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Clinical Cancer Research Vol. 11, 3191-3197, May 1, 2005
© 2005 American Association for Cancer Research

Human Cancer Biology

Differences in Smad4 Expression in Human Papillomavirus Type 16–Positive and Human Papillomavirus Type 16–Negative Head and Neck Squamous Cell Carcinoma

Adriana Báez1,2, Alan Cantor6, Sharon Fonseca2, Maria Marcos-Martinez3, Linda A. Mathews5, Carlos A. Muro-Cacho4 and Teresita Muñoz-Antonia4,5

Authors' Affiliations: Departments of 1 Otolaryngology-Head and Neck Surgery, 2 Pharmacology and 3 Pathology, University of Puerto Rico School of Medicine, San Juan, Puerto Rico and Departments of 4 Interdisciplinary Oncology, 5 Molecular Oncology Program, and 6 Biostatistics and Informatics Core, H Lee Moffitt Cancer Center Research Institute, Tampa, Florida

Requests for reprints: Adriana Báez, Department of otolaryngology, School of Medicine, University of Puerto Rico, P.O. Box 365067, San Juan, PR 00936-5067. Phone: 787-758-2525; Fax: 1-787-759-6722; E-mail: abaez{at}rcm.upr.edu.


   Abstract
Top
 Abstract
Materials and Methods
 Results and Discussion
 References
 
The SMADs are a group of interrelated proteins that mediate transforming growth factor ß (TGF-ß) signaling. Upon TGF-ß binding the TGF-ß type I receptor phosphorylates Smad2 and Smad3, which then complex with Smad4 and translocate to the nucleus, with subsequent activation of target genes. Disruption of TGF-ß signaling is thought to contribute to the development of head and neck squamous cell carcinomas (HNSCC). Alterations in the function of the DPC4/Smad4 tumor suppressor gene have been found to inactivate TGF-ß signaling in several tumor types. For example, DPC4/Smad4 is lost or mutated in colorectal, pancreatic, and esophageal cancers. In addition, DPC4/Smad4 transcriptional activity and TGF-ß ability to inhibit DNA synthesis is blocked by the E7 protein of the human papillomavirus type 16 (HPV16) in cervical carcinoma cell lines. HPV16 infection is a risk factor for the development of a subset of HNSCC. This study was undertaken to investigate a potential correlation between expression of components of the TGF-ß signaling pathway and HPV16 status in HNSCC tumors. We examined the expression of TGF-ß signaling proteins Smad2, Smad2-P, and Smad4 by immunohistochemistry in 27 HPV16-negative and 16 HPV16-positive HNSCCs. We compared the expression patterns and assessed their relationship to HPV16 status. No significant differences were detected between HPV16-positive and HPV16-negative tumors in the expression of Smad2 and Smad2-P. Smad4 expression, however, was decreased in 56% of the HPV16-positive tumors and in 39% of HPV16-negative tumors. This difference was statistically significant (P = 0.01) suggesting that loss of Smad4 expression may be involved in HPV16-induced carcinogenesis of HNSCC.

Key Words: Head and Neck Cancer • Smad4 • DNA tumor viruses • Signal transduction pathways • Head and neck/oral cancers

Transforming growth factor ß (TGF-ß) belongs to a multigene family with multiple biological activities (1). TGF-ß proteins are potent endogenous modulators of cellular growth, differentiation, migration, extracellular interactions, immune system function, and apoptosis. These activities are mediated by a ligand-induced cell surface complex formed by TGF-ß receptors type I (TßR-I) and type II (TßR-II; ref. 2). TGF-ß signals are transduced by the TGF-ß receptors and their substrates, the Smad proteins, which maintain the cell in a nonproliferative state in G1 (3, 4). Specific TGF-ß receptor kinases phosphorylate Smad2 and Smad3 upon ligand binding. These Smads then associate with Smad4 and the resulting complex translocates to the nucleus where it activates transcription of target genes.

The TGF-ß receptor complex, including the downstream signaling molecules, has been implicated in the pathogenesis of many human cancers including head and neck squamous cell carcinoma (HNSCC; refs. 5–12) and have been suggested to act as a tumor suppressor pathway (13). Alterations in genes involved in the TGF-ß growth inhibitory pathway would allow tumor cells to escape TGF-ß-mediated growth regulation. Previous studies have shown a direct relationship between reduced levels of TßR-II, resistance to the growth inhibitory control of TGFß-1, and tumorigenicity, thus defining a role for TßR-II as a tumor suppressor gene. The exact mechanisms for down-regulation of TßR-II expression are not fully understood; however, TßR-II is frequently mutated in a wide variety of tumor types including HNSCC (5, 7). Mutations in TßR-I are less frequent (11, 12). Consistent with a tumor suppressor role for the TGF-ß signaling pathway, alterations have been identified in other components of the pathway (14–24). For example, mutations in the DPC4/Smad4 (14–19) and MADR2/Smad2 (21, 22) genes have been reported in pancreatic, cervical, esophageal, and colon cancer.

In addition to genetic alterations, other mechanisms such as infection with human papillomavirus (HPV) have been suggested to inactivate the TGF-ß signaling pathway in certain tumor types (25–27). Transfection with HPV16 DNA immortalizes human keratinocytes in vitro, suggesting a role for HPV in carcinogenesis (28). This HPV-induced carcinogenesis is mediated by the E6 and E7 viral oncoproteins, which exert overlapping effects in cell cycle control (29, 30). Both proteins are consistently expressed in cervical tumors and in a subset of HNSCCs (29–32). E7 forms complexes with members of the retinoblastoma family, pRb, p107, and p130 and enhances degradation of pRb and release of E2F, which activates cell cycle progression (33, 34). The oncogenic potential of E6 is linked to its antiapoptotic effect through its binding to p53 and to activation of host-cell telomerases (35–37). Binding of E6 to p53 mediates its degradation by proteasomes, affecting G1-S checkpoint control (36) and leading to chromosomal instability (37). Yet, malignant transformation by E6/E7 seems to require further alterations of cell cycle regulatory genes, including components of the TGF-ß signaling pathway (38, 39). In early stages of cervical cancer, malignant progression in HPV-infected cells has been linked to the loss of sensitivity to TGF-ß (38–45). This resistance has been linked to alterations in the expression of cell cycle regulatory genes (38). Whereas TGF-ß1 has been shown to down-regulate transcription of E6 and E7 genes in nontumorigenic HPV-infected genital epithelial cell lines, the HPV16-infected cells, SiHa and CasKi, show resistance to TGF-ß regulation (41, 46). TGF-ß resistance in HPV16-transformed human keratinocytes has been associated with loss of expression of TßR-I (42). In addition, four different mutations in TßR-I and a polymorphism that may predispose to cervical cancer have been detected in HPV16-infected cervical cells (43). More recently, it has been shown that E7 blocks the Smad transcriptional activity by binding to the complex formed by Smad2, Smad3 and Smad4 and preventing them from binding with DNA (25). These results suggest an interaction between HPV16 E6 and E7 oncoproteins and TGF-ß signaling components in the tumorigenicity of certain tumor types.

Tobacco and alcohol uses are the major risk factors for HNSCC (47); however, recent epidemiologic and molecular studies provide evidence that HPV may be associated with a subset of HNSCC (30–32). Infection with high risk HPV16 DNA immortalizes oral keratinocytes, and the E6 and E7 oncoproteins are mutagenic in human oral keratinocytes, suggesting a role for HPV in oral carcinogenesis (48). Furthermore, HPV infection has been associated with benign, premalignant, and malignant lesions in the head and neck region with HPV16 being the most common subtype (31). Recent reports using PCR-based assays have estimated a frequency of HPV16 in HNSCC of 20% to 30% (32). However, using similar PCR-based assays we found in a series of 118 Puerto Rican HNSCC cases that 44% were infected with HPV16 (49).

To examine whether aberrant expression of TGF-ß signaling components could be contributing to oncogenesis in HPV-positive HNSCC, we examined by immunohistochemistry the differences in patterns of expression of several key molecules of the TGF-ß pathway in HPV16-negative and HPV16-positive HNSCC. Our results show that differences in expression of Smad4 between HPV16-positive and HPV 16-negative HNSCC are statistically significant, suggesting that in this subgroup of HNSCC, Smad4 may play a role in the development of HNSCC.


   Materials and Methods
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 Abstract
 Materials and Methods
Results and Discussion
 References
 
Patient and tissue specimens. Tissue samples were obtained from 43 patients with HNSCC who underwent curative surgery. Patients were accrued through the Otolaryngology-Head and Neck Surgery Cancer Clinics at the University of Puerto Rico School of Medicine. Before enrollment in the study, a signed consent form was obtained from all patients to allow the collection of tissues and demographic and clinical data. The study was approved by the Institutional Review Boards of the University of Puerto Rico Medical Sciences Campus and the Moffitt Cancer Center and Research Institute. The data recorded for each patient included age, sex, site, stage, and pathologic grade.

DNA isolation. High molecular weight DNA from the tumor was isolated using the DNA Isolation kit for cells and tissues (Roche Applied Sciences, Indianapolis, IN) according to the manufacturer's instructions. DNA concentration was measured using a CytoFluor Series 4000 (Applied Biosystems, Foster City, CA).

Detection of HPV16. DNA from tumors was tested with a glyceraldehyde-3-phosphate dehydrogenase primer set to confirm the integrity of the genomic DNA. Samples were screened for the presence of HPV16 DNA by amplification with HPV16 E7-type specific primer sets 5'-GATGAAATAGATGGTCCAGC-3' and 5'-GTACCTACGTGTGTGCTTTGT-3', which generate a 120-bp amplicon and E6-type specific primer sets 5'-CGAAACCGGTTAGTATAA-3' and 5'-GTATTCTCCATGCATGATT-3', which generate a 523-bp amplicon. Each reaction was carried out in a 25-µL reaction volume with final concentration of 10 mmol/L Tris-HCl (pH 9), 1.5 mmol/L MgCl2, 50 mmol/L KCl, 0.4 µmol/L of each primer, 200 µmol/L of each deoxynucleotide triphosphate, 0.1 to 0.2 µg of genomic DNA template, and 1.5 units of Taq polymerase. DNA was amplified in a GeneAmp 2700 System (Applied Biosystems). Conditions for the E6 primer set consisted of an initial denaturation step of 3 minutes at 94°C and 35 cycles of 1 minute at 94°C, 1 minute at 55°C, 1.5 minutes at 72°C, and one final extension step of 7 minutes at 72°C for the 120-bp amplicon. For the E7 primer set, after the initial denaturation step, conditions consisted of 35 cycles of 30 seconds at 94°C, 30 seconds at 55°C, 1 minute at 72°C, and a final extension step of 7 minutes at 72°C. PCR products were separated by gel electrophoresis in a 2% ethidium bromide agarose gel in Tris/borate buffer [89 mmol/L Tris-borate, 89 mmol/L boric acid (pH 7.8), and 0.8 mmol/L EDTA]. The amplified products were detected using the Gel Doc EQ System with Molecular Analysis Software (Bio-Rad Laboratories, Hercules, CA). Reaction mixtures with SiHa DNA and K562 DNA were used as positive control and negative controls, respectively.

Immunohistochemistry. To minimize heterogeneity and tissue variability across the different tissue sections, consecutive 3-µm sections were prepared without discarding intervening tissue. The first section was stained with H&E and the rest of the sections were used for immunohistochemistry. Formalin-fixed, paraffin-embedded tissue sections were dried at 37°C overnight. Sections were deparaffinized by an initial warming to 60°C, followed by two xylene changes 10 minutes each, two series of 30 dips in absolute alcohol, 30 dips in 95% alcohol, and 20 dips in deionized water. Antigen retrieval or enzyme digestion procedures were done as previously described (8, 9). Slides were placed for 5 minutes in TBS/Tween and processed on a Dako Autostainer using the Dako LSAB+ Peroxidase detection kit (Dako, Carpinteria, CA). Endogenous peroxidase was blocked with 3% aqueous hydrogen peroxide followed by two 20-dip washes in deionized water. Anti-Smad2P and anti-Smad4 primary antibodies (both from Upstate Biotechnology, Lake Placid, NY) were applied at room temperature at a 1:100 dilution. On two other sections, the primary antibody was replaced with an isotype-match antibody of unrelated specificity and the primary antibody was omitted. In both situations, signal was not observed. After 1 hour, slides were rinsed with PBS. Detection was made using the Vectastain Elite avidin-biotin complex kit series (Vector Laboratories, Burlingame, CA) using diaminobenzidine as chromogen. Counterstain was done with modified Mayer's hematoxylin. Slides were dehydrated through graded alcohol, cleared with xylene and mounted with resinous mounting medium. In each case, quantification of protein expression was done visually by an experience immunopathologist. Signal intensity was classified as 0 (no intensity), 1+ (weak intensity), 2+ (moderate), and 3+ (strong). The percentages of positive cells in each of the four intensity groups were estimated and multiplied by their corresponding intensity scores. The four partial values obtained in each section were added and expressed as a final score. The average of three of these estimations is provided.

Statistical analysis. The Wilcoxon rank sum (Mann-Whitney U) test was used to compare protein expression between the different groups.


   Results and Discussion
Top
 Abstract
 Materials and Methods
 Results and Discussion
References
 
Recent reports using PCR-based assays have estimated an HPV16 frequency in HNSCC of 20% to 30% (32). More recently, we reported a significantly higher HPV16 frequency of 44% in a series of 118 HNSCC Puerto Rican patients (49). In the present study, we investigated the presence of HPV16 in 43 HNSCC specimens selected from the Otolaryngology-Head and Neck Surgery Cancer Clinics at the University of Puerto Rico School of Medicine. High molecular weight DNA from the tumors was isolated and screened for the presence of HPV16 DNA by PCR amplification with HPV16 E6 and E7-type specific primer sets. The results of the PCR amplification can be seen in Fig. 1. Of the 43 samples tested by PCR, 27 samples (63%) were HPV16 negative and 16 samples (37%) were HPV16 positive. Eleven of these samples were positive for E6 and E7. In five of these samples, the DNA available was only sufficient to test for E6. This frequency of 37% positivity, although higher than those of other series, is lower than the 44% previously reported by us in the Puerto Rican population.



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  		Fig. 1. PCR detection of HPV16 E6 and E7 in HNSCC tissues. Representative results of PCR reactions using HPV16 E6- and E7-specific primers. DNA samples were electrophoresed on 2% agarose gel and viewed by ethidium bromide staining. Size of PCR products was measured relative to molecular weight marker (M). Positive control (SiHa DNA) and negative control (K567 DNA) gave the expected results. Lanes 2-18, results of E6 primers (523 bp) from 16 tumors with positive results (lanes 3, 6, 8, 12, 15, 16). The 121-bp amplification products with E7 primers of the same tumor samples in the same order. Samples 3, 6, 8, 12, 15, and 16 are HPV16 E7 positive.

 
To test whether aberrant expression of TGF-ß signaling components could be contributing to the tumorigenic mechanism of HPV16-positive HNSCC, we used immunohistochemistry to examine the expression of TGF-ß signaling molecules. Both HPV16-positive and HPV16-negative HNSCC cases were examined for the TßR-II expression and TGF-ß intracellular signal transducers (Smads). The HNSCC cases were evaluated using different antibodies on consecutive sections of the same tissue block as described in MATERIALS AND METHODS. The decrease in TßR-II expression observed was comparable to results obtained previously (8). Well to moderately differentiated carcinomas showed heterogeneous expression of variable intensity, and poorly differentiated carcinomas were completely devoid of TßR-II (data not shown), suggesting that aberrant TßR-II expression is a contributing factor to the pathogenesis of HNSCC in our series. This is in agreement with what has been reported by us and others, suggesting that reduced expression of TGF-ß receptors is an unfavorable prognostic factor in human HNSCC (8). However, we found no apparent relationship between loss of TßR-II and HPV16 positivity (data not shown).

To further examine if alterations in the TGF-ß signaling pathway are involved in the tumorigenicity of HPV16-positive HNSCC, we examined by immunohistochemistry the expression of Smads, the TGF-ß intracellular signal transducers. As stated before, 43 squamous cell carcinomas were included in this study (see Table 1). The age of the patients ranged from 38 to 84 years with an average of 62 for the HPV16-positive cases and of 60 for the HPV16-negative cases. Thirty-seven patients (86%) were male and six (14%) were female. The anatomic location of the tumors included larynx, hypopharynx, oropharynx, nasopharynx, and oral cavity. We found no statistically significant difference in expression between normal tissue and the corresponding carcinoma for Smad2, Smad2-P, or Smad3 (data not shown). This contrasts with our previous report of 13 HNSCC from non-Hispanic whites, where we found, in ~70% of the tumors, a loss in the expression of the activated and phosphorylated form of Smad2 (9).


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  		Table 1. Expression of smads in HPV16+ and HPV16– HNSCC

 
Interestingly, when the patterns of expression were analyzed in the context of HPV16 status, the average expression of Smad4 in the normal tissue of both HPV16-positive and HPV16-negative cases was similar (0.58 versus 0.59). However, the average expression of Smad4 in HPV16-positive carcinomas was reduced (0.36) compared with that of HPV16-negative carcinomas (0.75; Fig. 2; Table 1). The difference between normal and tumor was statistically significant only in HPV16-positive cells (P = 0.01). These differences were independent of age, sex, differentiation, or tumor stage. In contrast, there was no statistically significant difference in the average expression of other Smads. For example, as can be seen in Fig. 3 and Table 1, there is no statistically significant difference in the expression of Smad2-P in HPV16-positive cases between normal tissue (1.1) and tumor (1.3).



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  		Fig. 2. Smad4 expression in HPV16-positive (A-D) and HPV16-negative (E-H) squamous cell carcinoma. A and C, nonneoplastic squamous epithelium. Strong expression in basal, parabasal, and low intermediate layers. B and D, marked reduction in expression in carcinoma. E and G, nonneoplastic squamous. Prominent expression in epithelium in most of the cells. F and H, marked expression in carcinoma.

 


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  		Fig. 3. Expression of Smad2-P detected by immunohistochemistry in squamous cell carcinoma. Comparison between HPV-positive (A and C) and HPV-negative (B and D) tissue samples. A, HPV-positive, nonneoplastic squamous mucosa showing koilocytotic changes and strong nuclear expression of Smad2-P. B, HPV-negative, nonneoplastic squamous mucosa with strong nuclear expression of Smad2-P. C, corresponding squamous cell carcinoma with marked decrease in Smad2-P expression. D, corresponding squamous cell carcinoma with no decrease in Smad2-P expression.

 
Loss of Smad4 has been reported in several human cancer types, including esophageal squamous cell carcinoma (50), where expression of Smad4 seems to be associated with depth of invasion. The mechanism for this alteration in the expression of Smad4 in HNSCC is not fully understood. For example, even in those instances where the protein levels are decreased, the DPC4/Smad4 mRNA levels remain relatively steady, suggesting that the defect is not at the level of transcription (50). The DPC4/Smad4 and MADR2/Smad2 genes have been mapped to 18q.21, and genetic alterations in DPC4/Smad4 and MADR2/Smad2 genes have been reported in human pancreatic and colon cancers, in which 18q.21 is frequently lost (14, 16, 17, 21). In head and neck cancers, however, genetic analysis revealed minimal loss of this chromosomal region (51). Mutations in DPC4/Smad4, Smad 3, and MADR2/Smad2 genes have been reported in pancreatic (14–16), colon (17, 18, 21), lung (52), and cervical cancers (22). However, in studies of head and neck, hepatocellular, breast, and prostate cancers mutations in the TßR-II, MADR2/Smad2 and DPC4/Smad4 genes were found to be rare (9, 52–57). This suggests that genetic instability in the Smad genes does not account for the widespread resistance to TGF-ß in HNSCC, and that other mechanism(s) may be involved.

Decreased expression of Smad4 could further contribute to TGF-ß signaling abnormalities at a post-receptor level in tumors with abnormal receptor expression and/or be responsible for defective signaling in tumors with normal receptor expression. Decrease in Smad4 expression is associated with a defective TGF-ß response (14–20). Loss of sensitivity to the antiproliferative effects of TGF-ß in pancreatic cells has been associated with loss of Smad4 expression and subsequent loss of p21 (58). We are currently examining the status of p21 in these tumors.

It could be speculated that a step in the HNSCC tumorigenic mechanism is the acquisition of resistance to TGF-ß which, in turn, makes cells more susceptible to infection by HPV16 and more tumorigenic. Alternatively, it can be speculated that HPV16 infection is the first event in HNSCC, and that in analogy to what has been reported in cervical cancers (which also exhibit significantly low Smad4 expression), binding of E7 (or E6) inhibits Smad biological activity (25). Inhibition of Smad biological activity could be a mechanism of resistance to the antiproliferative effects of TGF-ß. Experiments are in progress in our laboratory to investigate whether TGF-ß resistance makes cells more susceptible to HPV16 infection, or whether HPV16 infection makes cells resistant to TGF-ß.


   Footnotes
 
Grant support: Department of Otolaryngology and NIH grant P20 CA91402.

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.

Received 7/ 1/04; revised 11/23/04; accepted 12/13/04.


   References
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 Abstract
 Materials and Methods
 Results and Discussion
 References
 

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