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To characterize estrogen receptor (ER) expression and signaling in head and neck squamous cell carcinoma (HNSCC) cell lines and patient tissues and evaluate ER and epidermal growth factor (EGF) receptor (EGFR) cross-activation in HNSCC.
ER expression and signaling in HNSCC cell lines were assessed by immunoblotting. In vitro proliferation and invasion were evaluated in HNSCC cell lines in response to ER and EGFR ligands or inhibitors. ER and EGFR protein expression in patient tissues was assessed by immunohistochemical (IHC) staining.
Phospho-MAP kinase (P-MAPK) levels were significantly increased following combined estrogen (E2) and EGF treatment. Treatment of HNSCC cells with E2 and EGF significantly increased cell invasion compared to either treatment alone while inhibiting these two pathways resulted in reduced invasion compared to inhibiting either pathway alone. EGFR (P=0.008) and nuclear ERα (ERαnuc) (P<0.001) levels were significantly increased in HNSCC tumors (n=56) compared to adjacent mucosa (n=30) while ERβnuc levels did not differ (P=0.67). Patients with high ERαnuc and EGFR tumor levels had significantly reduced PFS compared to patients low tumor ERαnuc and EGFR levels (H.R. = 4.09, P = 0.01; Cox proportional hazards). In contrast, high ERβnuc tumor levels were not associated with reduced PFS alone or when combined with EGFR.
ERα and ERβ were expressed in HNSCC and stimulation with ER ligands resulted in both cytoplasmic signal transduction and transcriptional activation. ER and EGFR cross-talk was observed. Collectively, these studies indicate ER and EGFR together may contribute to HNSCC development and disease progression.
Epidermal growth factor receptor (EGFR) is overexpressed in 40-90% of head and neck squamous cell carcinoma (HNSCC), and EGFR overexpression is associated with reduced HNSCC patient survival (1, 2). The EGFR-targeted chimeric monoclonal antibody cetuximab (C225, ImClone) has been FDA-approved for the treatment of HNSCC. Even though EGFR is overexpressed in many HNSCC, clinical response to cetuximab and other EGFR-targeted therapies has been modest in clinical trials (3-5). In addition, response to EGFR-targeted treatment has not positively correlated with tumor EGFR levels in several studies (5-7). These data suggest that signaling pathways working in parallel or in concert with EGFR may modulate tumor response to EGFR-targeted therapies.
The mechanisms of acquired or de novo resistance to EGFR-targeting in EGFR-expressing tumors are incompletely understood. Estrogen receptor (ER) signaling independent of EGFR and/or in concert with EGFR has been reported for cancers of the lung and esophagus (8-11), and combined EGFR- and ER-targeting in lung cancer has been previously reported by our group to be a more effective anti-tumor therapy than targeting either alone (9).
EGFR overexpression is common in HNSCC (12-14), and the role of EGFR signaling in HNSCC growth and invasion has been well established (15, 16). In contrast, reports of ERα and ERβ expression in HNSCC are conflicting, and reports characterizing ER function in HNSCC are scarce. In addition, reported activities of ER in breast cancers where ER activities have been most characterized suggest that results differ according to experimental system or ER may play a complex role in cancer, as both tumorigenic and anti-tumor properties have been associated with specific ER subtypes (17-19).
We sought to evaluate the expression of ER subtypes, to determine whether ER activation was associated with cell proliferation and/or invasion, and to examine the functional interaction between EGFR and ERs in HNSCC cell lines. To evaluate the putative role of ER in HNSCC, we characterized ER subtype expression in patient HNSCC tumors and paired adjacent mucosal tissues. We hypothesized that the ER and EGFR pathways interact in HNSCC and we would achieve greater cell proliferation and/or invasion with combined treatment with estrogen and epidermal growth factor (EGF), an EGFR ligand agonist. We further hypothesized that inhibition of HNSCC invasion and/or proliferation would be greater with combined inhibition of ER and EGFR than with targeting either pathway alone. In order to test these hypotheses, we biochemically evaluated ER and EGFR signaling in several HNSCC cell lines in vitro and assessed ER, EGFR and their combined expression in patient HNSCC tumors for correlations and association with survival.
Estrogen (E2) was purchased from Sigma-Aldrich (St. Louis, MO). Recombinant human EGF and TGFα neutralizing antibody (NA) were purchased from Oncogene Research Products (San Diego, CA). M225 was obtained from Imclone Systems, Inc. (New York, NY). Marimistat was obtained from British Biotech (Oxford, United Kingdom). TGFα Quantikine ELISA kit, human HB-EGF and amphiregulin (AR) DuoSet ELISA kits, and amphiregulin NA were from R&D Systems (Minneapolis, MN). HB-EGF NA was from Calbiochem (San Diego, CA). Gefitinib was purchased from ChemieTek (Indianapolis, IN). Fulvestrant was purchased from Tocris (Ellisville, MO).
HNSCC cell lines PCI-15B, PCI-37A, 1483, UM-22B, Detroit-562 and UPCI SSC-103 were maintained in DMEM with 10% fetal bovine serum (FBS) at 37°C with 5% CO2. MCF7 breast cancer cells were purchased from ATCC and maintained in BME with 10% FBS. HNSCC cell lines were of human origin and derived from an oropharyngeal tumor (1483), metastatic cervical lymph node (UM-22B and PCI-15B), metastatic pleural effusion (Detroit-562) or epiglottis (PCI-37A and UPCI SCC-103) as described previously (20-23). UM-22B, Detroit-562 and UPCI SSC-103 were derived from female patients while PCI-15B, PCI-37A and 1483 were derived from male patients.
Whole cell extracts from cultured HNSCC cells were prepared as described previously (9). Equal amounts of protein (25 μg) from each sample were analyzed by immunoblotting for ERα, ERβ, EGFR and β-actin. Proteins were fractionated using 10% SDS-Tricine gels and transferred to nitrocellulose membranes. Membranes were blocked by incubation in 1 X TBS-T/5% milk for 1 hr at room temperature, followed by incubation overnight at 4°C with the following primary antibodies: anti-ERα antibody, HC-20 (1:1000) (Santa Cruz Biotechnology, Santa Cruz, CA), anti-ERβ antibody, H-150 (1:1000) (Santa Cruz Biotechnology), anti-EGFR antibody, 1005 (1:500) (Santa Cruz Biotechnology) or anti-actin antibody (1:10,000) (Millipore Corporation, Billerica, MA). Blots were washed in 1 X TBS-T and incubated with horseradish peroxidase- (HRP-) conjugated anti-mouse or - rabbit IgG (1:2000) (Amersham, Piscataway, NJ). Immune complexes were detected using SuperSignal West Pico Chemiluminescent substrate (Pierce Biotechnology, Rockford, IL) and exposure to autoradiography film. Densitometry was done using Molecular Dynamics ImageQuaNT software version 5.2.
For induction of P-MAPK, HNSCC cells were grown to 75% confluency. Cells were serum deprived for 48 hr in phenol red-free media. E2 and/or EGF were added for 5 minutes. Inhibitors and NAs were added for 2 hr prior to ligand stimulation. Whole cell protein extracts were prepared, gel fractionated, transferred and blocked as described above. Membranes were probed with anti phospho-p44/p42 mitogen-activated protein kinase (P-MAPK) antibody (1:1000) (Cell Signaling Technology, Beverly, MA) or anti-total-p44/p42 MAPK (T-MAPK) antibody (1:1000). Secondary was HRP-conjugated anti-rabbit IgG (1:2000). Washes, detection and quantification were performed as described above. The quantified results represent the mean ± SE of 2 samples per experimental treatment for 3 independent experiments.
Cells were plated 3.5 ×103 cells per well in complete media on 96-well plates and allowed to attach overnight. The cells were serum-deprived in phenol red-free medium for 48 hr. Treatments were added for 72 hr with media replenishment every 24 hr. Samples were analyzed using the CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega, Madison, WI) as described previously (9). The quantified results represent the mean ± SE of two independent experiments, each with six samples per experimental treatment.
Cells were plated in complete media at 1 ×105 cells/well in 6-well plates. The next day, the medium was changed to one containing 10% charcoal-stripped serum and no phenol-red to deprive the cells of E2. Cells were transfected the following day, harvested and analyzed for luciferase activity as previously described (24). Values were corrected for protein concentration and are presented as the mean ± SE of three independent experiments, each with two samples per experimental treatment.
PCI-37A cells were grown to 75% confluency. The cells were then serum deprived in phenol red-free medium for 48 hr and treated with 1nM E2 for 10 minutes. Supernatants were collected and cells were centrifuged at 1,200 × g for 10 minutes. The resulting supernatants were concentrated to 300μl using Amicon ultrafilter devices and tested for levels of TGFα, HB-EGF and amphiregulin by ELISA following the manufacturers’ instructions. Results are expressed as the fold-increase with E2 treatment compared to controls. Results represent the average ± SE from five samples per experimental treatment assayed in duplicate.
For stimulation experiments shown in Figure 3A, PCI-37A cells that have been serum and phenol red deprived for 24 hr were plated at a density of 7.5 ×103 cells/well in a 24-well BD Biocoat Matrigel growth-factor reduced invasion chamber (BD Biosciences, San Jose, CA). E2 (1nM) and/or EGF (0.5ng/ml) was added to the media plus 10% charcoal-stripped serum in the lower chamber as indicated in the figures and incubated for 24 hr. For inhibition experiments shown in Figure 4B, PCI-37A cells grown in complete media were plated 7.5 × 103 cells/well in a 24-well BD Biocoat Matigel invasion chamber. Gefitinib (10μM) and/or fulvestrant (5μM) were added to the media in both the upper and lower chambers and incubated for 24 hr. The lower chamber also contained 10% FBS. For both experiments, non-invading cells were removed, and invading cells were fixed and stained with the Diff-Quik staining kit (VWR International, West Chester, PA). Invasion is expressed as the mean number of cells invading through the Matrigel matrix. Control treatment was set to 100 and all results expressed relative to control. Results are the mean ± SE of three independent experiments with two membranes per experimental treatment and four independent regions counted per membrane at 40X magnification.
Patients who were to undergo surgical resection with curative intent for the treatment of HNSCC with pathologically-confirmed cancer of the upperaerodigestive tract (oral cavity, oropharynx, hypopharyx, or larynx) gave written informed consent and donated tumor tissues and adjacent mucosa for study. Tumor specimens from 56 HNSCC patients, 30 with paired adjacent histologically normal mucosa, were incorporated into a tissue microarray (TMA). Tissues were collected under the auspices of a tissue bank protocol approved by the University of Pittsburgh Institutional Review Board. Subject smoking histories and body mass index (BMI) data were obtained for n=55 and n=44 subjects, respectively, through administered questionnaire or clinical chart review. A summary of subjects with tumor specimens incorporated into TMAs is provided in Table 1.
Cores were sampled from paraffin-embedded tissue blocks from surgical specimens by a head and neck cancer pathologist (RS). TMAs were constructed from 0.6 mm triplicate tissue cores extracted from HNSCC tumors or adjacent histologically normal tissues arrayed on two recipient paraffin blocks. The newly constructed array was then warmed to 37°C for 10 minutes to allow annealing of donor cores to the paraffin wax of the recipient block.
TMA sections were deparaffinized with successive ethanol and xylene treatments, re-hydrated, and stained for ERα, ERβ or EGFR. For ERα staining, sections underwent heat-induced antigen retrieval in citrate buffer. Following three washes with 3% hydrogen peroxide and one wash with TRIS-buffered saline (TBS) (25mM TRIS, 0.15 M NaCl, pH 7.5), slides were blocked with Dako Protein Block Serum (X0909, Dako, Denmark) for 5 minutes then incubated for 30 minutes with anti-ERα antibody (HC-20, SC-543, Santa Cruz) diluted 1:200 in antibody diluent (S0809, Dako). Signal amplification was performed using the Dako Envision kit (K1392, Dako). Immunoreactive cells were visualized following incubation with diaminobenzidine (DAB) chromogenic substrate (K3468, Dako) at room temperature for 10 minutes. For ERβ staining, heat induced antigen retrieval was performed with DivaDecloaker antigen retrieval buffer (DV2004, Biocare). Slides were treated with 3% hydrogen peroxidase for 10 minutes to block endogenous peroxidases and then treated with protein block for 10 minutes (BS966L, Biocare). Slides were incubated for 30 minutes with anti-ERβ (H-150, SC-8974, Santa Cruz) (1:100). Signal amplification was performed using the Dako Cytomation Envision Dual Link System Peroxidase (K4063, Dako); immunoreactive cells were visualized following incubation with Dako DAB chromogen substrate (K3468, Dako) at room temperature for 5 minutes. EGFR staining was performed without antigen retrieval using the anti-EGFR antibody (M3563, Dako) (1:500). Signal amplification was performed using an antibody-conjugated proprietary micropolymer peroxidase (ImmPRESS™, Vector). Immunoreactive cells were visualized as described for ERβ. All sections were counterstained with hematoxylin for 2.5 minutes. Staining intensity for each core was scored as 0 (none), 1+ (weak), 2+ (moderate), or 3+ (strong). The percentage of immunoreactive cells was recorded and rounded to the nearest 5th percentile. For ERα and ERβ, nuclear (nuc) and cytoplasmic (cyto) staining was evaluated independently. Tumor cores with more than 10% of cells staining +1 or greater were defined as positive. A composite score (IHC Score) was derived from the product of the percentage and intensity of staining, and these composite scores were averaged for the triplicate cores. Median IHC Scores were used to divide tumors into high versus low categories for each protein evaluated.
In vitro results are expressed as mean ± SE. The Student’s t-test or One-way ANOVA was used for all statistical analysis related to in vitro experiments. Two-sided significance was 0.05.
Differences between paired tumor and adjacent mucosal IHC scores were evaluated using the signed-rank test. Correlations between tumor IHC scores were evaluated by Spearman’s rank correlation coefficient. Progression-free survival (PFS) was defined as time from surgery to first new primary tumor, recurrence, metastasis or death. To evaluate association of ERα, ERβ and EGFR with PFS, the median HNSCC tumor IHC score was used to divide tumors into high and low categories for each marker. Hazards ratios (H.R.) for subjects with high versus low tumor IHC scores were estimated using univariate and multivariable Cox proportional hazards models, adjusted for age, sex, and clinical disease stage. Tumor combined ER and EGFR status was evaluated as a categorical variable in Cox proportional hazards models with categories of high ER, (ERH), low ER (ERL), high EGFR (EGFRH), and low EGFR (EGFRL) as follows: ERH-EGFRH, ERH-EGFRL, ERL-EGFRH, and ERL-EGFRL. The assumption of proportional hazards was tested for all models by evaluation of scaled Schoenfeld residuals. All P values presented are two-sided.
We first examined protein expression of EGFR and ERs in a panel of 6 HNSCC cell lines derived from both male and female patients (Figure 1). MCF7 breast cancer cells were used as a positive control for ERα and ERβ. All cell lines examined expressed full-length ERα (66KDa) and ERβ (59KDa) protein, although ERα was expressed at relatively lower levels compared to MCF7 cells (Figure 1A). There was no difference in ERα or ERβ expression between cell lines derived from males (PCI-15B, PCI-37A and 1483) versus females (UM-22B, Detroit-562 and UPCI SCC-103). Even though the highest ERα expression was consistently observed in the UM-22B cell line, the lowest ERα expression was also observed in a female derived cell line, UPCI SCC-103. EGFR expression was variable in the cell lines examined. PCI-15B, 1483 and UM-22B expressed high levels of EGFR while PCI-37A, Detroit-562 and UPCI SCC-103 expressed relatively low levels of EGFR. No relationship was observed between EGFR expression and ERα or ERβ expression. β-actin protein expression showed no differences between these cell lines. Reproducibility of protein expression levels was confirmed in at least two separate experiments for each cell line.
If estrogen influences HNSCC development, a stimulatory effect attributable to estrogen on the growth of HNSCC cells would be expected. In order to determine if estrogen could induce tumor growth in HNSCC cells, cell proliferation was assessed by MTS assay in 4 HNSCC cell lines. Figure 1B shows the effect of EGF and E2 on cell growth. EGF alone significantly stimulated cell growth by 1.4-to 1.8-fold in all cell lines examined. E2 stimulated cell proliferation to a lesser extent (1.1 —to 1.5- fold) than EGF compared to vehicle control, but was statistically significant. One mechanism of ligand-dependent nuclear ER action is through genomic responses whereby nuclear ERs are activated by estrogen binding at either estrogen responsive elements (EREs) or activator protein 1 (AP-1) sites in estrogen responsive genes. To verify a biologically functional role of ERs in HNSCC cells, we used a gene reporter assay with a single vitellogenin ERE upstream of a minimal thymidine kinase promoter and the firefly luciferase gene (pERE-TK-LUC) to determine if the endogenous ERs present in HNSCC cell lines, expressing different amounts of EGFR, could activate transcription in this manner. pRL-CMV was co-transfected to control for transfection efficiency. The results from three independent experiments are shown (Figure 1C). Estrogen consistently increased ER transcription with doses as low as 0.1nM E2, however the increase was not statistically significant in PCI-15B cells. No correlation was found between ERα or ERβ protein expression levels and extent of transcriptional response. An inverse correlation was observed between EGFR expression and transcriptional response. ERE-luciferase induction was highest in PCI-37A cells which also had the lowest EGFR expression of the cell lines tested while PCI-15B cells had the highest EGFR expression and the lowest transcriptional activation. This suggests that if EGF signaling is low, estrogen signaling is more functional and vice versa.
In order to determine the relative contributions of ERα and ERβ signaling in HNSCC, we transiently transfected an AP-1 luciferase construct into HNSCC cells. Estrogen activates transcription from AP-1 sites when complexed to ERα and inhibits transcription when complexed to ERβ, allowing for assessment of the relative activities of ERα and ERβ (25). MCF7 cells, which express high amounts of ERα and low amounts of ERβ, showed a 3.5-fold increase in luciferase activity. In UM-22B and PCI-37A HNSCC cells, luciferase activity remained the same or slightly decreased upon estrogen treatment (Figure 1D). These results suggest that the ERs present in HNSCC are functional and that ERβ is the predominant transcriptionally active ER in UM-22B and PCI-37A cells.
P-MAPK is a downstream signaling mediator of the EGFR pathway. We have previously shown that rapid activation of P-MAPK is a surrogate endpoint for EGFR activation (9). To determine if the ERs in head and neck cancer cell lines can transactivate EGFR downstream signaling pathways, PCI-37A cells (the cell line that responded best to E2 in cell proliferation and transcription assays) were treated for 5 min with E2 (1nM), EGF (.5ng/ml) or a combination of the two treatments and analyzed for P-MAPK expression (Figure 2A). Submaximal concentrations of E2 and EGF were used in order to observe a combined effect of the two ligands. Higher concentrations of EGF resulted in maximal stimulation of the P-MAPK pathway (data not shown). A 2.8-fold and 3.8-fold stimulation of P-MAPK was observed with E2 and EGF treatment alone, respectively. The combination achieved an almost additive effect of 6-fold compared to control-treated cells (P<0.001 for E2 versus E2 plus EGF and EGF versus E2 plus EGF). P-MAPK induction by E2 is maximal at 5-10 min and then returns to basal levels (data not shown).
To determine if estrogen-induced P-MAPK stimulation was dependent on EGFR, the cells were pretreated with an EGFR NA, M225, for 2 hr prior to ligand stimulation. Figure 2B shows that M225, almost completely abrogated P-MAPK induction by E2. It has previously been shown that transactivation of EGFR by other receptors involved extracellular release of EGFR ligands from the plasma membrane mediated by matrix metalloproteinases (MMPs) (26). Pretreatment of PCI-37A cells with the MMP inhibitor, marimastat, also completely inhibited the E2-induced P-MAPK (Figure 2B), indicating that MMP activity was required for this signaling.
We next examined which EGFR ligands were involved in this response. Pretreatment of cells with TGFα, AR and HB-EGF NAs followed by estrogen treatment diminished the P-MAPK response compared to estrogen treatment alone (Figure 2C). TGFα NA resulted in complete inhibition of estrogen-induced P-MAPK while AR NA and HB-EGF NA resulted in only partial inhibition (AR NA versus AR NA + E2, P>0.05, n.s.; HB-EGF NA versus HB-EGF NA + E2, P<0.005). Additionally, ELISA assays for each of these three ligands showed a 3.34-, 2.21- and 1.59-fold increase in secretion of TGFα, AR and HB-EGF, respectively, in the supernatant upon estrogen stimulation compared to no estrogen treatment (P<0.05) (Figure 2D). These results suggest that TGFα, followed by AR and HB-EGF are the primary ligands cleaved by estrogen stimulation and support a functional interaction between ER and EGFR in head and neck cancer cells.
We and others have previously shown that EGF can mediate invasion in HNSCC (27-29). To examine the effect of E2 alone and in combination with EGF on cell invasion, PCI-37A cells were grown on Matrigel invasion chambers and treated with either E2, EGF or the combination, and the number of invading cells was determined after 24 hr treatments. Both E2 and EGF alone significantly stimulated cell invasion by 3.8- and 4.2-fold, respectively (P<0.001) (Figure 3A). The combination of E2 and EGF further enhanced cell invasion significantly over single agent treatment with a 5.7-fold increase in invading cells observed (E2 versus E2 plus EGF P<0.01; EGF versus E2 plus EGF P<0.01) (Figure 3A).
We next examined the ability of the cells grown in complete media containing serum to respond to drugs that target either the ER or EGFR in an invasion assay. We used the pure antiestrogen, fulvestrant, and the EGFR tyrosine kinase inhibitor, gefitinib. Gefitinib and fulvestrant alone inhibited cell invasion by 52% and 46.7%, respectively (P<0.001), and the combination of gefitinib and fulvestrant inhibited cell invasion by 73.8% (gefitinib versus gefitinib plus fulvestrant P<0.01; fulvestrant versus gefitinib plus fulvestrant P<0.01) (Figure 3B). Using agents that target both the ER and EGFR signaling pathways together may have enhanced benefit compared to single pathway targeting.
In order to determine whether increased expression of ERα and/or ERβ was associated with tumorigenesis, TMA-arrayed HNSCC tumors and adjacent mucosal tissues were evaluated for ERα and ERβ protein levels by IHC staining. EGFR has been previously reported by us and others to be overexpressed in HNSCC compared to histologically normal tissues (13, 30). In order to evaluate EGFR in this cohort and assess correlations between ERs and EGFR, we also evaluated EGFR expression in these arrayed tissues. ERα and ERβ demonstrated predominantly nuclear staining, while EGFR staining was distributed through the plasma membrane and cytoplasmic compartments (Figure 4A). Of the HNSCC tumors evaluated, 52%, 95% and 44% were positive for EGFR, nuclear ERα (ERαnuc) and nuclear ERβ ERβnuc), respectively. Levels of EGFR and ERαnuc were found to be significantly higher in tumor than in paired adjacent mucosa while ERβnuc levels did not differ between tumors and adjacent tissues (Figure 4B). Of note, ERαnuc and ERβnuc expression levels in tumors and adjacent mucosal tissues did not differ by patient sex, P = 0.81 and P = 0.66, respectively, by the rank sum test (data not shown). Cytoplasmic ERα ERαcyto) and cytoplasmic ERβ (ERβcyto) were detected though staining was less robust than the nuclear compartment. 70% and 60% of HNSCC tumors were positive for ERαcyto and ERβcyto, respectively. Both ERαcyto and ERβcyto levels were elevated in HNSCC tumors compared to adjacent mucosa (P<0.001 and P =0.008, respectively) (data not shown). Neither ERα nor ERβ nuclear nor cytoplasmic levels differed by patient sex or tumor anatomical site (data not shown). Only paired adjacent mucosal tissues confirmed by our pathologist (RS) to be histologically normal were included in each analysis. The number of HNSCC tumors evaluated with paired adjacent mucosal tissues confirmed to be histologically normal for each protein evaluated is indicated in Figure 4B.
ERα, ERβ and EGFR high versus low tumor levels were independently evaluated for association with progression-free survival (PFS). Kaplan-Meier plots indicated that patients with high tumor levels of ERαnuc or EGFR tended to have shorter PFS than patients with low tumor levels (Figure 5A). High versus low tumor ERβnuc levels were not associated with differential PFS (Figure 5A). Neither ERαcyto nor ERβcyto levels were associated with differential PFS, P=0.20 and P=0.99, respectively (log rank test). PFS did not differ by subject sex (P=0.22) (Table 1). However, women with high tumor ERαnuc levels tended to have shorter PFS as assessed by Cox proportional hazards models than women with low ERαnuc (H.R.=6.32, P=0.09), a trend not observed for male cases (H.R.=0.65, P=0.25) or for female cases with high versus low EGFR tumor levels (H.R.=1.06, P=0.94). Our cohort had significantly more women than men never smokers at diagnosis, and women tended to smoke fewer pack-years than men (Table 1). BMI did not differ for subjects with high versus low ERαnuc tumor levels (P=0.09, rank sum test).
We confined analyses evaluating EGFR and ER tumor levels together to the ER nuclear component, the fraction associated with survival. Though ERαnuc levels were elevated in many tumors that expressed relatively low levels of EGFR, tumor ERαnuc and EGFR protein levels were found to be positively correlated (Figure 5B, left panel). Interestingly, patients with high ERαnuc and high EGFR tumor levels tended to have reduced PFS compared to patients with high tumor levels of either ERαnuc or EGFR or neither (Figure 5C, left panel).
Recently, the presence of human papilloma virus (HPV) in oropharyngeal HNSCC has been identified as an important prognostic indicator (31, 32). HPV detected by quantitative PCR or in situ hybridization has been reported to be present in 40-60% of oropharyngeal HNSCC and present in only small proportions of tumors from other sites (32). We performed a subset analysis excluding oropharyngeal tumors in order to evaluate whether our survival findings were likely related to HPV infection. Similar trends were observed for high versus low EGFR tumor levels (H.R.=1.73, P=0.18) and for high versus low tumor nuclear ERα levels (H.R.=2.15, P=0.08) in Cox univariate models excluding subjects with oropharyngeal tumors. Though the findings were not significant in these analyses with reduced power, the similarly increased hazards associated with high tumor EGFR or ERαnuc levels are likely not dependent on tumor HPV status.
EGFR expression in tumors is a proven prognostic factor for HNSCC. These findings indicate that the inclusion of ERαnuc tumor levels enhances the prognostic significance of EGFR tumor levels. In contrast, ERβnuc and EGFR tumor levels were not correlated (Figure 5B), and PFS for patients with high ERβnuc and high EGFR levels did not differ from patients whose tumors expressed high levels of either ERβnuc or EGFR (Figure 5C, right panel). Patients with EGFRH, ER-αnucH tumors were estimated to have significantly decreased PFS compared to patients with EGFRL, ER-αnucL (H.R. = 4.09; P = 0.01, univariate Cox proportional hazards) even after adjusting for age, sex, and clinical disease stage (H.R. = 4.19, P =0.03, Cox proportional hazards). The H.R. for patients with EGFRH, ERβnucH versus EGFRL, ERβnucL was estimated to be 2.27 (P=0.11) (Figure 5C, right panel) using univariate Cox proportional hazards models and 2.41 (P=0.12) after adjusting for age, sex, and clinical disease stage.
Our study was not adequately powered to determine whether patient PFS differed significantly for patients with both high EGFR and high ERαnuc compared to patients with tumors expressing high levels of only one of these markers. However, the approximate 2-fold increase in the H.R. when ERαnuc and EGFR status are both considered compared to either protein alone suggests that combining ERαnuc and EGFR tumor status likely improved predicted survival discrimination over ERαnuc or EGFR tumor status alone.
We have shown that ERs are expressed in HNSCC cell lines and tumors. We report here that the addition of exogenous estrogen stimulated HNSCC proliferation and invasion in vitro, indicating that ER activation contributes to HNSCC cell growth and invasion. ER has well-described genomic transcriptional and cytoplasmic signal transduction activities (33). Our findings are consistent with ER having both of these properties: we found that estrogen increased transcription from ERE and induced activation of MAPK in HNSCC cell lines. ER expression and function in HNSCC cell lines did not differ by sex of the patient from whom the cell lines were derived. In addition, ER expression was detected in the majority of HNSCC tumors, and expression levels did not differ by patient sex. Taken together, these data indicate that ER likely contributes to HNSCC growth and invasion in both men and women.
The literature regarding ER function in HNSCC is mixed. Our data support studies reporting a positive role for ERs in HNSCC growth and invasion. These reports include the findings that E2 treatment potentiated the growth of laryngeal xenograft tumors in nude mice and E2 was found to stimulate oral squamous cell carcinoma (OSCC) invasion in vitro (34, 35). Also, consistent with our data were the reported findings that the inhibition of ER activity by Tamoxifen reduced HNSCC cell growth in vitro (36) and that Tamoxifen treatment induced apoptosis and inhibited invasion of OSCC in vitro (35). However, the majority of the HNSCC cell lines found to be inhibited by Tamoxifen in the cell growth study were reported to not express ER (36).
Reports of the frequency of ER positive HNSCC vary widely. Expression of either ER subtype has been reported to be present in only 2.7% of HNSCC tumors by ER receptor assay and in 50.7% of HNSCC tumors by IHC (37, 38). There have been several reports that HNSCC tumors and cell lines do not express ER or the frequency of ER expression was less than 10% of tumors or HNSCC cell lines evaluated (37, 39, 40). In contrast, ER expression has been described in patient tumors with expression of the ERα subtype predominating over the ERβ subtype in an IHC study with PCR confirmation of ER subtype expression of 67 oral cavity and laryngeal/hypopharyngeal cancers (38). In a separate study of 15 primary HNSCC tumors, ERβ expression was observed in all HNSCC tumors while ERα expression was observed in only 2 of the 15 HNSCC tumors (35). Our data are consistent with more recent findings that ERs are expressed in the majority of HNSCC tumors and cell lines. In fact, all HNSCC cell lines that we evaluated expressed ERs. An earlier report suggested the ERs were more frequently expressed in tumors of larynx than other head and neck cancers (41); however, we found both ERα and ERβ were expressed in the majority of HNSCC tumors with no difference in expression level by anatomical tumor site. Importantly, we found high ERαnuc levels were associated with reduced progression-free survival (PFS) but no association of ERβ levels in either the nuclear or cytoplasmic compartment with PFS. Interestingly, we noted a trend in women for reduced PFS with high ERαnuc levels, which was not observed in men. Female cases tended to smoke less than male cases, suggesting the possibility that ER may be especially important for HNSCC etiologies less related to tobacco exposure. Though the patient tumor data suggest a more prominent role for ERαnuc in HNSCC for women, a larger cohort will be required to definitively assess the relationships between ER expression, gender and smoking.
Though we saw no sex-based ER expression level differences in HNSCC cell lines or tumors, it is possible that ER activity may play a role in the sex differences in tobacco-related susceptibility to HNSCC as has been proposed for lung cancer. Tobacco use is an identified risk factor for HNSCC. Though men were found to smoke more than women, hazards associated with smoking were reported to be higher for women than men in a large prospective cohort study of 476,211 participants (42). Smoking-related risk for oral cancers has also been reported to be higher for women than men in at least one independent study (43). ER-mediated events may be responsible for at least some of the increased tobacco-related HNSCC risk for women compared to men because women have higher circulating levels of estrogen. ER-mediated events may also contribute to HNSCC in men. Tissue levels of estrogen in men may be high enough to show biological effects because testosterone can be converted to estrogen through the action of aromatase, which has been shown to be expressed in head and neck tissue samples (44).
Our finding is the first evaluation of ER levels and HNSCC prognosis. To date, there are no reports evaluating ER expression and HNSCC disease progression. Several studies have evaluated the relationship between ER levels and disease prognosis in upper aerodigestive cancers including lung cancers (45-48). Of these, elevated ERαcyto level by IHC was associated with poorer overall survival in a study of 132 NSCLC (45). In this same study, loss of ERβnuc expression associated with poorer survival, and ERαnuc positive-ERβcyto negative patients had significantly reduced survival compared to ERαnuc negative-ERβcyto positive patients(45). Two of these lung cancer studies reported elevated nuclear ERβ levels were associated with better survival in men only (47, 48). These data and our data suggest that ER subtype and subcellular localization may differ for HNSCC and lung cancers. However, subtype and localization are important determinants of ER involvement in upperaerodigestive cancers including HNSCC. The ERα and ERβ antibodies used in our study are the same antibodies that we and others have used previously to detect ER expression (24, 45, 46).
It is important to note that our in vitro studies characterize ER function while our evaluation of ER protein expression in tumors does not, and assessment of ER levels in patient tumors included subcellular localization while in vitro studies evaluated whole cell lysates. At least one study has reported that ER expression level and ER activity were not correlated (49), and in this study of lung adenocarcinoma-derived cells, ER expression levels did not differ by patient sex but ER activity was higher in cells derived from females (49). Therefore, though we found that high ERαnuc levels by IHC were associated with reduced PFS, our data do not necessarily indicate that ERα activity is elevated in tumors with high ERαnuc protein. In addition, the in vitro analysis indicates that several possible signaling mechanisms occur in HNSCC, including both nuclear and cytoplasmic estrogen signaling. However, the predominant mechanism involved in the survival analysis appears to be the nuclear estrogen signaling.
We were especially interested in the evaluation of ER and EGFR cross-activation. We report here evidence that ER and EGFR cross-talk is present in HNSCC. The rapid activation of EGFR by non-nuclear ER was dependent upon matrix metalloproteinases (MMPs) and was present in HNSCC cell lines derived from both males and females. We found combined ER and EGFR inhibition in vitro reduced HNSCC invasion but not proliferation compared to single targeting. Though ER and EGFR ligand activation promoted both invasion and proliferation when administered separately, we did not observe enhanced inhibition of proliferation with dual inhibition. Our data suggest that there is likely redundancy in the pathways leading to proliferation for EGFR and ER while at least some independent contribution to invasion are provided by EGFR- and ER-mediated signaling events. Interestingly, in vitro we found that the highest estrogen induced transcriptional activity was observed in cells with low EGFR expression while low transcriptional activity was observed in cells with high EGFR expression. This is in agreement with a previously reported reciprocal control mechanism for estrogen-EGF signaling reported for lung cancer and breast cancer cells (9, 50). Our evaluation of EGFR and ER expression in HNSCC tumors indicated that in a subset of tumors, coordinated elevated expression of EGFR and ERαnuc was associated with poor prognosis. EGF and estrogen independently activate signaling pathways known to be involved in tumorigenesis, and these data combined with our in vitro data suggest that EGFR and ER cross-talk promote tumor invasion, which may contribute to poor patient prognosis. The cross-signaling between the ER-EGFR pathways in HNSCC provides rationale to combine anti-estrogen therapy with EGFR inhibition for head and neck cancer treatment. These results suggest that increased E2 and EGF signaling contribute to the invasive properties of HNSCC and that combined inhibition of these two pathways augment the inhibition of invasion compared with blockade of each pathway separately. These data provide rationale for further investigation of the mechanism of combined ER and EGFR targeting in HNSCC with expression of these proteins.
This work was supported from the Oral Cancer Center at the University of Pittsburgh awarded to LPS as well as from NIH P50CA097190 SPORE in Head and Neck Cancer awarded to JRG. We gratefully thank Jennifer Ridge Hetrick for her assistance with the patient data. We also thank Kim Fuhrer, Marianne Notaro and Marie Aquafondata for their technical assistance in the preparation of the TMAs and the immunohistochemical staining.
Supported by the Oral Cancer Center at the University of Pittsburgh and NIH P50CA097190 SPORE in Head and Neck Cancer.