835
Human mesothelioma cells exhibit tumor cell–specific
differences in phosphatidylinositol 3-kinase/AKT
activity that predict the efficacy of Onconase
Maria E. Ramos-Nino,1 Giovina Vianale,2
Tara Sabo-Attwood,1 Luciano Mutti,3
Camilo Porta,4 Nicholas Heintz,1
and Brooke T. Mossman1
1
Department of Pathology, University of Vermont College of
Medicine, Burlington, Vermont; 2Department of Oncology and
Neuroscience, Sections of Clinical and General Pathology,
G. D’Annunzio University, Chieti, Italy; 3Health Authority 11,
Piemonte-IRRCS S. Maugeri Foundation; and 4Matteo University
Hospital, Pavia, Italy
Abstract
Malignant mesothelioma is an aggressive cancer with no
known cure, which has become a therapeutic challenge.
Onconase is one of few chemotherapeutic agents that
have been studied in patients with malignant mesothelioma that has the advantage of low toxicity and limited
side effects. Here, we evaluate the effect of Onconase on
killing of malignant mesothelioma cells and how the
phosphatidylinositol 3-kinase/AKT (PI3-K/AKT) survival
pathway influences this effect. Our results show that
Onconase induces apoptosis in malignant mesothelioma
cell lines and that this effect is tumor cell specific.
Malignant mesothelioma cell lines with the highest AKT
activation, which correlated with the presence of the
SV40 large and small T antigen (SV40+
+), were the most
resistant to the drug. Finally, a cooperative effect was
observed between small molecule inhibitors of PI3-K and
Onconase in the killing of malignant mesothelioma cells.
Our results suggest that kinase screening of individual
malignant mesotheliomas for endogenous levels of activated PI3-K/AKT may be predictive of the efficacy of
Onconase and possibly other chemotherapeutic agents.
[Mol Cancer Ther 2005;4(5):835 – 42]
Received 9/13/04; revised 1/22/05; accepted 3/2/05.
Grant support: NIH grant R01s ES/HL02913 (B.T. Mossman), K01
CA104159-01 (M.E. Ramos-Nino), and the Italian Association of Cancer
Research (G. Vianale).
The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
advertisement in accordance with 18 U.S.C. Section 1734 solely to
indicate this fact.
Requests for reprints: Brooke T. Mossman, Department of Pathology,
College of Medicine, University of Vermont College of Medicine,
89 Beaumont Avenue, HSRF 215, Burlington, VT 05405.
Phone: 802-656-0382; Fax: 802-656-8892.
E-mail: [email protected]
Copyright C 2005 American Association for Cancer Research.
Introduction
The processes involved in the initiation and development
of malignant mesothelioma, a malignancy derived from
pluripotent mesothelial cells with a variety of tumor
phenotypes, including epithelial, sarcomatous, and mixed
varieties (1), are under intense investigation (2). This
unique tumor has been associated historically with occupational exposures to amphibole types of asbestos (1, 3). In
the past few years, SV40, a DNA virus, has been linked to
the etiology of mesothelioma. Multi-institutional studies
show that f50% of human mesotheliomas in the United
States contain SV40 large T-antigen DNA sequences (4, 5).
Although malignant mesothelioma is a relatively rare
cancer, its incidence is increasing in several countries (6).
More important, the prognosis of patients with mesothelioma is grim as most survive <1 year after initial diagnosis
(1, 3). Thus, effective therapeutic strategies are desperately
needed.
Current treatment modalities include surgery, chemotherapy, and radiation therapy (7) and several new
investigational approaches are now being tested, including
intrapleural IFNg, photodynamic therapy, immunotherapy,
and gene therapy. Whereas some believe that radical
surgical resection is the only chance of cure or meaningful
improvement in survival, the majority of patients presenting with malignant mesothelioma are not candidates for
radical surgical resection due to unresectable tumors or
medical illness (8). As alternatives or complements, many
chemotherapeutic agents have been studied in patients
with malignant mesothelioma, either as single or combined
agents, but low response rates have been found in most
studies with the highest response rates generally achieved
using combined treatment regimens (8). No clear standard
of care has emerged and in most cases palliative treatment
is used as the primary means of therapy (9).
One novel agent for the treatment of malignant mesothelioma is Onconase, a RNase derived from eggs and early
embryos of the leopard frog (Rana pipens) that has shown
activity against a variety of human tumors in vitro and
in vivo (10). Onconase has been tested in phase I and phase
II human clinical trials for treatment of numerous solid
tumors, including malignant mesothelioma (11). Phase III
trials of Onconase are still ongoing. Onconase is a
homologue of RNA A, which preferentially degrades tRNA
(12). It has been found that Onconase inhibits cell growth
and proliferation and induces apoptosis through proteinsynthesis inhibition-dependent and inhibition-independent
mechanisms (11). One advantage of Onconase as an
anticancer drug is that proliferating cells are more
susceptible to it than quiescent cells (13), which could be
one reason why Onconase is more toxic to cancer cells than
Mol Cancer Ther 2005;4(5). May 2005
Downloaded from mct.aacrjournals.org on June 17, 2017. © 2005 American Association for Cancer Research.
836 AKT-Dependent Resistance to Onconase in Mesothelioma
to noncancer cells (10). The potential of RNases as cancer
chemotherapeutic agents are enormous due to their low
toxicity in humans. However, more research is needed to
understand the mechanisms of resistance of malignant
mesothelioma to these drugs, increase their efficacy, and be
able to rationally combine them with other chemotherapeutic drugs.
In this study, we investigated the involvement of the
PI3-K/AKT survival pathway in malignant mesothelioma
resistance to Onconase. The PI3-K pathway is activated in
many cancers (14) and we have recently shown that is it
also frequently activated in human mesotheliomas where
it can be targeted to inhibit mesothelioma cell growth.5
Here, we show that malignant mesotheliomas have
different degrees of response to killing by Onconase.
Those malignant mesothelioma cell lines with the most
elevated AKT kinase activity, which correlates in most
cases with positive SV40 status (SV40+), were less
susceptible to inhibition of cell growth and survival by
Onconase. Our results suggest that screening of malignant
mesothelioma for cell survival – related kinases, particularly PI3-K/AKT, may be necessary in determining
effective treatment of malignant mesotheliomas with
Onconase.
Materials and Methods
Human Mesothelial and Mesothelioma Cell Lines
Human pleural mesothelioma cell lines (n = 9) were
isolated from patients at autopsy and were kindly
provided by Drs. Michele Carbone (Loyola University,
Chicago, IL; MPP, MHF, and MCAT), Luciano Mutti
(Maugeri Foundation, Pavia, Italy; MB and MMO), and
Harvey Pass (Wayne State University, Detroit, MI;
MGAR, MGAT, MMIL, M17). The MB, MMO, MGAR,
MGAT, MMIL, and M17 lines were negative for SV40
large and small T-antigen (SV40 ), and MHF, MPP, and
MCAT were SV40+. All cell lines were tested for the
mRNA expression of large and small T/t-antigen by PCR
before each experiment. Cells were retrieved from frozen
stocks and propagated in DMEM/F12 medium (Life
Technologies, Grand Island, NY) containing 10% fetal
bovine serum, hydrocortisone (100 ng/mL), insulin
(2.5 Ag/mL), transferrin (2.5 Ag/mL), and selenium
(2.5 ng/mL; Sigma, St. Louis, MO).
Small Molecule Inhibitors and Chemicals
Stock solutions of the PI3-K inhibitor LY294002 were
diluted in DMSO and used at nontoxic and specific
concentration (20 Amol/L; ref. 15). LY294002 was obtained
from Calbiochem (La Jolla, CA). Wortmannin (Sigma), an
inhibitor of PI3-K, was added at a final concentration of
50 nmol/L (16). Onconase, a gift from Dr. Stanislaw
Mikulski (AlfaCell, Corp., Bloomfield, NJ), was used at
5
Altamore DA, You H, Xiao G, et al. Human and mouse mesotheliomas exhibit
elevated AKT/PKB activity, which can be targeted pharmacologically to inhibit
tumor cell growth. Oncogene. In Press 2005.
1 and 10 Ag/mL medium and was prepared in medium
from a lyophilized stock solution. After resuspension,
aliquots were stored at 20jC. All untreated control cells
received DMSO in medium as a solvent control.
Growth Curves
Cells (n = 2-3 plates/group/time point) were plated at
f2 105 cells per 60-mm-diameter plate in complete
medium, allowed to attach for 24 hours, and then treated
with inhibitors at the indicated time points. Cells were
removed by trypsinization and aliquots counted using a
hemocytometer to determine total cell number.
Western Blot Analyses
Nearly confluent malignant mesothelioma cells were
washed thrice with cold PBS, scraped from the plates, and
collected by centrifugation at 14,000 rpm for 1 minute. The
pellet was resuspended in lysis buffer [20 mmol/L Tris
(pH 7.4), 1% Triton X-100, 10% glycerol, 137 mmol/L NaCl,
2 mmol/L EDTA, 25 mmol/L h-glycerophosphate,
1 mmol/L Na3VO4, 2 mmol/L PPi, 1 mmol/L phenylmethylsulfonyl fluoride, 10 Ag/mL leupeptin, 1 mmol/L
DTT, 10 mmol/L NaF, 1% aprotinin], incubated at 4jC for
15 minutes, and centrifuged at 14,000 rpm for 20 minutes.
Protein concentrations were determined using a Bio-Rad
assay (Bio-Rad, Hercules, CA). Thirty micrograms of
protein in sample buffer [62.5 mmol/L Tris-HCl (pH 6.8),
2% SDS, 10% glycerol, 50 mmol/L DTT, 0.1% w/v
bromophenol blue] was resolved by electrophoresis in
10% SDS-polyacrylamide gels and transferred to nitrocellulose using a semidry transfer apparatus (Ellard Instrumentation, Ltd., Seattle, WA). Blots were incubated in
blocking buffer (TBS containing 5% nonfat dry milk plus
0.1% Tween 20; Sigma) for 1 hour, washed thrice for
5 minutes each in TBS/0.1% Tween 20, and incubated at
4jC overnight with antibodies specific to p-AKT or AKT,
both at a 1:500 dilution (Santa Cruz Biotechnology, Inc.,
Santa Cruz, CA). Blots were then washed thrice with TBS/
0.1% Tween 20 and incubated with a specific peroxidaseconjugated secondary antibody for 1 hour. After washing
blots thrice in TBS/0.1% Tween 20, protein bands were
visualized with the LumiGlo enhanced chemiluminescence
detection system (Kirkgaard and Perry Laboratories,
Gaithersburg, MD) and quantitated by densitometry (17).
Blots were reprobed with an antibody to a-tubulin in a
dilution 1:1,000 (Santa Cruz Biotechnology) to validate
equal loading between lanes (18).
Kinase Activity Assays
Protein kinases were immunoprecipitated from whole
cell lysates (300 Ag) prepared from near-confluent
malignant mesothelioma cells using an AKT antibody
(2 Ag; Santa Cruz Biotechnology), then washed and
incubated for 20 minutes at 30jC in kinase buffer
[20 nmol/L HEPES (pH 7.5), 2 mmol/L 2-mercaptoethanol, 5 mmol/L MgCl2] containing 5 ACi [g-32P]ATP and
5 Ag protein kinase Ba substrate per reaction (Calbiochem). Incorporation of 32P into the substrate was
visualized by autoradiography following SDS-PAGE
and quantified using phosphoimaging (Bio-Rad MultiAnalysis program; ref. 18).
Mol Cancer Ther 2005;4(5). May 2005
Downloaded from mct.aacrjournals.org on June 17, 2017. © 2005 American Association for Cancer Research.
Molecular Cancer Therapeutics
Flow Cytometry
Nearly confluent cells were maintained in complete
medium containing 0.5% fetal bovine serum overnight
before addition of Onconase at 1 and 10 Ag/mL medium.
At 24, 48, and 72 hours, medium was removed and
adherent cells harvested by trypsinization. Cells were
resuspended at 106/mL in staining solution (50 Ag/mL
propidium iodide, 0.1% Triton X-100, and 32 Ag/mL RNase
A) in PBS and incubated for 30 minutes at 37jC before
analysis of 10,000 cells/group/time point in triplicate. The
distribution of cells, including cells with a hypodiploid
DNA content indicative of apoptosis or necrosis, was
determined using a Coulter Epics Elite flow cytometer and
appropriate software as previously described (19).
Methods for Detection of DNA Strand Breaks and
Apoptosis
Terminal deoxynucleotidyl transferase (TdT) – mediated
nick end labeling (TUNEL) assays for detection of DNA
strand breaks were done using a commercial kit following
the manufacturer’s instructions (Promega Corporation,
Madison, WI; ref. 20). Cells were plated on glass coverslips
and grown to near confluence. After exposure to agents,
slides were fixed in 10% buffered formalin for 25 minutes at
room temperature, washed with fresh PBS several times,
and cells permeabilized using 0.2% Triton X-100 solution in
PBS. Slides then were incubated with 100 AL of equilibration buffer, a biotinylated nucleotide mix, and the TdT
reaction mix at 37jC for 1 hour in a humidified chamber.
The reaction was terminated with 2 SSC for 15 minutes.
Endogenous peroxidases were blocked by immersing the
slides in 0.3% H2O2 for 5 minutes. Slides then were
incubated with streptavidin horseradish peroxidase complex for 30 minutes, stained with 3-3V diaminobenzidine
tetrahydrochloride, and counterstained with hematoxylin
to detect apoptotic/necrotic nuclei, which were analyzed
using transmission electron microscopy. Negative controls
included cells incubated in enzyme alone and positive
controls consisted of cells treated with DNase I (Promega).
To determine the number of apoptotic cells, cells were
stained with annexin V and propidium iodide in the dark
for 15 minutes, and 5,000 events per sample were scored by
flow cytometry as described above. For staining, cell pellets
were suspended in 93 AL of 1 binding buffer [10 mmol/L
HEPES/NaOH (pH 7.4), 140 mmol/L NaCl, 2.5 mmol/L
CaCl2], 5 AL of propidium iodide at a final concentration of
2.5 Ag/mL (Sigma), and 2 AL of FITC labeled-annexin V
(Alexis, Firenze, Italy). Cells with annexin V – positive
staining were scored as apoptotic.
3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide Assay for Cell Viability
Assays were done on 96-well microtiter plates after
plating of 7.7 104 cells/well. Malignant mesothelioma cell
lines were then cultured for 24 hours in complete medium
before changing to medium containing 0.5% fetal bovine
serum. Cells were then treated with LY294002 or DMSO
(solvent control) 1 hour before the addition of Onconase (10
Ag/mL). Cell viability was assessed using an 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) assay (R&D Systems, Minneapolis, MN), in which
0.5 mg/mL of MTT was added to the wells for 3 to 4 hours
before dissolution in DMSO and its absorbance read at 570
and 650 nm. DAbsorbance from these two wavelengths are
reported as the corrected viability. Fold changes were
calculated with respect to the control.
Statistical Analyses
In all experiments, duplicate or triplicate determinations
were conducted for each group (n = 2-3) per time point.
Experiments were done in duplicate. Results were evaluated by one-way ANOVA using the Student-NewmanKeuls procedure for adjustment of multiple pairwise
comparisons between treatment groups. Differences with
P V 0.05 were considered statistically significant.
Results
Onconase Differentially Affects Mesothelioma Cell
Growth
Figure 1 shows the effect of Onconase at 1 and 10 Ag/mL
on growth of 4 malignant mesothelioma cell lines (MB/
MMO as SV40 cell lines and MPP/MCAT as SV40+ cell
lines). The effect of Onconase was tumor-specific, with the
SV40+ cell lines showing an increased resistance to the
drug. These studies showed that Onconase at both 1 and 10
Ag/mL caused significant (P V 0.05) dose-related killing at
24 to 72 hours in MB cells (Fig. 1A) and at 48 and 72 hours
in MMO cells (Fig. 1B). Onconase at these same concentrations did not significantly affect the growth of MPP and
MCAT (SV40+) cell lines (Fig. 1C and D).
Onconase Causes Dose-Related Increases in Proportions of Hypodiploid (sub-G0/G1) Mesothelioma Cells
Over Time That Is Reflected in Increased Cell Killing
and TUNEL Positivity
To examine the effect of Onconase on cell growth, we
determined the effects of the drug (1 and 10 Ag/mL) on cell
cycle kinetics of these four malignant mesothelioma cell
lines over a 72-hour period using flow cytometry. In
comparison to the more resistant MPP and MCAT SV40+
cell lines, the MB and MMO lines exhibited significant
increases (P V 0.05) in the fraction of the cell population in
sub-G0/G1, which increased over time (Fig. 2A – D). These
changes occurred with concomitant decreases in cells in
G0/G1 (Fig. 2E – H).
To determine the mechanism of cell death by Onconase
in malignant mesothelioma, control and Onconase-treated
MB and MMO SV40 cell lines were examined using the
TUNEL assay, which detects apoptotic and necrotic cell
death. In comparison to sham control cells (Fig. 3A and C)
or cells incubated without TUNEL enzyme ( control in
Fig. 3C), TUNEL-positive cells were observed in both
mesothelioma cell lines exposed to Onconase (10 Ag/mL for
48 hours; Fig. 3B and D) and in MB cells pretreated with
DNase (+ control in Fig. 3F). Although TUNEL-positive
cells often exhibited smaller nuclei indicative of apoptosis
(Fig. 3B and D, arrows), transmission electron microscopy of
adherent cells treated with Onconase also indicated lytic
cell death (data not shown).
Mol Cancer Ther 2005;4(5). May 2005
Downloaded from mct.aacrjournals.org on June 17, 2017. © 2005 American Association for Cancer Research.
837
838 AKT-Dependent Resistance to Onconase in Mesothelioma
Figure 1. SV40 status correlates with cell killing by Onconase. Growth curves of human mesothelioma cell lines treated with Onconase [control (c ),
1 or 10 Ag/mL] for 8, 24, 48, or 72 h. A, MB (SV40 ). B, MMO (SV40 ). C, MPP (SV40+). D, MCAT (SV40+). *P V 0.05 compared with
untreated control.
AKT Activity Is Increased in SV40+ Malignant Mesothelioma Cell Lines
To establish whether the AKT survival pathway was a
factor in resistance to Onconase in SV40+ malignant
mesothelioma cells, we studied the status of AKT activity
in nine malignant mesothelioma cell lines. After a 24-hour
period in low serum (0.5% fetal bovine serum) medium and
addition of 10% fetal bovine serum for 4 hours, increased
phosphorylated AKT were observed in the SV40+ cell lines,
MCAT, MHF, and MPP, compared with the SV40 cell
lines (Fig. 4A). Using a kinase activity assay, we confirmed
in four malignant mesothelioma lines that AKT activity was
higher in SV40+ cell lines (Fig. 4B). These results suggested
that the efficacy of Onconase on killing of malignant
mesotheliomas may be dependent on the extent of
endogenous PI3-K/AKT pathway activity.
Inhibition of PI3K/AKT Increases Cell Death Induced
by Onconase
A wide range of extracellular signals can activate PI3K
and trigger the phosphorylation of protein kinase B (AKT),
providing increased cell survival. Based on results presented above, we hypothesized that AKT activation would
protect malignant mesothelioma cells from killing by
Onconase. Figure 5 shows the survival patterns, using the
MTT assay, of MB (SV40 ) and MPP (SV40+) cell lines over
a 72-hour period when treated with LY294002 alone
(20 Amol/L), Onconase alone (10 Ag/mL), or both in
combination. The MB (SV40 ) cell line, with the lower
basal AKT activity, had the higher response to Onconase
and a lower response to LY294002 (Fig. 5A). The MPP
(SV40+) cell line, with the higher basal AKT activity, had a
lower response to Onconase and the higher response to
LY299402 (Fig. 5B). Both cell lines exhibited decreased
survival with coexposure to LY294002 (20 Amol/L) and
Onconase (10 Ag/mL) at 48 and 72 hours. To verify if these
results reflected cooperative inhibition of apoptosis by
these agents, the effects of wortmannin (50 nmol/L) and
Onconase (1 and 10 mg/mL), alone and in combination,
were evaluated for annexin V – positive cells by flow
cytometry. Apoptosis was significantly higher (P V 0.01)
in the combined treatment groups (wortmannin + Onconase) at both concentrations of Onconase (Fig. 5C).
Discussion
Malignant mesothelioma is a major therapeutic challenge.
Many chemotherapeutic agents have been studied in
patients with malignant mesothelioma, either as single
agents or as part of a combination chemotherapy regime,
with the highest response rates generally achieved using
multiagent regimes (8). Malignant mesothelioma clinical
trials for Onconase, a drug with low toxicity and limited
side effects (13), have shown that some patients benefit
from it whereas others do not (11). The biological reasons
underlying differences in tumor response are unclear and
undoubtedly complex. However, the observation that
endogenous AKT activity correlates with malignant mesothelioma resistance to Onconase provides a plausible
explanation for the differences observed here. We are
currently validating acitvation of AKT pathway in SV40+
and SV40 primary tumors using immunofluorescence
approaches on tissue arrays and primary cell cultures.
Mol Cancer Ther 2005;4(5). May 2005
Downloaded from mct.aacrjournals.org on June 17, 2017. © 2005 American Association for Cancer Research.
Molecular Cancer Therapeutics
In this study, we evaluated the effect of Onconase on
killing of malignant mesotheliomas in cell culture, and
how the PI3-K/AKT survival pathway may influence
these responses. Onconase affected cell growth and
survival of malignant mesotheliomas in a differential
manner, with those cell lines with higher AKT activity
being the most resistant to the drug. One of the
intracellular signaling pathways frequently activated in
cancer is the PI3-K pathway (14). Downstream targets of
the PI3-K pathway include serine/threonine kinases, such
as the three isoforms of AKT (or PKB). In recent years,
increasing evidence has implicated the PI3-K/AKT
pathway in the regulation of cell size, proliferation, and
survival, as well as tumor metastasis, angiogenesis, and
Figure 2. Onconase causes dose-related increases in proportions of hypodiploid (sub-G0/G1) and decreases in G0/G1 in mesothelioma cells over time. Flow
cytometric analyses (A – H) showing cycle distribution in sub-G0/G1 (A, C, E, G) and G0/G1 (B, D, F, H) after exposure to Onconase (1 and 10 g/mL) in
human mesothelioma cell lines SV40 : MB (A, B) and MMO (C, D) and SV40+: MPP (E, F) and MCAT (G, H). *P < 0.05 compared with sham control
cells at the same time.
Mol Cancer Ther 2005;4(5). May 2005
Downloaded from mct.aacrjournals.org on June 17, 2017. © 2005 American Association for Cancer Research.
839
840 AKT-Dependent Resistance to Onconase in Mesothelioma
Figure 3. Onconase causes cell death that is
reflected in TUNEL positivity. TUNEL staining
of untreated MB (SV40 ; A ) and MMO
(SV40 ; C) mesothelioma cells and those
exposed to Onconase (10 Ag/mL; B and D)
for 48 h. E, untreated MB cells incubated
without TUNEL enzyme ( control); F, MB cells
pretreated with DNase before TUNEL staining
(+control). Arrowheads in (B) and (D) show
TUNEL-positive cells.
invasiveness (14, 21 – 23). Recently, we have shown that the
PI3-K/AKT pathway is frequently activated in human
mesotheliomas and can be targeted to inhibit mesothelioma
cell growth.5 Here, we present evidence that human
Figure 4. Levels of AKT activity differ in human mesotheliomas. A,
Western blot showing p-AKT/AKT expression. B, AKT kinase activity
assay.
mesothelioma cell lines with higher constitutive AKT
activity are more resistant to Onconase. High AKT
activity of mesothelioma cells correlated with SV40+
status. In recent years, it has been postulated that SV40
act as a cocarcinogen with asbestos in the pathology of
mesothelioma (2, 24 – 27), although the pathogenic mechanisms are unclear. We suggest that SV40 may cooperate
with asbestos in the development of malignant mesothelioma by increasing the activity of the PI3-K/AKT
survival pathway. The PI3-K/AKT pathway can be
activated by asbestos or SV40 Tag, which uses the PI3-K
pathway to increase cell survival and/or inhibit apoptosis (28). To further support our results, it was shown
recently in human mesotheliomas that the hepatocyte
growth factor receptor (c-Met) is activated by SV40 (29).
Previously, we also showed that hepatocyte growth
factor can activate the PI3-K/AKT pathway in malignant
mesotheliomas.5 These results suggest that SV40 might
activate the PI3-K pathway through c-Met or other
receptor-mediated mechanisms.
Our studies on the cooperative effects of cell killing
using PI3-K inhibitors and Onconase suggest that
Mol Cancer Ther 2005;4(5). May 2005
Downloaded from mct.aacrjournals.org on June 17, 2017. © 2005 American Association for Cancer Research.
Molecular Cancer Therapeutics
knowledge of the underlining signaling pathways involved in the survival of individual tumors might
increase the efficacy of cell killing by chemotherapeutics.
We surmise that initial screening of malignant mesotheliomas for activation of kinases important in tumor cell
survival may be exploited to inhibit growth and
development of malignant mesotheliomas and possibly
other tumors. An understanding of the signaling pathways critical to malignant mesothelioma development
and progression is critical for the design of more rational
treatment options for patients.
References
1. Mossman B, Gee J. Asbestos related disease. N Engl J Med 1989;320:
1721 – 30.
2. Carbone M, Rdzanek MA. Pathogenesis of malignant mesothelioma.
Clin Lung Cancer 2004;5 Suppl 2:S46 – 50.
3. Mossman B, Bignon J, Corn M, Seaton A, Gee J. Asbestos: scientific
developments and implications for public policy. Science 1990;247:
294 – 301.
4. Klein G, Powers A, Croce C. Association of SV40 with human tumors.
Oncogene 2002;21:1141 – 9.
5. Pass HI, Donington JS, Wu P, et al. Human mesotheliomas contain
the simian virus-40 regulatory region and large tumor antigen DNA
sequences. J Thorac Cardiovasc Surg 1998;116:854 – 9.
6. McDonald J, Wagner A. The epidemiology of mesothelioma in
historical context. Eur Respir J 1996;9:1932 – 42.
7. Vogelzang NJ. Multimodality therapy in mesothelioma: role of
chemotherapy. Thorac Surg Clin 2004;14:531 – 42.
8. Pistolesi M, Rusthoven J. Malignant pleural mesothelioma: update,
current management, and newer therapeutic strategies. Chest 2004;126:
1318 – 29.
9. Martino D, Pass HI. Integration of multimodality approaches in the
management of malignant pleural mesothelioma. Clin Lung Cancer 2004;5:
290 – 8.
10. Leland PA, Raines RT. Cancer chemotherapy-ribonucleases to the
rescue. Chem Biol 2001;8:405 – 13.
11. Mikulski SM, Costanzi JJ, Vogelzang NJ, et al. Phase II trial of a single
weekly intravenous dose of ranpirnase in patients with unresectable
malignant mesothelioma. J Clin Oncol 2002;20:274 – 81.
12. Iordanov MS, Ryabinina OP, Wong J, et al. Molecular determinants of
apoptosis induced by the cytotoxic ribonuclease onconase: evidence for
cytotoxic mechanisms different from inhibition of protein synthesis.
Cancer Res 2000;60:1983 – 94.
13. Smith MR, Newton DL, Mikulski SM, Rybak SM. Cell cycle-related
differences in susceptibility of NIH/3T3 cells to ribonucleases. Exp Cell Res
1999;247:220 – 32.
14. Vivanco I, Sawyers CL. The phosphatidylinositol 3-kinase AKT
pathway in human cancer. Nat Rev Cancer 2002;2:489 – 501.
15. Xiao GH, Jeffers M, Bellacosa A, Mitsuuchi Y, Vande Woude
GF, Testa JR. Anti-apoptotic signaling by hepatocyte growth factor/
Met via the phosphatidylinositol 3 kinase/Akt and mitogen activated
protein kinase pathways. Proc Natl Acad Sci U S A 2001;98:
247 – 52.
16. Muscella A, Elia MG, Greco S, Storelli C, Marsigliante S. Activation of
P2Y2 receptor induces c-FOS protein through a pathway involving
mitogen-activated protein kinases and phosphoinositide 3-kinases in HeLa
cells. J Cell Physiol 2003;195:234 – 40.
17. Scapoli L, Ramos-Nino ME, Martinelli M, Mossman BT. Src-dependent
ERK5 and Src/EGFR-dependent ERK1/2 activation is required for cell
proliferation by asbestos. Oncogene 2004;23:805 – 13.
Figure 5. Inhibition of PI3K/AKT increased cell death induced by
Onconase. MTT assay showing the effect of LY4002 (20 Amol/L),
Onconase (10 Ag/mL) alone and the combination on cell survival of MB
(SV40 ; A) and MPP (SV40+; B) malignant mesotheliomas. *P V 0.05
compared with sham control cells. +P V 0.05 compared with Onconase
alone and cP V 0.05 compared with Onconase alone and LY294002 alone
groups. C, evidence for the cooperative effect of the P13K inhibitor
wortmannin (wort , 50 nmol/L) and Onconase (1 and 10 Ag/mL) on the
percentage of annexin V – positive cells in the MPP (SV40 Tag+) cell line.
Cells were treated with Onconase with and without wort for 72 h before
staining with annexin V – propidium iodide and analyzed using flow
cytometry to determine the percentage of annexin V – positive cells.
*P V 0.01 compared with untreated control group. cP V 0.01 compared
with groups with Onconase or wort alone.
18. Scapoli L, Ramos-Nino M, Martinelli M, Mossman B. Src-dependent
ERK5 and Src/EGFR-dependent ERK1/2 activation is required for cell
proliferation by asbestos. Oncogene 2004;23:805 – 13.
19. BeruBe K, Quinlan T, Fung H, et al. Apoptosis is observed in
mesothelial cells after exposure to crocidolite asbestos. Am J Respir Cell
Mol Biol 1996;15:141 – 7.
20. Goldberg J, Zanella C, Janssen Y, et al. Novel cell imaging approaches
show induction of apoptosis and proliferation in mesothelial cells by
asbestos. Am J Respir Cell Mol Biol 1997;17:265 – 71.
21. Cantley LC. The phosphoinositide 3-kinase pathway. Science
2002;296:1655 – 7.
22. Brazil DP, Hemmings BA. Ten years of protein kinase B signalling:
a hard Akt to follow. Trends Biochem Sci 2001;26:657 – 64.
Mol Cancer Ther 2005;4(5). May 2005
Downloaded from mct.aacrjournals.org on June 17, 2017. © 2005 American Association for Cancer Research.
841
842 AKT-Dependent Resistance to Onconase in Mesothelioma
23. Leslie NR, Downes CP. PTEN: the down side of PI 3-kinase
signalling. Cell Signal 2002;14:285 – 95.
about the association of SV40 with human mesothelioma. Oncogene
2003;22:5173 – 80.
24. Aldieri E, Orecchia S, Ghigo D, et al. Simian virus 40 infection
down-regulates the expression of nitric oxide synthase in human
mesothelial cells. Cancer Res 2004;64:4082 – 4.
28. Yu Y, Alwine JC. Human cytomegalovirus major immediateearly proteins and simian virus 40 large T antigen can inhibit
apoptosis through activation of the phosphatidylinositide 3V-OH
kinase pathway and the cellular kinase Akt. J Virol 2002;76:
3731 – 8.
25. Carbone M, Fisher S, Powers A, Pass HI, Rizzo P. New molecular
and epidemiological issues in mesothelioma: role of SV40. J Cell
Physiol 1999;180:167 – 72.
26. Carbone M, Kratzke RA, Testa JR. The pathogenesis of mesothelioma. Semin Oncol 2002;29:2 – 17.
27. Carbone M, Pass HI, Miele L, Bocchetta M. New developments
29. Cacciotti P, Libener R, Betta P, et al. SV40 replication in human
mesothelial cells induces HGF/Met receptor activation: a model for
viral-related carcinogenesis of human malignant mesothelioma. Proc
Natl Acad Sci U S A 2001;98:12032 – 7.
Mol Cancer Ther 2005;4(5). May 2005
Downloaded from mct.aacrjournals.org on June 17, 2017. © 2005 American Association for Cancer Research.
Human mesothelioma cells exhibit tumor cell−specific
differences in phosphatidylinositol 3-kinase/AKT activity that
predict the efficacy of Onconase
Maria E. Ramos-Nino, Giovina Vianale, Tara Sabo-Attwood, et al.
Mol Cancer Ther 2005;4:835-842.
Updated version
Cited articles
Citing articles
E-mail alerts
Reprints and
Subscriptions
Permissions
Access the most recent version of this article at:
http://mct.aacrjournals.org/content/4/5/835
This article cites 27 articles, 9 of which you can access for free at:
http://mct.aacrjournals.org/content/4/5/835.full.html#ref-list-1
This article has been cited by 4 HighWire-hosted articles. Access the articles at:
/content/4/5/835.full.html#related-urls
Sign up to receive free email-alerts related to this article or journal.
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Department at [email protected].
To request permission to re-use all or part of this article, contact the AACR Publications
Department at [email protected].
Downloaded from mct.aacrjournals.org on June 17, 2017. © 2005 American Association for Cancer Research.