Download Chemosensitization of head and neck cancer cells by PUMA

3180
Chemosensitization of head and neck cancer cells
by PUMA
Quanhong Sun,1,2 Tsukasa Sakaida,1,3 Wen Yue,1,3
Susanne M. Gollin,1,2,4,5 and Jian Yu1,2
1
University of Pittsburgh Cancer Institute; Departments of
Pathology, 3Pharmacology, and 4Otolaryngology, University of
Pittsburgh School of Medicine; and 5Department of Human
Genetics, The University of Pittsburgh Graduate School of Public
Health, Pittsburgh, Pennsylvania
2
Abstract
Head and neck squamous cell carcinoma (HNSCC) ranks
the eighth most common cancer worldwide. The patients
often present with advanced disease, which responds
poorly to chemoradiation therapy. PUMA is a BH3-only
Bcl-2 family protein and a p53 target that is required for
apoptosis induced by p53 and various chemotherapeutic
agents. In this study, we found that PUMA induction by
chemotherapeutic agents is abrogated in most HNSCC cell
lines. Adenoviral gene delivery of PUMA induced apoptosis and chemosensitization more potently than did adenoviral delivery of p53 in HNSCC cells. Finally, we showed
that PUMA suppressed the growth of HNSCC xenograft
tumors and sensitized them to cisplatin through induction
of apoptosis. Our data suggest that absence of PUMA activation in HNSCC cells contributes to chemoresistance and
that gene therapy with PUMA might be an efficient substitute for p53 to enhance the responses of HNSCC cells
to chemotherapy. [Mol Cancer Ther 2007;6(12):3180 – 8]
Introduction
Head and neck cancer ranks the eighth most common
cancer worldwide (1). About 95% of head and neck tumors
are squamous cell carcinoma (HNSCC). Conventional
treatment options for HNSCC include surgery, radiation
therapy, and chemotherapy. Unfortunately, 70% or more of
the patients present with stage III or IV disease with a
5-year survival rate of 30% in spite of treatment (1). The
Received 4/11/07; revised 9/13/07; accepted 10/9/07.
Grant support: Flight Attendant Medical Research Institute (FAMRI), the
Alliance for Cancer Gene Therapy (ACGT), University of Pittsburgh Head
and Neck SPORE grant (P50CA097190) career development award
(J. Yu). SMG is supported in part by NIH R01DE014729 and University of
Pittsburgh Head and Neck SPORE grant (P50CA97190).
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: Jian Yu, University of Pittsburgh Cancer Institute,
Hillman Cancer Center Research Pavilion, Suite 2.26H, 5117 Centre
Avenue, Pittsburgh, PA 15213. Phone: 412-623-7786;
Fax: 412-623-7778. E-mail: [email protected]
Copyright C 2007 American Association for Cancer Research.
doi:10.1158/1535-7163.MCT-07-0265
mortality is mostly due to a high rate of tumor recurrence
and the development of second primary tumors (2).
Therefore, a better understanding of the molecular mechanisms of HNSCC pathogenesis is expected to facilitate the
development of novel therapies for this disease.
Emerging evidence suggests that deregulated programmed cell death or apoptosis is a major contributor to
tumor initiation, progression, and development of acquired
resistance to anticancer therapies (3 – 6). Apoptosis induced
by a wide range of anticancer agents is largely mediated
through the intrinsic pathway by the Bcl-2 family proteins
(7 – 9). An increased understanding of deregulated apoptosis in cancer has led to efforts to restore apoptosis in cancer
cells for therapeutic purposes (10). For example, a defective
p53 pathway is one of the most common signatures of
human cancer and is associated with poor response to
therapy in some cancers including HNSCC (11). Several
recent reports using sophisticated animal models showed
that restoration of p53 function induced tumor regression,
owing in part to p53-induced apoptosis (12 – 14). The
combination of p53 restoration with DNA-damaging agents
further increased the survival of tumor-bearing mice in a
lymphoma model (12). Moreover, p53 replacement gene
therapy has been tested extensively in preclinical and
clinical settings and shown to improve the therapeutic
responses of a number of solid tumors including HNSCC
(15, 16). Most of these studies also showed that the
enhanced therapeutic effect is associated with increased
apoptosis (15, 16). Several recent studies also showed that
small-molecule compounds that mimic the functions of the
BH3 domain or the mitochondrial apoptogenic protein
Smac sensitized cancer cells or xenograft tumors to conventional chemotherapies by enhancing mitochondriamediated apoptosis (17, 18).
We and others discovered PUMA as a transcriptional
target of p53 and a potent apoptosis inducer in cancer cells
(19 – 21). PUMA is a member of the BH3-only Bcl-2 protein
family, members of which are suggested to initiate
apoptosis in a tissue and stimulus-specific manner (6, 22).
Biochemically, PUMA functions through Bax/Bak by
antagonizing the antiapoptotic activities of the Bcl-2 like
proteins, including, but not limited to, Bcl-2 and Bcl-xL, to
trigger mitochondrial dysfunction and caspase activation
(23, 24). Mounting evidence suggests that PUMA is an
essential mediator of apoptosis induced by a wide range of
anticancer agents. For example, PUMA is induced by DNAdamaging agents in a p53-dependent manner (19, 23, 25).
Targeted deletion of PUMA resulted in resistance
to apoptosis induced by p53, DNA-damaging agents,
g-irradiation, and hypoxia in human colorectal cancer cells
(23). Moreover, studies with PUMA-deficient mice also
showed that PUMA is required for apoptosis induced by
oncogenes and DNA-damaging agents in several cell types
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Molecular Cancer Therapeutics
(26, 27). Although PUMA has been shown to regulate anticancer drug-induced apoptosis in a variety of cell types, its
role in the therapeutic response of HNSCC cells has not
been studied.
In this study, we examined PUMA expression in HNSCC
cells following treatment with anticancer agents and its role
in the induction of apoptosis and chemosensitization
in vitro and in vivo. Our results showed that PUMA is not
induced by commonly used chemotherapies in most
HNSCC cell lines. High levels of adenoviral delivered
PUMA expression alone induce extensive apoptosis in
HNSCC cells, whereas lower levels enhance the therapeutic
response of HNSCC cells to chemotherapy.
Materials and Methods
Cell Culture and DrugTreatment
The cell lines UPCI:SCC003, UPCI:SCC104,
UPCI:SCC105, UPCI:SCC116 (28, 29), 1483 (30), UPCI-15B
(15B; ref. 31), UM-SCC-22A, UM-SCC-22B, JHU-012 (32),
and two early-passage normal human keratinocyte primary
cultures, HN1771 and HN1871 (28), used in the study were
from the University of Pittsburgh Cancer Institute (UPCI)
Head and Neck cancer program. All cell lines were
maintained at 37jC in 5% CO2. Cell culture media included
DMEM (Mediatech) for 1483 and 15B cells, RPMI 1640
(Cellgro) for JHU-012 cells, and EMEM (Invitrogen) for the
UPCI:SCC cell lines and the primary cultures. The cell
culture media were supplemented with 10% fetal bovine
serum (HyClone), 100 units/mL penicillin, and 100 Ag/mL
streptomycin (Invitrogen). The anticancer drugs used in
the study, including Adriamycin, cisplatin, 5-fluorouracil
(5-FU), etoposide, mitomycin C, and Taxol, were purchased
from Sigma. Caspase inhibitors including the pan-caspase
inhibitor Z-VAD-fmk, caspase-3 inhibitor Z-DEVD-fmk,
and caspase-9 inhibitor Z-LEHD-fmk were purchased from
R&D Systems. All drugs were dissolved in DMSO and
diluted to the appropriate concentrations with cell culture
media. Some cells were exposed to g-irradiation at 8 Gy.
For combination treatments, cells were infected with adenoviruses for 24 h before drug treatment or g-irradiation
in virus-free media.
Adenoviruses
The recombinant adenoviruses, Ad-PUMA, Ad-DBH3,
and Ad-p53, constructed using the Ad-Easy system, were
previously described (23, 33). High-titer viruses were
produced in HEK-293 cells and purified by CsCl gradient
ultracentrifugation.
Apoptosis and Growth Assays
After treatment, adherent and floating cells were harvested and analyzed for apoptosis by nuclear staining with
Hoechst 33258. A minimum of 300 cells were analyzed for
each treatment. The Hoechst-stained cells were analyzed by
flow cytometry to determine the fraction of sub-G1 cells.
Cell growth was measured by the 3-(4,5-dimethyl-thiazol2yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H tetrazolium (MTS) assay in 96-well plates (2,500 cells per
well) using CellTiter 96 AQueous One Solution (Promega)
following the manufacturer’s instructions. A 490 nm was
measured with a Victor III (Perkin-Elmer/Wallace) plate
reader. Each experiment was done in triplicate and
repeated at least twice. For colony formation assays, equal
numbers of cells were subjected to various treatments and
plated into 6- or 12-well plates at different dilutions.
Colonies were visualized by crystal violet staining 11 to
14 days after plating as previously described (33).
Western Blotting and Antibodies
Western blotting was done as previously described (19).
The antibodies used for Western blotting included those
against caspase-3 (Stressgen), caspase-9 (Cell Signaling
Technology), a-tubulin, p21 (EMD Chemicals, Inc.), hemagglutinin (for Ad-PUMA and Ad-DBH3), p53, p27, c-Myc,
cyclin D1 (Santa Cruz Biotechnology), and PUMA (23).
Xenograft Tumors and Tissue Staining
All animal experiments were approved by the Institutional Animal Care and Use Committee at the University of
Pittsburgh. Xenograft tumors (f50 – 100 mm3) were established by s.c. injection of 5 106 1483 cells into both flanks
of 5- to 6-week-old female athymic nude mice (Harlan).
A total of three intratumoral injections of Ad-PUMA or
Ad-DBH3 in 100 AL of PBS were administered (on days 1, 3,
and 5). In the combination model, cisplatin (3 mg/kg/d)
was injected i.p. into the mice (on days 6 – 10). Tumor
growth was monitored every other day with calipers
to calculate tumor volumes according to the formula
(length width2) / 2. To avoid potential systemic effects
of different viruses, Ad-PUMA and Ad-DBH3 were injected
into separate tumors in the same animals. Six tumors were
analyzed for each group.
Two additional tumors in each group were harvested
during the experiments and embedded in optimum cutting
temperature compound (Tissue-Tek). In the single-agent
experiments, tumors were harvested 48 h after the second
Ad-PUMA treatment (day 5). In the combination experiments, tumors were harvested following the second
treatment of cisplatin (day 8). Mice were injected i.p. with
a single dose of bromodeoxyuridine (BrdUrd, dissolved in
PBS to a final concentration of 30 mg/mL) at 150 mg/kg of
body weight, 2 h before sacrifice to label cells in S phase.
Histologic and immunofluorescence analysis were done
using 5-Am frozen sections. The expression of transgene
was determined by fluorescence microscopic analysis of
green fluorescent protein. All images were acquired with a
Nikon TS800 fluorescence microscope using SPOT camera
imaging software.
Terminal deoxyribonucleotidyl transferase – mediated
dUTP nick end labeling (TUNEL) staining on frozen
sections was done with recombinant terminal transferase
(Roche) and dUTP-Alexa 594 (Invitrogen) according to the
manufacturers’ instructions and counterstained with 4¶,6diamidino-2-phenylindole. Apoptotic cells were counted
under a fluorescence microscope in randomly chosen fields,
and the apoptosis index was calculated as a percentage of
TUNEL-positive cells in at least 1,000 scored cells.
BrdUrd incorporation was visualized with anti – BrdUrdAlexa 594 antibody (Molecular Probes) on frozen sections
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Figure 1. PUMA expression in
head and neck cancer cells and
normal oral keratinocytes following
the treatment of chemotherapeutic
agents. PUMA, p53, and tubulin
(loading control) in total cell extract
were detected by Western blotting.
The two major isoforms of PUMA
(a and h) were indicated by arrows.
A, the indicated cancer cell lines
were treated with 5-FU (50 Ag/mL)
or untreated for 24 h. B, the indicated cell lines were treated with cisplatin (Cis, 25 Amol/L) for 48 h. C,
two normal oral keratinocyte primary
cultures were treated with 5-FU
(50 Ag/mL) or Adriamycin (Adr ;
0.2 Ag/mL). D, the relative levels of
PUMA in the cell lines or primary
cultures following 5-FU treatment
were analyzed and quantitated by
densitometry.
and nuclei were visualized with 4¶,6-diamidino-2-phenylindole. The proliferation index was calculated by counting
cells under a fluorescence microscope in several randomly
chosen fields. The BrdUrd index was calculated as a
percentage of BrdUrd-positive cells in at least 1,000 scored
cells.
Statistical Analysis
Statistical analysis was done with GraphPad Prism IV
software. P values were calculated with Student’s t test
or two-way ANOVA. P < 0.05 was considered significant.
The mean values F SD were displayed in the figures.
Results
PUMA Induction by Chemotherapeutic Agents Is
Abolished in Head and Neck Cancer Cells
PUMA is normally expressed at low basal levels and can
be induced by p53 or DNA-damaging agents (19, 20, 26,
27, 34). To investigate the regulation of PUMA by anticancer agents in head and neck cancer cells, eight HNSCC
cell lines were treated with two commonly used chemotherapeutic agents, 5-FU (50 Ag/mL) and Adriamycin
(0.2 Ag/mL), which are known to induce PUMA in colon
and lung cancer cells with wild-type p53 (19, 34). The
expression of PUMA was analyzed by Western blotting.
PUMA was not induced by 5-FU in six of eight cell lines,
and only slightly induced in two cell lines (UPCI:SCC104
and UM-SCC-22B; Fig. 1A). Similar results were obtained
with Adriamycin (data not shown). We then treated three
HNSCC cell lines with cisplatin, an alkylating agent
commonly used in HNSCC treatment. PUMA was not
induced in any of these lines by cisplatin even when
p53 was induced (15B; Fig. 1B). Four of the nine HNSCC
lines were reported to have wild-type p53 (Supplementary
Table S1; refs. 30, 32, 35, 36).6 Unlike in the cancer cell lines,
PUMA was found to be induced by 5-FU and Adriamycin
in two normal oral keratinocyte primary cultures, 1771N
and 1871N (Fig. 1C). PUMA expression was then directly
compared in several HNSCC cancer cell lines and these
primary cultures. We found that PUMA levels were
significantly lower in HNSCC cells following 5-FU treatment (Fig. 1D). These results indicate that PUMA induction
by chemotherapeutic agents is abolished in most HNSCC
cells.
PUMA Induces Extensive Apoptosis in HNSCC Cells
The lack of PUMA induction in HNSCC cells prompted
us to investigate the effects of PUMA expression on growth
and apoptosis in these cells. Six head and neck cancer cell
lines were infected with an adenovirus expressing PUMA
(Ad-PUMA) or a control adenovirus (Ad-DBH3) expressing
PUMA lacking the BH3 domain, which is essential for its
proapoptotic function (23). All of the cell lines were
infected by 40 MOI (multiplicity of infection) of adenovirus,
with efficiencies between 80% and 100% based on green
fluorescent protein – positive cells (Fig. 2B and data not
shown). Ad-PUMA, but not Ad-DBH3, caused massive
apoptosis in these cells 48 h after infection, as determined
6
Supplementary material for this article is available at Molecular Cancer
Therapeutics Online (http://mct.aacrjournals.org/).
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Molecular Cancer Therapeutics
by a nuclear fragmentation assay (Fig. 2A and B).
Measuring long-term cell viability by the colony formation
assay in 1483 and 15B cells showed that PUMA potently
inhibited cell growth (Fig. 2C and D).
PUMA Induces Apoptosis and Suppresses Growth
More Potently than Does p53 in HNSCC Cells
Numerous in vitro and in vivo studies have shown
that p53 adenovirus (Ad-p53) induces apoptosis and
growth suppression in cancer cells (16). To test the potential
use of PUMA in cancer gene therapy, Ad-PUMA and
Ad-p53, an adenovirus expressing p53 constructed in
the same system, were compared for their potency in
apoptosis induction and growth suppression in 1483 and
15B cells. These two cell lines were selected because of their
robust growth and ability to form xenograft tumors in nude
mice (1483). Using several titers of Ad-PUMA and Ad-p53,
we found that Ad-p53 was inefficient in apoptosis
induction in 1483 and 15B cells, even at 80 and 160 MOI,
compared with Ad-PUMA at 40 and 80 MOI (Fig. 3A and
data not shown). Ad-p53 was able to induce apoptosis at
titers >200 MOI in some HNSCC lines (data not shown).
This result was also confirmed by analyzing the sub-G1
fraction of cells using flow cytometry (Supplementary
Fig. S1A).6 Because p53 induces growth suppression and/
or apoptosis, depending on the cellular context, we compared the effects of Ad-PUMA and Ad-p53 on cell growth
by the MTS assay. Ad-PUMA seemed to suppress cell
proliferation better than Ad-p53 in 1483 and 15B cells at
24 and 48 h (Fig. 3B).
Consistent with the above observations, Ad-PUMA, but
not Ad-p53, was found to induce activation of caspase-3
and caspase-9 by Western blotting (Fig. 3C). PUMAinduced apoptosis was efficiently blocked by the pancaspase inhibitor Z-VAD in 1483 and 15B cells. The
caspase-3 and caspase-9 inhibitors also blocked PUMAinduced apoptosis, but to a lesser extent (Supplementary
Fig. S1B and C).6 In addition, Ad-p53 induced high levels of
p21, but not PUMA, consistent with its growth-suppressive
effects on HNSCC cells (Fig. 3C and B). We also examined
expression of several other cell cycle – related proteins and
found that p27 and cyclin D1 levels were suppressed by
PUMA but not by p53 (Fig. 3C). c-Myc levels were not
significantly affected by PUMA or p53 (Fig. 3C). These
results show that PUMA is more potent than p53 in
apoptosis induction and growth suppression in HNSCC
cells.
Figure 2. PUMA-induced apoptosis in HNSCC cells. A, the indicated HNSCC cell lines were infected with PUMA adenovirus (Ad-PUMA) or the BH3domain deleted PUMA adenovirus (Ad-DBH3) for 48 h. Apoptosis was analyzed by nuclear fragmentation assay. B, an example of PUMA-induced
apoptosis in UPCI:SCC116 cells that were subjected to the indicated treatments for 48 h. The cells were visualized with phase-contrast (phase ) or
fluorescence (GFP ) microscopy. The nuclei of the harvested cells were stained with 4¶,6-diamidino-2-phenylindole (DAPI ). C, 1483 and 15B cells were
infected with the indicated adenoviruses or left untreated for 24 h, then plated at 1:500 dilution (f400 cells per well) in a 12-well plate and allowed to form
colonies for 14 d. D, number of colonies in C containing >50 cells were scored.
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Figure 3. PUMA induces apoptosis and suppresses growth more
potently than p53 in HNSCC cells.
A, apoptosis was quantitated by the
nuclear fragmentation assay in 1483
(left ) and 15B (right ) cells following
Ad-PUMA, Ad-DBH3, and Ad-p53
infection at 24, 48, and 72 h. B,
the growth-suppressive effects of
Ad-PUMA and Ad-p53 were compared by MTS assays. The growth of
the cells infected with Ad-DBH3 was
considered to be 100%, which was
similar to that of uninfected cells
(data not shown). C, the expression
of PUMA, p21, p53, caspase-3,
caspase-9, c-Myc, p27, and cyclin
D1 was analyzed by Western blotting. Arrows, active forms of caspases.
PUMA Sensitizes HNSCC Cells to Chemotherapeutic
Agents and ;-Irradiation
The important role of PUMA in DNA damage – induced
apoptosis predicts that elevated PUMA expression can
sensitize cancer cells to some anticancer agents. To test this
hypothesis, 1483 cells were treated with a lower dose of
Ad-PUMA (10 MOI, which is not sufficient to induce
apoptosis) alone or in combination with g-irradiation or
chemotherapeutic agents including mitomycin C, Adriamycin, cisplatin, etoposide, Taxol, and 5-FU. PUMA, but
not PUMA-DBH3, was found to significantly enhance the
growth inhibitory effects of these agents (Fig. 4A).
Interestingly, PUMA seems to synergize best with several
agents that induce either DNA strand breaks or cross-links,
such as mitomycin C, Adriamycin, cisplatin, and girradiation (Fig. 4A). For example, growth suppression
increased >5-fold when Ad-PUMA was combined with
mitomycin C or Adriamycin compared with the drug alone
or in combination with Ad-DBH3. Furthermore, we found
that PUMA significantly enhanced the growth inhibitory
effects of Adriamycin at concentrations between 0.05 and
0.8 Ag/mL in both 1483 and 15B cells (Fig. 4B).
We then tested whether apoptosis induction is a major
mechanism of PUMA-mediated chemosensitization. AdPUMA (10 MOI) and Adriamycin (0.2 Ag/mL) alone did not
induce significant apoptosis in 15B cells, but their combination dramatically increased apoptosis (Fig. 4C). In
contrast, the combination of Ad-p53 (10 MOI) and Adriamycin did not result in an obvious increase in apoptosis.
Analyzing apoptosis using other approaches, including
cell cycle analysis and Annexin V staining, confirmed
these results (data not shown). As we expected, the
Adriamycin and Ad-PUMA combination resulted in significant activation of caspase-3 and caspase-9, whereas
the single agent or the Adriamycin and Ad-p53 combination did not (Fig. 4D). These data indicate that PUMA
synergizes with different chemotherapeutic agents and
g-irradiation to induce growth suppression and apoptosis
in HNSCC cells.
PUMA Suppresses HNSCC Tumor Growth In vivo
To determine whether PUMA confers antitumor activity
in vivo, established 1483 xenograft tumors (f100 mm3)
were treated with three intratumoral injections of AdPUMA (5 108 plaque-forming units), Ad-DBH3, or PBS
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(Fig. 5A). Ad-DBH3 did not have an effect on tumor growth
compared with PBS alone, with tumors reaching 12 times
their initial volumes in 26 days (Fig. 6A and B). In contrast,
Ad-PUMA treatment resulted in 67% growth suppression
(P < 0.001; Fig. 5A). Analyzing tissue sections from tumors
harvested 48 h after the second injection revealed that the
transgenes were highly expressed in the tumors (Fig. 5B,
GFP). Ad-PUMA, but not Ad-DBH3 treatment, significantly
increased apoptosis and decreased proliferation in the
tumors as assessed by TUNEL and BrdUrd staining,
respectively (Fig. 5B). These data show that PUMA
effectively inhibits the growth of established HNSCC
tumors through apoptosis induction.
PUMA Sensitizes HNSCC Xenografts to Cisplatin
PUMA was found to enhance growth suppression
induced by cisplatin in both short-term and long-term
growth assays in vitro (Figs. 4A and 6A). We wanted to
determine whether such effects can be obtained in the 1483
xenograft tumor model. To see the potential additive or
synergistic effects, we used suboptimal doses of Ad-PUMA
(1.25 108 plaque-forming units/d for 3 days) and
cisplatin (3 mg/kg/d for 5 days) in these experiments.
Ad-PUMA or cisplatin treatment alone resulted in f35%
(P < 0.01) and 16% (P < 0.01) growth inhibition compared
with Ad-DBH3 or PBS control, respectively (Fig. 6B and
data not shown). In contrast, Ad-PUMA combined with
cisplatin resulted in >60% growth suppression (P < 0.01),
indicating synergism (Fig. 6B). Analysis of tumor sections
after the second cisplatin injection (day 8) revealed that the
Ad-PUMA and cisplatin combination resulted in a significant increase in apoptosis and a decrease in cell proliferation compared with Ad-PUMA alone or the Ad-BH3 and
cisplatin combination (Fig. 6C and Supplementary Fig. S2).6
These results suggest that increased PUMA levels enhance
the therapeutic responses of HNSCC tumors to cisplatin
through apoptosis induction.
Discussion
PUMA is located in an area of chromosome 19q13.3 that
frequently undergoes loss of heterozygosity in HNSCC, but
the PUMA gene is not found to be mutated or silenced in
HNSCC (32). Our study showed that PUMA induction by
chemotherapeutic agents is abrogated in most HNSCC
cells. Perhaps it is deregulated due to the defects in
upstream signaling molecules including p53. Adenoviral
Figure 4.
PUMA sensitizes HNSCC to chemotherapeutic agents through apoptosis induction. In combination treatments, cells were infected by the
indicated adenovirus at 10 MOI, then treated by chemotherapeutic agents or radiation in virus-free medium. A, 1483 cells were treated with
chemotherapeutic agents and g-irradiation alone, or in combination with Ad-PUMA or Ad-DBH3. Cell growth was measured at 48 h by the MTS assay. The
ratios of growth inhibition conferred by the combinations over drug alone. MMC, mitomycin C; Oxa, oxaliplatin; Etp, etoposide; IR, g-irradiation. B, the
effects on the growth of cells treated with Adriamycin at different concentrations combined with Ad-PUMA or Ad-DBH3 were determined in 1483 and
15B cells. C, the cells were subjected to indicated treatments for 24 and 48 h. Apoptosis was quantitated by nuclear fragmentation assay. Adriamycin,
0.2 Ag/mL. D, the expression of PUMA (DBH3), p53, p21, caspase-3, and caspase-9 was analyzed by Western blotting. Arrows, active forms of caspases.
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gene delivery of PUMA resulted in apoptosis and enhanced
sensitivity to chemotherapeutic agents and g-irradiation in
HNSCC cells, suggesting that adequate levels of PUMA are
crucial for triggering apoptotic responses to these agents.
Lack of PUMA induction may contribute to the chemoresistant phenotype of HNSCC cells. Our data also suggest
that PUMA might be particularly useful when combined
with DNA-damaging agents, such as mitomycin C,
Adriamycin, cisplatin, or g-irradiation, to induce cell
killing, which is generally believed to be more effective in
cancer treatment than cytostasis (4, 5). Inhibition of cell
proliferation has been attributed to the antitumor effects of
chemotherapy. Our data indicate that Ad-PUMA negatively affects several cell cycle regulators, suggesting a possible
mechanism for enhanced growth suppression when combined with chemotherapy (Figs. 3C and 4A). These
observations are consistent with the findings that PUMA
plays a critical role in DNA damage – induced apoptosis
and sensitizes lung and esophageal cancer cells to
chemotherapeutic agents (23, 26, 27, 34, 37). It will be
interesting to further investigate whether PUMA is an
effective radiosensitizer and chemosensitizer in other
tumor types.
The prevalence of p53 dysfunction in cancer and the
importance of p53-mediated apoptosis in tumor initiation
and progression have made p53 a very appealing target for
cancer therapy (15). A recent study showed that restoration
of p53 function in vivo induced tumor regression through
apoptosis induction, and its combination with DNAdamaging agents enhanced therapeutic efficacy in a
lymphoma model (12). These observations complemented
the numerous earlier studies, in which nonreplicating
adenoviruses expressing p53 were shown to suppress
cancer cell growth and induce apoptosis (16). Combinations of p53 gene therapy with chemotherapy or radiation
exhibit synergistic antitumor effects in clinical trials as well
as in various in vitro and in vivo models (16). Particularly,
adenoviral p53 gene therapy has been explored as a new
modality in head and neck cancer, primarily due to its
anatomic location and the morbidity and mortality largely
related to local recurrence (2, 16). Just a few years ago,
Gendicine, a p53 adenovirus, became the first approved
cancer gene therapy product in the world to treat head and
neck cancer in China and has shown encouraging results
(38). Our data suggest that PUMA induces apoptosis and
chemosensitization more potently than p53 itself in
HNSCC cells, consistent with the findings reported in lung
and esophageal cancer cells (34, 37). Based on these
observations, PUMA might be a more effective radiosensitizer and chemosensitizer than p53 in solid tumors.
However, intratumoral injection of Ad-PUMA has limitations in clinical settings, and therefore it will be interesting
to explore other means of delivering PUMA gene to tumor
cells.
One limitation of p53 gene therapy is for tumors that are
positive for human papilloma virus. Studies indicate that
20% to 25% of head and neck cancers contain oncogenic
human papilloma viruses, whereas >95% of cervical
squamous cell carcinomas are linked to persistent human
papilloma virus infection (39). The human papilloma
virus – encoded E6 oncoprotein promotes p53 degradation
and inactivation and renders tumors resistant to p53
gene therapy (39). PUMA has not been shown to be
subjected to E6-mediated degradation. In addition, we
and others have shown that some tumor cells are resistant
to Ad-p53 – induced apoptosis due to high levels of the
Figure 5. PUMA suppresses the growth of HNSCC xenograft tumors through apoptosis induction. A, growth curve of 1483 tumors (n = 6 per group)
subjected to Ad-PUMA, Ad-DBH3, and PBS treatments. The three intratumoral treatments were indicated by arrows. B, frozen sections of 1483 tumors
48 h after the second injection (day 5) were analyzed by H&E staining. Apoptosis was analyzed by TUNEL staining (red ). Proliferation was analyzed by
BrdUrd (BrdU ) incorporation (red). The nuclei were counterstained by 4¶,6-diamidino-2-phenylindole (blue ). Magnification, 200. Bottom, apoptotic and
growth indices of TUNEL-positive or BrdUrd-labeled cells, respectively.
Mol Cancer Ther 2007;6(12). December 2007
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Molecular Cancer Therapeutics
Figure 6. PUMA sensitizes HNSCC xenograft tumors to cisplatin. A, PUMA enhanced cisplatin-induced growth suppression in long-term culture. An
equal number of 1483 cells were infected with Ad-PUMA or Ad-BH3 (10 MOI) and then treated with cisplatin for 48 h. Cells plated in a 12-well plate at
1:500 dilution were allowed to form colonies. Bottom, number of colonies with >50 cells. B, growth curve of 1483 tumors (n = 6 per group) subjected to
Ad-DBH3 alone, Ad-PUMA alone, Ad-DBH3 plus cisplatin, or Ad-PUMA plus cisplatin treatments. The three treatments of Adenovirus are indicated by black
arrows, whereas the five treatments of cisplatin are indicated by red arrows. *, P < 0.01. C, index of TUNEL-positive or BrdUrd-labeled cells in 1483
tumors 24 h after the second injection of cisplatin (day 8), determined as in B. *, P < 0.001.
cyclin-dependent kinase inhibitor p21, a p53 target primarily involved in cell cycle arrest (refs. 23, 40; Fig. 4D). On the
other hand, Ad-PUMA induces apoptosis in a variety of
cancer cells independent of their p53 status (refs. 23, 34, 37;
Fig. 2A). These observations suggest that selectively
activating apoptosis rather than cell cycle arrest is
therapeutically desirable. Based on its biological properties,
PUMA gene therapy might be particularly useful in tumors
that are resistant to p53 gene therapy.
Emerging evidence suggests that the BH3-only proteins
are critical initiators of apoptosis and function in a tissueand stimulus-specific manner (9, 22). Most BH3-only
proteins selectively antagonize a subset of the antiapoptotic
Bcl-2 family members, whereas PUMA, tBid, and Bim
antagonize all known members including Bcl-2, Bcl-xL,
Bcl-w, Mcl-1, and A1, some of which are frequently overexpressed in cancer (22, 41). This might explain why PUMA
and Bim are more potent in promoting apoptosis in a wide
range of cancer cells compared with other BH3-only proteins
(22). The studies with knockout animals also suggest that
PUMA seems to be a major mediator of apoptosis induced
by DNA damage, oncogenes, and growth factor deprivation
in several cell types, and it integrates death signals in both
a p53-dependent and a p53-independent manner (21, 26, 27).
Tumor cells overcome a great deal of apoptotic pressure
often by deregulating both antiapoptotic and proapoptotic
proteins, and thus become highly dependent on these
oncogenic changes (3). The so-called ‘‘oncogenic addition’’
of cancer cells might explain why proapoptotic genes like
p53 or PUMA seem to be less toxic to normal cells and offer
some degree of much needed therapeutic index in cancer
treatment.
Acknowledgments
We thank other members of our laboratories for helpful discussions and
comments and Drs. Jennifer Grandis and Robert Ferris at University of
Pittsburgh for providing some of the HNSCC cell lines.
References
1. Jemal A, Siegel R, Ward E, Murray T, Xu J, Thun MJ. Cancer statistics,
2007. CA Cancer J Clin 2007;57:43 – 66.
2. Vikram B. Adjuvant therapy in head and neck cancer. CA Cancer J Clin
1998;48:199 – 209.
3. Hanahan D, Weinberg RA. The hallmarks of cancer. Cell 2000;100:
57 – 70.
4. Coultas L, Strasser A. The molecular control of DNA damage-induced
cell death. Apoptosis 2000;5:491 – 507.
Mol Cancer Ther 2007;6(12). December 2007
Downloaded from mct.aacrjournals.org on June 18, 2017. © 2007 American Association for Cancer Research.
3187
3188 Chemosensitization by PUMA in HNSCC
5. Johnstone RW, Ruefli AA, Lowe SW. Apoptosis: a link between cancer
genetics and chemotherapy. Cell 2002;108:153 – 64.
PUMA-mediated apoptosis induced by DNA damage. Proc Natl Acad Sci
U S A 2007;104:4054 – 9.
6. Yu J, Zhang L. Apoptosis in human cancer cells. Curr Opin Oncol 2004;
16:19 – 24.
26. Villunger A, Michalak EM, Coultas L, et al. p53- and drug-induced
apoptotic responses mediated by BH3-only proteins Puma and Noxa.
Science 2003;302:1036 – 8.
7. Horvitz HR. Genetic control of programmed cell death in the nematode
Caenorhabditis elegans. Cancer Res 1999;59:1701 – 6s.
8. Korsmeyer SJ. BCL-2 gene family and the regulation of programmed
cell death. Cancer Res 1999;59:1693 – 700s.
9. Cory S, Huang DC, Adams JM. The Bcl-2 family: roles in cell survival
and oncogenesis. Oncogene 2003;22:8590 – 607.
10. Reed JC. Apoptosis-targeted therapies for cancer. Cancer Cell 2003;
3:17 – 22.
11. Vogelstein B, Kinzler KW. Cancer genes and the pathways they
control. Nat Med 2004;10:789 – 99.
12. Martins CP, Brown-Swigart L, Evan GI. Modeling the therapeutic
efficacy of p53 restoration in tumors. Cell 2006;127:1323 – 34.
13. Ventura A, Kirsch DG, McLaughlin ME, et al. Restoration of p53
function leads to tumour regression in vivo . Nature 2007;445:661 – 5.
14. Xue W, Zender L, Miething C, et al. Senescence and tumour clearance
is triggered by p53 restoration in murine liver carcinomas. Nature 2007;
445:656 – 60.
15. Lane DP, Lain S. Therapeutic exploitation of the p53 pathway. Trends
Mol Med 2002;8:S38 – 42.
27. Jeffers JR, Parganas E, Lee Y, et al. Puma is an essential mediator of
p53-dependent and -independent apoptotic pathways. Cancer Cell 2003;
4:321 – 8.
28. Huang X, Gollin SM, Raja S, Godfrey TE. High-resolution mapping of
the 11q13 amplicon and identification of a gene, TAOS1, that is amplified
and overexpressed in oral cancer cells. Proc Natl Acad Sci U S A 2002;99:
11369 – 74.
29. White JS, Weissfeld JL, Ragin CC, et al. The influence of clinical and
demographic risk factors on the establishment of head and neck squamous
cell carcinoma cell lines. Oral Oncol 2007;43:701 – 12.
30. Lin CJ, Grandis JR, Carey TE, et al. Head and neck squamous cell
carcinoma cell lines: established models and rationale for selection. Head
Neck 2007;29:163 – 88.
31. Heo DS, Snyderman C, Gollin SM, et al. Biology, cytogenetics, and
sensitivity to immunological effector cells of new head and neck
squamous cell carcinoma lines. Cancer Res 1989;49:5167 – 75.
32. Hoque MO, Begum S, Sommer M, et al. PUMA in head and neck
cancer. Cancer Lett 2003;199:75 – 81.
16. Roth JA. Adenovirus p53 gene therapy. Expert Opin Biol Ther 2006;
6:55 – 61.
33. Yu J, Zhang L, Hwang PM, Rago C, Kinzler KW, Vogelstein B.
Identification and classification of p53-regulated genes. Proc Natl Acad Sci
U S A 1999;96:14517 – 22.
17. Oltersdorf T, Elmore SW, Shoemaker AR, et al. An inhibitor of Bcl-2
family proteins induces regression of solid tumours. Nature 2005;435:
677 – 81.
34. Yu J, Yue W, Wu B, Zhang L. PUMA sensitizes lung cancer cells to
chemotherapeutic agents and irradiation. Clin Cancer Res 2006;12:
2928 – 36.
18. Li L, Thomas RM, Suzuki H, De Brabander JK, Wang X, Harran PG. A
small molecule Smac mimic potentiates TRAIL- and TNFa-mediated cell
death. Science 2004;305:1471 – 4.
35. Kim MS, Li SL, Bertolami CN, Cherrick HM, Park NH. State of p53, Rb
and DCC tumor suppressor genes in human oral cancer cell lines.
Anticancer Res 1993;13:1405 – 13.
19. Yu J, Zhang L, Hwang PM, Kinzler KW, Vogelstein B. PUMA induces
the rapid apoptosis of colorectal cancer cells. Mol Cell 2001;7:673 – 82.
36. Yin XY, Smith ML, Whiteside TL, Johnson JT, Herberman RB, Locker J.
Abnormalities in the p53 gene in tumors and cell lines of human squamouscell carcinomas of the head and neck. Int J Cancer 1993;54:322 – 7.
20. Nakano K, Vousden KH. PUMA, a novel proapoptotic gene, is induced
by p53. Mol Cell 2001;7:683 – 94.
21. Han J, Flemington C, Houghton AB, et al. Expression of bbc3, a proapoptotic BH3-only gene, is regulated by diverse cell death and survival
signals. Proc Natl Acad Sci U S A 2001;98:11318 – 23.
22. Labi V, Erlacher M, Kiessling S, Villunger A. BH3-only proteins in cell
death initiation, malignant disease and anticancer therapy. Cell Death
Differ 2006;13:1325 – 38.
23. Yu J, Wang Z, Kinzler KW, Vogelstein B, Zhang L. PUMA mediates the
apoptotic response to p53 in colorectal cancer cells. Proc Natl Acad Sci
U S A 2003;100:1931 – 6.
24. Ming L, Wang P, Bank A, Yu J, Zhang L. PUMA dissociates Bax and
BCL-XL to induce apoptosis in colon cancer cells. J Biol Chem 2006;281:
16034 – 42.
25. Wang P, Yu J, Zhang L. The nuclear function of p53 is required for
37. Wang H, Qian H, Yu J, et al. Administration of PUMA adenovirus
increases the sensitivity of esophageal cancer cells to anticancer drugs.
Cancer Biol Ther 2006;5:380 – 5.
38. Peng Z. Current status of gendicine in China: recombinant human Adp53 agent for treatment of cancers. Hum Gene Ther 2005;16:1016 – 27.
39. Ragin CC, Modugno F, Gollin SM. The epidemiology and risk factors
of head and neck cancer: a focus on human papillomavirus. J Dent Res
2007;86:104 – 14.
40. Polyak K, Waldman T, He T-C, Kinzler KW, Vogelstein B. Genetic
determinants of p53 induced apoptosis and growth arrest. Genes Dev
1996;10:1945 – 52.
41. Chen L, Willis SN, Wei A, et al. Differential targeting of prosurvival
Bcl-2 proteins by their BH3-only ligands allows complementary apoptotic
function. Mol Cell 2005;17:393 – 403.
Mol Cancer Ther 2007;6(12). December 2007
Downloaded from mct.aacrjournals.org on June 18, 2017. © 2007 American Association for Cancer Research.
Chemosensitization of head and neck cancer cells by PUMA
Quanhong Sun, Tsukasa Sakaida, Wen Yue, et al.
Mol Cancer Ther 2007;6:3180-3188.
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