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. 2002 May 16;35(3):183–192. doi: 10.1046/j.1365-2184.2002.00237.x

Transforming growth factor β1 dysregulation in a human oral carcinoma tumour progression model

S Hsu 1,✉, J L Borke 1, J B Lewis 1, B Singh 1, A C Aiken 1, C T Huynh 1, G S Schuster 1, G B Caughman 1, D P Dickinson 1, A K Smith 1, T Osaki 2, X F Wang 3
PMCID: PMC6496909  PMID: 12027954

Abstract

Abstract. A human oral tumour progression model was established that consists of normal epithelial cells and three cell lines representing stages from dysplastic to metastatic cells. To investigate the impact of exogenous transforming growth factor‐β1 on this model system, we analysed the responsiveness of those cells to transforming growth factor‐β1 and explored the potential mechanism underlying the transforming growth factor‐β1 activity. We found that the growth of all cell types, regardless of their stage of tumour progression, is inhibited by transforming growth factor‐β1, although to different degrees. Transforming growth factor‐β1 induced the expression of cyclin‐dependent kinase inhibitors p15INK4B, p21WAF1/CIP1 and p27KIP1. In contrast, transforming growth factor‐β1 was found to stimulate the invasive potential of one cell type that represents the most advanced stage of tumour phenotype, suggesting that the impact of transforming growth factor‐β1 on functional features of tumour cells other than cellular proliferation may play a significant role in the process of oral tumour progression.

Introduction

Transforming growth factor (TGF)‐β is a multifunctional cytokine that regulates cell proliferation, differentiation, adhesion, mobility, programmed cell death and immune response (Moses et al. 1991). TGF‐β belongs to a structurally and functionally related polypeptide superfamily, which also includes bone morphogenic proteins (BMP), activin, inhibin, and mullerian inhibiting substance, factors that are known to be involved in regulation of various types of cellular functions and physiological processes (Kingsley 1994). TGF‐β1 has been shown to be a potent growth inhibitor for a wide range of cell types and it acts by binding to receptors (TGF‐βR) that are found on the surface of most cell types (Tucker et al. 1984; Kimchi et al. 1988). The ligand–receptor interaction induces the formation of a stable heteromeric complex containing the type I and type II receptors, leading to activation of the serine–threonine kinase of the type I receptor and phosphorylation of downstream effectors (Wells et al. 1999). Recent studies have demonstrated that the Smad family of proteins plays an important role as the main effectors phosphorylated by the receptor kinases of the TGF‐β superfamily, transmitting signals from the cytoplasm into the nucleus (Ulloa et al. 1999; Wong et al. 1999). Specifically, the activated TGF‐β type I receptor phosphorylates either Smad2 or Smad3, which then associates with the common Smad4 and translocates into the nucleus. In the nucleus, the Smad complex can associate with either specific DNA binding sites or other sequence‐specific DNA binding transcription factors to regulate the expression of target genes (Massague 1998 for review, Datto et al. 1999; Liberati et al. 1999).

While the Smad proteins have emerged as the main effectors in the TGF‐β signal transduction pathway, other signalling pathways may also be involved in modulating the biological responses induced by TGF‐β. For example, it has been shown that the mitogen‐activated protein kinase (MAP kinase) mediated signalling pathway is activated in certain cell types in response to TGF‐β (Ohtsuki & Massague 1992; Wells et al. 1999). The mechanism by which TGF‐β inhibits cell proliferation has been studied extensively, and it appears that TGF‐β may act through multiple mechanisms to block cell cycle progression. In various cell types, TGF‐β inhibits c‐myc expression (Pietenpol et al. 1990) and Rb phosphorylation (Laiho et al. 1990). Suppression of Rb phosphorylation appears to be achieved through either an inhibition of cyclin‐dependent kinase‐4 (CDK4) (Datto et al. 1995) or an activation of CDK inhibitors (CKIs) p15INK4B, p21WAF1/CIP1 and p27KIP1 (Reynisdottir et al. 1995; Datto et al. 1997).

Because of the important role of TGF‐β in controlling cell proliferation, disruption of TGF‐β signalling has been postulated to contribute to tumorigenesis in multiple systems. For example, mutations in the type II TGF‐β receptor have been found in tumours derived from patients who have developed non‐polyposis colon carcinomas. Loss of TGF‐β responsiveness has also been correlated with loss of p15 expression in human gliomas. Thus, disruption in the TGF‐β‐mediated growth‐inhibitory signalling pathway may be commonly associated with the development of various types of human cancers. Indeed, one study has shown that TGF‐β signalling is negatively affected in an oral carcinoma cell line through a reduction in the numbers of TGF‐β receptors (Game et al. 1990). In a separate study, the growth of rat oral tumour cells induced by treatment with 4‐nitroquinoline N‐oxide was suppressed by exogenous TGF‐β1 overexpression (Davies et al. 1997). However, it remains to be determined if disruption of TGF‐β signalling plays a role in the development of oral cancers due to the lack of a model system in which the potential association between the progression of oral carcinomas and dysregulation of TGF‐β signal transduction can be evaluated.

The purpose of the current study is to determine the regulatory impact of TGF‐β1 in an oral tumour progression model and evaluate the behaviour and intracellular response from each cell type in this system. We established a model of oral tumour progression consisting of three cell lines representing different stages of oral tumour progression: DOK, a precancerous oral dysplastic cell line, which resembles normal oral epithelial cells but is immortal (Chang et al. 1992); SCC25, an oral squamous carcinoma cell line isolated from primary oral tumour site, which resembles primary oral cancer status without metastatic property (Rheinwald & beckett, 1980); and OSC2, an advanced metastatic oral squamous cell carcinoma cell line, which resembles the metastatic and aggressive stage of oral cancers (Osaki et al. 1994). Utilizing this model system, combined with the use of normal human keratinocytes as a control for the status of normal epithelial cells, we performed a comprehensive analysis by examining cellular functions including cell growth, invasion, and the expression of specific components involved in G1 cell cycle regulation. Those components include p53, cyclin D1, cdk4, p15, p21, p27 and p57KIP2. We also examined the responsiveness of those different types of cells to exogenous TGF‐β1 and evaluated the activity of the TGF‐β1 signalling pathway by determining the nuclear translocation activity of Smad3. We found that disruption of Smad‐mediated TGF‐β1 signal transduction is not an early requirement for oral tumour progression. Interestingly, while TGF‐β1 stimulates the invasive activity of the metastatic cell line OSC2, it still inhibits the growth of all cell lines, even in serum‐containing media, through induction of p15 and p21. We conclude that the multiple signal transduction pathways of TGF‐β1 enable two cell lines with disrupted Smad‐mediated signal transduction pathway to respond to exogenous TGF‐β1 by growth inhibition and reduction of invasiveness.

Materials and methods

Cell lines/type

The oral tumour progression model consisted of normal human keratinocytes (Clonetics/BioWhittaker, Walkersville, MD, USA); a dysplastic, precancerous oral epithelial cell line DOK; a primary, early stage oral squamous carcinoma cell line SCC25 (American Type Culture Collection, Manassas, VA, USA); and an advanced, metastatic oral squamous carcinoma cell line OSC2. The DOK cell line was isolated from dysplastic tongue mucosa from a 57‐year‐old‐man. The doubling time for DOK cells is approximately 40 h in Dulbecco’s modified Eagle’s minimal essential medium (DMEM), and the DOK cell line does not induce tumours in the nude mouse (Chang et al. 1992). DOK cells have elevated p53 and harbour a deletion at 188–191 in the p53 gene (Burns et al. 1994). The SCC25 cell line was isolated from a squamous cell carcinoma of the tongue of a 70‐year‐old male (Rheinwald & Beckett 1980). The OSC2 cell line was isolated from a submandibular lymph node metastasis of a 68‐year‐old‐female. The primary tumour was located in the gingiva of this patient (Osaki et al. 1994). OSC2 cells have a p53 mutation at exon 8, site 280, resulted in Arg → Thr substitution (Yoneda et al. 1999). The doubling time for OSC2 is 22.9 h.

Cell culture and treatment

The cell lines were maintained in DMEM/Ham’s F12 medium, with 10% fetal calf serum, penicillin/streptomycin (100 IU/mL, 100 µg/mL) and 5 µg/mL hydrocortisone. The normal human keratinocytes were maintained in keratinocyte growth medium 2 (KGM‐2). For Western blot analysis, TGF‐β1 treatment of the three cell lines was carried out in insulin/transferrin/selenous acid (ITS) medium following overnight incubation in ITS medium (DMEM/F12 with 20 ng/mL gentamicin, 4 mm glutamine, 1 mg/mL fatty acid free bovine serum albumin (BSA), 0.1 unit/mL insulin, 1.7 µg/mL transferrin, 0.1 µm selenous acid, and 0.29 µm linoleic acid). Treatment of the keratinocytes was carried out in KGM‐D medium following overnight incubation in KGM‐D. Cells were either treated with 5 ng/mL TGF‐β1 or without treatment as controls.

Cell growth analysis

Cell growth analysis was performed on exponentially growing cells in complete DMEM/Ham’s F12 medium with or without the presence of 5 ng/mL TGF‐β1. Cell quantification was achieved by cell counting using a haemocytometer and trypan blue exclusion. This analysis was conducted on all cell lines for 24‐, 48‐ and 96‐h treatment periods. For each cell line, four tissue culture flasks (25 cm2) were seeded with 5 × 105 cells in each flask. 5 ng/mL TGF‐β1 was added to three flasks separately at time point 0, 48 and 72 h, with the fourth flask as control. At the 96 h time point, all cells in each flask were trypsinized and then counted. The cell numbers from the treatment flasks were then expressed as a percentage of the number of cells in the control flask. Three identical experiments were performed with similar results.

Cell migration assay

These assays were conducted using a Transwell apparatus with wells 6.5 mm in diameter and with membranes that contain a pore size of 8 µm (Costar/Corning, Corning, NJ, USA). The migration of each cell line was tested with or without treatment with TGF‐β1 in complete DMEM/F12 medium immediately following the cell count procedure described above. Cells (105) were seeded in each transwell in complete DMEM/F12 medium. Evidence of migration was detected microscopically by counting the cells after incubation for 48 h followed by removing the transwells and maintaining in DMEM/F12 media for another 12 h. Three identical experiments were performed with similar results.

Western blot analysis

Following 24‐h treatment with TGF‐β1 or no treatment, protein extracts were prepared by cell lysis in RIPA buffer (10 mm sodium phosphate, pH 7, 1% Triton X‐100, 0.1% SDS and 2 mm edta) containing proteinase inhibitors (0.2 mm phenylmethylsulphonyl fluoride (PMSF), 10 µg/mL leupeptin, and 10 µg/mL pepstatin A). The protein concentration for each sample was then determined using the Bio‐Rad DC Protein Assay Kit (Hercules, CA, USA). The expression of targeted proteins involved in cell cycle regulation was determined using standard 10 or 15% sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and blotting onto nitrocellulose membranes. An aliquot of 50 µg of protein from each sample was separated. Detection of each protein band was accomplished using a primary polyclonal antibody specific for each protein and a secondary antibody with ECL chemiluminescence X‐ray film detection (Amersham Biosciences, Piscataway, NJ, USA).

Immunocytochemistry

The cell lines were grown on four‐well chamber slides (Nagle Nunc International, Rochester, NY, USA). A specific antirabbit Smad3 polyclonal antibody (Santa Cruz Biotechnology, Inc. Santa Cruz, CA, USA) was used for the presence of Smad proteins by means of a modified avidin–biotin–peroxidase technique (Hsu & Raine 1981; Borke & Nau 1987). Cells were fixed to the slides by immersing in cold acetone (4 °C) for 10 min, air‐dried for 5 min, and rinsed twice in phosphate‐buffered saline (PBS) for 3 min each. To block endogenous peroxidases, cells were incubated for 3 min in 0.03% hydrogen peroxide. The cells were then incubated in 1.4% normal goat serum (Vector Laboratories, Burlingame, CA, USA) for 1 h. The Smad3 antibody was then applied at a 1 : 50 dilution and incubated for 1 h. The cells were then rinsed with PBS, and a rabbit biotinylated antibody (Vector Laboratories) was applied to the cells, which were incubated for 30 min. The cells were rinsed in PBS and then incubated for 30 min in avidin–biotin complex (Vector Laboratories). Cells were then rinsed, reacted with diaminobenzidine (DAB) in the presence of hydrogen peroxide and then dehydrated in ascending alcohol to xylene then were coverslipped for visualization.

Antibodies

All antibodies used were purchased from Santa Cruz. All incubations in Western blotting were performed in 10 mL of 5% non‐fat milk–PBST (phosphate‐buffered saline with 0.1% Tween‐20) for 1 h. The only exception was that the incubation times for p53 were 45 min for both primary and secondary antibody.

Results

To establish a profile for the responsiveness to the growth inhibitory effect of TGF‐β1 by the various cell lines in our oral tumour progression model system, we treated each cell line with 5 ng/mL of TGF‐β1 for a different length of time. As shown in Fig. 1, all three cell lines, representing different stages of tumour progression, showed a significant response to the treatment of exogenous TGF‐β1. By the 96‐h time point, the proliferation of all three cell lines had been reduced by TGF‐β1 treatment in comparison to the untreated control. The precancerous dysplastic DOK cells were growth inhibited by about 70%, while the growth of advanced metastatic oral squamous carcinoma OSC2 cells was inhibited to a similar level. Interestingly, the non‐metastatic SCC25 cells were growth inhibited to a lesser extent in comparison to the other two cell types. To establish a molecular profile for the expression of various components in the TGF‐β signalling pathways, we next performed Western blot analysis. Not surprisingly, we found that all cell lines/type express the signalling TGF‐β receptors type I and type II and their expression was not altered by TGF‐β1 treatment in a 24‐h period (Fig. 2). In contrast, significant differences are present among these cell lines/type in regard to p53 expression levels. SCC25 cells do not have detectable levels of p53 protein; the mechanism for this is unknown. The normal human keratinocytes, DOK, and OSC2 cell lines expressed different amounts of p53. DOK cells expressed higher level of p53 than normal keratinocytes, possibly a result of mutation in the p53 gene. OSC2 cells are known to contain a p53 mutation at exon 8, site 280, resulted in Arg → Thr conversion, consequently expressing a large amount of p53. In keratinocytes and DOK cells, TGF‐β1 slightly down‐regulated p53 expression. The p53 expression in OSC2 cells, with a higher basal level, was not modulated by TGF‐β1.

Figure 1.

Figure 1

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Cell growth assay result in three cell lines treated with 5 ng/mL TGF‐ β 1 for 24, 48 and 96 h. DOK and OSC2 cell lines show 74% and 64% growth inhibition in 96 h. SCC25 cell line showed less growth inhibition during the same period.

Figure 2.

Figure 2

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Western analysis result from four cell lines/type treated with 5 ng/mL TGF‐ β 1 for 24 h in serum free media. Kera: Human neonatal pooled keratinocytes. C: control without treatment. T: 5 ng/mL TGF‐β1 for 24 h.

To determine if the growth inhibitory effect of TGF‐β on those three cell types is mediated by a modulation of the expression of CKIs, we analysed the expression levels of a number of cell cycle components in response to TGF‐β treatment. As shown in Fig. 2, the expression of three CKIs, p15, p21 and p27, were all up‐regulated by TGF‐β in DOK, SCC25, and OSC2 cells, suggesting that the induction of those inhibitors may be directly involved in TGF‐β induced inhibition of cell proliferation in these cells. Interestingly, while there was no change in the expression levels of p21 and p27 in normal keratinocytes, p15 was induced significantly, suggesting that different mechanisms may be employed by different cell types to inhibit cell cycle progression in response to TGF‐β1. The levels of Cdk4 and cyclin D1 were previously shown to be down‐regulated by TGF‐β in specific cell types. Consequently, we also analysed the expression profiles of these two proteins. As also shown in Fig. 2, each cell type expressed similar levels of Cdk4, although keratinocytes and DOK cells showed a slight down‐regulation of Cdk4 after TGF‐β1 treatment, while SCC25 and OSC2 cells showed slight up‐regulation. All cell types expressed cyclin D1 protein, but the levels differed quantitatively. Keratinocytes have the lowest level; DOK and OSC2 possess medium amount; SCC25 cells have the highest level. The DOK, SCC25, and OSC2 cells all showed an up‐regulation in cyclin D1 level after TGF‐β1 treatment, while keratinocytes showed no change in cyclin D1 expression (Fig. 2).

To determine if there is a difference in response to TGF‐β mediated cell invasion by the three cell types in our model system, we performed cell migration assay following established protocols. As shown in Fig. 3, TGF‐β1 stimulated the invasive behaviour of the metastatic oral carcinoma OSC2 cells, despite the fact that these cells are growth inhibited by the same treatment to a significant extent. The OSC2 cell line was stimulated by TGF‐β1 initially, to more than 250% of invasive cells in comparison to the untreated control. This stimulation became weaker when 5 ng/mL TGF‐β1 treatment was extended to 96 h, but still higher than the control even at that late time point. On the other hand, migration of the precancerous DOK cells was inhibited by TGF‐β1 during the first 48‐h period and the cells recovered to normal level at 96 h (Fig. 3). SCC25 cells did not have the capability to migrate across the membrane with 8 µm pores as well as the keratinocytes (data not shown).

Figure 3.

Figure 3

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Result of cell migration assays from TGF‐ β 1 treated DOK cells (left) and OSC2 (right) with controls as 100%. The keratinocytes and SCC25 cells failed to migrate through the transwell membrane (data not shown).

Finally, as a measure of TGF‐β signalling activity, we evaluated the status of Smad3 translocation in these three cell types in response to TGF‐β1 treatment. As shown in Fig. 4, the Smad3 nuclear localization in OSC2 cells was rapidly and efficiently induced by TGF‐β1 within a 2‐h period. On the other hand, Smad3 nuclear translocation in DOK and SCC25 cells was not detected, probably due to lack of Smad response in these cell lines.

Figure 4.

Figure 4

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Immunocytochemistry result showing TGF‐β 1 signal transduction through Smad3 nuclear translocation in three cell lines. Left panel: control without TGF‐β1, nuclei are not stained with Smad3 antibody. Right panel: cells treated with 5 ng/TGF‐β1 for 2 h, only the nuclei of OSC2 cells were stained with Smad3 antibody, indicating Smad3 nuclear translocation. From top: DOK, SCC25 and OSC2 cells.

Discussion

As a potent epithelial growth inhibitor, TGF‐β1 induced significant growth inhibition in the oral tumour progression model. This result demonstrated that although the degree of response differs for each of the cell lines, there is no significant alteration in TGF‐β1‐mediated growth inhibition to correlate with the progression of tumour development states. This result is in contrast to previous findings that showed a general loss of responsiveness to TGF‐β‐mediated growth inhibition with the progression of tumours to a more advanced stage and a switch from growth inhibitory to stimulatory response to TGF‐β often associated with certain most advanced metastatic tumours.

From the Western analysis, a common feature for the mechanism of TGF‐β1 mediated growth inhibition emerges for all three tumour cell lines: they all showed TGF‐β1 mediated induction of CKIs, p15, p21 and p27. Furthermore, the induction of these inhibitors is mediated through a p53‐independent signalling pathway in this tumour progression model, since none of the three cell lines possesses wild type p53. In addition to the up‐regulation of p15, p21 and p27, DOK cells showed a down‐regulation of Cdk4 after treatment with TGF‐β1 (Fig. 2). This combined impact of exogenous TGF‐β1 on both the induction of CKIs and inhibition of Cdk4 expression probably contributed to a higher degree of growth inhibition of DOK cells by TGF‐β1 in comparison to the other two cell lines. The metastatic OSC2 cells showed a significant level of over‐expression of p53, indicating the loss of normal p53 function. However, OSC2 cells responded to TGF‐β1 by a significant up‐regulation of p15 and p21, resulting in a sharp growth inhibition in 96 h (Fig. 1). Taken together, these results suggest that, in this cell‐line based oral epithelial tumour progression model, TGF‐β1 is a potent growth inhibitor to cells representing all stages of tumour development through a p53‐independent, p15‐, p21‐ and p27‐mediated signalling pathway. This growth inhibition pathway does not directly involve quantitative changes in the expression level of Cdk4 and cyclin D1 when the expression of p15, p21 and p27 is induced. Another CKI, p57, is not likely to be involved in the TGF‐β1 effect since p57 protein levels remained low and stable (Fig. 2). Previous studies have shown that TGF‐β may stimulate the invasive feature of certain advanced tumour cells, even if those cells maintain their response to the TGF‐β growth inhibitory effect. In colon carcinoma models (Hsu et al. 1994), one oral carcinoma cell line IF (Hasina et al. 1999), and in clinical studies (Osaki et al. 2000), TGF‐β1 was shown to have a stimulating effect on invasion. Our result indicated that a similar pattern exists for the metastatic oral carcinoma OSC2 cells.

Surprisingly, the signal transduction pathway through Smad was intact in the most advanced oral carcinoma OSC2 cells but not in DOK or SCC25 cells, as shown in Fig. 4. This observation suggests that induction of p15, p21 and p27 expression by TGF‐β1 may be mediated through pathways that involve Smad activity, as well as other signalling components, and this inhibition of cell growth may not have a correlation with TGF‐β1‐mediated stimulation of cell invasion.

Recent and previous investigations have shown that reduction in TGF‐β receptor II (TGF‐β RII) expression was associated with oral tumour progression in human (Paterson et al. 2001) and animal cell line models (Game et al. 1990). This reduction in TGF‐β RII resulted in loss of growth inhibition. Combined with data in the current study, it is suggested that, at least in oral squamous carcinomas, TGF‐β RII is more responsible for growth inhibition, either through Smad or alternative pathways. All cell lines/type in this human oral tumour progression model express TGF‐β RII (Fig. 2) and none of the cell lines lost growth inhibition. However, when oral carcinoma cells advanced to metastatic status, inhibitory signals still function if the TGF‐β RII and Smad systems are intact, as shown in OSC2 cells.

In conclusion, our data indicate that during oral cancer progression, loss of normal p53 function may be a common event. TGF‐β1‐induced growth inhibition is independent from p53 status and likely mediated by the induction of cyclin kinase inhibitors p15/p21/p27. The induction of p15/p21/p27 may be mediated through multiple signal transduction pathways when TGF‐β RI and TGF‐β RII are present.

In early stages of tumour progression, TGF‐β1 not only inhibits the growth but also invasiveness of the tumour; but when tumour cells become metastatic, TGF‐β1 may stimulate their invasive potential. The clinical significance for this observation is that treatments or therapies employing TGF‐β1 as a growth inhibitor may lead to stimulation of metastasis of advanced tumour cells. Furthermore, the healing process following surgical removal of oral cancer involves local elevation of TGF‐β1 expression, which may promote the migration of residual advanced metastatic tumour cells that evaded surgical procedures.

Acknowledgements

The authors thank Dr Patricia Schoenlein and Dr Carol Lapp for their suggestions and Laura Drake for proof‐reading the manuscript. This work was supported by a grant from Medical College of Georgia Research Institute to S. Hsu.

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