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[Cancer Research 61, 5919-5925, August 1, 2001]
© 2001 American Association for Cancer Research

Tumor Biology

Abrogation of Transforming Growth Factor-{alpha}/Epidermal Growth Factor Receptor Autocrine Signaling by an RXR-selective Retinoid (LGD1069, Targretin) in Head and Neck Cancer Cell Lines 1

John I. Song, Miriam N. Lango, James D. Hwang, Stephanie D. Drenning, Qing Zeng, William W. Lamph and Jennifer Rubin Grandis2

Departments of Otolaryngology [J. I. S., M. N. L., J. D. H., S. D. D., Q. Z., J. R. G.] and Pharmacology [J. R. G.], University of Pittsburgh School of Medicine, and the University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania 15213 [W. W. L.], and Ligand Pharmaceuticals, Inc., San Diego, California 92121 [W. W. L.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Clinical studies have demonstrated that retinoids, including retinol (Vitamin A) and its synthetic derivatives, can eradicate leukoplakia and suppress the formation of squamous cell carcinoma of the head and neck (SCCHN). Nonselective retinoids have been shown to abrogate transcriptional activation of transforming growth factor-{alpha} (TGF-{alpha}) and epidermal growth factor receptor (EGFR), which characterize SCCHN. LGD1069 (Targretin) is a potent RXR-selective retinoic acid agonist with a reduced toxicity profile compared with other nonselective retinoids. We examined the effect of LGD1069 (10 µm) on cellular proliferation and expression of putative intermediate biomarker genes including TGF-{alpha}, EGFR, and RAR-ß in seven SCCHN cell lines. A quantitative reverse transcription-PCR assay using a novel "primer dropping" method was used to determine expression levels of EGFR, TGF-{alpha}, and RAR-ß before and after treatment with LGD1069 (10 µM). SCCHN proliferation was reduced by a mean of 50% at 4 days in seven SCCHN cell lines after LGD1069 treatment (P <= 0.05). EGFR expression levels were decreased by a mean of 58.4% (P = 0.007), TGF-{alpha} levels were decreased by a mean of 28.8% (P = 0.01), and RAR-ß levels were increased by a mean of 60% (P = 0.03). TGF-{alpha} stimulation of EGFR is associated with constitutive signal transducer and activator of transcription 3 (Stat3) activation in SCCHN. Abrogation of constitutive Stat3 activation was seen with LGD1069 treatment. These results suggest that an RXR-selective retinoic acid decreases SCCHN proliferation in part by interfering with TGF-{alpha}/EGFR autocrine signaling.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
SCCHN 3 is the sixth most common neoplasm worldwide with >500,000 new cases diagnosed each year (1) . Despite the use of combined surgical, radiation, and chemotherapy regimens, the outcome for patients with advanced SCCHN has not improved significantly over the past 25 years (2 , 3) . The 5-year survival for patients with resectable tumors remains 40% (4) . If the patient is cured of their primary tumor, there is a constant and continuing risk, from 2.7 to 4% per year, of second primary tumor (i.e., aerodigestive tract) formation each year after initial treatment (5) . Few treatment modalities are available to patients with recurrent head and neck cancer, and those who develop a second malignancy have only a 25% 5-year survival rate (6) . Therefore, effective chemoprevention of second primary tumors is needed to improve long-term survival in patients with SCCHN.

Retinoids, including vitamin A and its analogues, regulate the growth and differentiation of a wide variety of cells (7) . In some developing countries, vitamin A deficiency has been associated with an increased incidence of cancer (8 , 9) . Retinoids suppress tumor formation in animals and have been shown to be effective chemopreventive and therapeutic agents in many cancers, including SCCHN (10) . 13-cis-RA treatment can eradicate premalignant lesions such as oral leukoplakia and reduce the incidence of second primary tumors in patients cured of their initial SCCHN (11 , 12) . However, the chronic administration of retinoids is limited by vitamin A toxicity, which can include chelitis, hypertriglyceridemia, and hepatosplenomegaly (13) .

Retinoids act by binding to intracellular RARs, which interact with specific DNA response elements to regulate the transcriptional activity of retinoid target genes (14) . There are two classes of retinoid receptors, RARs and RXRs, each with three subtypes, {alpha}, ß, and {gamma}. 9-cis-RA, the natural ligand for RXRs, has been shown to have superior antitumor efficacy compared with 13-cis-RA in acute promyelocytic leukemia, and in an experimental breast cancer model, with a lower toxicity profile because of its selective RXR binding (15) . LGD1069 (Targretin) is a highly selective synthetic RXR ligand devoid of significant RAR binding or transactivation of RAR-responsive genes (16) . Studies have shown that LGD1069 may serve as an effective chemopreventive compound against nitrosomethylurea-induced mammary tumors and demonstrates antitumor efficacy in animal models of established mammary carcinomas (16 , 17) . There is some evidence to suggest that LGD1069 may be an effective chemopreventive agent against aerodigestive tract tumors including NSCLC (18 , 19) .

We have demonstrated previously that RA reduces SCCHN proliferation and down-modulates TGF-{alpha} and EGFR levels in SCCHN by decreasing the transcription rate of these two genes (20) . Activation of TGF-{alpha}/EGFR gene transcription appears to be an early event in SCCHN carcinogenesis, as evidenced by TGF-{alpha}/EGFR up-regulation in nontransformed squamous epithelium of SCCHN patients as well as in premalignant dysplasias (21 , 22) . Further investigation demonstrated that TGF-{alpha}/EGFR mitogenic signaling in SCCHN is mediated by constitutive activation of Stat3 (23 , 24) . In this study, we examined the hypothesis that an RXR-selective retinoid (LGD1069) can suppress the proliferation of SCCHN cells in vitro by interference with TGF-{alpha}/EGFR autocrine signaling.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cells and Growth Assays.
SCCHN cell lines were established at the University of Pittsburgh Cancer Institute (PCI-15b and PCI-37a; Ref. 25 ). Several SCCHN cell lines (SCC-66, SCC-103, SCC-104, and SCC-105) were kindly provided by Dr. Susanne M. Gollin (University of Pittsburgh, Pittsburgh, PA). The 1483 cell line was established at M. D. Anderson Cancer Center (Houston, TX) and was generously provided by Dr. Ruben Lotan. The cell line UM-22B was kindly provided by Dr. Thomas Carey (University of Michigan, Ann Arbor, MI). Cell lines were grown in DMEM (Cellgro, Washington, DC) with 15% FBS (Life Technologies, Inc., Grand Island, NY), plus 100 units/ml of penicillin and 100 units/ml of streptomycin (Life Technologies, Inc.). The compound, LGD1069, was a generous gift from Dr. William W. Lamph (Ligand Pharmaceuticals). It was received as a lyophilized powder and dissolved in DMSO at a concentration of 1:1000. To test the inhibitory growth effects of LGD1069, proliferation rates of SCCHN cell lines were measured by cell count experiments using vital dye (erythrosin B) exclusion. At least three separate growth curves were generated for each SCCHN cell line using triplicate wells.

To determine whether the addition of exogenous TGF-{alpha} could "rescue" SCCHN cells from the inhibitory effects of LGD1069, recombinant TGF-{alpha} (30 ng/ml) was added daily for a total of 6 days, followed by cell counts using vital dye exclusion. To determine the direct role of Stat3, a dominant active Stat3 construct, Stat3C, was stably transfected into a representative SCCHN cell line, UM-22B, as described previously (26) .

Animal Studies.
1483 cells in log phase were harvested and resuspended in culture medium at a concentration of 1 x 107 cells/ml prior to s.c. implantation into mice. Female athymic nu/nu mice, 6–7 weeks of age (20 ± 1–2 g; Taconic Labs, Germantown, NY), were quarantined for 1 week prior to study and allowed access to food (Purina Chow 5010; Purina, St. Louis, MO) and water ad libitum. Mice were implanted with 1 x 106 cells into axial regions with a 24-gauge needle/1-ml tuberculin syringe (Becton Dickinson, Rutherford, NJ). Animals were randomized 48 h after tumor implantation into treatment groups or after tumors reached ~100 mm3 volumes. Treatment with LGD1069 was begun after established tumors had formed. LGD1069 was administered with a 20-gauge intragastric feeding tube (Pepper and Sons, New Hyde Park, NY) daily, 5 days/week, in 0.1 ml of super-refined sesame oil (Croda, Inc., Parsippany, NJ). Animals were treated for 24 days, and tumors were harvested 6 h after the last treatment. RNA was extracted, and quantitative RT-PCR was performed.

Quantitative RT-PCR.
RT-PCR reactions were performed using two pairs of primers in each reaction with the SuperScript one-step RT-PCR system (Life Technologies, Inc., Gaithersburg, MD). One pair of primers was specific for the target intermediate biomarker gene, and the other pair was specific for ß-actin to be used as an internal control for quantitation (see Ref. 26 for primer sequences and PCR product lengths). Because all of the target genes were less abundant than ß-actin and coamplification of mRNAs present at different levels might result in competitive interference, we preamplified the target genes with several cycles of PCR after the reverse transcriptase reaction before "dropping in" the sense ß-actin primer to coamplify both genes for the remaining PCR cycles. The RT-PCR reaction volume was scaled down to 20 µl. A total of 0.01 µl of total RNA was used as a template in the reaction mixture with 1 x RT-PCR buffer, 0.5 µl of 20 µM sense and antisense primers of the target gene, 0.5 µl of 20 µM ß-actin antisense primer, 0.3 µl of [{alpha}-32P]dCTP (600 Ci/mmol, 20 mCi/ml; New England Nuclear, Boston MA), and 0.5 µl of SuperscriptII RT/Taq mix. The RT reaction was performed for 30 min at 50°C and ended by incubating the reaction at 94°C for 2 min. PCR conditions for TGF-{alpha} were 94°C for 30 s, 60°C for 30 s, and 72°C for 30 s. Conditions for EGFR and RAR-ß were 94°C for 30 s, 60°C for 30 s, and 72°C for 45 s. The ideal primer dropping point for each target gene (TGF-{alpha}, 6 cycles; EGFR, 8 cycles; and RAR-ß, 14 cycles) was established so that the target gene amplification was in the linear portion of the exponential phase for each PCR amplification as described previously (27) . Five µl of a solution containing 1x RT-PCR buffer and 0.5 µl of the 20 µM ß-actin sense primer were added to each reaction, followed by 18 additional cycles.

Quantitation of PCR Products.
Ten µl of each PCR product were electrophoresed on a 7.5% polyacrylamide gel containing 0.5x TBE (0.045 M Tris-borate, 0.001 M EDTA, pH 8.0) and 2.5% glycerol with 0.5x TBE as running buffer using the Mighty Small II SE250 vertical gel electrophoresis apparatus (Hoefer-Amersham Life Science, Arlington Heights, IL). After electrophoresis, the gel was dried in a Gel Dryer (Bio-Rad model 1583) on 3M Whatman paper, then loaded into a phosphor screen cassette (Molecular Dynamics, Sunnyvale, CA) and exposed for 0.5–5.0 h. The absorbed radioactive signals on the phosphor screen (derived from incorporated [{alpha}-32P]dCTP in the PCR products, which were in proportion to the density of each electrophoresed band) were scanned using a computerized PhosphorImager and analyzed by ImageQuant software (Molecular Dynamics, Sunnyvale, CA). The ratio of the target gene to ß-actin was used to designate gene expression levels. Designated gene expression levels in SCCHN cell lines before LGD1069 treatment were compared with expression levels after treatment.

ELISA.
Cells were grown to 80–90% confluence in 100 x 20-mm Petri dishes. Medium was changed ±10 µM LGD1069. After 24 h, cells were washed once with PBS, scraped, and pelleted. Protein was extracted and quantified. Protein extracts were tested for TGF-{alpha} and EGFR protein expression using commercially available assays according to the manufacturer’s instructions (Oncogene Research Products, Cambridge, MA).

EMSA.
EMSAs were performed on 4% polyacrylamide gels from whole cell extracts. Stat3 activation was evaluated using a radiolabeled high-affinity serum inducible element duplex oligonucleotide as described previously (28 , 29) . Stat3 activation may be assessed by determining SIF activity, where SIF-A represents Stat3 homodimers. Quantitation of the Stat3 signal was performed by scanning the SIF-A band using a Molecular Dynamics Personal Densitometer SI and ImageQuant software. Blots were normalized by running 5-µg aliquots of U937 cell lysates that demonstrate Stat3 activation on each gel and calculating the designated value relative to the positive control on each blot as described previously (24) . For supershift experiments, extracts were preincubated with a Stat3{alpha} polyclonal antibody (C-20; Santa Cruz Biotechnology).

Statistics.
Statistical analyses were performed using a statistical software package (InStat; GraphPad Software, San Diego, CA). Comparisons between LGD1064-treated and untreated groups were made using unpaired Student’s t test with two-tailed comparison. Differences of P < 0.05 were considered significant.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
LGD1069 Inhibits SCCHN Cell Line Growth in Vitro.
Using a well-characterized SCCHN cell line (1483), we established a dose-response curve and demonstrated an IC50 of 10 µM for LGD1069 (data not shown). Treatment of SCCHN cells with solvent alone did not affect proliferation (Fig. 1A)Citation . Seven representative SCCHN cell lines were treated with this dose of LGD1069 for 6 days, where the medium was replenished at days 2 and 4. Growth inhibition was detected in all seven cell lines after 2–3 days of treatment. The linear phase of the SCCHN cell line growth was generally reached on day 4, and the percentage of growth inhibition between the control and LGD1069-treated groups was calculated at this time point (Fig. 1)Citation . The mean percentage of growth inhibition after 4 days of treatment ranged from 24.7% (P = 0.05) to 80.2% (P = 0.03; Table 1Citation ).



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  		Fig. 1. LGD1069 decreases proliferation of SCCHN cell lines in vitro. Cells were treated with 10 µM LGD1069 for 6 days ({diamond}) or were treated with diluent alone ({square}). Each point is the mean of three experiments performed in triplicate wells; bars, SE. A, diluent control. B, 1483. C, SCC 103; D, SCC 105.

 

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  		Table 1 Mean percentage of inhibition of SCCHN cell line proliferation after 4 days of treatment with LGD1069

 
LGD1069 Modulates Intermediate Biomarker Expression Levels in Vitro.
Previous work in our laboratory has demonstrated that levels of EGFR and TGF-{alpha} are increased early in SCCHN and that treatment with nonselective RAs normalizes EGFR and TGF-{alpha} gene transcription rates (20 , 30) . Cumulative evidence suggests that levels of RAR-ß are decreased in premalignant and malignant lesion of the upper aerodigestive tract (31) and are restored (i.e., increased) after treatment with RAs (11) . To determine whether an RXR-specific RA could modulate putative intermediate biomarker gene expression levels in SCCHN cell lines, we developed a semiquantitative RT-PCR assay using the ß-actin gene as an internal standard (27) . In this method, the target gene in question (i.e., EGFR, TGF-{alpha}, or RAR-ß) was preamplified prior to the addition of ß-actin primers by selective "primer dropping." EGFR expression was found to be down-modulated in all seven cell lines after 24 h of treatment with 10 µM LGD1069 (Fig. 2a)Citation . The mean EGFR expression level in the seven cell lines was decreased by 58.4% after 24 h of LGD1069 treatment (P = 0.007; Fig. 2bCitation ; Table 2Citation ). Similar to EGFR, TGF-{alpha} gene expression was found to be down-modulated by a mean percentage decrease of 28.8% after LGD1069 treatment in all seven cell lines tested (Fig. 3, A and BCitation ; Table 2Citation ; P = 0.01). Northern blot analysis also demonstrated reduced EGFR and TGF-{alpha} and mRNA expression (data not shown). ELISA assay of SCCHN cells treated with LGD1069 showed decreased levels of EGFR and TGF-{alpha} protein after treatment with LGD1069 (Figs. 2CCitation and 3CCitation ). To establish that down-modulation of TGF-{alpha}/EGFR autocrine growth was a plausible mechanism for the inhibitory effects of the compound, proliferation assays were performed in the presence and absence of exogenous TGF-{alpha}. As demonstrated in Fig. 3DCitation , the addition of TGF-{alpha} (30 ng/ml) abrogated the growth-inhibitory effects of LGD1069. RAR-ß gene expression was up-regulated in all seven SCCHN cell lines after LGD1069 treatment with a mean percentage increase of 60.0% (P = 0.03; Fig. 4, A and BCitation ; Table 2Citation ).



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  		Fig. 2. LGD1069 abrogates EGFR expression. A, autoradiograph of representative SCCHN cell lines (SCC-103, PCI-37a, and 1483), where EGFR is preamplified prior to ß-actin coamplification. B, cumulative results of RT-PCR EGFR expression levels in seven SCCHN cell lines before and after treatment with LGD1069. Cell lines were incubated with LGD1069 (10 µM) for 24 h. Results are expressed as the ratio of EGFR to ß-actin expression levels. C, decreased EGFR protein expression levels after 24 h of treatment with LGD1069 (10 µM) in two representative cell lines (PCI-37a and SCC-104).

 

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  		Table 2 Mean percentage of change of intermediate biomarker gene expression in seven SCCHN cell lines after 24 h of LGD1069 treatment

 


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  		Fig. 3. LGD1069 down-modulates TGF-{alpha} expression. A, autoradiograph of TGF-{alpha} and ß-actin RT-PCR products in representative SCCHN cell lines (1483 and SCC-104). B, cumulative results of TGF-{alpha} expression levels in seven SCCHN cell lines showing down-modulation after 24 h of LGD1069 treatment. Results are expressed as the ratio of TGF-{alpha}:ß-actin expression levels; bars, SE. C, decreased TGF-{alpha} protein expression levels of TGF-{alpha} after 24 h of treatment with LGD1069 (10 µM) in two representative SCCHN cell lines (PCI-37a and SCC-104). D, the growth-inhibitory effects of (LGD1069; {diamond}) are reversed by the addition of exogenous TGF-{alpha} (30 ng/ml; {square}).

 


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  		Fig. 4. LGD1069 increases RAR-ß expression. A, autoradiograph of RAR-ß RT-PCR products after LGD1069 treatment of representative SCCHN cell lines (PCI-37a, 1483, SCC-105, and PCI-15b). Bars, SE. B, cumulative results of RAR-ß expression in seven SCCHN cell lines after 24 h of LGD1069 treatment (10 µM). Results are expressed as the ratio of RAR-ß:ß-actin expression levels.

 
LGD1069 Modulates TGF-{alpha} and EGFR in Vivo.
To establish the physiological significance of TGF-{alpha}/EGFR abrogation by RXR-selective blockade, SCCHN tumor-bearing mice were treated with LGD1069. Mice received 30 mg/kg of LGD1069 via an intragastric feeding tube 5 days/week for a total of 24 days. The last treatment was given 6 h before the mice were sacrificed, and the tumors were harvested, followed by RNA extraction. Quantitative RT-PCR demonstrated decreased TGF-{alpha} and EGFR expression in the LGD1069-treated tumors compared with control (untreated) tumors (Fig. 5)Citation .



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  		Fig. 5. LGD1069 down-modulates TGF-{alpha}/EGFR in vivo. A, autoradiograph of representative SCCHN xenografts (1483) treated with LGD1069 followed by coamplification of TGF-{alpha} and ß-actin in a quantitative RT-PCR reaction. B, the same tumor RNA analyzed for EGFR expression using EGFR and ß-actin coamplification.

 
Constitutive Stat3 Activation Is Down-Modulated by LGD1069.
Previous work in our laboratory has linked TGF-{alpha}/EGFR signaling to constitutive Stat3 activation in vitro and in vivo (23 , 24) . In these earlier studies, abrogation of TGF-{alpha} or EGFR was found to specifically decrease the SIF-A band on EMSA representing the Stat3 homodimer. EMSAs were performed to determine the effects of RA treatment on Stat3 activation in SCCHN cell lines. Two SCCHN cell lines (PCI-15b and PCI-37a), shown previously to demonstrate decreased TGF-{alpha}/EGFR expression levels after nonselective RA treatment, were treated with 10 µM ATRA, followed by EMSA. Activation of Stat3 was found to be decreased in both cell lines within the first 2 h of ATRA treatment (Fig. 6A)Citation . The predominant complex formed was SIF-A representing Stat3 homodimer. The presence of Stat3 in the gel shift complex was verified by supershift analysis using specific antisera. We next examined the effects of the RXR-selective retinoic acid (LGD1069) on constitutive Stat3 activation. Treatment of the majority (five of seven) SCCHN cell lines with LGD1069 resulted in decreased constitutive Stat3 activation (Fig. 6B)Citation .



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  		Fig. 6. ATRA and LGD1069 abrogate constitutive Stat3 activation in SCCHN cell lines. A, constitutive Stat3 activation in representative SCCHN cell lines (PCI-15b and PCI-37a) after ATRA treatment (10 µM). The position of the SIF-A complex (Stat3 homodimer) is indicated on the right, and the presence of Stat3 in the gel shift complex was verified by supershift analysis. B, constitutive Stat3 activation in representative SCCHN cell lines (PCI-15b, PCI-37a, and SCC-104) before and after 2 h of treatment with LGD1069 (10 µM). Right, position of the SIF-A complex (Stat3 homodimer). C, to demonstrate the role of Stat3 activation in the growth-inhibitory effects of LGD1069, a representative SCCHN cell line (UM-22B) was stably transfected with a dominant active Stat3 construct, Stat3C. A representative clone ({triangleup}) was treated with LGD1069 (10 µM), and cell growth was assessed by vital dye exclusion compared with a representative vector-transfected control clone ({square}).

 
To determine that Stat3 activation directly contributes to the growth-inhibitory effects of LGD1069, SCCHN cells were stably transfected with a dominant active Stat3 construct, Stat3C (26) . Stat3C-transfected cells were treated with LGD1069 (10 µM) and were resistant to the growth-inhibitory effects of LGD1069 compared with vector-transfected control cells (Fig. 6c)Citation .


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
These results demonstrate that an RXR-selective RA, LGD1069, has marked antiproliferative activity in a series of SCCHN cell lines. Furthermore, RXR stimulation resulted in down-modulation of both EGFR and TGF-{alpha} gene expression levels in all seven SCCHN cell lines tested. Abrogation of constitutive Stat3 activation, an EGFR signaling protein, was seen after treatment with either a nonselective RA (ATRA) or the RXR selective RA (LGD1069).

Retinoids play critical roles in normal development by modulating cell growth, differentiation, and inducing apoptosis (32 , 33) . Several studies support the potential role of retinoids in preventing and/or treating SCCHN (20 , 34, 35, 36) . Oral administration of 13-cis-RA, which binds to both RARs and RXRs, reverses oral leukoplakia and decreases the incidence of second primary cancers in the adjuvant setting (12 , 37) . However, the use of these nonselective retinoids has been limited by their toxicity, which can include skin, ocular, and muscle abnormalities, pseudo-tumor cerebri, hepatotoxicity, and hypertrigyleridemia (38) . Some of these toxic effects have been specifically attributed to the binding of these nonselective retinoids to the RAR-{gamma} receptor subtype (16) .

RXR-selective compounds have been shown to demonstrate antitumor effects in mammary carcinoma, NSCLCs, myeloid leukemic cells, and prostate cancer cells without the dose-limiting toxicities associated with other retinoids (16 , 17 , 19 , 39, 40, 41) . In a rat mammary carcinoma model, LGD1069 in combination with tamoxifen was found to be an effective treatment for tumors that failed to respond to tamoxifen alone (17) . The minimal toxicity profile of LGD1069 has prompted examination of this agent in the clinical setting (18 , 19) . Initial results of clinical trials reveal that selected patients with advanced metastatic NSCLCs demonstrate stable disease for up to 13 months while receiving LGD1069 (19) . Clinical responses have also been seen using LGD1069 for cutaneous T-cell lymphoma (18) .

Several studies have reported that selective retinoids can suppress squamous cell differentiation in vitro (38 , 42) . Growth of SCCHN xenografts was inhibited in a dose-dependent manner using an RAR-selective retinoid, ALRT1550 (43) . The present study is the first to examine the consequences of an RXR-selective retinoid on growth and autocrine signaling in a series of SCCHN cell lines. The mechanisms by which RXR-selective compounds abrogate SCCHN proliferation are not completely understood. Activation of TGF-{alpha}/EGFR autocrine growth is an early event in SCCHN tumorigenesis where TGF-{alpha} and EGFR mRNA and protein are up-regulated in both histologically normal tissue and in tumors from patients with SCCHN (21 , 30) . This increase in TGF-{alpha} and EGFR mRNA is primarily attributable to activated gene transcription (20) . We have demonstrated previously that ATRA, a pan-agonistic retinoic acid, can abrogate increased steady-state levels of TGF-{alpha} and EGFR in SCCHN cells by decreasing the rates of transcription of these two genes (20) . Other nonselective retinoic acids, including 13-cis-RA and 9-cis-RA, also inhibit tumorigenesis by down-modulating EGFR and TGF-{alpha} levels (44, 45, 46) . As early markers of SCCHN carcinogenesis in SCCHN, EGFR and TGF-{alpha} represent potential therapeutic targets and evaluable intermediate biomarker end point for chemoprevention or therapeutic agents, such as retinoic acids. Our finding that EGFR and TGF-{alpha} expression levels are significantly reduced after LGD1069 treatment suggests that RXR-selective retinoids, such as the nonselective RA agonists, inhibit SCCHN tumorigenesis by interrupting TGF-{alpha}/EGFR autocrine signaling.

RAs can modulate cytokine-specific transcription factors such as IFN regulatory factors and Stat. In addition to IFN, Stats are activated by other cytokines and growth factors, including EGFR and TGF-{alpha} (47) . To date, seven Stats have been identified (Stats 1, 2, 3, 4, 5a, 5b, and 6). In myeloid leukemic cell lines, ATRA-induced differentiation and growth inhibition is associated with up-regulation of both Stat1 and Stat2 gene expression (48) . Human breast cancer cell line growth is inhibited by treatment with all-trans-RA and 9-cis-RA, in part by down-regulation of the prolactin-dependent activation of Stat5 (49) . Evidence is emerging that Stat3 functions as an oncogene and may contribute to resistance to apoptosis (26 , 50) . We reported previously that constitutive activation of Stat3 is associated with TGF-{alpha}/EGFR autocrine stimulation of SCCHN in vitro and in vivo (23 , 24) . Down-modulating Stat3 resulted in growth inhibition and increased apoptosis, suggesting that Stat3 signaling is critical in SCCHN proliferation (24) . The present study suggests that the antiproliferative effects of both nonselective and RXR-selective RAs in SCCHN are associated with abrogation of constitutive Stat3 activation.

Defective retinoid receptors can directly alter endogenous retinoid regulation of gene expression. The RXR-{alpha} receptor has been shown to be important in mediating the growth-inhibitory effects of RAs in some human carcinoma cells including squamous carcinoma cells (51) . RXR-selective retinoids generally inhibit DNA synthesis in SCCHN cells. However, a point mutation in the ligand binding domain of RXR-{alpha} will initiate retinoid-induced growth inhibition, and a constitutively active RXR-{alpha} receptor mutant will inhibit DNA synthesis even in the absence of ligand (51) . In the upper aerodigestive tract, down-modulation of RAR-ß expression is characteristic of premalignant and malignant lesions, and the loss of RAR-ß expression has been associated with development of squamous cell carcinoma (11 , 52) In human lung cancer cells, activation of either RARs or RXRs contributes to the induction of RAR-ß, which in turn plays a role in mediating retinoid-induced growth inhibition and apoptosis (53) . Failure to induce RAR-ß expression has been associated with RA resistance of RA treatment (53) . We examined a series of SCCHN cell lines and found that RAR-ß was detected in all SCCHN cell lines in the absence of retinoids and was increased by a mean of 60% after treatment with LGD1069.

These results suggest that the new class of RXR-selective retinoids, which have shown promise against leukemia, breast, and prostate cancers in early clinical trials, may also be useful in the prevention and treatment of head and neck cancer. Down-modulation of TGF-{alpha}/EGFR expression levels, up-regulation of RAR-ß, and abrogation of constitutive Stat3 activation by LGD1069 suggest a possible mechanism by which an RXR-selective retinoid may regulate SCCHN proliferation.


   FOOTNOTES
 
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 in part by the Eye and Ear Foundation, the Pittsburgh Foundation, NIH Grant RO1CA77308-01 (to J. R. G.), and the American Academy of Otolaryngology–Head and Neck Surgery Foundation Young Investigator Award (to J. I. S.). Back

2 To whom requests for reprints should be addressed, at The Eye and Ear Institute, Suite 500, 200 Lothrop Street, Pittsburgh, PA 15213. Back

3 The abbreviations used are: SCCHN, squamous cell carcinoma of the head and neck; RA, retinoic acid; RAR, RA receptor; RXR, retinoid X receptor; TGF, transforming growth factor; EGFR, epidermal growth factor receptor; Stat3, signal transducer and activator of transcription 3; RT-PCR, reverse transcription-PCR; EMSA, electrophoretic mobility shift assay; SIF, sis inducible factor; ATRA, all-trans RA; NSCLC, non-small cell lung cancer. Back

Received 7/24/00. Accepted 5/24/01.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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