Loss of transforming growth factor-?
type II receptor promotes metastatic
head-and-neck squamous cell carcinoma
Shi-Long Lu,1Heather Herrington,1Douglas Reh,1Stephen Weber,1Sophia Bornstein,1
Donna Wang,1Allen G. Li,1,3Chin-Fang Tang,1Yasmin Siddiqui,1Jo Nord,1Peter Andersen,1
Christopher L. Corless,2and Xiao-Jing Wang1,3,4,5
1Department of Otolaryngology,2Department of Pathology,3Department of Dermatology, and4Department of Cell and
Developmental Biology, OHSU Cancer Institute, Oregon Health and Science University, Portland, Oregon 97239, USA
The prognosis of head-and-neck squamous cell carcinoma (HNSCC) has not been improved in the past 20
years. Validation of HNSCC biomarkers for targeted therapy has been hindered by a lack of animal models
mimicking human HNSCC at both the pathological and molecular levels. Here we report that overexpression
of K-ras or H-ras and loss of transforming growth factor-? type II receptor (TGF?RII) are common events in
human HNSCC. Activation of either K-ras or H-ras in combination with TGF?RII deletion from mouse
head-and-neck epithelia caused HNSCC with complete penetrance, some of which progressed to metastases.
These tumors displayed pathology indistinguishable from human HNSCCs and exhibited multiple molecular
alterations commonly found in human HNSCCs. Additionally, elevated endogenous TGF?1 in these lesions
contributed to inflammation and angiogenesis. Our data suggest that targeting common oncogenic pathways
in tumor epithelia together with blocking the effect of TGF?1 on tumor stroma may provide a novel
therapeutic strategy for HNSCC.
[Keywords: HNSCC; head-and-neck-specific knockout; metastasis; Ras; TGF?RII; TGF?1]
Supplemental material is available at http://www.genesdev.org.
Received January 25, 2006; revised version accepted March 17, 2006.
Head-and-neck cancer refers to cancers that develop
from the nasal and oral cavities, the throat, and the upper
esophagus. More than 90% of head-and-neck cancer
cases are head-and-neck squamous cell carcinomas
(HNSCCs) (Forastiere et al. 2003). HNSCCs represent
the sixth most common cancer in the United States (Je-
mal et al. 2004). Unlike other cancers in which lethality
is associated with metastasis, primary HNSCCs can
cause death as a result of internal bleeding, airway ob-
struction, and malnutrition related to difficulty with
food intake. Among the multiple common genetic alter-
ations identified in HNSCCs, alterations that specifi-
cally play a role in initiation and/or promotion in
HNSCC have yet to be defined. K-ras or H-ras gene mu-
tation, a common initiation event in human cancers
(Hanahan and Weinberg 2000), occurs in >50% of oral
cancer cases in south Asian populations (Saranath et al.
1991), but varies from 5% to 20% of oral cancer cases in
Western countries (Anderson et al. 1994; Hardisson
2003; Weber et al. 2003). However, increased wild-type
K-ras or H-ras protein levels occur at a much higher rate
than ras mutation in HNSCC cases in the United States
(McDonald et al. 1994; Hoa et al. 2002). Similar to mu-
tant ras, wild-type ras overexpression is sufficient to in-
duce hyperproliferation of HNSCC cells (Hoa et al.
2002). In mice, K-ras activation initiates benign papil-
loma formation in head-and-neck epithelia, but is insuf-
ficient to induce invasive HNSCCs (Caulin et al. 2004;
Vitale-Cross et al. 2004), even though one study observed
skin SCC formation (Vitale-Cross et al. 2004).
HNSCC, somatic mutations in the gene encoding trans-
forming growth factor-? type II receptor (TGF?RII) and
reduction of TGF?RII protein have been identified in hu-
man HNSCC samples (Garrigue-Antar et al. 1995; Wang
et al. 1997; Fukai et al. 2003). Although the role of
TGF?RII in SCC development has been extensively
studied (for reviews, see Reiss 1999; Wang 2001; Prime et
al. 2004), the role of TGF?RII in HNSCC pathogenesis
has yet to be determined. It is commonly accepted that
TGF?-mediated tumor-suppressive effects require func-
tional TGF?RII. However, TGF? also promotes tumor
invasion at later stages of carcinogenesis (Reiss 1999;
Wang 2001; Prime et al. 2004), and the results related to
TGF?RII loss in TGF?-associated tumor promotion are
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conflicting in both clinical studies (Tateishi et al. 2000;
Watanabe et al. 2001; Fukai et al. 2003) and experimental
systems (Yang et al. 2002; Siegel et al. 2003; Forrester et
al. 2005; Han et al. 2005). In the current study, we focus
on assessing the role and mechanisms of TGF?RII loss in
HNSCC development and progression.
Overexpression of ras and loss of TGF?RII expression
are common events in human HNSCCs
Previously, overexpression of Ras protein in human
HNSCC has been reported to reach ∼70% of cases for
H-ras, and 45% of cases for K-ras (McDonald et al. 1994).
To determine whether ras is activated at the transcrip-
tional level in HNSCC, we examined K-ras and H-ras
transcripts in 32 pairs of human HNSCCs and adjacent
tissues. Oropharyngeal samples from sleep apnea pa-
tients were included as normal controls. In comparison
with the average K-ras or H-ras expression level in nor-
mal tissue, 18/32 (56%) HNSCC samples and 10/32
(31%) adjacent tissue samples exhibited twofold to 14-
fold greater levels of K-ras mRNA, and 12/32 (38%)
HNSCC samples and 15/32 (47%) adjacent tissue
samples exhibited twofold to 25-fold greater levels of H-
ras mRNA (Fig. 1A,B). Sequencing analyses revealed that
three (9%) HNSCC samples without K-ras overexpres-
sion possessed a glycine (G)-to-aspartic acid (D) mutation
at codon 12 of the K-ras gene, a rate that is similar to
previous reports (Hardisson 2003; Weber et al. 2003). In
contrast to oral cancer cases in South Asia, in which the
frequency of H-ras mutations exceeds that of K-ras mu-
tations (Saranath et al. 1991), no mutation of H-ras was
found in these HNSCC samples. This result suggests
that exposure to different types of oral carcinogens could
affect specific molecular alterations in HNSCCs. Never-
theless, 81% of the human HNSCC samples we analyzed
exhibited either overexpression of wild-type K-ras or H-
ras or, albeit less frequently, mutation of K-ras. Immu-
nohistochemistry to detect Ras protein in these samples
revealed that Ras protein was barely detectable in nor-
mal oropharyngeal epithelia of sleep apnea patients, but
stained strongly in the mucosa adjacent to HNSCCs and
HNSCC lesions in which elevated transcripts were de-
tected (Supplementary Fig. 1). The overall cases of Ras-
positive staining correlated with the increased mRNA
levels. These data suggest that ras overexpression in hu-
man HNSCCs occurred predominantly at the transcrip-
Previous reports indicated that loss of TGF?RII pro-
tein is more frequent than disruption at the genetic level
in HNSCC (Garrigue-Antar et al. 1995; Wang et al. 1997;
Fukai et al. 2003). To determine whether TGF?RII loss
occurs mainly at the pre- or post-translational level, we
examined TGF?RII transcripts in human HNSCCs and
adjacent tissues. The average expression level in the nor-
mal control samples was arbitrarily set as 100%. The
average mRNA level of TGF?RII in the normal control
group was 100% ± 29% (Fig. 1C). The average mRNA
level of TGF?RII in tissue samples adjacent to HNSCCs
was 108% ± 14%, which is similar to the levels in nor-
mal controls (Fig. 1C). However, the average level of
TGF?RII mRNA in HNSCC samples was 36% ± 11%,
which was significantly lower than that in the adjacent
mucosa or normal controls (p < 0.01) (Fig. 1C). Among 32
pairs of HNSCC samples, 22 (69%) HNSCC samples and
two (6.3%) adjacent tissue samples exhibited a >50% de-
crease in TGF?RII mRNA level in comparison with the
average expression level of normal tissue samples (Fig.
1C). We then performed TGF?RII immunohistochemis-
try on these samples. TGF?RII staining exhibited a simi-
lar intensity in both the normal oropharyngeal epithelia
of sleep apnea patients and the mucosa adjacent to
HNSCCs (Supplementary Fig. 1), but was significantly
reduced or lost in HNSCC cells (Supplementary Fig. 1).
of TGF?RII expression in human HNSCC samples examined by
qRT–PCR. Each pair of bars in the main graph represents the
fold change of tumor and adjacent mucosa from a single case
relative to a normal control group. The case ID and numbers
were identical and in the same order in all three panels. The
average fold change for each tissue type is shown in the inset.
Seven normal oropharyngeal samples from sleep apnea patients
and 32 pairs of HNSCC and adjacent tissue samples were ex-
amined. The dotted horizontal line in each of the main graphs
represents the average expression level of each molecule from
the normal control group. (A) Expression levels of K-ras. The
average K-ras mRNA levels in HNSCC samples and adjacent
tissue samples are presented in the inset. (†) Tumors with a
G-to-D mutation at codon 12 of the K-ras gene. (B) Expression
levels of H-ras. The average H-ras mRNA levels in HNSCCs
and adjacent tissue samples are presented in the inset. (C)
TGF?RII mRNA levels in human HNSCC samples. The average
levels of TGF?RII mRNA in HNSCC and adjacent tissue
samples are presented in the inset. (*) p < 0.01 in comparison
with normal control.
Ras overexpression and mutation, and reduction/loss
Lu et al.
1332GENES & DEVELOPMENT
filing Resource of the Departments of Otolaryngology and Der-
matology, the surgeons in Otolaryngology for collecting
HNSCC samples, and Drs. John Scott and Hua Lu for comments
on the manuscript. This research was supported by NIH grants
DE015953, CA87849, CA105491, and CA79998 to X.J.W. H.H.
is a recipient of the NIH training grant.
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