Affiliations of authors: M. N. Lango, K. F. Dyer, V. W. Y. Lui (Department of Otolaryngology), C. Gubish, J. M. Siegfried (Department of Pharmacology), J. R. Grandis (Departments of Otolaryngology and Pharmacology), University of Pittsburgh School of Medicine, PA; W. E. Gooding, Department of Biostatistics, University of Pittsburgh Cancer Institute.
Correspondence to: Jennifer Rubin Grandis, M.D., 200 Lothrop St., Suite 500, Pittsburgh, PA 15213 (e-mail: jgrandis{at}pitt.edu).
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ABSTRACT |
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INTRODUCTION |
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Anti-GRP murine monoclonal antibody 2A11 binds the carboxyl-terminal region of the GRP peptide, functionally sequestering the ligand. This antibody has undergone a phase I clinical trial in patients with SCLC and patients with NSCLC (13). Treatment with this antibody has been associated with a tumor growth inhibition of more than 90% in a murine lung cancer model (9). Thus, GRPR is implicated in an autocrine or paracrine growth pathway in SCLC and NSCLC cells, where GRPR appears to be an early marker of susceptibility to tobacco-related cancers.
GRPR signaling appears to mediate tobacco-related injury to the lungs and the autocrine growth of human lung carcinomas (2,79). Although tobacco exposure is a well-characterized risk factor for the development of squamous cell carcinoma of the head and neck (SCCHN), to our knowledge, expression of neither GRP nor GRPR in SCCHN has been reported previously. To investigate whether GRPR-mediated autocrine growth is involved in SCCHN carcinogenesis, we investigated whether GRP stimulated growth in SCCHN cell lines and tumors, whether GRPR messenger RNA (mRNA) was expressed in SCCHN tissues and normal control mucosa, and whether blocking the interaction of GRP and GRPR affected the growth of SCCHN cells in vitro and in vivo.
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MATERIALS AND METHODS |
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Tumor tissue was collected between 1990 and 1995 at the University of Pittsburgh Medical Center (PA) from 25 patients with SCCHN who were undergoing surgical resection with curative intent and had adequate tissue for analysis. A sample of normal-appearing mucosa at the margin of the surgical resection several centimeters from the tumor was also collected and designated histologically normal mucosa. Patients with pathologically confirmed SCCHN of the upper aerodigestive tract (oral cavity, oropharynx, hypopharynx, or larynx) were eligible. Tissue was collected under the auspices of a tissue-bank protocol that was approved by the Institutional Review Board. Written informed consent was obtained from all patients. Clinical characteristics of the SCCHN patients are presented in Table 1. Previous radiation therapy failed for four patients. Of the 19 patients whose smoking histories were available, 18 patients were current smokers or former smokers and one female with an oral cavity cancer was a nonsmoker. The six female smokers in our study reported a mean smoking history of 20 pack-years, and the 13 male smokers reported a mean smoking history of 80 pack-years. Pack-years are defined as the number of packs smoked per day multiplied by the number of years over which that amount was smoked. Eight patients were alive with no evidence of disease on follow-up from 39 to 91 months after surgical resection. Eleven patients died of their disease, either from recurrence or second primary SCCHN tumors, five patients died of other causes, and one patient was lost to follow-up. The median follow-up for surviving patients was 55 months. The median survival of patients who died of disease was 20 months. Mucosa from six control noncancer patients who did not have SCCHN was also harvested. These control patients underwent unrelated head and neck surgical procedures (e.g., uvulopalatopharyngoplasty). Patients were matched with the study group with respect to age (mean age ± 5 years) and sex.
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Fourteen cell lines derived from patients with SCCHN were grown in Dulbecco's modified Eagle medium (DMEM; Cellgro, Washington, DC), supplemented with 12% fetal bovine serum (Life Technologies, Inc. [GIBCO BRL], Rockville, MD), penicillin (0.5 µg/mL), and streptomycin (0.5 µg/mL; Life Technologies, Inc.). These cell lines are part of a large collection established in the Department of Otolaryngology, University of Pittsburgh (15). Cell lines UM-22A and UM-22B were derived from a primary SCCHN (22A) and a metastatic cervical lymph node (22B) (16). Cell line 1483 was derived from an oropharyngeal tumor (17). SCCHN cell lines UPCI:SCC066, UPCI:SCC078, UPCI:SCC111, UPCI:SCC105, UPCI:SCC068, UPCI:SCC104, UPCI:SCC143, UPCI:SCC099, UPCI:SCC089, and UPCI:SCC103 were gifts from Dr. Susanne M. Gollin (University of Pittsburgh Graduate School of Public Health, PA). Cell line OSC-19 was derived from a squamous cell carcinoma of the tongue (18). Primary mucosal cultures established from oropharyngeal mucosa harvested from control noncancer patients were used within the first three passages, as described previously (19). Cell line 201T is an NSCLC cell line that was used as a positive control for GRPR immunoblotting.
RNA Extraction and Quantitative Reverse TranscriptasePolymerase Chain Reaction
Total RNA was extracted from primary tissues and SCCHN cell lines with TRIZOL reagent (Life Technologies, Inc.), as described by the manufacturer. GRPR was amplified in a one-step reverse transcriptasepolymerase chain reaction (RTPCR) with Superscript/Taq enzyme (Life Technologies, Inc.) and 0.05 µg of total RNA in each reaction. The sequence of the 5` GRPR primer was 5`-CTCCCCGTGAACGATGACTGG-3`, and the sequence of the 3` GRPR primer was 5`-ATCTTCATCAGGGCATGGGAG-3`. This reaction gave a 390-base-pair (bp) PCR product. The sequence of the 5` -actin primer was 5`-GGCGGCACCACCATGTACCCT-3`, and the sequence of the 3`
-actin primer was 5`-AGGGGCCGGACTCGTCATACT-3`. This reaction gave a 202-bp PCR product. The sequence of the 5` GRP primer was 5`-GGGACCATGCGCGGCAGTGA-3`, and the sequence of the 3` primer was 5`-TGCAAGGAATTTGCTGGGTCTC-3`. This reaction gave a 405-bp product. RTPCR conditions for GRP were identical to those used for GRPR. The RT reaction was carried out at 50 °C for 30 minutes, followed by PCR for 40 cycles at 95 °C for 30 seconds, 58 °C for 45 seconds, and 72 °C for 45 seconds. A semiquantitative RTPCR protocol for GRPR mRNA quantitation was developed in our laboratory. Expression levels were based on a ratio of GRPR or GRP mRNA expression to
-actin mRNA expression. Preamplification of GRPR or GRP for 17 cycles before adding ("dropping in") a 5`
-actin primer was followed by coamplification for an additional 18 cycles. The preamplification was determined empirically but required both PCR products to be in the linear phase of amplification. Deoxycytidine 5`-[32P]triphosphate was incorporated into the PCR products. The products were separated by electrophoresis on a 7.5% polyacrylamide gel, and the gel was dried onto Whatman paper (Whatman, Inc., Clifton, NJ). PCR products were exposed to X-Omat film (Eastman Kodak, Rochester, NY) and subsequently exposed to a phosphor screen for 0.55 hours. Intensities of the GRPR or GRP bands and
-actin bands were quantified by use of a computerized PhosphorImager (Molecular Dynamics, Sunnyvale, CA) and analyzed by ImageQuant software (Molecular Dynamics) as described previously (20).
Sequencing
The amplified GRPR PCR product from a representative cell line, UM-22B, was isolated from the agarose gel and purified by use of a Qiagen DNA purification column (Qiagen, Valencia, CA), as described by the manufacturer. Ten nanograms of PCR product was cycle sequenced (AmpliTaq; PerkinElmer, Foster City, CA) with 3.4 pmol of 3` GRPR primer in an automated sequencing reaction and an ABI Prism 377 sequencer. Base-specific, laser-induced fluorescent emissions data were collected and processed by computer software after dye-labeled fragments were separated by size on a polyacrylamide gel.
Western Blotting
Lysis buffer (1% Nonidet P-40, 150 mM NaCl, 1 mM EDTA, 10 mM sodium phosphate [pH 7.2], aprotonin [10 µg/mL], and leupeptin [10 µg/mL]) was used to extract total cellular protein. Cells were incubated with the lysis buffer for 5 minutes on ice, and the lysate was centrifuged for 30 minutes at 12 000g. The protein concentration in the supernatant was measured with the Bio-Rad Protein Assay (Bio-Rad Laboratories, Hercules, CA). Fifteen micrograms of total protein was loaded onto a sodium dodecyl sulfate-10% polyacrylamide gel. After electrophoresis, the protein was transferred to a Protran membrane (Schleicher & Schuell, Keene, NH) for immunoblot analysis. GRPR antiserum was generated as described previously (21).
Growth Stimulation by GRP
The 1483 SCCHN cells were plated at 2.5 x 104 cells per well in a 24-well plate in serum-free DMEM and incubated overnight. Recombinant GRP (Sigma-Aldrich, St. Louis, MO) in 0.01 M glacial acetic acid (Sigma-Aldrich) was added to the cells as indicated. Cells were then incubated at 37 °C in an atmosphere of 5% CO2/95% air. After 48 hours, a vital dye (erythrosin B) was added, and living cells, excluding the dye, were counted.
In Vitro Studies With Anti-GRP Antibody 2A11
The 1483 SCCHN cells were plated at 6 x 104 cells per well in a 24-well plate in DMEM supplemented with 12% fetal bovine serum, 1% penicillin, and 1% streptomycin. Twenty-four hours later, cells were treated with either 50 µg or 100 µg of GRP-neutralizing antibody 2A11 (Abbott Biotech, Inc., Needham Heights, MA) (in 100 µL) or an equivalent volume of saline (100 µL) in DMEM supplemented with 12% fetal bovine serum, 1% penicillin, and 1% streptomycin. All assays were performed in triplicate. The GRP-neutralizing antibody binds to the carboxyl-terminal end of GRP and neutralizes the biologic effects of GRP. Every 48 hours for 8 days, cells were then trypsinized, resuspended, and counted with a hemocytometer by vital dye exclusion.
In Vivo Tumor Xenograft Studies
The 1483 SCCHN cells grow well as xenografts in nude mice. For subcutaneous implantation into mice, cells 75%80% confluent were harvested by trypsinization, resuspended in DMEM supplemented with 10% fetal bovine serum, centrifuged at 400g for 10 minutes, and resuspended in culture medium at 1 x 107 cells per milliliter. Female athymic nude mice (nu/nu, 46 weeks old; 20 ± 2 g [mean ± standard deviation]; Harlan Sprague-Dawley, Inc., Indianapolis, IN) were implanted with 1 x 106 cells into the right flank and 1 x 106 cells into the left flank in volumes of 200 µL by using a 26-gauge needle and a 1-mL tuberculin syringe. One day later, mice were randomly assigned to a treatment group of 0.5 mg of 2A11 antibody per dose or no treatment. In each experiment, each treatment group had 810 mice. Experiments were repeated three times to ensure reproducibility. Anti-GRP-neutralizing monoclonal antibody 2A11 was administered intraperitoneally twice a week for 5 weeks, for a total of 10 doses. Treatment was initiated 24 hours after tumor cell implantation. Bilateral tumors were measured with calipers before each injection (twice a week), and the average tumor volume for each mouse was calculated (tumor volume = length x [width]2/2) by averaging the volumes of both tumors. Mice were killed when the tumors ulcerated or reached a maximum diameter of 2 cm. Animal care was in strict compliance with institutional guidelines established at the University of Pittsburgh, the Guide for the Care and Use of Laboratory Animals (National Academy of Sciences, 1996), and the Association for Assessment and Accreditation of Laboratory Animal Care International.
Statistical Analysis
GRPR mRNA levels in tumors or histologically normal adjacent mucosa from patients with SCCHN were compared with those in normal mucosa from noncancer patients. Statistically significant differences were tested with two-tailed t tests with a Welch modification for unequal variances. Statistically significant differences in GRPR mRNA levels among patient subgroups having various clinical parameters were tested with the Wilcoxon test (gender and extracapsular spread) or the KruskalWallis test (N stage and disease site). The correlation between levels of GRPR mRNA in tumor and normal mucosa was examined, and a Spearman correlation coefficient was calculated. The effect of the GRP-neutralizing antibodies on SCCHN cell growth in vitro was tested with the exact two-sided JonckheereTerpstra test. The antitumor effect of the anti-GRP antibody in mice was tested by fitting mixed linear models to the log-transformed tumor growth curves. Before averaging the tumor volumes from both flanks, a flank-by-treatment interaction and a random effect of flank were tested and found to be no different from zero. The association between dose levels of exogenous GRP and SCCHN proliferation was tested by fitting a linear regression model for the regression of the logarithm of cell count on the logarithm of dose. Overall survival was measured in months from the date of surgery to the date of death or to the last follow-up. Survival analysis was performed by use of a median split method in which patients were divided into two groups on the basis of whether GRPR mRNA levels were greater than or less than the median. Differences between the survival of the two groups were examined with the log-rank test. All statistical tests were two-sided. Confidence intervals (CIs) were computed by assuming that estimates were normally distributed.
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RESULTS |
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We have demonstrated that GRPR is expressed in NSCLC (10). To determine whether GRPR mRNA was expressed in SCCHN, we developed a quantitative RTPCR assay based on the primer-dropping method as described previously by our laboratory (20) and compared the levels of GRPR mRNA expression in tumor specimens and adjacent normal mucosa from 25 patients with SCCHN with those in mucosal samples from six noncancer patients matched for age (±5 years) and sex. In primary tumors and adjacent normal mucosa from patients with SCCHN, the level of GRPR mRNA expression (quantitated as a ratio of GRPR mRNA to -actin mRNA) was sixfold and fourfold higher, respectively, than in normal mucosa from noncancer patients (P<.001, t test, Welch modification for unequal variances). Means and 95% CIs for the means for these ratios were as follows: for tumor level, 0.414 (95% CI = 0.285 to 0.543); for normal mucosa from SCCHN patients, 0.566 (95% CI = 0.411 to 0.712); and for normal mucosa from noncancer patients, 0.090 (95% CI = 0.0 to 0.188) (Fig. 1, A,
and data not shown).
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When 14 SCCHN cell lines were examined for GRPR mRNA expression, all lines expressed GRPR mRNA. The amplified GRPR fragment from a representative cell line (UM-22B) was isolated and sequenced to confirm the presence of GRPR mRNA (data not shown). No evidence of mutations was found in the amplified sequence from UM-22B cells. Polymorphisms of the GRPR gene have been previously reported, but it is not known whether these polymorphisms segregate to cancer patients (12). When GRPR expression levels (reported as a ratio of GRPR mRNA to -actin mRNA) in these 14 SCCHN cell lines were compared with levels in primary cultures of normal mucosal epithelial cells from six noncancer patients, steady-state levels of GRPR mRNA expression were fivefold higher in the SCCHN cells, indicating that GRPR is expressed by squamous epithelial cells and is expressed at an increased level in transformed cells in vitro (cell line mean = 0.320 [95% CI = 0.460 to 1.100]; normal mucosal epithelial cells mean = 1.791 [95% CI = 1.081 to 2.501]) (P = .005). High levels of GRPR protein were observed by western blotting in SCCHN cell lines but not in cultured control mucosal epithelial cells from noncancer patients (Fig. 1, B
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GRP Expression in SCCHN Cells
To determine whether an autocrine pathway involving GRP and GRPR was active in SCCHN, SCCHN cell lines were examined for GRP expression with quantitative RTPCR. The level of GRP expression was higher in SCCHN cell lines than in normal mucosal epithelial cells (Fig. 1, C). To investigate whether stimulation of GRPR induces SCCHN cells to proliferate, exogenous GRP was added to cultured SCCHN cells in the absence or presence of 12% fetal bovine serum. Linear regression of the logarithm of cell count on the logarithm of the dose of (GRP + 1) was statistically significant (
= 0.114, 95% CI for
= 0.063 to 0.165; P = .006), indicating that the addition of GRP stimulated the growth of SCCHN cells in a dose-dependent manner. A similar degree of growth stimulation was observed in other SCCHN cell lines (data not shown).
Antitumor Effect of GRP-Blocking Antibody In Vitro and In Vivo
To determine whether disruption of the GRP/GRPR autocrine pathway in vitro would affect SCCHN tumor growth, 1483 SCCHN cells were treated with murine GRP-neutralizing monoclonal antibody 2A11, which binds the carboxyl-terminal region of the GRP peptide and functionally sequesters GRP (13). After 8 days, proliferation of 1483 SCCHN cells was reduced by anti-GRP 2A11 in a dose-dependent manner compared with untreated cells (JonckeereTerpstra test, P = .001; Fig. 2, A). An isotype-matched IgG1 control mouse monoclonal antibody had no effect on the growth rate of these cells (data not shown).
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Elevated GRPR Expression in the Normal Mucosa of Patients with SCCHN
Because of the high incidence of second primary tumors in patients with SCCHN, alterations detected in the histologically normal mucosa from these patients may provide indirect evidence of early changes in SCCHN carcinogenesis. Consequently, we determined levels of GRPR mRNA expression in primary tumor tissues and adjacent normal mucosa from patients with SCCHN and found that the levels of GRPR mRNA in these tissues correlated with each other (Spearman correlation coefficient = .652, P = .001). These results support the concept of field cancerization and suggest that increased GRPR expression is an early event in SCCHN formation.
GRPR Expression and Clinical and Pathologic Parameters
Metastasis to the lymph nodes of the neck is the strongest predictor of poor outcome in patients with SCCHN. In this study, such patients had a poorer outcome than did those without clinical or pathologic evidence of such metastasis. All patients with extracapsular invasion of the cervical lymph nodes died of their disease, consistent with reports that extracapsular spread is associated with poor prognosis (2224). The level of GRPR mRNA in primary tumor tissues from patients with SCCHN was not associated with tumor stage, tumor recurrence, or a history of prior radiation therapy. Elevated levels of GRPR mRNA in the SCCHN tumor were associated with a distal aerodigestive tract site (P = .014). Although there were few proximal tumors for comparison, tumors in the oral cavity had lower levels of GRPR mRNA expression than did tumors in more distal aerodigestive tract sites, such as the oropharynx, hypopharynx, or larynx. We observed a trend toward increased levels of GRPR mRNA expression in tumors with increased lymph node stage (P = .077). Although most patients had well-differentiated tumors, there was an association between tumor grade and level of GRPR expression (P = .037). The few patients with extracapsular spread after pathologic examination of the cervical lymph nodes had high levels of GRPR in their primary tumor (P = .026) and in their histologically normal mucosa (P = .034) (Fig. 3).
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To determine whether levels of GRPR mRNA in patients with SCCHN were associated with overall survival, KaplanMeier analyses were performed for 24 patients with complete follow-up and vital status information. Levels of GRPR mRNA suggested a tendency toward decreased survival but with insufficient power to detect differences (Fig. 4).
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DISCUSSION |
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The level of GRPR mRNA expression in cultured bronchial epithelial cells from smokers has been shown previously to correlate with smoking history (11). There is further evidence that regulation of GRPR expression is sex specific. Female smokers with NSCLC had higher levels of GRPR mRNA expression at a lower mean number of pack-years of tobacco exposure than male smokers, and female nonsmokers with NSCLC had higher levels of GRPR expression than their nonsmoking male counterparts (12). The increased susceptibility of women to the carcinogenic effects of tobacco has been attributed to higher levels of GRPR in female patients when stratified for smoking history (12,2729). The increased baseline levels of GRPR in females has been attributed to the location of the GRPR gene in a pseudoautosomal region on the X chromosome that escapes inactivation when the rest of the X chromosome is inactivated. Sex differences also exist in the risk for head and neck cancer, with females at higher risk than males when stratified for tobacco use, suggesting that a relationship among sex, tobacco exposure, and level of GRPR mRNA expression may increase the risk of developing SCCHN (30). We found that the level of GRPR mRNA expression correlated with the site of the aerodigestive tract tumor. Tumors from more distal locations including the oropharynx, hypopharynx, and larynx had higher levels of GRPR expression than tumors from the oral cavity. One potential explanation for this finding may be the closer link of tumor formation in distal aerodigestive tract sites to tobacco use, whereas the more proximal sites, including the oral tongue, buccal mucosa, and hard palate, have been linked to other etiologic agents, such as human papillomavirus (31). Alternatively, tobacco-mediated induction of GRP expression in neuroendocrine cells in distal regions of the aerodigestive tract may stimulate tumor growth through a paracrine mechanism. Our resultsthat both GRP and GRPR mRNAs are overexpressed in SCCHN cells lines, that exogenous GRP stimulates growth, and that anti-GRP-neutralizing monoclonal antibodies inhibit cell proliferation in vitro and in vivoare consistent with a GRP/GRPR autocrine growth pathway in SCCHN. However, the mechanism of GRP-mediated mitogenesis may be more complex in vivo than in vitro. Tobacco appears to induce GRP expression in pulmonary neuroendocrine cells. Exposure of hamsters to tobacco smoke causes hyperplasia of pulmonary neuroendocrine cells and correlates with increased levels of GRP in these animals (2). In another study, induction of pulmonary neuroendocrine cell hyperplasia in an animal model of preneoplastic lung injury was associated with increased levels of GRP mRNA expression by pulmonary neuroendocrine cells (32). Although SCCHN cells can promote their own growth through an autocrine mechanism in vitro, paracrine-mediated stimulation of SCCHN growth by neuroendocrine cells from distal aerodigestive tract sites may also contribute to tumor growth in vivo.
When human tumor cells express high levels of growth factor receptors, receptor-directed therapies may be useful anti-cancer strategies. Expression of bombesin-like peptide ligands and receptors has been reported in several cancers including glioblastomas (33) and carcinomas of the ovary (34), colon (35), kidney (36), breast (37), and lung (10). Mammalian forms of bombesin-like peptides have been identified as mitogens and morphogens for a variety of cancers, including NSCLC. Although classically associated with cells of neuroendocrine origin, several transformed cell types, including airway epithelial cells, respond to bombesin-like peptides (38). Immortalized human bronchial epithelial cells engineered to overexpress GRPR responded to exogenous bombesin by increasing calcium influx and cell proliferation (39). We reported previously that GPRP expression in lung epithelium was associated with a history of prolonged tobacco exposure and responsiveness to the mitogenic effects of bombesin-like peptides (11). In an animal model of chemical-induced oral cancer, a bombesin antagonist prevents the formation of preinvasive mucosal lesions (40). Many studies have demonstrated the antitumor effect of GRPR-specific inhibitors in preclinical animal models (41,42). A phase I clinical trial using the anti-GRP antibody 2A11 was conducted in patients with SCLC or NSCLC, with no evidence of toxicity noted (13). An antitumor effect was observed with this anti-GRP antibody in patients with SCLC where one of 12 patients who could be evaluated was disease-free for 4 months (43). In this study, we have demonstrated that treatment with anti-GRP antibody 2A11 inhibited the growth of SCCHN cells in vitro and in vivo. Therefore, strategies that specifically target GRP and/or GRPR may prove to be effective therapies for SCCHN.
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NOTES |
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Manuscript received May 4, 2001; revised January 3, 2002; accepted January 22, 2002.
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