Vol. 8, 885– 892, March 2002
Clinical Cancer Research 885
Local Recurrence in Head and Neck Cancer: Relationship to
Radiation Resistance and Signal Transduction1
Anjali K. Gupta,2 W. Gillies McKenna,
Charles N. Weber, Michael D. Feldman,
Jeffrey D. Goldsmith, Rosemarie Mick,
Mitchell Machtay, David I. Rosenthal,
Vincent J. Bakanauskas, George J. Cerniglia,
Eric J. Bernhard, Randal S. Weber, and
Ruth J. Muschel
Departments of Radiation Oncology [A. K. G., W. G. M., C. N. W.,
M. M., D. I. R., V. J. B., G. J. C., E. J. B.], Pathology and Laboratory
Medicine [M. D. F., J. D. G., R. J. M.], Biostatistics and
Epidemiology [R. M.], and Otolaryngology and Head and Neck
Surgery [R. S. W.], University of Pennsylvania, Philadelphia,
Pennsylvania 19104
ABSTRACT
Purpose: Locoregional recurrence is the dominant form
of treatment failure in head and neck (H&N) cancer. The
epidermal growth factor receptor (EGFR) is frequently amplified in this disease (<80%) and can lead to activation of
phosphatidylinositol-3-kinase (PI3K), both directly and indirectly through Ras. We have shown previously that radioresistance could be conferred via the Ras-PI3K pathway.
Here we investigate the contribution of EGFR to this pathway and its impact on treatment outcome.
Experimental Design: In a series of 38 H&N cancer
patients, overexpression of EGFR by immunohistochemical
staining was assessed. PI3K signaling was evaluated by
staining for phosphorylated Akt (P-Akt), a downstream target of PI3K. Both EGFR and P-Akt were then related to
outcome. Radiation survival was determined in the SQ20B
cell line, a radioresistant squamous cell line derived from a
recurrent laryngeal cancer, after pharmacological blockade
of EGFR with Iressa, of Ras by the FTI L744,832, or of PI3K
by LY294002.
Results: A significant association was found between
P-Akt staining and local control in the patient series. Twoyear local control was 100% for patients staining 0 –1ⴙ for
P-Akt as compared with 70.6% for patients staining 2–3ⴙ
Received 10/5/01; revised 12/6/01; accepted 12/10/01.
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.
1
Supported by NIH Grants 1 PO-1 CA75138 (to W. G. M.), RO-1
GM47439 (to R. J. M.), Radiation Therapy Oncology Group Seed Grant
(to A. K. G.), and P30-CA16520 (to R. M.).
2
To whom requests for reprints should be addressed, at Department of
Radiation Oncology, 195 John Morgan Building, 3620 Hamilton Walk,
University of Pennsylvania, Philadelphia, PA 19104-6072. E-mail:
gupta@xrt.upenn.edu.
(P ⴝ 0.04). In our series of 38 H&N cancers, 30 (78.9%) of
the specimens were strongly (3ⴙ) positive for EGFR,
whereas 25 (65.8%) were moderately to strongly (2–3ⴙ)
positive for P-Akt. Pharmacologically inhibiting EGFR,
Ras, and PI3K led to radiosensitization of SQ20B cells.
Conclusions: Evaluation of PI3K activation by Akt
phosphorylation might be a prognostic marker for response
to therapy, and PI3K could be a useful target for therapy.
These results also suggest that signaling from EGFR to PI3K
can lead to radioresistance.
INTRODUCTION
Locoregional disease recurrence remains the dominant
form of treatment failure for patients with advanced SCC3 of the
H&N. For patients treated with primary radiation therapy, attempts to enhance locoregional disease control have included
altered fractionation schemes (1) and incorporation of chemotherapy (2, 3). Although in a select group of patients these
strategies result in improvement in local control, treatment toxicity is high.
Sensitivity of tumor cells to radiation therapy is a critical
determinant of the probability of local control and, ultimately, of
cure (4, 5). Thus, one approach to improving the outcome of
therapy depends on determining which factors lead to tumor cell
resistance to therapy. Overexpression of the EGFR receptor has
been shown to accompany development and growth of malignant tumors, including those of the H&N (6). There is also
increasing evidence that high expression of EGFR is associated
with aggressive tumor growth and poor clinical outcome in
these cancers (7). A number of studies has shown a positive
relationship between EGFR expression and tumor resistance to
radiation (8). Experimentally, Milas et al. (9) demonstrated in
mice with H&N carcinoma xenografts enhanced tumor radiosensitivity after combined treatment with C225 (monoclonal
anti-EGFR antibody) and radiation. Similarly, Bonner et al. (10)
have shown that combining C225 and radiation results in greater
cell killing of SCCs than with either treatment alone. Signaling
through the erb family of receptors similarly led to radioresistance in a glioma cell line (11). The results of these studies have
provided the basis for proceeding with clinical trials (reviewed
in Ref. 12).
The EGFR family consists of four closely related growth
factor receptors, including EGFR or HER-1 (erb-B1), HER-2
(erb-B2/neu or p185neu), HER-3 (erb-B3), and HER-4 (erb-B4).
EGFR binds several distinct ligands, including EGF, transforming growth factor-␣, and ampheregulins. Heregulins and neu-
3
The abbreviations used are: SCC, squamous cell carcinoma; H&N,
head and neck; EGFR, epidermal growth factor receptor; PtdIns, phosphatidylinositol; PI3K, phosphatidylinositol-3-kinase; PTEN, ; P-Akt,
phosphorylated Akt; FTI, farnesyltransferase inhibitor.
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886 Local Recurrence and Signal Transduction
regulins bind to erb-B3 and erb-B4. Erb-B2 (HER-2/neu)
does not directly bind to any known ligand. Instead, it forms
heterodimers with the three other family members and, in so
doing, enhances ligand-binding affinity and reduces the rate
of ligand dissociation. HER-2/neu heterodimers also amplify
growth factor signals through activation of the HER-2/neu
intracellular kinase domain and auto-cross phosphorylation
(reviewed in Ref. 13).
The detailed mechanism by which EGFR signaling leads to
radiation resistance is unknown. EGFR receptors initiate cytoplasmic signaling through autophosphorylation of their intracellular domains (14). EGFR has a number of effectors that include
Ras and PI3K. Transfection with oncogenic ras itself has also
been shown to increase radioresistance (15). We have shown
that Ras-mediated radiation resistance is mediated through PI3K
(16) and that P-Akt was a good marker for this effect. PI3K
activity is stimulated by Ras activation as a result of direct
interaction (17). PI3K phosphorylates PtdIns-4,5-P2 to yield
PtdIns-3,4,5-P3. PtdIns-3,4,5-P3 in turn causes membrane localization of protein kinase B (Akt) and the phosphoinositidedependent kinase phosphoinositide-dependent kinase 1 (18).
phosphoinositide-dependent kinase 1 phosphorylates one of two
sites on Akt (19), whereas a second PI3K-activated kinase,
ILK-1, phosphorylates a second site (20), resulting in full activation of Akt. Akt has been shown to act as an inhibitor of
apoptosis (21). One mechanism for the antiapoptotic activity of
Akt appears to be the phosphorylation and inactivation of the
proapoptotic BAD protein, although multiple other proteins are
also substrates for Akt phosphorylation (reviewed in Ref. 22).
These findings together implicate Akt as a possible regulator of
cell survival. Because Akt is downstream of PI3K, Ras, PTEN,
and EGFR, they also raise the possibility, explored here, that
there might be common pathways mediating radiation resistance
in tumors carrying multiple types of oncogenic mutation.
In this study, we asked whether EGFR expression and Akt
phosphorylation measured both in human H&N cancers and
tissue culture are associated with the response to radiation. If the
EGFR-Ras-PI3K pathway mediates radiation resistance in human H&N cancers, then examination of activity in this pathway
might predict outcome in these patients. Association between
immunohistochemical staining of EGFR, Pan Akt, and P-Akt
and clinical outcome was tested in patients with H&N cancer
treated similarly with chemotherapy and radiation. We found
P-Akt to be a significant predictor for local control, further
indicating that the EGFR-Ras-PI3K pathway might play an
important role in mediating radiation resistance.
Further exploring this idea using the H&N cancer cell line
SQ20B that has constitutively active EGFR and wild-type Ras,
we found that Akt was phosphorylated constitutively. Treatment
of SQ20B cells with the EGFR inhibitor Iressa, the Ras processing inhibitor, FTI L744,832, or the PI3K inhibitor LY294002
resulted in both reduced Akt phosphorylation and caused radiosensitization.
PATIENTS AND METHODS
Patient Selection. Fifty-three patients with advanced
(Stage III/IV) oropharyngeal cancer were treated in a Phase II
trial (23) at the University of Pennsylvania between August
1997 and June 2000. All patients were treated by the same team
of physicians. The patients were treated with combined modality
therapy consisting of induction chemotherapy with carboplatin
and paclitaxel followed by chemoradiotherapy with planned
neck dissection for N2 disease or greater. If patients did not have
at least a partial response to induction chemotherapy, they were
referred for radical surgery followed by postoperative radiation.
Additional treatment details and results have been published
previously (23). Of the 53 patients originally entered in this trial,
we were able to obtain sufficient paraffinized tissue from 38 of
the 53 patients for immunohistochemical staining. In all cases,
the tissue was from the time of original diagnosis and before any
treatment. All patients were followed at 1–2 month intervals by
the radiation oncologist, surgeon, and the medical oncologist.
Follow-up for survival is through June 2001.
Immunohistochemical
Staining. Paraffin-embedded
tissue sections were stained with antibody to total EGFR clone
H11 (DAKO Corp.), using the DAKO Envision ⫹ System, and
IHC-specific phosphorylated Ser 473 Akt and pan Akt antibodies (New England Biolabs), as described by Zhou et al. (24).
Immunohistochemically stained slides were interpreted blindly
and independently by two pathologists (M. D. F. and J. D. G.)
using a four-tiered grading system based on staining pattern and
intensity. Grading was based on examination of invasive tumor
only. Because keratinized tumor cells often caused artifactual
staining, these keratinized areas were excluded from the analysis. EGFR-stained slides were graded as negative, 1⫹, 2⫹, and
3⫹ based on the intensity of the membrane staining and pattern.
If the general staining was weak with a pattern of incomplete
circumferential staining of each tumor cell, this qualified as 1⫹.
The 2⫹ showed complete circumferential staining with a weak
or intermediate intensity, and 3⫹ showed complete circumferential staining with strong intensity. Pan Akt and P-Akt immunohistochemical stains were interpreted using identical grading
schemes. The 1⫹ pattern was defined as weak, homogeneous
cytoplasmic positivity without a granular staining pattern. The
2⫹ and 3⫹ patterns both had strong granular cytoplasmic
staining with the 2⫹ having it in ⬍20% of the tumor cells and
the 3⫹ having it in ⬎20% of the tumor cells.
Statistical Consideration. Descriptive statistics were
used to characterize the distribution of patient variables. For
categorical variables, frequency and percentage were used. For
continuous variables, mean, SD, minimum, and maximum were
used. The association between EGFR and P-Akt was tested by
Fisher’s exact test (25). Survival and time to local failure distributions were estimated by the method of Kaplan and Meier
(26). Survival was defined as months from diagnosis to death
because of any cause or last patient contact. Time to local failure
was defined as months from diagnosis to documented local
failure. Patients experiencing other events, such as distant failure or death without documented local failure, were censored.
Survival and time to local failure were compared between
groups of patients by the Log-rank test (27). The degree of
agreement between the two pathologists classifying the same
sample using the same ordered scale (e.g., 0 –3 scale for EGFR
and P-Akt) was assessed by a weighted statistic (28). A
one-sided Exact Linear trend test for ordered populations
(StatXact v. 4.0) was used to look at T Stage and P-Akt positivity and local failure. All Ps quoted are two sided. A P of
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Clinical Cancer Research 887
⬍0.05 is considered statistically significant. Statistical analyses
performed in either StatXact v. 4.0 (Cytel, Corp., Cambridge,
MA) or SPSS v. 9.0 (SPSS, Inc., Chicago, IL).
Cells. SQ20B cells were obtained from American Type
Culture Collection (Rockville, MD). Cells were cultured in
DMEM (Fisher Scientific, Pittsburgh, PA) supplemented with
10% fetal bovine serum (Atlanta Biologicals, Norcross, GA),
penicillin (100 units/ml), and streptomycin (100 mg/ml; Life
Technologies, Inc., Gaithersburg, MD) at 37°C in humidified
5% CO2-95% air.
Inhibitors. The PI3K inhibitor LY294002 and the mitogen-activated protein/extracellular signal-regulated kinase inhibitor PD98059 were obtained from Alexis Corp. The EGFR
inhibitor Iressa was obtained from Astra Zeneca. The FTI
L744,832 was obtained from Merck Pharmaceuticals. All inhibitors were dissolved as concentrated stock solutions in DMSO
and diluted at the time of treatment in medium. Control cells
were treated with medium containing the same concentration of
DMSO.
Cell Growth Curves. Cultures in log growth phase were
counted, and 3 ⫻ 105 cells were plated in each T25 flask. The
cells were allowed to attach, and the inhibitors were added. At
various times, total cell number was assessed in triplicate.
Radiation Survival Determination. Cultures in log
growth phase were counted and plated in 60-mm dishes containing 2 ml of media. The cells were allowed to attach, and
inhibitors were added to cultures ⱖ1 h before radiation.
L744,832 treatment was initiated 24 h before irradiation. Treatment was continued for 24 h after irradiation, at which time 3 ml
of additional drug-free media were added to the kill curves
involving LY294002, PD98059, and L744,832. For the survival
curves with Iressa and the control for the Iressa-treated curves,
the media were completely replaced at 24 h with fresh media so
no Iressa remained in the dishes. Cells were irradiated with a
Mark I cesium irradiator (J. L. Shepherd, San Fernando, CA) at
a dose rate of 1.6 Gy/min. Colonies were stained and counted
10 –14 days after irradiation. A light box was used to assist in
counting colonies. The surviving fraction was calculated by
dividing the number of colonies formed by the number of cells
plated times plating efficiency. Each point on the survival curve
represents the mean surviving fraction from at least three replicate dishes.
Western Blotting. Cells were lysed without trypsinization by rinsing culture dishes once with PBS followed by lysis
with reducing Laemeli sample buffer. Samples were boiled,
sheared, and clarified by centrifugation and stored at ⫺20°C.
Samples containing equal amounts of protein were separated on
a 12% SDS polyacrylamide gel and blotted onto nitrocellulose
membranes. Membranes were blocked in PBS containing 0.1%
Tween 20 and 5% powdered milk before primary antibody
addition. Monoclonal H-Ras antibody LA069 (Quality Biotech)
was used at a dilution of 1:5000; monoclonal antiphosphorylated EGFR (HER-1; Upstate Biotechnology), polyclonal antiphosphorylated Ser 473 Akt, and polyclonal pan Akt (New
England Biolabs) were all used at 1:2000 dilution. Antibody
binding was detected using the enhanced chemiluminescence kit
(Amersham, Arlington Heights, IL). Images were digitized using an Arcus II scanner, and figures were assembled using
Adobe Photoshop 3.0 and Microsoft Power Point.
Table 1 Patient characteristics
Total number of patients
Sex
Male
Female
Age
Mean
Median
Range
Stage
III
IV
T stage
T2
T3
T4
N stage
N0
N1
N2a
N2b
N2c
N3
#
%
38
100
29
9
76.3
23.7
57.7
57.0
33–75
5
33
13.2
86.8
8
14
16
21.2
36.8
42.1
3
4
2
21
6
2
7.9
10.5
5.3
53.3
15.8
5.3
RESULTS
EGFR and Akt Phosphorylation in Human H&N Cancer. Patient characteristics are described in Table 1. All of the
patients had stage III/IV disease with 33 of the 38 having stage
IV disease. At the time of analysis, 12 patients had died. The
median follow-up for the 26 living patients was 28.2 months.
Fig. 1A demonstrates examples of the staining obtained. H&E
staining of typical SCCs is shown. The staining distribution is
shown in Table 2. The distribution of EGFR staining was 0 (4
patients), 1⫹ (1 patient), 2⫹ (3 patients), and 3⫹ (30 patients).
All of the tumors stained using the pan Akt antibody. The
distribution for P-Akt staining was 0 (3 patients), 1⫹ (10 patients), 2⫹ (16 patients), and 3⫹ (9 patients). Fig. 2 shows
examples of different intensities and densities of staining with
P-Akt. In the 30 patients with strongly positive EGFR staining,
20 or 67% had positive P-Akt staining. In the 8 patients with
weak or moderate EGFR staining, 5 were positive for P-Akt
(Table 3). No association between EGFR and P-Akt was evident
(P ⫽ 1). The agreement between our two pathologists was very
strong; weighted statistic was 0.92 for EGFR and 0.71 for
P-Akt (P ⬍ 0.0001 for each). It should be noted that false
positive staining was frequently noted on keratin both in keratin
pearls (Fig. 1B) and the epithelium (Fig. 1C) but that this
staining was excluded from consideration. In addition, inflammatory cells stained intensely positive for P-Akt and served as
an internal control (Fig. 1D). Neither EGFR nor P-Akt staining
was associated with T stage, N stage, or differentiation of
disease (data not shown). The numbers, however, were too small
to do meaningful multivariate analysis. Of the 8 patients that
were T2, 5 were P-Akt positive, and 0 had local failure; of the
14 patients that were T3, 8 were P-Akt positive, and 2 or 25%
had local failure; of the 16 patients that were T4, 12 were P-Akt
positive, and 5 or 42% had local failure (Table 4). Higher T
stage was not associated with increased P-Akt positivity (P ⫽
0.29). Higher T stage was associated with increased local failure
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888 Local Recurrence and Signal Transduction
Fig. 1 Immunostaining. A, staining of a typical tumor with H&E, EGFR, PanAkt, and
P-Akt. The pictures are in ⫻200. Nonspecific
staining in a keratin pearl, B (⫻200) and surface epithelium, C (⫻100). D, intensely positive staining of inflammatory cells (⫻200).
The top picture in B–D is H&E staining, and
the bottom is P-Akt.
Table 2
EGFR
P-Akt
Staining distribution
0
1⫹
2⫹
3⫹
4 (10.5%)
3 (7.9%)
1 (2.6%)
10 (26.3%)
3 (7.9%)
16 (42.1%)
30 (78.9%)
9 (23.7%)
(P ⫽ 0.05). Of the 7 patients with local failure, none had
evidence of distant disease at the time of relapse.
EGFR and P-Akt Correlated to Outcome. At the time
of this analysis, 26 of the 38 patients (68.4%) were alive. The
median survival time for all patients was 41.2 months (95%
confidence interval, 26.9 –55.5 months). Survival was not associated significantly with EGFR (P ⫽ 0.41) or P-Akt (P ⫽ 0.16)
staining (Fig. 3). There was a total of seven local failures in this
group. All of the local failures were in the EGFR-positive (3⫹)
and P-Akt-positive (2–3⫹) groups. Time to local failure was
significantly associated with P-Akt (P ⫽ 0.04; Fig. 4B). EGFR
expression was not a statistically significant predictor of time to
local failure (P ⫽ 0.14; Fig. 4A), but it should be noted that there
were 8 EGFR-negative to moderate staining specimens in this
series.
Tissue Culture Analysis. Our data on this patient group
raise the possibility that evaluation of signaling through this
pathway might predict treatment outcome. This led us to study
the H&N cell line SQ20B. SQ20B is a human H&N cancer cell
line with a constitutively active mutation in EGFR and wildtype ras. This cell line was derived from a locally recurrent
laryngeal cancer and is highly radioresistant. The activity of the
EGFR-Ras-PI3K-Akt signaling pathway was verified in the
SQ20B cells. Phosphorylated EGFR was detected in SQ20B as
expected from its active EGFR. The downstream target Akt was
also phosphorylated (Fig. 5). Inhibition of EGFR with Iressa (1
M) resulted in reduced levels of phosphorylated EGFR and
P-Akt (Fig. 5). Inhibition of Ras processing by the FTI
L744,832 (5 M) or of PI3K with LY294002 (10 M) also
inhibited Akt phosphorylation.
Clonogenic Survival after Inhibition of EGFR Signaling. To test the effects of EGFR-Ras-PI3K signaling on radiation survival, we determined the time course of action for a
single application of each of the pharmacological agents. Cells
treated with Iressa had decreased P-Akt in ⱕ10 min of application, maximal inhibition by 1 h, and the effect was maintained
for 72 h (Fig. 6A). LY294002 resulted in complete absence of
P-Akt by 1 h, but at 24 h, P-Akt could again be detected (Fig.
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Clinical Cancer Research 889
Fig. 2 Grading of P-Akt. Intensities of staining
of four different tumors with P-Akt. The left pictures are the ⫻100 view, and the right pictures
demonstrate the square box within the ⫻100 view
magnified to ⫻400. The grading of the tumors as
to 0 –3⫹ are listed on the left side.
Table 3 Association of EGFR by P-Akta
EGFR negative
(0–2⫹)
EGFR positive
(3⫹)
Total
a
Table 4 P-Akt and T Stagea
P-Akt negative
(0–1⫹)
P-Akt positive
(2–3⫹)
Total
T Stage
No.
3 (37.5%)
5 (62.5%)
8 (21.1%)
10 (33.3%)
20 (67.7%)
30 (78.9%)
T2
T3
T4
8
14
16
13 (34.2%)
25 (65.8%)
38 (100%)
a
P-Akt positive
(2–3⫹)
Local failure
5 (62.5%)
8 (57.1%)
12 (75.0%)
P ⫽ 0.29
0
2 (25%)
5 (42%)
P ⫽ 0.05
One-sided Exact Linear trend test for ordered populations.
P ⫽ 1 by Fisher’s exact test.
6A). We have shown previously that inhibition of H-Ras prenylation by FTI treatment is detected after 2–5 h and reaches
50% by 24 h. Inhibition persists for ⱖ21 h after inhibitor
removal (29). Effects on cell growth by these drugs were also
examined (Fig. 6B) because clonogenic survival assays depend
on the ability of cells to grow and form colonies. Treatment of
SQ20B with Iressa caused a pronounced growth delay for ⱕ7
days consistent with the time course showing long-term inhibi-
tion by Iressa. Treatment with LY294002 revealed an initial lag
in growth followed by recovery to a growth rate equivalent to
that of control cells.
We designed clonogenic assays to evaluate the effect of
blocking signaling in the EGFR pathway on radiation survival.
To test the effect of Iressa while limiting the effect of its growth
inhibition, we completely replaced the media in these experiments in ⱕ24 h of irradiation. Fig. 7A shows the clonogenic
survival curve of SQ20B cells with and without Iressa (1 M).
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890 Local Recurrence and Signal Transduction
Fig. 3 Survival of patients with
tumors that were A, EGFR positive
(3⫹) or B, P-Akt positive (2–3⫹).
Fig. 4 Local control of patients
with tumors that were A, EGFR
positive (3⫹) or B, P-Akt positive (2–3⫹).
sitization of SQ20B cells, we also did clonogenic assays with
the mitogen-activated protein/extracellular signal-regulated kinase inhibitor PD98059 (25 M), which did not result in sensitization (Fig. 7B).
DISCUSSION
Fig. 5 Inhibitors known to block EGFR signaling pathways. Signaling
through EGFR was evaluated by immunoblot analysis using antibodies
with activity against the active or phosphorylated forms of EGFR and
Akt. H-Ras was used as a loading control. SQ20B cells were exposed to
the indicated inhibitors.
Control curves in which media was replaced at 24 h (Fig. 7A)
showed greater apparent radiosensitivity than those that did not
undergo this manipulation (Fig. 7B). Nonetheless, Iressa treatment reproducibly lowered survival (Fig. 7A). Disruption of Ras
signaling using L744,832 (5 M) and inhibition of PI3K with
LY294002 (10 M) also sensitized SQ20B cells (Fig. 7B) to
radiation. To ascertain that all inhibitors did not result in sen-
The results of this retrospective study evaluating EGFR
and Akt in H&N cancer patients treated with multimodality
therapy have found a significant association between P-Akt and
treatment failure. These results implicate P-Akt in radiation
resistance because failures were all local. Moreover, these data
are strengthened by in vitro studies showing that inhibition of
EGFR, PI3K, and Akt radiosensitized the H&N squamous cancer cell line SQ20B.
We have shown previously that PI3K is an important
mediator of Ras-induced radiation resistance (16). In the present
study, we show that EGFR, which is upstream of PI3K, may
also mediate resistance through this common pathway. It should
be noted, however, that there was no direct association between
EGFR and P-Akt. Of the eight tumors that were EGFR negative,
five had P-Akt-positive disease, implying that mutations downstream of EGFR can also result in activation of Akt. Similarly,
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Clinical Cancer Research 891
Fig. 7 Clonogenic survival in SQ20B cells after treatment with inhibitors of the EGFR signal transduction pathway. Survival after treatment
with A, Iressa (Œ) and the control with drug washout (F). This curve
was repeated three times with similar results. B, survival after treatment
with LY294002 (Œ), L744,832 (f), PD98059 (), and the control
without drug manipulation (F). Error bars are shown and, if not visible,
were contained within the point of the graph.
Fig. 6 Time course of inhibition by the various pharmacological
agents. A, inhibition of P-Akt by Iressa and LY294002 over time was
assessed in SQ20B cells using immunoblot analysis. The same gels were
stripped and probed with PanAkt. B, cell growth in the presence of the
inhibitors over time was determined.
EGFR overexpression did not guarantee that the PI3K pathway
would be activated, because 10 of the 30 tumors that were
EGFR positive were P-Akt negative. In addition to EGFR and
Ras, PTEN can also regulate the PI3K pathway. PTEN is a
phosphatase that antagonizes PI3K by converting its active
product PI(3,4,5)P3 to PI(4,5)P2 (30). In the tumors that were
EGFR positive yet P-Akt negative, PTEN may be modulating
PI3K activity. Mutations in PTEN that cause it to be functionally inactive are also frequently found in many human cancers.
Tumor cells with these mutations may have augmented PI3K
activity and, hence, be susceptible to radiosensitization by PI3K
inhibition. Wick et al. (31) have shown that PTEN gene transfer
in human malignant gliomas sensitized cells to radiation, although in this case, PTEN transfer was associated with growth
suppression, which may complicate the interpretation.
The estimated incidence of new H&N cancers in the United
States is 41,000 (32). Early stage disease (stages T1-T2 and N0)
can be cured with either surgery or radiation therapy alone or
with a combination of both. However, more advanced tumors
(T2– 4 and N1–3) have a local failure rate of ⱖ50% (33). All
patients in this study were locally advanced. The numbers were
too small to do a multivariate analysis in terms of EGFR, P-Akt
staining, T stage, or N stage. There was no increase in P-Akt
positivity with more advanced T stage, yet more advanced
tumors did have increased local failure. All of the local failures
were in the P-Akt-positive patients. Of the tumors that were
P-Akt positive and had local failure, 0% were T2, 25% were T3,
and 42% were T4. This points toward P-Akt being a confounding variable along with T stage as a predictor of local failure. All
seven of the local failures were isolated local failure without
evidence of distant disease. There was not a statistically significant association between P-Akt and survival (P ⫽ 0.16), but the
patient numbers were too small for any conclusion. Identifying
a common signal for EGFR, Ras, or PTEN that results in
radiation resistance may uncover targets for developing molecular-based radiosensitization protocols for tumors resistant to
radiation and, thus, improve the local control that can be obtained after radiation therapy. Additional work is needed to
identify the subset of patients that will benefit most from this
treatment.
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Local Recurrence in Head and Neck Cancer: Relationship to
Radiation Resistance and Signal Transduction
Anjali K. Gupta, W. Gillies McKenna, Charles N. Weber, et al.
Clin Cancer Res 2002;8:885-892.
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