Epithelial cell nicotinic acetylcholine receptor expression in head and neck squamous cell carcinoma pathogenesis.

Abstract

Molecular events following nicotinic acetylcholine receptor (nAChR) activation by nicotine are poorly understood. The phosphatidylinositol 3-kinase (PI3-K)/Akt/PTEN pathway has been suggested to play a role in the antiapoptotic responses to nicotine.
To elucidate the possible role of aalpha3, alpha5 and alpha7 nAChR subunit mediated PI3-K/Akt/PTEN pathway activation in squamous cell carcinoma of the head and neck (HNSCC) development, mRNA was isolated from 30 HNSCC tissues of known Akt activation state and were analyzed by reverse transcription polymerase chain reaction (RT-PCR).
alpha3, alpha5 and alpha7 nAChR subunits were expressed in 1/30 (3.33%), 15/30 (50%) and 10/30 (33.33%), respectively. These results did not correlate with pAkt levels, previously assessed in our laboratory, or any of the clinicopathological parameters considered.
This is the first report on nAChR subunit expression in human HNSCC surgical specimens of known pAkt levels. Our results suggest that nAChRs might exert their function through pathways different from PI3-K/Akt/PTEN and that alpha3, alpha5 or alpha7 nAChR subunit expression might not be useful prognostic markers in HNSCC.

Full text

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Abstract. Background: Molecular events following nicotinic
acetylcholine receptor (nAChR) activation by nicotine are
poorly understood. The phosphatidylinositol 3-kinase (PI3-
K)/Akt/PTEN pathway has been suggested to play a role in the
antiapoptotic responses to nicotine. Materials and Methods: To
elucidate the possible role of ·3, ·5 and ·7 nAChR subunit
mediated PI3-K/Akt/PTEN pathway activation in squamous
cell carcinoma of the head and neck (HNSCC) development,
mRNA was isolated from 30 HNSCC tissues of known Akt
activation state and were analyzed by reverse transcription
polymerase chain reaction (RT-PCR). Results: ·3, ·5 and ·7
nAChR subunits were expressed in 1/30 (3.33%), 15/30 (50%)
and 10/30 (33.33%), respectively. These results did not
correlate with pAkt levels, previously assessed in our laboratory,
or any of the clinicopathological parameters considered.
Conclusion: This is the first report on nAChR subunit
expression in human HNSCC surgical specimens of known
pAkt levels. Our results suggest that nAChRs might exert their
function through pathways different from PI3-K/Akt/PTEN
and that ·3, ·5 or ·7 nAChR subunit expression might not be
useful prognostic markers in HNSCC.
Squamous cell carcinoma of the head and neck (HNSCC)
remains a significant cause of morbidity and mortality,
afflicting approximately 500,000 new cases worldwide each
year (1). The complex development of HNSCC is thought
to progress through a series of well-defined clinical and
histopathological stages, which correlate with the
accumulation of multiple genetic events (2). The recognized
risk factors for head and neck carcinoma (heavy smoking
and high alcohol consumption in developed countries) and
the clinical appearance of premalignant lesions are poor
predictors of risk of tumor development so molecular
markers for cancer-risk assessment are needed (3).
Among the thousands of chemicals present in tobacco
smoke, nicotine is one of the main ones responsible for the
deleterious consequences of cigarette smoking and has been
shown to be involved in the inhibition of apoptosis (4).
Moreover, its presence interferes with cell death induced by
a variety of DNA-damaging agents, affecting the efficacy of
cancer therapy, specifically in head and neck carcinomas
(5). Nicotine exerts these effects by displacing the
endogenous neurotransmitter acetylcholine (ACh) from the
neuronal nicotinic acetylcholine receptors (nAChR) (6).
The mammalian neuronal nAChR are a family of proteins
each formed by five homologous or identical subunits,
arranged symmetrically around a central ion channel. Seven
of the subunits harbor the principal components of the
ligand binding site (·2, ·3, ·4, ·6, ·7, ·9, and ·10) while
four are structural proteins (·5, ‚2, ‚3, and ‚4) that confer
unique functional and pharmacological properties on the
receptors. Although originally found in neural tissue,
nAChRs have recently been reported in non-excitable cell
types (6-11), where they might contribute to the
development of tobacco-associated morbidity. West et al.
(2003) have shown that nicotinic activation of Akt depends
upon phosphatidylinositol 3-kinase (PI3-K) and specific
nAChRs. The signaling pathways from calcium influx to
PI3-K/Akt activation have not been well characterized. It
has been suggested that the Src family tyrosine kinases may
regulate calcium-mediated signaling to the PI3-K/Akt
pathway (12). These findings are related to lung cancer, only
one of many tobacco-related carcinomas. Nicotinic
activation of the PI3-K/Akt pathway might contribute to the
biology of other types of tobacco-related tumors. Our
laboratory recently identified the PI3-K/Akt/PTEN signaling
835
Correspondence to: Maria Victoria Gonzalez, Oncología Molecular,
Residencia Ntra. Sra. Covadonga 1ª planta, centro, c/ Celestino
Villamil s/n, 33006 Oviedo, Asturias, Spain. Tel: +34 98 5108000 (ext
38828), Fax: +34 98 5107954, e-mail: gonzalezvictoria@uniovi.es /
mvmeana@hotmail.com
Key Words: HNSCC, nicotinic acetylcholine receptors, PI3-K/Akt
pathway.
ANTICANCER RESEARCH 27: 835-840 (2007)
Epithelial Cell Nicotinic Acetylcholine Receptor Expression
in Head and Neck Squamous Cell Carcinoma Pathogenesis
DARIO GARCIA CARRACEDO1, JUAN PABLO RODRIGO1,2,
CARLOS SUAREZ NIETO1,2and MARIA VICTORIA GONZALEZ1
1Instituto Universitario de Oncologia del Principado de Asturias, Obra Social CajAstur (IUOPA), and
2Servicio de Otorrinolaringología Hospital Universitario Central de Asturias (HUCA),
c/ Celestino Villamil s/n, 33006 OVIEDO, Asturias, Spain
0250-7005/2007 $2.00+.40

Page 2

pathway as one of the most frequently altered in head and
neck squamous cell carcinoma (13), showing that 17/36
(47%) cases for which most of the targets of our study were
analyzed harboured at least one molecular alteration of the
PI3-K-initiated signaling pathway. Furthermore, it was
altered in some normal mucosa paired with our HNSCC
series. The role of the nAChR in HNSCC development and
its relationship to the PI3-K/Akt pathway nicotine-
dependent activation is still unclear. In the present study,
the ·3, ·5 and ·7 (previously described to be present in
epithelial cell lines) nAChR subunit expression level were
investigated in 30 head and neck squamous cell carcinomas,
in an effort to determine whether there was any relationship
between nAChR expression levels and the PI3-K/Akt
pathway activation and whether such expression could be
used as molecular markers with any prognostic value.
Materials and Methods
Samples and controls. Surgically derived tissue specimens from 30
patients with HNSCC who consecutively underwent resection of
their tumors at the Hospital Universitario Central de Asturias
(HUCA) were prospectively obtained for our study, after ethical
approval following institutional review board guidelines. None of
them had received radio/chemotherapy prior to intervention or
were thought to have distant metastasis at the time of diagnosis.
Samples were sharply excised, placed in sterile tubes and frozen
immediately in liquid nitrogen. All tissue samples were stored at
–80ÆC until RNA extraction and analysis. All cases were confirmed
to be neoplastic by the pathologist. The characteristics of the
studied patients and the clinicopathological features of their
tumors are shown in Table I. All the patients were regular tobacco
and alcohol consumers.
The SH-SY5Y neuroblastoma cell line was grown to 80%
confluence and total RNA was isolated using Tri-reagent (Sigma,
St Louis, MO, USA) following the manufacturer’s protocol and was
used as positive control for the ·3, ·5 and ·7 nAChR subunits (14).
RNA isolation. RNA was isolated and purified from 30 samples of
fresh frozen tumor tissue using the Micro-to-Midi Total RNA
Purification System (Invitrogen Life Technologies, Carlsdad, CA,
USA) as specified by the manufacturer, including DNase
treatment. The RNA concentration and purity were evaluated by
spectrophotometric analysis. The RNA structural integrity was
assessed by gel electrophoresis referring to the appearance of
rRNA 28S and 18S bands, corresponding to lengths 4.5 and 1.9 kb,
respectively, as indicators of absence of RNA degradation.
Detection of nAChR Subunit mRNA by Reverse Transcription
Polymerase Chain Reaction (RT-PCR). The total RNA (1 Ìg) was
reverse transcribed using the Thermoscript RT-PCR System
(Invitrogen Life Technologies) as specified by the manufacturer.
The primers for genes encoding the human nicotinic acetylcholine
receptor ·3, ·5 and ·7 subunits were designed with the assistance
of the Primer Express software version 2.0 computer program
(Applied Biosystems, Foster City, CA, USA). The forward and
reverse oligonucleotide sequences and the expected product sizes
(in parentheses) were as follows: ·3: 5’-AGAGTTCATGCG
TGTCCCTG-3’ and 5’-AGGCTTTGGTCTTGTCGTCC-3’ (101
bp), ·5: 5’-GCGCTCGATTCTATTCGCTA-3’ and 5’-CGATCA
AGAACCTGGGCTATG -3’ (101 bp) and ·7: 5’ ¬CCAATGA
CTCGCAACCACTC-3’ and 5’-CAGCCAAATGTTGGTGGTT
AA-3’ (101 bp). Primers forming a pair were placed in different
exons to avoid amplification of contaminating genomic DNA. All
of them were purchased from Invitrogen Life Technologies. The
PCR conditions used were as follows: a 10 min step at 95ÆC
(enzyme activation) followed by 32 cycles of 95ÆC for 1 min, 60ÆC
1 min 72ÆC for 1 min and a final step at 72ÆC. PCR products were
separated by 2% agarose gel electrophoresis and visualized by
ethidium bromide gel staining.
Statistical analysis. For statistical purposes, clinicopathological
features were dichotomized as follows: pT category: 1-3 vs. 4; pN
category: 0 (free lymph node) vs. 1-3 (affected lymph node); TNM
stage: I-III vs. IV.
The molecular results data distributed among the different
clinical groups of tumors were tested for significance employing the
¯2test (with Yate's correction where appropriate) with the help of
the statistical software package SPSS 14.0 (SPSS Inc., Chicago, IL,
USA). Values for p<0.05 were considered statistically significant.
Results
·3 subunit mRNA expression was only found in 1/30 cases.
Expression of ·5 nAChR subunit mRNA was detected in 15/30
(50%) and ·7 subunit in 10/30 (33.33%) of the tumors studied
ANTICANCER RESEARCH 27: 835-840 (2007)
836
Table I. Patient characteristics and clinicopathological features. TNM
stage: according to the International Union Against Cancer (26).
FeatureFrequency (%)
Mean age at
resection (median) 59.6 (61.5)
Gender
Male
Female
Site
Pharynx
Larynx
pT category
T1
T2
T3
T4
pN category
N0 (free margins)
N1-N3
TNM stage
II
III
IV
Histopathological grade
Well-differentiated
Moderately-differentiated
Poorly-differentiated
27/30
3/30
(90)
(10)
18/30
12/30
(60)
(40)
3/30
5/30
13/30
9/30
(10)
(16.67)
(43.33)
(30)
14/30
16/30
(46.67)
(53.33)
6/30
7/30
17/30
(20)
(23.33)
(56.67)
14/30
8/30
8/30
(46.66)
(26.67)
(26.67)

Page 3

(Figure 1). Interestingly, 7/30 cases (23.3%) co-expressed ·5
and ·7 subunits. The internal control gene, GAPDH, was
expressed at the same relative level in all subjects.
There was no correlation between these results and the
pAkt accumulation that had been previously assessed in our
laboratory (13) (Table II).
Clinical correlations. No statistical correlation was found
between nAChR subunits and any of the clinicopathological
features considered (Table III). As could be expected, there
was a trend towards patients with tumors arising at the
pharynx showing lymph node metastasis more frequently
than those with laryngeal tumors (2.983, p=0.084). In
addition, patients with pharyngeal tumors were diagnosed
with more advanced TNM stages than those with laryngeal
tumors (10.677, p=0.005).
Discussion
To date, analysis of nAChR subunit expression has been
mainly addressed in cell culture or mouse model systems (6,
15-17). This is the first report of the expression of nAChR
subunits in a series of human tumor samples (HNSCC) of
known Akt activation status. It is well established that
nACHRs of different subunit composition exhibit very
different pharmacological and functional properties. The fact
that the case expressing the ·3 subunit also displayed ·5
subunit expression is in good agreement with the previous
idea that neuronal nicotinic receptors formed by the ·3
subunit generally include the ·5 and the ‚2 or ‚4 subunits
(18). The three cases that expressed only the ·7 subunit are
good candidates to harbour homooligomers of this subunit
that have been described to be dramatically different from
their heteromeric counterparts. They are more permeable to
calcium than other nAChRs which suggests that the
activation of the nAChR·7-type receptors, impacts upon free
intracellular calcium and calcium-dependent mechanisms in a
manner quite distinct from other nAChRs (19). Although ·5
subunits are apparently unnecessary for the assembly of
functional receptors and cannot yield functional receptors
when expressed alone or in combination with ‚ subunits only,
they can alter the properties of nAChRs. In addition,
functional heteropentamers derived from ·7 and ·5 have
been also reported (19). Our observation that 7/30 of the
HNSCC tissues coexpress ·5 and ·7 subunits raises the
possibility that they coassemble, probably with ‚2, to form
functional nAChRs. Seven cases expressed only the ·5
subunit. It could participate in receptors containing the ·4
and ‚2 subunits, as has been proposed to explain the central
role of ·5 in mediating nicotinic-induced seizures arising in
the hippocampus of ·5 –/– mice after short-term exposure to
nicotine (20). No significant correlation was found between
the expression of any of the studied subunits and any of the
clinicopathological parameters considered. Thus, ·3, ·5 and
·7 might not be useful pronostic markers for HNSCC
development. Smoking is related to changes in oncogenes or
tumor suppressor genes, however, activation of signal
transduction pathways that promote cellular survival might
also contribute to tobacco-related tumor development. The
PI3-K/Akt pathway contributes to tumorigenesis and
resistance to therapy and has recently been identified as one
of the more frequently altered pathways in HNSCC in our
laboratory (13). Many reports have pointed to this pathway
as being the mediator of nicotine antiapoptotic effects upon
nAChR activation (11, 21). However, the mechanisms
through which stimulation of the nAChRs results in PI3-
K/Akt activation remain obscure. In neuronal systems it
appears that nicotine stimulation of the ·7 nAChR
transduces signals in a cascade to Akt via Janus kinase 2
Carracedo et al: Nicotinic Acetylcholine Receptor in Head and Neck Squamous Cell Carcinoma
837
Figure 1. ·3, ·5 and ·7 nAChR subunit mRNA expression in head and
neck tumors (T), blank (--), positive control, SH-SY5Y cell line and
internal control gene (GAPDH).
Table II. Distribution of ·3/pAkt, ·5/pAkt, ·7/pAkt positive and negative
cases (p-value, ¯2test).
pAkt ·3·5·7
Positive Negative Positive Negative Positive Negative
Positive
Negative
p-value
0
1
6241
9
5
231311 15
0.6110.361 0.333

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(JAK2) (22). Other studies have provided evidence for a novel
signaling route coupling the stimulation of the ·7 nAChR to
the activation of extracellular-signal-regulated-kinase (ERK)
(23). One study has demonstrated that in human airway
epithelial cells Akt is activated upon short-term exposure to
nicotine, which antagonizes apoptosis. This effect has been
suggested to contribute to tobacco-related carcinogenesis. In
addition, Akt activation was found in ten human lung cancer
specimens from smokers, but the expression of the nAChR
subunits in those samples was not assessed (11). Thus, linking
pAkt accumulation to nAChR activation in human lung cancer
samples is indirect, as it is based on observations obtained from
cultured cells or mouse models. The consequences of nAChR
activation are likely to be both cell-type and context specific
(24). In our HNSCC series no evidence for any relationship
between ·3, ·5 or ·7 expression and Akt pathway activation
(previously assessed in our laboratory (13)) was found. The
apparent discrepancy between our results and those reported
by others could be a consequence of the different effects of
transient vs. sustained exposure to nicotine, the latter being the
case of every HNSCC patient in our study. In fact, the
molecular mechanisms of long-term nicotine exposure in the
development of cancer are not clear. The data by Chu et al.
(25) have revealed that nicotine potentiates the lung
carcinogenesis process, mainly by up-regulating Ras activity.
However, they showed that PI3-K, was transiently activated
and this effect was only detected after 1 h of nicotine exposure,
diminishing after longer exposure periods. Thus, our results
suggest that biochemical pathways other than the PI3-K/Akt
pathway could be responsible for the adverse effects of nicotine
on HNSCC development. Alternatively, differences in
technical approaches (western blot vs. immunohistochemical
detection of pAkt) could account at least in part for the
differing results.
In conclusion, our findings suggest that the adverse long-
term effects of tobacco components acting through nAChR
activation on the development of HNSCC do not seem to
be mediated by the PI3-K/Akt biochemical pathway and that
the expression patterns of ·3, ·5 and ·7 might not have
prognostic value.
Acknowledgements
Supported by Programa Ramon y Cajal, Ministerio de Educacion y
Ciencia SPAIN (MVG) and FICYT, Consejeria de Educacion y
Cultura del Principado de Asturias Plan I+D+I 2001-2004 (DGC).
Conflict of interest statement: none declared
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ANTICANCER RESEARCH 27: 835-840 (2007)
838
Table III. Associations between the expression of ·3, ·5 and ·7 subunits and clinicopathological characteristics (p-value calculated by ¯2test).
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Carracedo et al: Nicotinic Acetylcholine Receptor in Head and Neck Squamous Cell Carcinoma
839