Phospho-Akt overexpression is prognostic and can be used to tailor the synergistic interaction of Akt inhibitors with gemcitabine in pancreatic cancer
Massihnia et al. Journal of Hematology & Oncology
Phospho-Akt overexpression is prognostic and can be used to tailor the synergistic interaction of Akt inhibitors with gemcitabine in pancreatic cancer
Daniela Massihnia 0 3
Amir Avan 2
Niccola Funel 1
Mina Maftouh 0
Anne van Krieken 0
Carlotta Granchi 6
Rajiv Raktoe 0
Ugo Boggi 5
Babette Aicher 4
Filippo Minutolo 6
Antonio Russo 3
Leticia G. Leon 1
Godefridus J. Peters 0
Elisa Giovannetti 0 1
0 Department of Medical Oncology VU University Medical Center, Cancer Center Amsterdam , CCA room 1.52, De Boelelaan 1117, 1081 HV Amsterdam , The Netherlands
1 Cancer Pharmacology Lab, AIRC Start Up Unit, University of Pisa , Pisa , Italy
2 Metabolic syndrome Research center, School of Medicine, Mashhad University of Medical Sciences , Mashhad , Iran
3 Department of Surgical, Oncological and Oral Sciences, Section of Medical Oncology, University of Palermo , Palermo , Italy
4 terna Zentaris GmbH , Frankfurt am Main, Frankfurt , Germany
5 Department of Surgery, University of Pisa , Pisa , Italy
6 Department of Pharmacy, University of Pisa , Pisa , Italy
Background: There is increasing evidence of a constitutive activation of Akt in pancreatic ductal adenocarcinoma (PDAC), associated with poor prognosis and chemoresistance. Therefore, we evaluated the expression of phospho-Akt in PDAC tissues and cells, and investigated molecular mechanisms influencing the therapeutic potential of Akt inhibition in combination with gemcitabine. Methods: Phospho-Akt expression was evaluated by immunohistochemistry in tissue microarrays (TMAs) with specimens tissue from radically-resected patients (n = 100). Data were analyzed by Fisher and log-rank test. In vitro studies were performed in 14 PDAC cells, including seven primary cultures, characterized for their Akt1 mRNA and phospho-Akt/Akt levels by quantitative-RT-PCR and immunocytochemistry. Growth inhibitory effects of Akt inhibitors and gemcitabine were evaluated by SRB assay, whereas modulation of Akt and phospho-Akt was investigated by Western blotting and ELISA. Cell cycle perturbation, apoptosis-induction, and anti-migratory behaviors were studied by flow cytometry, AnnexinV, membrane potential, and migration assay, while pharmacological interaction with gemcitabine was determined with combination index (CI) method. Results: Immunohistochemistry of TMAs revealed a correlation between phospho-Akt expression and worse outcome, particularly in patients with the highest phospho-Akt levels, who had significantly shorter overall and progression-freesurvival. Similar expression levels were detected in LPC028 primary cells, while LPC006 were characterized by low phospho-Akt. Remarkably, Akt inhibitors reduced cancer cell growth in monolayers and spheroids and synergistically enhanced the antiproliferative activity of gemcitabine in LPC028, while this combination was antagonistic in LPC006 cells. The synergistic effect was paralleled by a reduced expression of ribonucleotide reductase, potentially facilitating gemcitabine cytotoxicity. Inhibition of Akt decreased cell migration and invasion, which was additionally reduced by the combination with gemcitabine. This combination significantly increased apoptosis, associated with induction of caspase-3/6/8/9, PARP and BAD, and inhibition of Bcl-2 and NF-kB in LPC028, but not in LPC006 cells. However, targeting the key glucose transporter Glut1 resulted in similar apoptosis induction in LPC006 cells. (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.
(Continued from previous page)
Conclusions: These data support the analysis of phospho-Akt expression as both a prognostic and a predictive
biomarker, for the rational development of new combination therapies targeting the Akt pathway in PDAC. Finally,
inhibition of Glut1 might overcome resistance to these therapies and warrants further studies.
Pancreatic ductal adenocarcinoma (PDAC) is among the
most lethal solid tumors. Despite extensive preclinical
and clinical research, the prognosis of this disease has
not significantly improved, with a 5-year survival rate
around 7% . This dismal outcome can partially be
explained by the lack of biomarkers for screening and
diagnosis at earlier stages, and by the resistance to most
currently available chemotherapy regimens. This
resistance has been attributed to both the desmoplastic tumor
microenvironment and to the strong inter- and
intratumor heterogeneity in terms of complexity of genetic
aberrations and the resulting signaling pathway
activities, as well as to resistance mechanisms that quickly
adapt the tumor to drugs .
Oncogenic KRAS signaling is the main driving force
behind PDAC. Activating KRAS mutations occur early,
followed by loss of p16, and then later, inactivation of
TP53 and SMAD4 [3, 4]; however, targeting these events
has proven to be very difficult. Conversely, the
phosphatidylinositol-3 kinase (PI3K)/Akt downstream
pathway represents an exciting new target for
therapeutic intervention, especially because it emerged among
the core signaling pathways in PDAC [5, 6], and several
known inhibitors are currently in clinical trials
In particular, the serine/threonine kinase Akt, which is
coded in three highly homologous isoforms (Akt1, Akt2,
and Akt3), is overexpressed in more than 40% of PDAC
patients . Mechanisms underlying aberrant Akt
activation in cancer include direct alterations such as mutations,
amplification, or overexpression, but also activation of
upstream signaling events, such as activation of HER-2/
neu signaling or PTEN mutation/loss [8–11].
The PI3K/Akt pathway plays a key role in cell
proliferation, survival, and motility . Deregulation of
components involved in this pathway could confer resistance
to chemotherapy [13, 14], while blockage of Akt
signaling results in programmed cell death and inhibition of
tumor growth [15, 16]. Activation of Akt is a frequent
event in PDAC and has been correlated to its poor
prognosis [17, 18].
Several inhibitors of Akt are under investigation, but
three are the farthest along and showed the most
promise in early clinical research: the pan-Akt and PI3K
inhibitor perifosine (KRX-0401, Aeterna Zentaris/Keryx),
the allosteric pan-Akt inhibitor MK-2206 (Merck), and
the dual PI3K/mTOR inhibitor dactolisib (NVP-BEZ235,
In particular, the synthetic oral alkylphospholipid
perifosine [19, 20] has been evaluated in clinical trials for
several tumors, including colon , breast , head
and neck, and prostate cancer [23, 24]. Unfortunately, it
failed the phase III clinical trials for treatment of colon
cancer and relapsed refractory multiple myeloma
(www.clinicaltrials.gov). These failures, together with the
disappointing response rates to perifosine as a single
agent in most solid tumors, including PDAC, prompt
further studies into its mechanism of action  as well
as on synergistic combinations.
Perifosine prevents translocation of Akt to the cell
membrane by blocking the pleckstrin homology (PH)
domain of Akt  leading to inactivation of downstream
pathway and inhibition of cell proliferation. Previous
studies demonstrated perifosine activity against different
cancer types, in vitro and in vivo . Recently, Pinton
and collaborators showed that perifosine inhibited cell
growth of malignant pleural mesothelioma cells by
affecting EGFR and c-Met phosphorylation . Another study
showed that perifosine decreased the AEG-1 gene
expression along with inhibition of Akt/GSK3/c-Myc signaling
pathway in gastric cancer . Perifosine and curcumin
synergistically increased the intracellular level of reactive
oxygen species and ceramide, and downregulated the
expression of cyclin-D1 and Bcl-2 in colorectal cancer
cells . Finally, perifosine also inhibits the
antiapoptotic mitogen-activated protein kinase (MAPK)
pathway and modulates the balance between the MAPK and
pro-apoptotic stress-activated protein kinase (SAPK/JNK)
pathways, thereby inducing apoptosis .
The aims of current study were to investigate the
expression of phospho-Akt in PDAC tissues and cells,
and to evaluate the effects of growth inhibition by Akt
inhibitors, using PDAC cell lines and primary cultures
growing as monolayer or as spheroids. Moreover, we
characterized several key factors, affecting cell cycle
perturbation, apoptosis induction, as well as inhibition
of cell migration and invasion and modulation of key
factors in glucose metabolism in PDAC cells exposed to
perifosine and perifosine/gemcitabine combination.
Tissue microarrays (TMAs), immunohistochemistry (IHC),
and immunocytochemistry (ICC)
Phospho-Akt protein expression was evaluated in slides
from four formalin-fixed, paraffin-embedded
PDACspecific TMAs build with neoplastic cores from a cohort
of radically resected patients (n = 100), using the TMA
Grand Master (3DHistec, Budapest, Hungary)
instrument, and stained according to standard procedures with
the EP2109Y rabbit monoclonal antibody (1:50 dilution;
Abcam, Cambridge, UK). Visualization was obtained
with BenchMark Special Stain Automation system
(Ventana Medical Systems, Tucson, AZ). Two
pathologists reviewed all the slides, assessing the amount of
tumor and tissue loss, background staining, and overall
interpretability before the phospho-Akt reactivity
evaluation. Staining results were evaluated using a
computerized high-resolution acquisition system (D-Sight,
Menarini, Florence, Italy), including the analysis of
positive cells number and staining intensity which
resulted in values expressed as arbitrary units (a.u.). All
patients have provided a written informed consent. This
study was approved by the Local Ethics Committee of
the University of Pisa. Date of approval: July 3, 2013 (file
For ICC, the cells were grown in a Chamber Slides
System (Lab-Tek, Collinsville, IL). After 24 h, the cells
were fixed with 70% ethanol for 10 min, followed by
incubation with the antibody described above (4 °C
overnight, 1:30 dilution in PBS). Cells were stained with the
avidin-biotin-peroxidase complex (UltraMarque HRP
Detection, Greenwood, AR). Negative controls were
obtained by replacing the primary antibody with PBS.
The sections were reviewed and scored using a digital
system based on staining intensity and on the number of
positively stained cells, as described above.
Drugs and chemicals
Perifosine was provided by Æterna Zentaris Inc.
(Frankfurt am Main, Germany), NVP-BEZ235 was purchased
from Selleck Chemicals (Houston, TX), while
gemcitabine and MK-2206 were generous gifts from Eli-Lilly
(Indianapolis, IN) and Merck (Whitehouse Station, NJ),
respectively. The drugs were dissolved in Dimethyl
sulfoxide (DMSO) or sterile water and diluted in culture
medium before use. RPMI-1640 medium, foetal bovine
serum (FBS), penicillin (50 IU/ml), and streptomycin
(50 μg/ml) were from Gibco (Gaithersburg, MD). All
other chemicals were purchased from Sigma-Aldrich
(Zwijndrecht, The Netherlands).
Eight PDAC cell lines (PL45, MIA-PaCa2, HPAF-II,
CFPAC-1, Bxpc3, HPAC, and PANC-1) and the human
immortalized pancreatic duct epithelial-like cell line
hTERT-HPNE were obtained from the American Type
Culture Collection, whereas seven primary PDAC
cultures (LPC006, LPC028, LPC033, LPC067, LPC111,
LPC167, and PP437) were isolated from patients at the
University Hospital of Pisa (Pisa, Italy), as described
previously . The cell lines were tested for their
authenticity by PCR profiling using short tandem repeats
by BaseClear (Leiden, The Netherlands). The cells were
cultured in RPMI-1640, supplemented with 10%
heatinactivated FBS and 1% streptomycin/penicillin at 37 °C,
and harvested with trypsin- EDTA in their exponentially
Total RNAs were extracted from cells using the TRI
REAGENT-LS (Invitrogen, Carlsbad, CA), according to
the manufacturer’s protocol. RNA was also extracted
from seven primary tumors, after laser micro-dissection
with a Leica-LMD7000 instrument (Leica, Wetzlar,
Germany), using the QIAamp RNA Micro Kit (Qiagen,
Hilden, Germany), as described .
RNA yield and purity were checked at 260 to 280 nm
with NanoDrop-1000 Detector (NanoDrop
Technologies, Wilmington, DE). One microgram of RNA was
reverse-transcribed using the DyNAmo Synthesis Kit
(Thermo Scientific, Vantaa, Finland). qRT-PCR was
performed with specific TaqMan® primers and probes for
Akt1, human equilibrative nucleoside transporter-1
(hENT1), deoxycytidine kinase (dCK), cytidine
deaminase (CDA), ribonucleotide reductase subunit-M1
(RRM1), and subunit-M2 (RRM2), E-cadherin, and the
glucose transporter 1 (SLC2A1/Glut1) which were
obtained from Applied Biosystems TaqMan Gene
expression products (Hs00920503_m1, Hs01085706_m1,
Hs00984403_m1, Hs01040726_m1, Hs00156401_m1,
Hs00168784_m1, Hs01072069_g1, Hs01023894_m1, and
Hs00892681_m1). The cDNA was amplified using the
ABI-PRISM 7500 instrument (Applied Biosystems,
Foster City, CA). Gene expression values were
normalized to β-actin, using a standard curve of cDNAs
obtained from Quantitative PCR Human Reference RNA
(Stratagene, La Jolla, CA), as described earlier .
Growth inhibition studies
The cell growth inhibitory effects of perifosine,
MK2206 and NVP-BEZ235 were evaluated in the PANC-1,
LPC028, and LPC006 cells. Further studies evaluated
perifosine and gemcitabine combination in CFPAC-1,
PANC-1, LPC028, and LPC006 cells. These cells were
treated for 72 h with perifosine (1–500 μM), gemcitabine
(1–500 nM), and simultaneous combination at a fixed
ratio based on IC50 (i.e., concentration of a drug
required for 50% inhibition of cell growth) of each drug.
The plates were then processed for the
sulforhodamineB assay, as described .
Evaluation of synergistic/antagonistic interaction with
The pharmacological interaction between perifosine and
gemcitabine was evaluated by the median drug effect
analysis method as described previously . In this
regard, the combination index (CI) was calculated to
compare cell growth inhibition of the combination and
each drug alone. Data analysis was carried out using
CalcuSyn software (Biosoft, Oxford, UK).
Effects on multicellular spheroids
LPC006 and LPC028 spheroids were established by
seeding 104 cells per ml in DMEM/F12 + GlutaMAX-I
(1:1) with insulin-transferrin-selenium (1:1000,
Invitrogen), in 24-well ultra-low attachment plates (Corning
Incorporated, NY). The cytotoxic effects were evaluated by
measuring the size and number of spheroids with the
inverted phase contrast microscope Leica-DMI300B
(Leica, Wetzlar, Germany), taking 9 pictures for each
well. Spheroid volume (V) was calculated from the
geometric mean of the perpendicular diameters D = (Dmax
+ Dmin)/2, as follows: V = (4/3) × π (D/2)3.
In order to evaluate the modulation of Akt1,
phosphoAkt1, PARP, BAD, Bcl-2, NF-kB, and Glut1 protein
expression in PDAC cells treated for 24 h with
perifosine, gemcitabine, and their combination, Western blot
analyses were executed as described previously using the
Akt1 sc-5298 mouse monoclonal (Santa Cruz,
Biotechnology, Santa Cruz, CA) and the EP2109Y rabbit
monoclonal antibody (1:500 dilution; Abcam), PARP
sc-8007 mouse monoclonal (1:500 dilution; Santa Cruz),
BAD sc-8044 mouse monoclonal (1:500 dilution;Santa
Cruz), Bcl-2 sc-7382 mouse monoclonal (1:500 dilution;
Santa Cruz), NF-kB sc-114 rabbit polyclonal (1:500
dilution; Santa Cruz), and Glut1 sc-1605 goat polyclonal
(1:500 dilution; Santa Cruz) . Briefly, 40 μg of
proteins was separated on a 10% SDS-polyacrylamide gel
and transferred onto polyvinylidene difluoride (PVDF)
membrane (Immobilion®-FL, Millipore, Billerica, MA).
The membrane was incubated overnight with mouse
and rabbit anti-Akt1, anti-phospho-Akt1, as described
above, as well as with mouse anti-BAD, anti-Bcl-2,
antiPARP, with rabbit anti-NF-kB (1:1000, diluted in the
blocking solution; all from Santa Cruz Biotechnology,
Santa Cruz, CA), goat anti-Glut-1 (ab652, 1:500, diluted
in the blocking solution, from Abcam, Cambridge, UK),
and mouse anti-β-actin (1:10000; Sigma–Aldrich). The
secondary antibodies were goat anti-rabbit-InfraRedDye®
800 Green and goat anti-mouse InfraRedDye® 680 Red
(1:10000, Westburg, Leusden, The Netherlands).
Fluorescent proteins were monitored by an Odyssey Infrared
Imager (LI-COR Biosciences, Lincoln, NE), equipped
with Odyssey 2.1 software to perform a
semiquantitative analysis of the bands.
Akt and phospho-Akt analysis by enzyme linked
immunosorbent (ELISA) assay
To investigate the inhibitory effects of perifosine on Akt
[pS473] and [Thr308] phosphorylation, specific ELISA
assays were performed using the Pierce AKT
Colorimetric In-cell ELISA Kit (Thermo Scientific, Rockford, IL),
which has a sensitivity approximately twofolds greater
than Western blotting. The levels of Akt and
phosphoAkt were measured in cells seeded in a 96-well-plate at a
density of 105 cells per well, and treated for 4 or 24 h
with perifosine, gemcitabine, and their combination at
IC50 values. The absorbance was measured in a Synergy
HT Multi-Detection Microplate Reader (BioTek, Bad
Friedrichshall, Germany) at a wavelength of 450 nm.
In vitro migration and invasion assays
The ability of perifosine and its combination with
gemcitabine and MK-2206 and its combination with
gemcitabine to inhibit the migratory behaviour of PDAC cells
was investigated by in vitro migration assay, as described
. The cells were exposed to the drugs at their IC50s.
Images were taken at the beginning of the exposure
(time 0), with those taken after 4, 6, 8, 20, and 24 h.
Transwell chambers with polycarbonate membranes,
and 8 μm pores were used for invasion assays. These
assays were carried out through coated transwell filters,
with 100 μl of 0.1 mg/mL collagen I solution. A total of
105 cells were plated on the upper side of the filter and
incubated with the drugs at IC50 concentrations in
RPMI-1640 medium. After 24 h, cells migrated into the
lower side were fixed with paraformaldehyde and stained
with Giemsa in 20% methanol. The filters were
photographed and cells were counted.
Analysis of cell-cycle and cell death
To investigate the effect of drugs on modulation of cell
cycle, LPC028, LPC006, CFPAC-1, and PANC-1 cells
were treated for 24 h with gemcitabine, perifosine, and
their combination at IC50 concentrations. Cells were
stained by propidium iodide (PI) and cell cycle
modulation was evaluated using a FACSCalibur flow cytometer
(Becton Dickinson, San José, CA), equipped with the
CELLQuest software for data analysis.
The ability of gemcitabine, perifosine, and its
combination with gemcitabine to induce cell death was
evaluated by measuring sub-G1 regions during cell cycle
analysis, as described above. Apoptosis induction was
also assessed by 3,3′-dihexyloxacarbocyanine iodide
(DiOC) labelling. DiOC is a lipophilic and green
fluorescent dye, which can pass the plasma membrane, without
being metabolized by the cell, and accumulate at the
membrane of mitochondria of living cells. Shortly, the
cells were stained with DiOC for 30 min, and analysed
by FACSCalibur, as described . Additional studies
were performed with the Annexin-V/PI assay, plating
the cells in 6-well-plates at a density of 1.5 × 105. After
24 h, the cells were treated with the drugs at their IC50,
followed by 24-h incubation. Then, the cell pellets were
re-suspended in 100 mL of ice-cold binding buffer
(0.1 M Hepes/NaOH (pH = 7.4), 1.4 M NaCl, 25 mm
CaCl2). The staining was performed according to the
manufacturer’s instructions (Annexin-V/PI detection
Kit-I, Becton Dickinson). Cells were stained by 5 μL
Annexin V-FITC and 5 μL PI. Samples were gently
vortexed and incubated for 15 min at room temperature.
Then, 400 μL of binding buffer was added to the cells.
The samples were analyzed by FACSCalibur using
excitation/emission wavelengths of 488/525 and 488/675 nm
for Annexin-V and PI, respectively.
Caspase activity assay
The effects of perifosine, gemcitabine and their
combination on the activity of caspase-3, -6, -7, -8, -9 were
determined by specific fluorometric assay kits (Zebra
Bioscience, Enschede, The Netherlands), according to
the manufacturer’s instructions. Briefly, 106 LPC006,
LPC028, CFPAC-1, and PANC-1 cells were exposed to
the drugs for 24 h at their IC50s. Fluorescence was
measured at 350 nm excitation and 460 nm emission
(Spectrafluor Tecan, Salzburg, Austria). Relative caspase
activity was normalized with respect to the untreated
Analysis of modulation of Glut1 by flow cytometry
To quantitatively detect the expression of
membranebound Glut1, cells were fixed with 80% ethanol,
incubated with anti-Glut1 antibody (Abcam), and then
stained with the appropriate FITC-conjugated
antirabbit IgG antibody (BD Pharmingen™, BD Biosciences,
San Jose, CA). Quantification of FITC fluorescence
intensity was performed using a FACSCanto flow
cytometer (BD Biosciences).
Evaluation of the cytotoxic and pro-apoptotic effects
inhibition of Glut1 inhibition combined with Akt inhibitors
The Akt signaling is involved in the modulation of Glut1
expression/localization, and a recent study showed that
increased glucose metabolism was associated to resistance
to the tyrosine kinase inhibitor axitinib, and this resistance
was overcame by Glut1 silencing . Therefore, we
performed additional cytotoxicity studies using the novel
Glut1 inhibitor PGL13. This compound was tested in the
LPC006 cells, at a concentration of 30 μM, which
effectively reduced glucose influx in previous studies [36, 37].
The cells were exposed to PGL13 for 72 h, alone or in
combination with IC50 concentration values of perifosine,
gemcitabine, and their combination. Cell growth
inhibition was then assessed by counting the cells after staining
with trypan blue, in comparison to untreated cells. Parallel
evaluation of apoptosis induction was performed by
fluorescence microscopy with bisbenzimide staining, as
described previously .
All experiments were performed in triplicate and repeated
at least twice. Data were expressed as mean values ± SEM
and analyzed by Student’s t test or ANOVA followed by
Tukey’s multiple comparison test. For the analysis of the
correlation of phospho-Akt expression and clinical data,
the overall survival (OS), and progression-free-survival
(PFS) were calculated from the date of pathological
diagnosis (i.e., the date of surgery) to the date of death and
tumor progression, respectively. OS and PFS curves were
constructed using Kaplan-Meier method, and differences
were analyzed using log-rank test. Data were analyzed
using SPSS v.20 statistical software (IBM, Chicago).
Statistical significance was set at P < 0.05.
Correlation with outcome and phospho-Akt and Akt1
mRNA expression in PDAC tissues and cells
The protein expression of phospho-Akt was successfully
evaluated by IHC in 100 human PDACs collected in two
TMAs. The main clinical characteristics of these patients
are reported in the Table 1. IHC showed a variable
protein expression with some specimens characterized by a
strong and diffuse staining, while other tissues had only
a few scattered positive cells with a weak staining (as
exemplified by the middle and lower panels in the Fig. 1a,
respectively). Patients were categorized according to
their high versus low phospho-Akt expression compared
to the median value (30 a.u.) calculated by digital scoring
(Fig. 1b, black line). No association was observed
between phospho-Akt and age, sex, grading, resection,
and lymph node infiltration (data not shown). Patients
with low phospho-Akt expression had a median OS of
16.2 months (95% CI, 14.8–20.1), while patients with a
high expression had a median OS of 12.0 months (95%
CI, 9.0–14.9, P = 0.03, Fig. 1c, upper panel). However,
only a trend toward a significant association was found
between phospho-Akt expression and PFS (P = 0.08,
Additional file 1: Figure S1a).
An additional analysis was performed categorizing the
patients with respect to a threshold expression of
57 a.u., which identified 14 cases with higher expression
Table 1 Outcome according to clinical characteristics in the 100
PDAC patients enrolled in the present study
Characteristics N (=%) OS months P
No. patients All 100 14.0 (12.1–15.8)
Fig. 1 Akt/phospho-Akt expression in PDAC tissues and cells. a Representative examples (original magnification, ×40) showing the variable
expression of phospho-Akt in paraffin-embedded PDAC samples collected in four TMAs (with 4 cores for each of the 100 patients). N.C., negative
control. b Expression values of phospho-Akt observed across the cohort of PDAC patients, obtained by digital quantification. Phospho-Akt showed
positive cytoplasmic and nuclear staining in most tissue sections, with intense staining in 14 out of 100 samples. The staining intensities of the
LPC028 and LPC006 cells were included in the very high and low Akt expression groups, respectively. c Kaplan–Meier survival curves according to
the expression of phospho-Akt in 100 radically resected PDACs, showing that patients with high expression (upper panel) and very high expression
(lower panel) of phospho-Akt had a significantly shorter survival compared to patients with low phospho-Akt expression. d Akt1 mRNA expression in
ATCC cell lines (black bars), primary tumor cultures (white bars), and their originator tissues (gray bars). Dashed bars identify the cells that were selected
for further in vitro studies; e Representative Western blot pictures of phospho-Akt1 and Akt1 expression in LPC006, CFPAC-1, PANC-1, and LPC028 cells.
Columns, mean values obtained from three independent experiments, bars, SEM
compared to all the others (defined as very high
phospho-Akt expression, Fig. 1b, blue square). Using
these categories, we observed a significant correlation
between high phospho-Akt protein expression and both
significantly shorter OS (P < 0.01, Fig. 1c, lower panel),
and PFS (Additional file 1: Figure S1b).
Parallel ICC studies revealed that the LPC006 cells
had a significantly lower phospho-Akt expression
compared to LPC028 cells, which were indeed included in
the category of low and very high phospho-Akt
expression, respectively (Fig. 1b, blue and red circles). The
mRNA expression of Akt1 was detectable in all PDAC
cells by qRT-PCR, as well as in the originator tissues of
the primary tumor cell cultures. This expression value
differed among the cells, ranging from 0.9 arbitrary unit
(a.u.) in LPC006 cells to 24.0 a.u. in LPC028 and
PANC1 cells (Fig. 1d). The mean and median expression in the
tumor cells (8.7 ± 0.2 and 8.4 a.u., respectively) were
significantly higher (P < 0.01) than the expression detected
in hTERT-HPNE cells (0.3 a.u.). Notably, Akt1 gene
expression in the seven primary tumor cells and their
laser-microdissected originator tumors showed a similar
pattern and were highly correlated with Spearman analysis
(R2 > 0.9, P < 0.05), suggesting that these cells represent
optimal preclinical models for our pharmacological
studies. Moreover, Western blot analysis revealed that the
LPC006 and CFPAC-1 cells had a lower phospho-Akt1/
Akt1 ratio (0.3 and 0.6 a.u., respectively) expression
compared to PANC-1 (0.8) and LPC028 (1.1) cells (Fig. 1e).
Therefore, we selected for further studies two primary
cell cultures (LPC006 and LPC028) which were
representative of low and very high expression values, as well as
two cell lines, PANC-1 and CFPAC-1, with high and
intermediate expression values of Akt1 mRNA, respectively.
Perifosine inhibits cell growth and interacts synergistically
with gemcitabine in PDAC cells with high expression of
The cytotoxic activity of three different Akt inhibitors
(perifosine, MK-2206, and NVP-BEZ235) was evaluated
in the PANC-1 cell line (Fig. 2a). All these compounds
caused a concentration-dependent inhibition of
proliferation, with IC50 values ranging from 5.1 (perifosine) to
15.8 μM (NVP-BEZ235). Higher IC50 values were
obtained in the LPC006 cells, i.e., 22.5, 31.7 and 45.5 μM
for perifosine, NVP-BEZ235, and MK-2206 (Additional
file 1: Figure S2), respectively. According to the lowest
IC50 values detected in these assays, we selected
perifosine for the following studies on the pharmacological
interaction of Akt inhibitors with gemcitabine.
The cell growth inhibitory effects of perifosine,
gemcitabine, and their combination in LPC028 and
LPC006 cells are shown in Fig. 2b, while the data for
CFPAC-1 and PANC-1 are reported in the Additional
file 1: Figure S3. Since the CI method recommends a
ratio of concentrations at which drugs are equipotent,
combination studies were performed using fixed ratios
with IC values at IC50s. Perifosine enhanced the
antiproliferative activity of gemcitabine, especially in the
LPC028 and PANC-1 cells, by decreasing the IC50s of
gemcitabine from 4.3 ± 1.1 and 17.2 ± 2.1 nM to 1.4 ± 0.5
and 4.0 ± 1.1 nM, respectively. The median drug-effect
analysis revealed a slight-to-moderate synergism in
CFPAC-1, and a strong synergism in the PANC-1 and
LPC028 cells, with CI values of 0.8, 0.5, and 0.2,
respectively (Fig. 2c). Conversely, the combination of perifosine
and gemcitabine was antagonistic in the LPC006 cells
(CI > 1.2). To evaluate whether these effects were
observed also in three-dimensional (3D) models and
Volume Day3-Day0 (mm3)
Fig. 2 Inhibition of cell proliferation in PDAC cells. a Growth inhibitory effects in PANC-1 cells after 72 h exposure to perifosine, MK-2206 and
NVP-BEZ235. b Growth inhibitory effects after 72 h exposure to perifosine, gemcitabine, or their combination at a fixed ratio based on IC50 values in
LPC028 and LPC006 cells. On the X axis, the drug concentrations for the combination are referred to gemcitabine. c Mean CI of the perifosine/gemcitabine
combination. CI values at FA of 0.5, 0.75. and 0.9 were averaged for each experiment, and this value was used to calculate the mean between experiments,
as explained in the Methods section. d Effect of perifosine and gemcitabine. and their combination, at IC50 values, on the volumes of PDAC spheroids after
72 h exposure. e Representative images of untreated spheroid versus spheroid treated with perifosine and gemcitabine (original magnification, ×40).
Columns and points mean values obtained from three independent experiments, bars, SEM; *Significantly different from controls
investigate the mechanisms underlying these different
interactions, several biochemical analyses were performed,
as detailed below.
Perifosine and its combination with gemcitabine reduce
the size of PDAC spheroids
Previous studies illustrated that 3D culture models are
generally more chemo-/radio-resistant than two-dimensional
monolayer cell cultures, supporting their use for drug
testing . In order to explore whether perifosine would be
active in 3D PDAC models, we evaluated this drug in
spheroids of LPC006, LPC028, and PANC-1 cells.
Perifosine remarkably increased the disintegration of
LPC028 and PANC-1 spheroids, which were significantly
(P < 0.05) reduced in size compared to the untreated
spheroids (Fig. 2d–e). The combination of perifosine
with gemcitabine additionally reduced the size of the
LPC028 and PANC-1 spheroids with respect to the
spheroids treated with the single drugs. In contrast, no
changes were observed in the LPC006 spheroids, further
supporting the antagonistic interaction of perifosine with
gemcitabine in this PDAC model.
Modulation of phospho-Akt and gemcitabine determinants
in PDAC cells
Perifosine inhibits the phosphorylation of Akt by blocking
the PH-domain in different cancer cell lines , but no
data have been reported yet on PDAC cells. Therefore, we
evaluated the expression of phospho-Akt (at serine residue
473 (Ser473) and at threonine residues 308 (Thr308)),
normalized to the total Akt levels, both in untreated cells
and in cells treated with Akt inhibitors (perifosine and
MK-2206), gemcitabine, and their combination. We
observed a similar inhibition of the phosphorylation status
after 4 or 24 h (Fig. 3a and Additional file 1: Figure S4) as
well as in both residues (Additional file 1: Figure S5a, b).
Perifosine significantly reduced the expression of p-Akt in
LPC028, CFPAC-1, and PANC-1 cells (e.g., 40, 25, and
30% reduction, respectively). Regarding Ser473
phosphorylation, the combination of perifosine and gemcitabine
was also able to significantly suppress Akt
phosphorylation, with a degree of inhibition ranging from −35
(CFPAC-1 cells) to −45% (LPC028 cells). Conversely, both
Ser473 and Thr308 phospho-Akt levels were not affected
by perifosine, MK-2206, and their combination with
gemcitabine in the LPC006 cells.
RRM1 and RRM2 encode for the catalytic and the
regulatory subunits of ribonucleotide reductase and is a
key molecular target of gemcitabine . Previous
studies demonstrated that the expression of RRM2 is
modulated by the Akt/c- MYC pathway . However, the
alterations in the expression or function of other
enzymes, involved in the transport, metabolism, and
catabolism of gemcitabine can also lead to resistance
(e.g., decreased dCK or increased CDA expression
). Therefore, we evaluated the mRNA expression of
several gemcitabine determinants in the LPC006,
LPC028 and PANC-1 cells. As shown in Fig. 3b, the
expression of RRM1 and RRM2 was significantly reduced
(approximately 2-fold) in LPC028 and also in PANC-1
cells (Additional file 1: Figure S6) treated with
perifosine versus untreated cells, while only minimal
variations were observed for hCNT1, hENT1, dCK, and
CDA expression. No significant changes were observed
in the LPC006 cells (Fig. 3b). These results can at least
in part explain the synergistic interaction of perifosine
with gemcitabine in PDAC cells with high phospho-Akt
Perifosine and its combination with gemcitabine inhibit
cell migration/invasion and upregulate the expression of
To determine the effects of perifosine, gemcitabine, and
their combination on migratory behavior, a scratch
mobility assay was performed in LPC028, LPC006 (Fig. 4a),
CFPAC-1, and PANC-1 (Additional file 1: Figure S7).
LPC028 showed a significant reduction of migration
starting after 8 h exposure to perifosine with a reduction
of the scratch-area of about 50%, and the perifosine/
gemcitabine combination additionally reduced cell
migration (P < 0.05; Fig. 4a left panel), while gemcitabine
alone did not affect cell migration. No modulation of cell
migration was observed in the LPC006 cells (Fig. 4a
right panel). Similarly, the migration of these cells was
not affected by MK-2206 alone and in combination with
gemcitabine (Additional file 1: Figure S8).
LPC028, CFPAC-1, and PANC-1 cells treated with
perifosine showed also a significantly reduced invasive
potential, compared to untreated cells (Fig. 4b). In
particular, the perifosine/gemcitabine combination was
more effective in inhibiting invasion than
perifosinealone in LPC028 and PANC-1 cells, as shown by the
significantly lower number of invading cells with
Giemsa’s stain. However, no modulation of cell invasion
was observed in the LPC006 cells.
Since previous studies suggested that the Akt signaling
pathway suppressed E-cadherin expression , we
investigated whether perifosine could affect the level of
this target at both mRNA and protein level. Perifosine
and its combination with gemcitabine significantly
enhanced E-cadherin mRNA expression in LPC028,
CFPAC-1, and PANC-1 (P < 0.05; Fig. 4c), while no
changes were detected in LPC006 cells. Similarly,
immunocytochemistry analysis in LPC028 cells
illustrated a significant increase of E-cadherin protein
staining after exposure to both perifosine and perifosine/
gemcitabine combination (data not shown).
RRM1 RRM2 dCK CDA hENT1 hCNT1
Fig. 3 Modulation of phospho-Akt and gemcitabine determinants. a Effect of 24-h exposure to gemcitabine, perifosine or their combination, at
IC50 values, on the expression of phospho-Akt, normalized to the expression of total Akt, as determined by ELISA. b Expression of gemcitabine
key determinants in LPC028 (left panel) and LPC006 (right panel) cells treated with perifosine at IC50 versus untreated cells, as determined by
qRT-PCR. Columns mean values obtained from three independent experiments, bars, SEM. Dashed line, values in untreated samples (Control).
*Significantly different from controls
Perifosine and its combination with gemcitabine affect
Perifosine, gemcitabine and their combination affected
cycle distribution of PDAC cells, as summarized in
Additional file 2: Table S1. Perifosine significantly (P <
0.05) increased the percentages of LPC028 cells in S and
G2/M phases (e.g., from 18.7 in the control to 26.1% in
the S phase) after 72 h, while reducing the percentage of
the cells in G0/G1. Similarly, the perifosine/gemcitabine
combination significantly decreased the cells in G1
phase, while increasing the cells in S phase, up to 48.9%.
Comparable perturbations of cell cycle were observed in
the CFPAC-1 and PANC-1 cells, suggesting that
perifosine might favor gemcitabine activity through a
significant increase of cells in the S phase. Opposite
modulation of cell cycle was observed in LPC006 cells,
with only a slight increase of the cells in the G0/G1
phase and minimal modulations of the S and G2/M phase
in cells exposed to perifosine/gemcitabine combination.
Perifosine and its combination with gemcitabine enhance
cell death and apoptosis
Analysis of the sub-G1 region of cell cycle perturbation
demonstrated that the treatment with perifosine
enhanced cell death (Additional file 2: Table S1). In
particular, the LPC028 cells treated with the
combination exhibited the largest sub-G1 signal (e.g., ≈20% in
cells treated with perifosine/gemcitabine combination
versus untreated cells).
Moreover, we evaluated the variation of mitochondrial
membrane potential in LPC028, LPC006, PANC-1, and
Fig. 4 Effects of perifosine, gemcitabine and their combination on PDAC cells migration and invasion. a Results of wound-healing assay in
LPC028 and LPC006 cells exposed to perifosine, gemcitabine or to their combination, at IC50 values for 24 h. b Results of invasion studies in the
PDAC cells exposed for 24 h to perifosine, gemcitabine, or to their combination, at IC50 values (insert: representative pictures of LPC028 cells at
24 h, original magnification ×40). c Modulation of E-cadherin mRNA levels in LPC028, LPC006, PANC-1, and CFPAC-1 cells after 24-h exposure to
perifosine, gemcitabine, or to their combination, at IC50 values, as determined by qRT-PCR. Columns or points mean values obtained from three
independent experiments; bars, SEM. *Significantly different from controls
CFPAC-1. As shown in Fig. 5a, the combination
perifosine gemcitabine causes an increase of
mitochondrial membrane potential in LPC028, PANC-1, and
Further analysis of cell death by the Annexin-V/PI
assay confirmed the induction of apoptosis by perifosine.
Perifosine increased both early and late apoptosis, as
shown in Fig. 5b (left panel) for the LPC028 cells.
Moreover, the combination of perifosine and gemcitabine
significantly increased the percentage of late apoptotic
cells up to 26%. Similar results were observed in
CFPAC-1 and PANC-1 cells (Additional file 1: Figure S9),
whereas no apoptosis induction was detected in LPC006
cells (Fig. 5b right panel).
Perifosine and its combination with gemcitabine activate
caspases and pro-apoptotic factors, and downregulate
Bcl-2 and NF-kB
In order to investigate the molecular mechanisms
underlying apoptosis induction, we explored several potential
cellular targets of perifosine, focusing on activation of
the initiator caspases, caspase-8 and -9, and the effector
caspases, caspase-3, and -6. Moreover, we studied the
expression of various pro-apoptotic and anti-apoptotic
proteins. As shown in Fig. 5c, perifosine and its
combination with gemcitabine were able to increase the activity
of caspase-3/-6/-8/-9 in LPC028 as well as CFPAC-1
and PANC-1 (Additional file 1: Figure S10) but not in
the LPC006 cells, as determined by specific fluorometric
Fig. 5 Apoptosis induction by perifosine, gemcitabine and their combination. a Mitochondrial membrane potential (as assessed by (DiOC) labelling) in
LPC028, LPC006, PANC-1, and CFPAC-1 cells. b Annexin-V assay in LPC028 and LPC006 cells. c Modulation of caspase-3, caspase-6/-8/ and caspase-9 in
LPC028 and LPC006 cells, as determined by a specific fluorometric assay described in the Methods section. d Representative Western blot pictures of
apoptosis determinants in LPC006 and LPC028 cells. All these results refer to cells exposed for 24 h to perifosine, gemcitabine. or their combination at
IC50s. Columns, mean values obtained from three independent experiments; bars, SEM. *Significantly different from controls
caspase activity assays. However, Western blot analyses
demonstrated the modulation of other important
apoptotic markers. In particular, perifosine and perifosine/
gemcitabine combination increased the expression of
PARP and BAD, while reducing Bcl-2 and NF-kB
expression in LPC028 cells. Conversely, none of these proteins
was affected by the exposure to perifosine and its
combination with gemcitabine in the LPC006 cells (Fig. 5d).
Glut1 is overexpressed in the cells resistant to Akt
inhibition, while its inhibition significantly reduces cell
growth and induces apoptosis after gemcitabine/
Since major oncogenic signaling pathways have been
linked to increased glucose metabolism, and previous
studies showed that stimulation of Akt1 induces Glut1
mRNA and protein accumulation,  we evaluated the
expression of this key glucose transporter in the LPC028
and LPC006 cells. As shown in the Fig. 6a, Glut1 mRNA
levels were significantly reduced after treatment with
perifosine alone and in combination with gemcitabine in
the LPC028 and PANC-1 cells, whereas no modulation
was detected in the LPC006 cells. However, since PI3K/
AKT/mTOR signaling seems to play an essential role in
trafficking of Glut1 from recycling endosomes and/or
retention of Glut1 at the plasma membrane , we
performed further studies to evaluate the amount of
membrane-bound Glut1 with FACS analysis (Fig. 6b).
In the LPC028 cells, we observed a significant
reduction (P < 0.05) of the membrane-bound expression of
Glut1 after treatment with perifosine (56% compared to
untreated cells). Further studies with Western blot
clearly demonstrated the overexpression of Glut1 in the
LPC006 compared to the LPC028 and PANC-1 cells. A
high expression of Glut-1 was also observed in PANC-1
cells (Fig. 6c). Moreover, Glut1 expression was not
reduced by Akt inhibition (Fig. 6c). We therefore
investigated whether inhibition of Glut1 by the novel specific
compound PGL13 (Fig. 6d) can at least in part
overcome the inherent resistance of the LPC006 cells
to perifosine and other Akt inhibitors. Remarkably, the
Glut1 inhibitor alone caused only a slight reduction of
cell growth (<10%), but its combination with perifosine
reduced significantly the percentage of surviving cells
compared to perifosine alone (Fig. 6e). Furthermore,
the combination of PGL13 with both perifosine and
gemcitabine led to a more dramatic drop in the number
of surviving cells, up to −81%, compared to control
which was associated with strong apoptosis induction, as
detected by characteristic apoptotic nuclear morphological
Fig. 6 Role of Glut1 expression and inhibition in cell growth and apoptosis induction by perifosine, gemcitabine, and their combination. a
Modulation of Glut1 mRNA levels in LPC028, LPC006, and PANC-1 cells after 24-h exposure to perifosine, gemcitabine, or to their combination, at
IC50 values, as determined by qRT-PCR. b Representative of Glut1 membrane-bound expression in LPC006 and LPC028 cells exposed for 24 h to
perifosine at IC50s; c Representative Western blot pictures of Glut1 expression in LPC006, LPC028, and PANC-1 cells exposed for 24 h to perifosine,
gemcitabine, or their combination at IC50s. d Structure of the compound PGL13. e Cell growth inhibition in LPC006 cells after 72-h exposure to
perifosine, gemcitabine, or to their combination, at IC50 values, together with DMSO or with the Glut1 inhibitor PGL13, at 30 μM. f Apoptosis
induction by perifosine, gemcitabine, and their combination as assessed by bisbenzimide staining as described in the Methods section (insert:
representative pictures of apoptotic LPC006 cells after treatment with perifosine and gemcitabine, original magnification ×40). Columns, mean
values obtained from three independent experiments; bars, SEM. *Significantly different from cells treated with DMSO
features with fluorescence microscopy. In particular the
LPC006 cells exposed to PGL13 with both perifosine and
gemcitabine had an apoptotic index of 27%, which was
similar to the apoptotic index of the LPC028 cells treated
with perifosine and gemcitabine (Fig. 6f ). The effect of a
combined Akt inhibitor/anti-Glut1 treatment was further
tested with MK-2205 and NVP-BEZ235, where it led to a
−14% and −20% decrease in cell viability compared with
these drugs alone (Additional file 1: Figure S11). Thus,
inhibition of Glut1 promoted anti-Akt-mediated cell
death, and this combined treatment shows promise for
future investigation in the treatment of PDAC.
The present study supports a role for phospho-Akt as a
prognostic factor in PDAC patients, and unravels its
potential role as a target for the synergistic interaction
of anti-Akt agents and gemcitabine through modulation
of apoptotic and invasive processes.
Several studies demonstrated that PDAC tissues have
increased activation of the PI3K/Akt, as assessed with
the phosphorylation of Akt, and this has been associated
with higher histological tumor grade  and worse
prognosis [46, 47]. In the present study, we further
explored the clinical relevance of phospho-Akt by
screening its expression in a homogeneous cohort of 100
surgically resected PDACs. In agreement with the
previous studies, even considering several clinicopathological
parameters, phospho-Akt expression was the only factor
correlated to differential clinical outcome. However, a
systematic review and meta-analysis of prognostic tissue
biomarkers for PDAC, including phospho-Akt among
the 22 markers associated with limitless replicative
potential eligible for examination, showed that only
Ki-67 maintained statistically significant associations
with outcome . These discrepancies might be
attributed to the different experimental procedures used,
including antigen retrieval technique, antibody
characteristics, and dilution, as well as observer variability in
staining pattern description and cutoff point selection.
Therefore, in the present study we have chosen an
antibody that was previously validated in an
immunohistochemical study on 102 colorectal cancer FFPE samples
 and we have used image analysis software to
calculate expression as a continuous parameter, in order to
facilitate the identification of cutoff points. This method
allowed the assignment of the specimens to different
categories, including a subset of tissues (about 14%)
characterized by extremely high expression of
phosphoakt, which could clearly influence the prognostic value.
Indeed this cutoff point identified a group of patients
with very poor outcome, who should be treated with
more aggressive, novel therapeutic approaches.
Of note, recent genomic studies showed that the PI3K/
Akt signaling is among the core signaling pathways
leading the intrinsic aggressiveness of PDAC, suggesting that
in the PDAC actionable genome about 9 and 6% of the
cases are Akt- and PI3K-dependent, respectively .
These data underline the potential importance of specific
inhibitors of PI3K/Akt as novel effective therapeutics in a
selected subpopulation of PDAC patients. Moreover,
activation of this signaling pathway is associated with PDAC
chemoresistance [13, 51], supporting the hypothesis that
Akt inhibitors might also be used to overcome resistance
towards conventional cytotoxic agents.
Several Akt/PI3K inhibitors are being developed. The
first generation of these inhibitors includes LY294002 and
wortmannin, which were tested to elucidate the value of
Akt/PI3K as therapeutic target . However, due to the
unfavorable pharmaceutical properties, toxicity, and
crossover inhibition of other lipid and protein kinases, these
compounds were not used in clinical studies .
More recently, several small molecules that inhibit the
PI3K/Akt signaling entered clinical development, but
more information on their activity in the preclinical
setting is warranted. For instance, a recent study showed
the potential inhibition of autophagy by perifosine
demonstrating that this drug impairs the autophagic flux in
HepG2 and U87 MG cells, which is related to defects in
intracellular cholesterol transport . These results
might be relevant for PDAC because some research lines
point at autophagy as a tumor-promoting mechanism.
Although a better understanding of the complexity of
autophagy is needed, the modulation of this process
might therefore open new opportunities for the
therapeutic use of autophagy inhibitors . Further
research to identify the precise mechanisms of
autophagy maturation may therefore provide a new insight into
the antiproliferative action of perifosine.
Our results demonstrate that perifosine is the targeted
anti-PI3K/Akt antitumor agent demonstrating the most
potent growth inhibitory effects in a panel of human
PDAC cells characterized by distinct molecular
properties. Limited published preclinical research focusing on
this issue in PDAC reported similar cytotoxic activity of
perifosine in PANC-1, MIA PaCa-2, and AsPC-1 cells
. Sensitivity to perifosine in the PDAC cells also fell
within the range of IC50 values previously reported in
PDAC cell lines and spheroids for other Akt inhibitors,
such as NVP-BEZ-235 [56, 57].
Furthermore, perifosine interacted synergistically with
gemcitabine in PDAC cells with high phospho-Akt
expression, but antagonistic in cells with low
phosphoAkt expression. Synergism was associated with inhibition
of migration/invasion and induction of apoptosis. These
results are in agreement with previous studies showing
synergistic interaction of gemcitabine with perifosine in
PANC-1 cells and xenografts  as well as enhanced
apoptotic cell death after combined treatment with
paclitaxel in chemoresistant ovarian cancer cells .
However, most previous studies were performed in
ATCC cell lines, which showed similar results , while,
to more effectively develop targeted compounds, it will be
helpful to understand why these agents fail when they do.
Thus, in the present study, cell growth inhibitory effects
of perifosine, gemcitabine, and their combination were
evaluated in several representative PDAC cells, including
primary PDAC cell cultures. For the LPC028 model, we
demonstrated that perifosine inhibited cell growth, both
in monolayer cell cultures and in cells growing as
spheroids, whereas LPC006 cells and spheroids were not
affected. Similarly, the perifosine/gemcitabine
combination had synergistic effects only in the cells with high
phospho-Akt or intermediate/high values of Akt1 mRNA,
as determined by RT-PCR. Conversely, this combination
was antagonistic in the cells with low Akt1, and
phosphoAkt1 expression. An important limitation of our findings
is the use of a single-cell culture (LPC006) as a model of
low phospho-Akt1. However, the results in two PDAC
models (LPC028 and PANC-1) with high phospho-Akt1
levels were similar. These data suggest that the expression
and activation of Akt might therefore be used to tailor
Importantly, two specific ELISA for the Akt Ser473
and the Thr308 phosphorylation showed that perifosine
effectively reached and inhibited its targets in the
LPC028 and PANC-1 cells, and the combination with
gemcitabine additionally inhibited Akt activation in
these cells. The present study demonstrated also that
perifosine interfered with pivotal determinants for the
activity of gemcitabine. In particular, we observed that
perifosine and its combination with gemcitabine
significantly reduced the expression of RRM1 and RRM2 in
the cells with a high expression of Akt, while this effect
was not statistically significant in the cells with low Akt
expression. RR is a key target of gemcitabine activity and
previous studies correlated the expression of its subunits
to gemcitabine sensitivity in PDAC cells [60, 61].
Therefore, the synergistic interaction between perifosine and
gemcitabine might be explained, at least in part, by the
modulation of gemcitabine sensitivity through RRM1
and RRM2 suppressions.
However, our results suggested that the synergistic
interaction of perifosine with gemcitabine is associated
with other important molecular mechanisms affecting
PDAC aggressiveness (Fig. 7). In agreement with
previous observations showing the reduction of cell
migration/invasion through Akt inhibition [16, 62], we
observed that perifosine and its combination with
gemcitabine markedly reduced cell migration and invasion in
PDAC cells. Several classes of proteins are involved in
this invasive behavior, including cell-cell adhesion
molecules like members of immunoglobulin and
calciumdependent cadherin families and integrins. In line with
previous evidence on inverse relationship between Akt
and E-cadherin expression , we demonstrated that
perifosine increased the expression of E-cadherin in
LPC028, CFPAC-1, and PANC-1 cells. This can at least
in part explain our findings on the reduction of
migration determined by perifosine. Furthermore, Toll et al.
 showed that decreased E-cadherin was associated
with poor prognosis of PDAC patients, supporting the
studies on novel compound which can modulate the
expression of this protein.
Since the Akt signaling pathway plays an important
role in cell survival process, its blockage can result in
activation of programmed cell death . Thus, we
GEMCITABINE (dFdC) Gemcitabine (dFdC)
Fig. 7 Molecular mechanisms involved in the synergistic interaction of perifosine with gemcitabine. The main upstream activator of Akt is
phosphatidylinositol-3 kinase (PI3K), which is activated in the response to a variety of growth stimuli through receptor tyrosine kinases and G
protein-coupled receptors. This kinase phosphorylates phosphatidylinositol-4,5-diphosphate (PIP2), which results in generation of
phosphatidylinositol-3,4,5-triphosphate (PIP3). PIP3 interacts with the pleckstrin homology (PH) domain of Akt, leading to translocation of Akt to
the cell membrane, and phosphorylation at Thr308 and Ser473. Perifosine inhibits Akt activation and enhances the growth inhibitory effects of
gemcitabine through its pronounced pro-apoptotic, anti-invasive effects, as well as by inhibiting the cell proliferation, followed by modulation of
ribonucleotide reductase (RR), potentially facilitating gemcitabine cytotoxicity. Moreover, Akt inhibition reduce Glut1 activity reducing glucose
influx and thereby favouring apoptosis induction and sensitizing PDAc cells to treatment with cytotoxic agents
further evaluated the effect of perifosine on cell cycle
perturbation and apoptosis induction. Previous findings
on the effect of perifosine after 24 h treatment showed
induction of G2/M arrest, potentially favoring the
activity of 6-thioguanine . Our results showed that after
72 h, perifosine treatment was associated with an
increase in the percentage of cells in the G0/G1, and S
phase, potentially favoring the cytotoxic activity of
gemcitabine. This modulation of the cell cycle was associated
with significant induction of apoptosis, as determined by
multiple methods, such as analysis of sub-G1,
mitochondria membrane potential and Annexin-V/PI. In order to
investigate the mechanisms underlying the activation of
programmed cell death, we checked the modulation of
critical factors involved in the apoptotic cascades.
Previous studies showed that drug-induced Akt deactivation
was associated with activation of pro-apoptotic factors,
including caspase-9 and BAD, as well as with a parallel
decrease in the expression of the anti-apoptotic factors
Bcl-2 and NF-kB [22, 65]. Our studies showed similar
results after exposure of the PDAC cells to perifosine.
Despite increasing evidence on the pivotal role of
PI3K/Akt signaling in cancer, the strategies to hit PI3K/
Akt/mTOR pathway have failed to demonstrate
therapeutic activity in most ongoing clinical trials, and a
previous phase II study testing perifosine in previously
untreated patients with locally advanced, unresectable,
or metastatic PDAC, was terminated as a result of
unacceptable adverse events .
It is already known that in PDAC cells, dual
PI3KmTOR inhibition induces rapid overactivation of MAPK
pathway through a PI3K-independent pathway , and
that drug resistance may be overcome by inhibition of
parallel oncogenic-dependent pathways, such as with the
dual MEK and PI3K/mTOR blockade .
One strategy to overcome resistance consists into
identifying key molecular differences in the tumors that
are less likely to respond. Oncogenic KRAS drives
metabolic reprogramming in tumor cells by increasing
aerobic glycolysis, and recent studies showed that subtypes
of PDAC cells with distinct metabolite levels associated
with glycolysis, lipogenesis, and redox pathways,
confirmed at the transcriptional level. The glycolytic and
lipogenic subtypes showed striking differences in glucose
and glutamine utilization, as well as mitochondrial
function, and corresponded to differences in cell sensitivity
to inhibitors of glycolysis, glutamine metabolism, lipid
synthesis, and redox balance . In the present study
we demonstrated that the resistant LPC006 cells were
characterized by overexpression of Glut1. Remarkably,
the inhibition of Glut1 dramatically enhanced perifosine
and perifosine/gemcitabine-induced cell death,
suggesting a cooperativity between Akt inhibitors and Glut1
inhibition. Agents directly inhibiting Glut1 are in early
phase evaluations, and a few preclinical studies have
demonstrated that Glut inhibitors led to diminish tumor
growth in vitro and in vivo . However, the altered
expression of Glut1 might also influence the sensitivity of
tumor cells to chemotherapy, since a recent study
showed that the knockdown of Glut1 sensitizes head
and neck cancer cells to the chemotherapy drug cisplatin
. To our knowledge, this is the first study showing
that Glut1 inhibitors can restore the repression of
aerobic glycolysis induced by PI3K/mTOR inhibitors in
resistant cells, and favor their synergistic interaction with
cytotoxic compounds. These results should prompt
further studies to understand how PDAC cell metabolism
might affect sensitivity to new anti-signaling therapies
and to identify promising therapeutic targets that might
be exploited by combination therapies.
Our data support the analysis of phospho-Akt expression as
both a prognostic and a predictive biomarker, for the rational
development of novel therapies targeting the Akt pathway in
PDAC. In particular, we observed that phospho-Akt
expression levels influence the antitumor activity of perifosine, as
well as the synergistic interaction with gemcitabine, through
its ability to attack key mechanisms involved in the
proliferation, cell cycle control, apoptosis and migration/invasion
properties. Finally, we demonstrated that inhibition of Glut1
overcame resistance to this combination treatment and might
provide the basis for the development of new therapeutic
approaches with Akt inhibitors in patients with PDAC.
Additional file 1: Figure S1. PFS curves according to phospho-Akt
expression in radically-resected PDACs, showing that patients with high
and “very high” phospho-Akt (right panel) had a significantly worse PFS.
Figure S2. Growth inhibitory effects after MK-2206 exposure in LPC006
(72-hours). Figure S3. Growth inhibitory effects after 72 hours exposure
to perifosine, gemcitabine or their combination at a fixed ratio based on
IC50 values in CFPAC-1 and PANC-1 cells. On the X-axis the drug
concentrations for the combination are referred to gemcitabine.
Figure S4. Phospho-Akt (serine residue-473) expression, normalized to
total Akt, after 4-hour exposure, as determined by ELISA. Figure S5. A
Phospho-Akt (serine residue-473) expression, normalized to total Akt, after
24-hour exposure. B Phospho-Akt (threonine residue-308) expression,
normalized to total Akt, after 24-hour exposure, as determined by ELISA.
Figure S6. Expression of gemcitabine determinants in PANC-1 cells
treated with perifosine, as determined by qRT-PCR. Dashed line, values in
untreated samples. Figure S7. Wound-healing assay in CFPAC-1 and
PANC-1 exposed to perifosine, gemcitabine or their combination (IC50
values, 24 hours). Figure S8. Wound-healing assay in LPC006 exposed to
MK-2206, gemcitabine or to their combination (IC50 values, 24 hours).
Figure S9. Annexin-V assay in LPC028 and LPC006. Figure S10:
Modulation of caspase-3, caspase-6/-8/ and caspase-9 in CFPAC-1 and
PANC-1, as determined by a specific fluorometric assay. Figure S11. Cell
growth inhibition in LPC006 cells after 72-hour exposure to MK-2205,
NVP-BEZ235 at IC50 values, together with DMSO or with the Glut1
inhibitor PGL13, at 30 μM. Points, or Columns, mean values obtained from
three independent experiments; bars, SEM. *Significantly different from
controls. (PPTX 328 kb)
Additional file 2: Table S1. Effects of gemcitabine and perifosine and
their combination on cell cycle distribution and on cell death (sub-G1).
(DOCX 14 kb)
The Authors would like to thank Professor A Griffioen (Department Medical
Oncology, VUmc, Amsterdam VUmc, Amsterdam) for the migration station
used to perform wound-healing assays, Dr. Abolfazl Avan (VUmc) for the
useful discussions on the role of Akt in chemoresistance, Dr. Sara Caponi
(University of Pisa) for her assistance in the collection of the clinical data,
and Dr. Kaamar Azijli (Vumc) for her support for the caspase activity assays.
This work was partially supported in the collections and analysis of data by
grants from Netherlands Organization for Scientific Research, NWO-Veni grant
(Elisa Giovannetti), CCA Foundation 2012 (Amir Avan, Godefridus J Peters, Elisa
Giovannetti), AIRC/Start-Up (Elisa Giovannetti), Istituto Toscano Tumori grant
(Ugo Boggi, Niccola Funel, Elisa Giovannetti), European Union iCARE Marie Curie
grant (Leticia G. Leon), CCA Foundation 2015 (Elisa Giovannetti), Tuscany Region
grant FAS Health (Ugo Boggi, Niccola Funel, Elisa Giovannetti).
Availability of data and materials
All data generated or analysed during this study are included in this
published article (and its supplementary information files).
EG was the principal investigator and takes primary responsibility for the
paper; UB, NF, FM, CG, BA provided patient samples, clinical data, and drugs;
DM, AA, MM, AVK, RR, and CG performed the research; EG, AR, and GJP
designed the research; DM and EG wrote the paper; AR, GJP, FM, and LGL
edited the paper. All authors read and approved the final manuscript.
The authors have no conflict of interest to disclose. B. Aicher is an employee
and stock option holder of Aeterna Zentaris GmbH.
Consent for publication
Consent to publish has been obtained from all the participants (or legal
parent or guardian for children) to report individual patient data.
Ethics approval and consent to participate
This study was approved by the Local Ethics Committee of the University of
Pisa. Date of approval: July 3, 2013 (file number 3909).
All patients have provided a written informed consent to participate to this study.
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