Crucial role of phospholipase Cepsilon in chemical carcinogen-induced skin tumor development.

Abstract

Mutational activation of the ras proto-oncogenes is frequently found in skin cancers. However, the nature of downstream signaling pathways from Ras involved in skin carcinogenesis remains poorly understood. Recently, we and others identified phospholipase C (PLC) epsilon as an effector of Ras. Here we have examined the role of PLCepsilon in de novo skin chemical carcinogenesis by using mice whose PLCepsilon is genetically inactivated. PLCepsilon(-/-) mice exhibit delayed onset and markedly reduced incidence of skin squamous tumors induced by initiation with 7,12-dimethylbenz(a)anthracene followed by promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA). Furthermore, the papillomas formed in PLCepsilon(-/-) mice fail to undergo malignant progression into carcinomas, in contrast to a malignant conversion rate of approximately 20% observed with papillomas in PLCepsilon(+/+) mice. In all of the tumors analyzed, the Ha-ras gene is mutationally activated irrespective of the PLCepsilon background. The skin of PLCepsilon(-/-) mice fails to exhibit basal layer cell proliferation and epidermal hyperplasia in response to TPA treatment. These results indicate a crucial role of PLCepsilon in ras oncogene-induced de novo carcinogenesis and downstream signaling from TPA, introducing PLCepsilon as a candidate molecular target for the development of anticancer drugs.

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[CANCER RESEARCH 64, 8808–8810, December 15, 2004]
Advances in Brief
Crucial Role of Phospholipase C? in Chemical Carcinogen-Induced Skin
Tumor Development
Yunfeng Bai,1Hironori Edamatsu,1Sakan Maeda,2Hiromitsu Saito,3Noboru Suzuki,3Takaya Satoh,1and
Tohru Kataoka1
1Division of Molecular Biology, Department of Molecular and Cellular Biology and2Division of Molecular Pathology, Department of Biomedical Informatics, Kobe University
Graduate School of Medicine, Kobe, Japan; and3Department of Animal Genomics, Functional Genomics Institute, Mie University Life Science Research Center, Mie, Japan
Abstract
Mutational activation of the ras proto-oncogenes is frequently found in
skin cancers. However, the nature of downstream signaling pathways
from Ras involved in skin carcinogenesis remains poorly understood.
Recently, we and others identified phospholipase C (PLC) ? as an effector
of Ras. Here we have examined the role of PLC? in de novo skin chemical
carcinogenesis by using mice whose PLC? is genetically inactivated.
PLC??/?mice exhibit delayed onset and markedly reduced incidence of
skin squamous tumors induced by initiation with 7,12-dimethylbenz(a)an-
thracene followed by promotion with 12-O-tetradecanoylphorbol-13-
acetate (TPA). Furthermore, the papillomas formed in PLC??/?mice fail
to undergo malignant progression into carcinomas, in contrast to a ma-
lignant conversion rate of approximately 20% observed with papillomas
in PLC??/?mice. In all of the tumors analyzed, the Ha-ras gene is
mutationally activated irrespective of the PLC? background. The skin of
PLC??/?mice fails to exhibit basal layer cell proliferation and epidermal
hyperplasia in response to TPA treatment. These results indicate a crucial
role of PLC? in ras oncogene-induced de novo carcinogenesis and down-
stream signaling from TPA, introducing PLC? as a candidate molecular
target for the development of anticancer drugs.
Introduction
The ras proto-oncogenes are mutationally activated in about 15%
of human neoplasms (1). Their products, Ras small GTPases, control
cell proliferation and differentiation through interaction with multiple
effector proteins, among which Raf kinases have been implicated in
carcinogenesis from studies on in vitro transformation of fibroblast
cell lines (2) and on genomic mutations in malignant melanoma (3).
However, downstream signaling pathways from Ras involved in ep-
ithelial cell carcinogenesis remain poorly understood, despite the fact
that ras mutations are more frequently found in epithelial cell-derived
neoplasms (1). Likewise, the role of phosphoinositide-specific phos-
pholipase C (PLC) in carcinogenesis remains obscure (4). PLC pro-
duces two vital intracellular second messengers, diacylglycerol and
inositol 1,4,5-trisphosphate, which induce activation of protein kinase
C and mobilization of Ca2?from intracellular stores, respectively.
Among 12 mammalian PLC isoforms classified into 5 classes (?, ?, ?,
?, and ?), PLC? is characterized by possession of the Ras-associating
domains, which are responsible for PLC? activation through direct
association with the GTP-bound active forms of the small GTPases
Ras (5, 6), Rap1 (7), and Rap2 (8). PLC? was also reported to be
regulated by ?12, ?13, and ?1?2subunits of heterotrimeric G proteins
and Rho small GTPase (9). Identification of PLC? as a Ras effector
has prompted us to examine the role of PLC? in carcinogenesis. Here
we show that PLC?-deficient mice are resistant to chemical carcino-
gen-induced skin tumor formation, suggesting a crucial role of PLC?
in tumor development downstream of Ras signaling.
Materials and Methods
PLC?/?Mice. Targeted inactivation of the PLC? gene was performed by a
standard embryonic stem cell-based method.4The targeted allele (PLC??)
expresses a mutant PLC? with an in-frame deletion of amino acids 1333 to
1408 corresponding to the NH2-terminal part of the catalytic X domain. This
mutant completely lost its PLC catalytic activity. PLC??/?mice were main-
tained on a mixed 129/Sv ? C57BL/6 background.
Reverse Transcription-Polymerase Chain Reaction Analysis. Reverse
transcription-polymerase chain reaction (RT-PCR) was performed as described
previously (10). Primers used for amplification of PLC? were 5?-TCAGTGC-
CTGGAGCAGCAG-3? and 5?-CTTGAAGGGGATCTTGGTTG-3?.
Skin Tumor Formation. A dorsal area of skin of 8-week–old mice was
shaved and treated with a single application of 7,12-dimethylbenz(a)anthra-
cene [DMBA (25 ?g in 100 ?L of acetone; Sigma, St. Louis, MO] and
subsequently treated with 12-O-tetradecanoyl-phorbor-13-acetate [TPA (0.2
mmol/L in 100 ?L of acetone; Sigma] twice a week for 20 weeks (11). Tumors
were assessed weekly for up to 30 weeks and defined as raised lesions with a
minimumdiameterof1mm.PvaluesweredeterminedbyunpairedStudent’sttest
using GraphPad InStat software (GraphPad Software, Inc., San Diego, CA).
Histologic Analysis. Paraffin-embedded sections were prepared and
stained with hematoxylin and eosin or with a specific antibody against mouse
PLC? (10), keratin 14 (PRB-155P; BAbCO, Berkeley, CA), or keratin 1 (PRB-
165P; BAbCO). Detection of immunoreactive signals was performed with Histo-
Mouse Plus kit (Zymed Laboratories, South San Francisco, CA) or with a fluo-
rescein isothiocyanate-conjugated secondary antibody (AP182F; Chemicon,
Temecula, CA).
12-O-Tetradecanoylphorbol-13-acetate–Induced Skin Hyperplasia. A
dorsal area of skin of 10-week–old mice was treated with TPA (0.2 mmol/L in
100 ?L of acetone). The mouse skin was analyzed by staining with an
anti-proliferating cell nuclear antigen (PCNA) antibody (M0879; Dako Cyto-
mation, Copenhagen, Denmark) or hematoxylin and eosin. The thickness of the
epidermis was measured at a minimum of five different points on the speci-
mens and averaged.
Analysis of Ha-ras Gene Mutations. Ha-ras gene mutations at the 61st
codon of the tumors were analyzed as described previously (12).
Results and Discussion
RT-PCR analysis of skin RNA detected two amplified products
whose sizes were identical to those predicted from the wild-type and
mutant PLC? mRNAs (Fig. 1A). Immunohistochemical analysis
Received 8/30/04; revised 10/12/04; accepted 10/21/04.
Grant support: Grant-in-Aid for Priority Areas 12215098 (T. Kataoka); Grants-in-
Aid for Scientific Research 15390093 (T. Kataoka), 16790187 (H. Edamatsu), and
15570117 (T. Satoh); and 21st Century COE Programs (T. Kataoka and T. Satoh) from the
Ministry of Education, Science, Sports and Culture of Japan.
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.
Note: Y. Bai and H. Edamatsu contributed equally to this work.
Requests for reprints: Tohru Kataoka, Division of Molecular Biology, Department of
Molecular and Cellular Biology, Kobe University Graduate School of Medicine, 7-5-1
Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan. E-mail: kataoka@kobe-u.ac.jp.
©2004 American Association for Cancer Research.
4M. Tadano, H. Edamatsu, S. Minamisawa, U. Yokoyama, Y. Ishikawa, N. Suzuki, H.
Saito, D. Wu, M. Masago-Toda, Y. Yamawaki-Kataoka, T. Setsu, T. Terashima, S.
Maeda, T. Satoh, and T. Kataoka. Congenital semilunar valvulogenesis defect in mice
deficient in phospholipase C?, submitted for publication.
8808

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showed that PLC? is expressed in the epidermis (Fig. 1B), including
keratin 14-positive proliferative keratinocytes and keratin 1-positive
differentiating keratinocytes, but not in the dermis, except for hair
follicles (Fig. 1C). To address the role of PLC? in de novo skin
carcinogenesis, we applied the skin two-stage chemical carcinogene-
sis protocol (11) on PLC??/?mice. Initiation was carried out with a
single application of DMBA, which almost invariably introduced
oncogenic mutations on the Ha-ras gene (11, 12). Subsequent pro-
motion by repeated treatment with TPA for 20 weeks caused the
selective clonal outgrowth of the initiated cells to produce benign
squamous tumors (Fig. 2A). PLC??/?mice showed significant delay
in the average time of tumor onset compared with PLC??/?mice
[average ? SE: 12.63 ? 0.42 weeks (PLC??/?; 21 mice analyzed)
versus 10.14 ? 0.47 weeks (PLC??/?; 14 mice); P ? 0.001; Fig. 2B].
PLC??/?mice showed an intermediate phenotype (11.79 ? 0.31
weeks; 23 mice; P ? 0.01), indicating the existence of an apparent
gene-dosage effect. The time to develop the first tumor also showed
a significant difference [PLC??/?, 6.06 ? 0.36 weeks; PLC??/?,
7.87 ? 0.30 weeks (P ? 0.001); PLC??/?, 9.86 ? 0.43 weeks
(P ? 0.0001)]. The number of tumors reached a maximum at 15
weeks. At this point, the average number of tumors per mouse was
reduced by approximately 70% in PLC??/?mice (4.14 ? 0.40;
P ? 0.0001) compared with PLC??/?mice (14.36 ? 1.25). Again,
PLC??/?mice showed an intermediate phenotype (10.22 ? 0.65;
P ? 0.0001; Fig. 2B). In PLC??/?mice, no tumor greater than 6 mm
in diameter was observed at 20 weeks (Fig. 2C). In the two-stage
protocol, a population of papillomas undergo progression into squa-
mous cell carcinoma (SCC) (11). At 30 weeks after initiation, tumors
of at least 2 mm in diameter were isolated and subjected to histologic
analysis (Table 1; Fig. 2D). In PLC??/?mice, approximately 20% of
the tumors were carcinomas. In contrast, essentially no carcinoma was
found in PLC??/?mice. PLC??/?mice showed a partial resistance to
malignant progression. Thus, PLC? deficiency strongly suppressed
malignant progression. All of the tumors tested carried the activating
mutations at the 61stcodon of the Ha-ras gene, irrespective of the
PLC? genetic background (data not shown).
We next investigated the effect of PLC? deficiency on TPA-
induced proliferation of the skin epidermis. Before or after treatment
Table 1 Histological analysis of tumors
PLC? genotypes
?/? (n ? 6)
?/? (n ? 14)
?/? (n ? 9)
Hyperplasias
Papillomas
Carcinomas
Carcinomas/tumors (%)
Total no. of tumors analyzed
NOTE. n represents the number of mice analyzed.
686
32
10
20.8
48
60 20
2
2.9
70
0
0
26
Fig. 1. Analysis of PLC? expression. A, RT-PCR analysis of PLC? mRNA in the skin.
?-Actin mRNA was used as an internal control. B and C, immunohistochemical analysis
of the expression of PLC?, keratin 14 (K14), and keratin 1 (K1) in the PLC??/?mouse
skin. Detection was performed with a fluorescein isothiocyanate-conjugated secondary
antibody (B) or the HistoMouse Plus kit (C). Scale bars, 100 ?m.
Fig. 2. Skin tumor formation. A, representative tumors developed in PLC??/?(?/?), PLC??/?(?/?), and PLC??/?(?/?) mice at 30 weeks after initiation. B, time course of
tumor formation. The average number of tumors per mouse (average ? SE) is shown. C, size distribution of tumors at 20 weeks after initiation. D, photomicrographs of hematoxylin
and eosin-stained sections of a representative SCC in a PLC??/?mouse (?/?) and a papilloma in a PLC??/?mouse (?/?) at 30 weeks. The SCC exhibits tumor invasion (black
arrow) and a cancer pearl with parakeratosis (white arrow). Scale bars, 1 mm.
8809
ROLE OF PLC? IN RAS-INDUCED SKIN CARCINOGENESIS

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with acetone, there was no apparent difference between PLC??/?and
PLC??/?mice in the skin architecture and the number of proliferating
cells positive for PCNA (Fig. 3). On TPA treatment, PLC??/?mouse
skin showed a marked increase in the number of PCNA-positive cells
in the basal layer cells (Fig. 3A). In striking contrast, PLC??/?mouse
skin showed only a moderate increase (Fig. 3A). TPA-induced epi-
dermal hyperplasia was also suppressed in PLC??/?mice (Fig. 3B).
The average thickness of the epidermis after 48 hours of TPA treat-
ment was 98.4, 66.3, and 31.3 ?m in PLC??/?, PLC??/?, and
PLC??/?mice, respectively, whereas that after acetone treatment was
27.7, 25.4, and 24.6 ?m, respectively.
We have shown here that PLC? plays a crucial role in skin papil-
loma formation and malignant progression, which are induced by ras
activation followed by TPA treatment. Furthermore, PLC? is shown to
function downstream of TPA to induce hyperproliferation of the basal
layer cells and skin hyperplasia. Thus, it is likely that PLC? functions
in TPA-induced tumor promotion of the initiated cells carrying the
activated ras genes. There are two possible mechanisms linking TPA
to PLC? activation. TPA may activate PLC? through Ras activation,
which is mediated by RasGRP1, a TPA-regulated Ras-specific gua-
nine nucleotide exchange factor (GEF) expressed in keratinocytes
(13). Rap1, whose activation is mediated by TPA-responsive Rap
GEFs including CalDAG-GEFI (14) and RasGRP2 (15), may also be
responsible for PLC? activation. Alternatively, TPA may activate
PLC? through secretion of tumor necrosis factor (TNF)-? from kera-
tinocytes (16) and subsequent TNF-?–induced Ras activation (17).
TNF-? has been implicated in both two-stage skin carcinogenesis and
TPA-induced skin hyperplasia (16).
Because targeted inactivation of protein kinase C (PKC) ? resulted
in enhancement of both papilloma formation and TPA-induced skin
hyperplasia, TPA-induced down-regulation of PKC? is thought to
play a crucial role in induction of these phenomena (18). In the present
study, TPA treatment failed to compensate for the deficiency in
papilloma formation and skin hyperplasia of PLC??/?mice, although
TPA is known to mimic diacylglycerol, a product of PLC?, in regu-
lating PKC?. The result indicates that the PLC? pathway has an
intrinsic role in skin hyperplasia and carcinogenesis, which is inde-
pendent of the PKC? pathway. This intrinsic function may be medi-
ated by another of its products, inositol 1,4,5-trisphosphate. On the
other hand, activation of PLC? in DMBA-initiated cells, which must
be induced by constitutively active Ras and produce diacylglycerol,
could not substitute for TPA treatment in promoting papilloma for-
mation. This suggests that TPA possesses another target that is also
required for tumor promotion. In addition, papillomas developed in
PLC??/?mice failed to undergo malignant conversion. It was re-
ported that prostaglandins are involved in skin tumor progression in
addition to promotion (19) and play a key role in intestinal polyposis
(20). Considering that arachidonic acid, a precursor of prostaglandins,
can be produced from diacylglycerol, it is possible that the role of
PLC? may be mediated through prostaglandin signaling.
Our present results have shown that PLC? plays a crucial role in ras
oncogene-induced de novo carcinogenesis of skin epithelial cells.
They also provide the first concrete evidence for the importance of the
PLC signaling in carcinogenesis. This leads to the idea that specific
inhibitors of PLC? may be useful for treatment and prevention of
certain types of cancer.
Acknowledgments
We thank Dr. Atsu Aiba, Dr. Ushio Kikkawa, Dr. Makoto Tadano, Shuzo
Ikuta, and Tadashi Murase for helpful discussion and Shuichi Matsuda for
excellent technical assistance.
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Fig. 3. Suppression of TPA-induced epidermal cell proliferation in PLC??/?mouse.
Mouse skin was treated with acetone alone (Vehicle) or with TPA in acetone (TPA), or left
untreated (No treatment). The sections were examined by staining with the anti-PCNA
antibody at 24 hours (A) or by staining with hematoxylin and eosin at 48 hours (B).
Representative photomicrographs of at least three independent experiments are shown.
The frequency of PCNA-positive basal layer cells is shown as percentage in A. Scale bars,
100 ?m.
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ROLE OF PLC? IN RAS-INDUCED SKIN CARCINOGENESIS