of RTK downstream signaling [23–26]—to
counteract the antiproliferative/proapoptotic effects of miR-34a.
However, such a mechanism was not found to protect
DMPM cells from the anti-invasive effect of the miRNA.
Noteworthy, the cytoprotective mechanism based on
ERK1/2 and AKTactivation was mainly evident in the DMPM
cell line MP115, derived from a biphasic subtype tumor. Such
subtype is known to be more aggressive and associated with a
reduced patient survival compared to the epithelioid ,
although differences in specific relevant biological properties
between the two DMPM subtypes are currently unknown. In
addition, the delayed pro-apoptotic and cytotoxic effects
observed in MesoII cells following miR-34a reconstitution are
consistent with the finding that, unlike other epithelioid cell
lines, they carry a mutant p53 .
A novel mechanism of miR-34a-dependent AKT inhibition
has been recently proposed by Wang et al. . In this study,
miR-34a is reported to inhibit Bmi-1 by targeting c-Myc in
gastric cancer cells, resulting in a PTEN-dependent reduction
of phospho-AKT. The observation that in DMPM cell lines
more susceptible to the cytotoxic effects of miR-34a (STO,
MP4, MP8), a decrease in phospho-AKT abundance is
observed early after miRNA reconstitution would suggest the
possibility that the above-described mechanism is also
operating in our models. However, results of phenocopy
experiments showing that siRNA-mediated silencing of c-MET and
AXL was able to decrease AKT activation in sensitive cells
(STO) but not in those less susceptible to miR-34a (MesoII)
would suggest that the main mechanism controlling AKT
phosphorylation status relies on RTK activity.
Fig. 5 Silencing AXL and c-MET phenocopies miR-34a effects. STO and
MesoII cells were treated with transfection reagent (Ctrl), siNeg (siRNA
with a nonsense/scrambled sequence) or AXL- and c-MET-directed
siRNA (siAXL, siMET) for 24 hours. a Effect of AXL and/or c-MET
knockdown on cell growth, as detected by cell counting at different
times after transfection. The antiproliferative effect induced by miR-34a
reconstitution is reported for comparative purposes. b Induction of
apoptosis at 96 h after transfection, as assessed by TUNEL assay
(**p < 0.01 by Student’s t test). Data are expressed as percentage (mean
± SD) of the proliferation of siAXL-/siMET- versus siNeg-transfected cells.
c Effect of RTK-siRNA on DMPM cell matrix degrading/invasive activities.
Cells were silenced and, after 72 h, subjected to Matrigel invasion assay
in serum-free medium. The number of invading cells per field is reported.
Histogram bars represent mean values ± SD of at least three independent
experiments (***p < 0.001 by Student’s t test)
Interestingly, miR-34a induced a remarkable antitumor
activity in the three cell lines (STO, MesoII and MP8) able
to generate tumors following xenotransplantation into
immunodeficient mice. Although to a different extent,
miR-34a reconstitution significantly reduced the growth
of the three s.c. xenograft models. Highly relevant to the
disease, miRNA ectopic expression also impaired the
growth of STO and MP8 orthotopic xenografts, which
properly recapitulate the dissemination pattern in the
peritoneal cavity of human DMPM [11, 12], thus
representing improved models to investigate novel therapeutic
approaches. Specifically, miR-34a significantly inhibited
the take of STO cells, with only one mouse developing
small tumor nodules in the abdominal cavity. Although
the miRNA did not influence the take of MP8 cells, a
significantly reduced tumor growth was observed.
Unfortunately, the inability of MP115—the only biphasic
DMPM model in our panel—to grow in vivo prevented us
to assess whether the in vitro cytostatic effect consequent
to miR-34a reconstitution, which was paralleled by the
induction of a senescence-like phenotype possibly sustained
by ATK activation, may result or not in tumor growth
impairment. However, the significantly reduced invasive
potential induced by miR-34a in DMPM cell lines through
the inhibition of FAK signaling could primarily contribute
to the antitumor effect observed in the xenograft models.
Moreover, the occurrence of miR-34a-induced inhibition
of cell invasion in the absence of appreciable
antiproliferative and proapoptotic effects that we observed in MP115
is not surprising since the same phenotype has been
previously reported by Li et al.  for miR-34a reconstituted
HepG2 hepatocellular carcinoma cells.
Interestingly, our evidence indicating that miR-34a
ectopic expression impairs the secretion of
angiogenesisrelated factors by MesoII cells strongly suggests that the
antitumor effect observed in both s.c. and ortothopic
xenograft models can also rely on miRNA-induced modification
of tumor microenvironment, making it less favorable to
In summary, the impressive inhibitory effects induced
by miR-34a on DMPM cell proliferation, invasion, and
growth in immunodeficient mice suggest a possible utility
of the clinically available miR-34a as novel therapeutic
option for DMPM patients who are not eligible for or relapse
after CRS+HIPEC. In addition, the evidence that miR-34a
reconstitution positively modulates the activity of
antitumor drugs in experimental models of different human
tumor types [8, 45–47] highlights the possibility that the
miR-34a mimic could have an important role also in
combined strategies for treating DMPM patients.
DMPM is a rapidly fatal tumor with scanty therapeutic
options. Here, we demonstrated for the first time that
Fig. 6 miR-34a inhibits the invasion of DMPM cells. Cells were transfected with Ctrl, Neg, or miR-34a for 24 h. a Cells were subjected to Matrigel
invasion assay in serum-free medium 72 h after transfection. Top: the number of invading cells per field is reported. Histogram bars represent
mean values ± SD of three independent experiments (**p < 0.01; ***p < 0.001 by Student’s t test). Bottom: micrographs from one experiment
representative of three. Original magnification, × 40. b Whole-cell lysates were analyzed by western blot with anti-phospho-FAK. Protein extraction
was performed 96 h after transfection with Ctrl, Neg, or miR-34a. Vinculin was used to confirm equal protein loading. A representative experiment
of three was reported. Cropped blots are shown
Fig. 7 Effect of miR-34a restoration on the secretion of angiogenesis-related factors. a Secreted angiogenesis-related molecules were examined
by antibody arrays in conditioned medium obtained from Neg- or miR-34a-transfected MesoII cells grown for 72 h in the absence of serum. C+,
internal standards. The same conditioned media were used to assess the expression of b uPa precursor by western blot and c maspin protein by
ELISA. Each bar represents mean ± SD of triplicate samples from a representative experiment (**p < 0.01 by Student’s t test)
Fig. 8 miR-34a inhibits DMPM tumorigenicity. a STO, MesoII, and MP8 cells were transfected with miR-34a or Neg and, on day 0, implanted
subcutaneously into the right flank of SCID mice. Micrographs show the mean tumor volumes ± SD measured at different time points after DMPM
cell injection. (*p < 0.05, **p < 0.01 by two-tailed Student’s t test). b Cropped western blot for c-MET and AXL expression in frozen tumors derived
from two additional mice sacrificed at different times (STO, 10 days; MesoII, 19 days; MP8, 25 days) after cell inoculum. Actin was used to confirm
equal protein loading
reconstitution of miR-34a in relevant models of the
disease induced a significant antitumor effect, which mainly
relied on c-MET and AXL down-regulation and
impairment of their downstream signaling. In vivo results were
complemented by in vitro data showing significant
antiproliferative, proapoptotic, and anti-invasive activities.
Taken together, our results provide evidence that (i)
cMET and AXL signaling pathways are critical
determinants of DMPM cell survival, growth, and invasiveness
and that miR-34a reconstitution can impair all these
functions and (ii) persistent activation of AKT and ERK1/2
downstream signaling pathways represents a
cytoprotective mechanism against miRNA-induced proapoptotic
effects, though not preventing its anti-invasive activity,
which instead mainly relies on FAK inhibition.
Overall, our preclinical data form a solid foundation
that could promote the clinical translation of clinically
available miR-34 mimic for the treatment of a still
incurable disease such as DMPM and, on the other hand,
provide the first evidence of a possible
cytoprotective/resistance mechanism that may arise towards
Additional file 1: Figure S1. Expression of miR-34a upon restoration in
DMPM cells. Cells were transfected with either Neg or miR-34a for 24 h. (A)
qRT-PCR analysis in the panel of DMPM cell lines 24 h after transfection. (B)
miR-34a expression by qRT-PCR in MesoII at different times after transfection.
Data are reported as −ΔΔCt between miR-34a- and Neg-transfected cells. A
representative experiment of three was reported. (TIF 236 kb)
Additional file 2: Figure S2. Effects of silencing AXL and c-MET on RTK
and downstream signaling pathways. DMPM cells were lysed 72 h after
transfection with RNAimax (Ctrl), control siRNA (siNeg), or AXL- and c-MET-directed
siRNAs (siAXL, siMET) for 24 h. RTK levels and activation status of ERK 1/2 or
AKT were assessed by western blot analysis. Cropped images of the protein
expression are reported. Vinculin was used to confirm equal protein loading.
(TIF 559 kb)
Additional file 3: Figure S3. a and b Effect of EGFR modulation by
miR-34a or siEGFR on DMPM cells. (a) EGFR protein expression was
assessed by western blot analysis at 72 and 96 h after 24 h transfection
with transfection reagent (Ctrl), Neg or miR-34a. Actin was used to confirm
equal protein loading. Cropped blots are presented. (b) EGFR protein
expression was determined by western blot analysis 72 h after 24 h
transfection with siNeg or EGFR-directed siRNA (siEGFR). Vinculin was used
to confirm equal protein loading. (c) DMPM cell proliferation was assessed
by cell counting at different time points after 24 h transfection with siNeg
or siEGFR. (d) DMPM cell invasion was determined in a Matrigel-based assay
at 72 h after 24 h transfection with transfection reagent (Ctrl), siNeg, or
siEGFR. (TIF 606 kb)
DMPM: Diffuse malignant peritoneal mesothelioma; ERK1/2: Extracellular signal
regulated kinase ½; FAK: Focal adhesion kinase; MAPK: Mitogen-activated protein
kinase; miRNA: MicroRNA; PI3K: Phosphoinositide 3-kinase; RTK: Receptor tyrosine
kinase; TVI: Tumor volume inhibition; TWI: Tumor weight inhibition;
uPA: Urokinase-type plasminogen activator
Availability of data and materials
All data generated or analyzed during this study are included in this
published article and its supplementary information files.
NZ and VZ conceived the study. RE, MDC, MP, and VZ carried out the
experiment. MD provided the tumor sample collection. RE, MDC, MP, PG, NZ,
and VZ analyzed the data. All authors were involved in writing the
manuscript. All authors read and approved the final manuscript.
Ethics approval and consent to participate
This study was approved by the Institutional Review Board and Ethical
Committee and each patient provided written informed consent to donate
to INT the leftover tissue after diagnostic and clinical procedures.
1Molecular Pharmacology Unit, Department of Experimental Oncology and
Molecular Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, 20133
Milan, Italy. 2Colon-Rectal Cancer Surgery Unit, Department of Surgery,
Fondazione IRCCS Istituto Nazionale dei Tumori, 20133 Milan, Italy.
1. Deraco M , Baratti D , Hutanu I , Bertuli R , Kusamura S. The role of perioperative systemic chemotherapy in diffuse malignant peritoneal mesothelioma patients treated with cytoreductive surgery and hyperthermic intraperitoneal chemotherapy . Ann Surg Oncol . 2013 ; 20 : 1093 - 100 .
2. Baratti D , Kusamura S , Cabras AD , Bertulli R , Hutanu I , Deraco M. Diffuse malignant peritoneal mesothelioma: long-term survival with complete cytoreductive surgery followed by hyperthermic intraperitoneal chemotherapy (HIPEC) . Eur J Cancer . 2013 ; 49 : 3140 - 8 .
3. Bartel DP . MicroRNAs: target recognition and regulatory functions . Cell . 2009 ; 136 : 215 - 33 .
4. Jansson MD , Lund AH . MicroRNA and cancer . Mol Oncol . 2012 ; 6 : 590 - 610 .
5. Li XJ , Ren ZJ , Tang JH. MicroRNA-34a: a potential therapeutic target in human cancer . Cell Death Dis . 2014 ; 5 : e1327 .
6. Schmid G , Notaro S , Reimer D , Abdel-Azim S , Duggan-Peer M , Holly J , et al. Expression and promotor hypermethylation of miR-34a in the various histological subtypes of ovarian cancer . BMC Cancer . 2016 ; 16 : 102 .
7. Henrich KO , Schwab M , Westermann F. 1p36 tumor suppression-a matter of dosage? Cancer Res . 2012 ; 72 : 6079 - 88 .
8. Misso G , Di Martino MT , De Rosa G , Farooqi AA , Lombardi A , Campani V , et al. Mir-34: a new weapon against cancer? Mol Ther Nucleic Acids . 2014 ; 3 : e194 .
9. Mirna Therapeutics , Inc; Cancer Prevention Research Institute of Texas. A multicenter phase I study of MRX34, microRNA miR-RX34 liposomal injection . In: ClinicalTrials.gov [Internet]. Bethesda: National Library of Medicine (US) . Available from: https://clinicaltrials.gov/ct2/show/ NCT01829971 NLM Identifier: NCT01829971 . Accessed 20 July 2016 .
10. Perrone F , Jocollè G , Pennati M , Deraco M , Baratti D , Brich S , et al. Receptor tyrosine kinases and downstream signaling analysis in diffuse malignant peritoneal mesothelioma . Eur J Cancer . 2010 ; 46 : 2837 - 48 .
11. De Cesare M , Cominetti D , Doldi V , Lopergolo A , Deraco M , Gandellini P , et al. Anti-tumor activity of selective inhibitors of XPO1/CRM1-mediated nuclear export in diffuse malignant peritoneal mesothelioma: the role of survivin . Oncotarget . 2015 ; 6 : 13119 - 32 .
12. De Cesare M , Sfondrini L , Pennati M , De Marco C , Motta V , Tagliabue E , et al. CpG-oligodeoxynucleotides exert remarkable antitumor activity against diffuse malignant peritoneal mesothelioma orthotopic xenografts . J Transl Med . 2016 ; 14 : 25 .
13. Spanò V , Pennati M , Parrino B , Carbone A , Montalbano A , Cilibrasi V , et al. Preclinical activity of new [1,2]oxazolo[5,4-e]isoindole derivatives in diffuse malignant peritoneal mesothelioma . J Med Chem . 2016 ; 59 : 7223 - 38 .
14. Zuco V , Supino R , Favini E , Tortoreto M , Cincinelli R , Croce AC , et al. Efficacy of ST1968 (namitecan) on a topotecan-resistant squamous cell carcinoma . Biochem Pharmacol . 2010 ; 79 : 535 - 41 .
15. Zuco V , Benedetti V , Zunino F. ATM- and ATR-mediated response to DNA damage induced by a novel camptothecin, ST1968 . Cancer Lett . 2010 ; 292 : 186 - 96 .
16. De Cesare M , Lauricella C , Veronese SM , Cominetti D , Pisano C , Zunino F , et al. Synergistic antitumor activity of cetuximab and namitecan in human squamous cell carcinoma models relies on cooperative inhibition of EGFR expression and depends on high EGFR gene copy number . Clin Cancer Res . 2014 ; 20 : 995 - 1006 .
17. Cox DR . Regression models and life tables . J R Stat Soc Series B Stat Methodol . 1972 ; 34 : 187 - 220 .
18. Glehen O , Gilly FN . Quantitative prognostic indicators of peritoneal surface malignancy: carcinomatosis, sarcomatosis and peritoneal mesothelioma . Surg Oncol Clin N Am . 2003 ; 12 : 649 - 71 .
19. Miyauchi H , Minamino T , Tateno K , Kunieda T , Toko H , Komuro I. Akt negatively regulates the in vitro lifespan of human endothelial cells via a p53/p21-dependent pathway . EMBO J . 2004 ; 23 : 212 - 20 .
20. Astle MV , Hannan KM , Ng PY , Lee RS , George AJ , Hsu AK , et al. AKT induces senescence in human cells via mTORC1 and p53 in the absence of DNA damage: implications for targeting mTOR during malignancy . Oncogene . 2012 ; 31 : 1949 - 62 .
21. Agostini M , Knight RA . miR-34: from bench to bedside . Oncotarget. 2014 ; 5 : 872 - 81 .
22. Hermeking H. The miR-34 family in cancer and apoptosis . Cell Death Differ . 2010 ; 17 : 193 - 9 .
23. Bader AG . miR-34 - a microRNA replacement therapy is headed to the clinic . Front Genet . 2012 ; 3 : 120 .
24. Zhou S , Liu L , Li H , Eilers G , Kuang Y , Shi S , et al. Multipoint targeting of the PI3K/mTOR pathway in mesothelioma . Br J Cancer . 2014 ; 110 : 2479 - 88 .
25. Donev IS , Wang W , Yamada T , Li Q , Takeuchi S , Matsumoto K , et al. Transient PI3K inhibition induces apoptosis and overcomes HGF-mediated resistance to EGFR-TKIs in EGFR mutant lung cancer . Clin Cancer Res . 2011 ; 17 : 2260 - 9 .
26. Suzuki VZM , Abe A , Imagama S , Nomura Y , Tanizaki R , Minami Y , et al. BCR-ABL-independent and RAS/MAPK pathway-dependent form of imatinib resistance in Ph-positive acute lymphoblastic leukemia cell line with activation of EphB4 . Eur J Haematol . 2010 ; 84 : 229 - 38 .
27. Ercan D , Xu C , Yanagita M , Monast CS , Pratilas CA , Montero J , et al. Reactivation of ERK signaling causes resistance to EGFR kinase inhibitors . Cancer Discov . 2012 ; 2 : 934 - 47 .
28. Brevet M , Shimizu S , Bott MJ , Shukla N , Zhou Q , Olshen AB , et al. Coactivation of receptor tyrosine kinases in malignant mesothelioma as a rationale for combination targeted therapy . J Thorac Oncol . 2011 ; 6 : 864 - 74 .
29. Organ SL , Tsao MS . An overview of the c-MET signaling pathway . Ther Adv Med Oncol . 2011 ; 3 ( Suppl 1 ): S7 - 19 .
30. Cho O , Hwang HS , Lee BS , Oh YT , Kim CH , Chun M. Met inactivation by S-allylcysteine suppresses the migration and invasion of nasopharyngeal cancer cells induced by hepatocyte growth factor . Radiat Oncol J . 2015 ; 33 : 328 - 36 .
31. Pénzes K , Baumann C , Szabadkai I , Orfi L , Kéri G , Ullrich A , et al. Combined inhibition of AXL, Lyn and p130Cas kinases block migration of triple negative breast cancer cells . Cancer Biol Ther . 2014 ; 15 : 1571 - 82 .
32. Ammoun S , Provenzano L , Zhou L , Barczyk M , Evans K , Hilton DA , et al. Axl/ Gas6/NFκB signalling in schwannoma pathological proliferation, adhesion and survival . Oncogene . 2014 ; 33 : 336 - 46 .
33. Graham DK , DeRyckere D , Davies KD , Earp HS . The TAM family: phosphatidylserine sensing receptor tyrosine kinases gone awry in cancer . Nat Rev Cancer . 2014 ; 14 : 769 - 85 .
34. Puri N , Khramtsov A , Ahmed S , Nallasura V , Hetzel JT , Jagadeeswaran R , et al. A selective small molecule inhibitor of c-Met, PHA665752, inhibits tumorigenicity and angiogenesis in mouse lung cancer xenografts . Cancer Res . 2007 ; 67 : 3529 - 34 .
35. Li Y , Ye X , Tan C , Hongo JA , Zha J , Liu J , et al. Axl as a potential therapeutic target in cancer: role of Axl in tumor growth, metastasis and angiogenesis . Oncogene . 2009 ; 28 : 3442 - 55 .
36. Stepanova V , Jayaraman PS , Zaitsev SV , Lebedeva T , Bdeir K , Kershaw R , et al. Urokinase-type plasminogen activator (uPA) promotes angiogenesis by attenuating Proline-rich homeodomain protein (PRH) transcription factor activity and de-repressing vascular endothelial growth factor (VEGF) receptor expression . J Biol Chem . 2016 ; 291 : 15029 - 45 .
37. Qin L , Zhang M. Maspin regulates endothelial cell adhesion and migration through an integrin signaling pathway . J Biol Chem . 2010 ; 285 : 32360 - 9 .
38. Zhang M , Volpert O , Shi YH , Bouck N. Maspin is an angiogenesis inhibitor . Nat Med . 2000 ; 6 : 196 - 9 .
39. Saito Y , Nakaoka T , Saito H. microRNA-34a as a therapeutic agent against human cancer . J Clin Med . 2015 ; 4 : 1951 - 9 .
40. Lodygin D , Tarasov V , Epanchintsev A , Berking C , Knyazeva T , Korner H , Knyazev P , Diebold J , Hermeking H. Inactivation of miR-34a by aberrant CpG methylation in multiple types of cancer . Cell Cycle . 2008 ; 7 : 2591 - 600 .
41. Li Y , Guessous F , Zhang Y , Dipierro C , Kefas B , Johnson E , et al. MicroRNA-34a inhibits glioblastoma growth by targeting multiple oncogenes . Cancer Res . 2009 ; 69 : 7569 - 76 .
42. Yan TD , Deraco M , Baratti D , Kusamura S , Elias D , Glehen O , et al. Cytoreductive surgery and hyperthermic intraperitoneal chemotherapy for malignant peritoneal mesothelioma: multi-institutional experience . J Clin Oncol . 2009 ; 36 : 6237 - 42 .
43. Wang X , Wang C , Zhang X , Hua R , Gan L , Huang M , et al. Bmi-1 regulates stem cell-like properties of gastric cancer cells via modulating miRNAs . J Hematol Oncol . 2016 ; 9 : 90 .
44. Li N , Fu H , Tie Y , Hu Z , Kong W , Wu Y , et al. miR-34a inhibits migration and invasion by down-regulation of c-Met expression in human hepatocellular carcinoma cells . Cancer Lett . 2009 ; 275 : 44 - 53 .
45. Li H , Yu G , Shi R , Lang B , Chen X , Xia D , et al. Cisplatin-induced epigenetic activation of miR-34a sensitizes bladder cancer cells to chemotherapy . Mol Cancer . 2014 ; 13 :8.
46. Zhao Y , Tu MJ , Yu YF , Wang WP , Chen QX , Qiu JX , et al. Combination therapy with bioengineered miR-34a prodrug and doxorubicin synergistically suppresses osteosarcoma growth . Biochem Pharmacol . 2015 ; 98 : 602 - 13 .
47. Fujita Y , Kojima K , Hamada N , Ohhashi R , Akao Y , Nozawa Y , et al. Effects of miR-34a on cell growth and chemoresistance in prostate cancer PC3 cells . Biochem Biophys Res Commun . 2008 ; 377 : 114 - 9 .