T he R ole of TGF-β in E pithelial M alignancy and its R elevance to the P athogenesis of O ral C ancer (P art II)

Abstract

The role of transforming growth factor-β (TGF-β) in epithelial malignancy is complex, but it is becoming clear that, in the early stages of carcinogenesis, the protein acts as a potent tumor suppressor, while later, TGF-β can function to advance tumor progression. We review the evidence to show that the pro-oncogenic functions of TGF-β are associated with (1) a partial loss of response to the ligand, (2) defects of components of the TGF-β signal transduction pathway, (3) over-expression and/or activation of the latent complex, (4) epithelial-mesenchymal transition, and (5) recruitment of signaling pathways which act in concert with TGF-β to facilitate the metastatic phenotype. These changes are viewed in the context of what is known about the pathogenesis of oral cancer and whether this knowledge can be translated into the development of new therapeutic modalities.

(I) Introduction

The role of TGF-β in epithelial malignancy has been controversial for some time, but it is now recognized that TGF-β acts as a potent tumor suppressor in the early stages of tumor progression, while later functioning more to enhance the malignant phenotype. Tumor-suppressor activities have been attributed to growth inhibition and the stimulation of apoptosis (reviewed by Siegel and Massagué, 2003). More recently, the maintenance of genomic stability and the induction of replicative senescence (Glick et al., 1996; Tremain et al., 2000), together with the suppression of telomerase activity (Yang et al., 2001), have been suggested as additional tumor-suppressor effects of TGF-β. By contrast, the tumor-promoting capabilities of TGF-β have been described in terms of a loss of the growth-inhibitory response to TGF-β, increased expression and/or activation of the ligand, and a change in the signal transduction pathways, with emphasis toward the pro-oncogenic activities of the ligand and the acquisition of a more invasive/metastatic phenotype (Fig.; Derynck et al., 2001; Roberts and Wakefield, 2003).

(II) Defects in Signal Transduction

Defects of signaling components at various points in the TGF-β signaling pathway have been described in a variety of cancers (Table) and are described below.

(A) Receptor anomalies

Inactivating mutations in TβRII occur frequently in a subset of human colorectal and gastric carcinomas, especially those exhibiting microsatellite instability associated with a deficient DNA mismatch repair system (Markowitz et al., 1995). The defect is classically a frameshift mutation in a 10-base-pair polyadenine tract (BAT-RII), resulting in functionally inactive truncated receptors lacking a serine-threonine kinase domain (Grady et al., 1999). In contrast, TβRII mutations in cancers other than those of the gastro-intestinal tract are rare (Abe et al., 1996; Vincent et al., 1996). The data implicating TβR-II as a tumor suppressor are consistent with results obtained with transgenic mice, where targeted deletion of TβR-II in specific tissues resulted in accelerated tumorigenesis following carcinogen treatment (Bottinger et al., 1997; Amendt et al., 1998; Go et al., 2000; Im et al., 2001; Kanzler et al., 2001; Hahm et al., 2002). Mutations or deletions of TβR-I have also been reported, but they are uncommon (Y Chen et al., 1998; Goggins et al., 1998; T Chen et al., 2001).

Analysis of recent data has shown that there is an alternative mechanism by which tumor cells escape the negative effects of TGF-β, and this is by the transcriptional repression of TGF-β receptors (Kim et al., 2000). TβR-II transcription, for example, has been shown to be down-regulated by mutations in the TβR-II promoter (Munoz-Antonia et al., 1996; Seijo et al., 2001), oncogenic Ras (Zhao and Buick, 1995), over-expression of cyclin D (Okamoto et al., 1994), and epigenetic alterations such as histone deacetylation of the TβR-II promoter (Osada et al., 2001; Zhao et al., 2003) and methylation-mediated repression of the transcription factor, Sp1 (Jennings et al., 2001; Venkatasubbarao et al., 2001). Similarly, transcriptional repression of TβR-I has been shown to be due to hyper-methylation of the TβR-I promoter (Kang et al., 1999).

The biological consequences of attenuation of TGF-β receptor activity are starting to be elucidated. Analysis of early data demonstrated that decreased TβR-II signaling conferred resistance to the growth-inhibitory effects of TGF-β, with retention of other transcriptional responses, suggesting that the signal transduction pathway(s) leading to cell-cycle arrest were distinct from those that mediated other effects of TGF-β (Chen et al., 1993). More recently, it has been shown that different signaling pathways and biological responses require different thresholds of TGF-β activation (Yingling et al., 1995; McEarchern et al., 2001). Specifically, higher levels of TβR-I signaling are required to phosphorylate Smad2 than to activate the PI3K and MAPK pathways (Dumont et al., 2003), and, presumably, the pro-oncogenic effects of TGF-β predominate in circumstances of low thresholds of TGF-β activity. In some studies, TGF-β signaling has been shown to be required for invasion and metastasis of carcinoma cells (Oft et al., 1998; McEarchern et al., 2001; Tang et al., 2003), and analysis of recent data shows that this is dependent upon an intact Smad pathway (Oft et al., 2002; Tian et al., 2003). Analysis of these data suggests that, in circumstances in which the tumor-suppressor properties of TGF-β have been compromised, the Smad pathway can function to promote a more aggressive phenotype. Interestingly, colon cancers that contain mutant TβR-II—and therefore the complete absence of both tumor-suppressor and pro-oncogenic TGF-β pathways—have a better prognosis than their counterparts with wild-type TGF-β receptors (Bubb et al., 1996). Further, decreased expression of TβR-II correlates with high tumor grade in a variety of human and experimental tumors (S Kim et al., 2000; J Kim et al., 2001).

A recent development is the observation that the accessory receptor NMA (BAMBI) is a downstream target of the Wnt signaling pathway, and that NMA is aberrantly over-expressed in a high proportion of colorectal and hepatocellular carcinomas (Sekiya et al., 2004). Over-expression of NMA in tumor cells inhibits their response to TGF-β (Sekiya et al., 2004), suggesting that NMA may play a role in tumorigenesis, particularly in those tumors with constitutive β-catenin signaling.

(B) Smad protein defects

Smad4 was originally isolated as a tumor-suppressor gene on chromosome 18q21, a site that is frequently deleted or mutated in a large proportion of human pancreatic cancers (Hahn et al., 1996). Genetic inactivation of Smad4, however, is largely restricted to pancreatic and gastro-intestinal tumors and is uncommon in a broad spectrum of other solid tumors (Schutte et al., 1996). Transgenic mouse models of colorectal carcinogenesis have given some clues as to the biological significance of Smad4 inactivation and indicate that haploid insufficiency of Smad4 may be sufficient for tumor initiation, and that bi-allelic loss is important for tumor progression (de Caestecker et al., 2000). In support of this proposal, complete genetic inactivation of Smad4 appears to be a late event in human colorectal cancer (Miyaki et al., 1999). Immunocytochemical studies have shown that loss of Smad4 protein in human cancer may occur more frequently than would be predicted from genetic analyses, indicating that epigenetic mechanisms (Natsugoe et al., 2002; Salovaara et al., 2002) and/or other anomalies (Saha et al., 2001) may be involved in Smad4 silencing. Interestingly, loss of Smad4 protein expression is often associated with the development of the invasive/metastatic phenotype and, concomitantly, a worse clinical outcome (Maitra et al., 2000; Natsugoe et al., 2002; Xie et al., 2002).

The gene encoding Smad2 is also located at 18q21 and is the target of inactivating mutations in a small subset of colorectal and lung cancers only (Eppert et al., 1996; Riggins et al., 1996). The majority of mutations in Smad2 and Smad4 disrupt Smad-dependent TGF-β signaling either by blocking receptor-dependent phosphorylation or by preventing heteromeric interactions between Smad proteins (Eppert et al., 1996; Shi et al., 1997). Certain mutations of Smad2 and Smad4, however, do not disrupt Smad activation or function, but rather target the mutant protein for ubiquitination and proteosome degradation (Xu and Attisano, 2000). Interestingly, loss of Smad4 has been shown to enhance the activity of the Ras/Erk pathway and to promote tumor progression in transformed keratinocytes containing mutant Ras (Iglesias et al., 2000).

Smad3 is located on chromosome 15q21-22 (Riggins et al., 1996), and this, again, is a frequent site of allelic loss in a broad spectrum of tumors (Hahn et al., 1995; Park et al., 2000). There is no evidence to suggest that Smad3 is mutationally inactivated in human cancer. However, certain strains of mice with a homozygous deletion of Smad3 develop aggressive metastatic colorectal cancer at an early age (Zhu et al., 1998). Further, loss of Smad3 protein has been reported recently in gastric tumors, possibly via the transcriptional repression of the Smad3 gene, and the re-introduction of Smad3 in gastric cell lines restored TGF-β responsiveness and suppressed tumorigenicity in vivo (Han et al., 2004).

Smad7 is located on chromosome 18q21.1 (Roijer et al., 1998) and, as an inhibitor of TGF-β signaling, is a putative oncogene. Reports implicating Smad7 in human cancer, however, are limited (Kleeff et al., 1999), but analysis of recent data indicates that Smad7 gene amplification is associated with a worse prognosis in patients with colorectal cancer (Boulay et al., 2003).

(III) Abnormalities in Smad Co-activators and Co-repressors

The transcriptional adapter proteins, p300 and CBP, can interact with Smad2, Smad3, and Smad4 and are required for transcriptional activation of a variety of TGF-β-dependent promoters. Mutations in p300 have been reported in a small proportion of cancers/cell lines of diverse origin (Muraoka et al., 1996; Gayther et al., 2000), and by inhibiting TGF-β-dependent transcriptional activation, they represent an alternative mechanism by which tumor cells escape the negative effects of TGF-β. Similarly, the oncoproteins Evi-1 (Kurokawa et al., 1998), Ski (Luo et al., 1999), SnoN (Stroschein et al., 1999), and BF-1 (Rodriguez et al., 2001) interact directly with Smad proteins and repress Smad signaling, which, in turn, leads to loss of TGF-β-mediated growth inhibition (Medrano, 2003).

(IV) Ligand Expression and Activation

In 1993, Glick and colleagues showed that the transition from benign papillomas to malignant squamous cell carcinomas in murine skin carcinogenesis was associated with a diminished expression of TGF-β1 (Glick et al., 1993). These findings were confirmed by the demonstration that loss of TGF-β1 expression in benign skin tumors of p53^null mice resulted in high risk of malignant conversion (Cui et al., 1994), and mice with inactivation of the gene encoding TGF-β1 showed an increased propensity for developing carcinomas (Glick et al., 1994; Tang et al., 1998; Engle et al., 1999). The use of transgenic mice that constitutively over-expressed TGF-β1, however, significantly altered the view that TGF-β was simply a tumor suppressor. After tumor exposure to a standard initiation/promotion protocol, over-expression of epidermal TGF-β1 suppressed the development of early, benign tumors, but, in the later stages of carcinogenesis, TGF-β1 enhanced the progression of benign tumors to more aggressive spindle cell carcinomas (Cui et al., 1996), suggesting that the peptide could also function to facilitate tumor progression.

Classically, both alleles of a tumor-suppressor gene must be inactivated for a tumor to form. The TGF-β pathway, however, does not appear to act in this on/off manner, and threshold effects are now known to be important in ligand-receptor interactions (Tang et al., 1998). This raises the possibility that TGF-β1 levels might affect susceptibility to cancer in humans, and there is some evidence to support this proposal (Ziv et al., 2001). Further, it is now recognized that the effect of TGF-β is context-dependent, and what is observed in one tumor type does not always apply to another. For example, studies to examine the function of TGF-β in regulating tumor behavior in vivo have produced conflicting results. Using a variety of different strategies, including the transfection of tumor cells with sense or antisense TGF-β expression constructs, investigators have demonstrated that TGF-β1 has the capacity to promote (Steiner and Barrack, 1992; Huang et al., 1995; Park et al., 1997) or inhibit (Wu et al., 1993; Pierce et al., 1995) tumor development and/or progression. Furthermore, high levels of TGF-β expression characterize carcinomas of the breast, colon, and pancreas (Dalal et al., 1993; Friess et al., 1993; Picon et al., 1998), whereas, in tumors of the integument, ligand expression is either decreased or remains unchanged (Schmid et al., 1996; Paterson et al., 2001).

An interesting hypothesis has recently been proposed which suggests that the mechanism by which TGF-β is activated may influence whether the ligand acts to suppress or promote tumor progression (Dumont and Arteaga, 2002). Through activation of the latent TGF-β complex, molecules such as thrombospondin and the integrins αvβ8 and αvβ5 have been linked with tumor suppression, while MMP 2 and MMP 9, the integrin αvβ6, and uPA/plasmin have been associated with tumor promotion. Clearly, these molecules are likely to be critical spatial and temporal determinants of the effects of TGF-β on tumorigenesis. In future work, however, it will be essential to examine the expression of these molecules in the context of both the autocrine and paracrine effects of TGF-β.

(V) Epithelial-Mesenchymal Transition

(A) Morphological changes

The process by which epithelial cells adopt a mesenchymal phenotype is termed epithelial-mesenchymal transition (EMT). EMT involves the disruption of epithelial cell-cell junctions and the remodeling of the actin cytoskeleton, leading to the formation of stress fibers. The process can be described biochemically as a delocalization of proteins such as E-cadherin, ZO-1, vinculin, and desmoplakin, a down-regulation of certain cytokeratins, and an increase in mesenchymal markers such as fibronectin and vimentin (Grunert et al., 2003).

TGF-β-mediated EMT was first reported by Miettinen et al.(1994) in cultured murine epithelial cells, but the phenomenon has now been described in a broad spectrum of cell lines (Caulin et al., 1995; Oft et al., 1996; Piek et al., 1999; Gotzmann et al., 2002), including those of human origin (Geng et al., 1999; Janji et al., 1999; Zavadil et al., 2001; Yi et al., 2002). TGF-β1 appears to induce EMT through autocrine mechanisms. TGF-β1-mediated EMT, for example, is reversed in vitro and in vivo by the addition of dominant-negative TβRII cDNA in murine keratinocytes (Oft et al., 1998; Portella et al., 1998), and elevated levels of endogenous TGF-β1 are thought to maintain the mesenchymal phenotype after the initial induction of EMT in MDCK and EpRas cells (Lehmann et al., 2000; Janda et al., 2002).

Significantly, TGF-β1 requires a co-factor to induce EMT, and in the majority of cells studied, this is the presence of activated Ras or Raf (Oft et al., 1996; Lehmann et al., 2000; Janda et al., 2002). Current thinking suggests that Ras enhances the tumor-promoting effects of TGF-β1 while at the same time attenuating its tumor-suppressor activities (Park et al., 2000; Yan et al., 2001). With the recognition that activated Ras is important in EMT, there has followed a plethora of studies that have examined the downstream targets of TGF-β1 and Ras signaling and their involvement in EMT; a review of this work is outside the scope of this report but has been described in detail by Grunert et al.(2003). It is cautionary to note, however, that EMT appears to involve different signaling pathways in different cell types, that a variety of experimental conditions has been used in different studies, that experiments involving the use of dominant-negative constructs do not always completely abrogate protein expression, and that different workers have used various definitions of EMT (morphological change as opposed to scattering). Perhaps more importantly, however, is that while there is a large body of work regarding the pathways and mediators of EMT, the majority of evidence is derived from murine carcinoma cell lines, and very little has been published on studies wherein cells of human origin were used. The scientific community is necessarily aware of the differences between cells of rodent and human origin (Rangarajan and Weinberg, 2003).

(B) Evidence for tumor promotion

It has been argued that EMT is phenomenological only and unlikely to be an end-point of epithelial tumor progression in humans, not least because spindle cell tumors of the integument are extremely rare. Spindle cells, however, are found commonly at the invasive front of carcinomas, and it is widely held that this phenotype facilitates both tumor invasion and metastases (Gabbert et al., 1985). Support for the proposal that TGF-β-induced EMT is important biologically has come from several sources. First, several studies have shown that transdifferentiated cells following TGF-β-induced EMT are more motile and more invasive in vitro (Lehmann et al., 2000; Ellenrieder et al., 2001; Bakin et al., 2002; Yi et al., 2002). Second, cells that have undergone EMT are more tumorigenic and form less-differentiated tumors, with an elevated metastatic capacity following transplantation to athymic mice (Oft et al., 1998; Portella et al., 1998; Janda et al., 2002). And third, studies with cyclosporin have shown that the drug induces EMT and promotes tumor progression in a TGF-β-dependent manner (Hojo et al., 1999). Interestingly, patients who have been treated with cyclosporin following organ transplantion appear to be susceptible to highly invasive squamous cell carcinomas of the skin (Marcil and Stern, 2001); in a clinical context, however, the potent immunosuppressive characteristics of cyclosporin cannot be excluded.

Indirect evidence to indicate that EMT has biological relevance is derived from studies of E-cadherin expression in malignancy. Cell lines with low levels of E-cadherin have increased invasive and metastatic capabilities (Frixen et al., 1991; Hoteiya et al., 1999), and in breast, gastric, bladder, and head and neck cancer, there is a clear correlation between decreased expression of E-cadherin and the more invasive/metastatic phenotype (Schipper et al., 1991; Oka et al., 1993; Wakatsuki et al., 1996). Recent studies have shown that E-cadherin expression is regulated by Snail, Slug, and SIP, and it is now known that over-expression of these transcription factors leads to down-regulation of E-cadherin, EMT, and increased invasion in vitro (Comijn et al., 2001; Bolos et al., 2002; Guaita et al., 2002; Yokoyama et al., 2003). Furthermore, Peinado et al.(2003) have recently showed that TGF-β induces the Snail transcription factor in epithelial cell lines, resulting in repression of E-cadherin expression and the induction of EMT in vitro.

(VI) Metastases

The relationship of TGF-β to tumor progression has been known for some time and has always been explained in terms of the generic effects of the ligand, leading to angiogenesis, immune suppression, and ECM elaboration. The effect of TGF-β on metastases has been recognized for some time (Welch et al., 1990; McEearchen et al., 2001), but more recently, the subject has attracted significant attention. Using a bi-transgenic mouse model of mammary carcinogenesis, Siegel et al.(2003) demonstrated that, in the context of an activated Neu oncogene (the equivalent of an activated ErbB-2 proto-oncogene in humans) driven by the mouse mammary virus long-terminal repeat (MMTV), TGF-β delayed the appearance of primary mammary tumors, consistent with a tumor-suppressor function, but promoted spontaneous metastases to the lung after activation of the TGF-β receptor, indicating a pro-oncogenic effect of TGF-β. Specifically, activation of TGF-β signaling in the tumor cells enhanced the proportion of extravasated metastases (Siegel et al., 2003). While previous observations showed that TGF-β/Smad and p38 signaling cooperate to promote metastases of human breast cancer cells (Yin et al., 1999; Kakonen et al., 2002), recent studies have demonstrated that TGF-β switches from tumor-suppressor to pro-metastatic factor in a model of breast cancer progression with cells of common genetic origin (Tang et al., 2003). Significantly, in this model, the Smad2/3 signaling pathway was shown to mediate both the tumor-suppressor and pro-metastatic effects of TGF-β (Tian et al., 2003). These results demonstrate that the role of TGF-β in carcinogenesis is contextual, and the importance of the work by Siegel et al.(2003) and Tang et al.(2003) is that it will now be possible to dissect the signaling networks that are associated with the pro-oncogenic effects of TGF-β, a development that should have therapeutic implications.

(VII) Oral Cancer

(A) Rat-4,Nitroquinoline N oxide (4NQO) model of oral carcinogenesis

The topical application of the water-soluble carcinogen 4NQO to the palates of Sprague-Dawley rats leads to the development of invasive squamous cell carcinomas of the palatal and lingual oral mucosae. In cell cultures of 4NQO-induced SCCs, two different cell phenotypes are evident, namely, well-differentiated (cytokeratin-positive/vimentin-negative) polygonal cells and undifferentiated (cytokeratin-negative/vimentin-positive) spindle-type cells. Following treatment with TGF-β1, well-differentiated and normal cells are similarly growth-inhibited by the ligand, while the undifferentiated spindle cells partially lose their growth-inhibitory response to the peptide (Game et al., 1991). When endogenous TGF-β1 is over-expressed in either the differentiated or undifferentiated tumor cells, however, the ligand acts to inhibit metastatic dissemination in vivo and, at the same time, promotes local bone resorption at the site of the primary tumor (Davies et al., 1997, 1999). We believe that the spindle cell phenotype is not simply a more advanced stage of polygonal cell development but, rather, may reflect alternative pathways of differentiation. It is not known whether there is a differential expression of oncogenic Ras in the polygonal and spindle cell phenotypes of the rat-4NQO model, but the presence of oncogenic Ras in the spindle cells would be likely to facilitate morphological change in the context of TGF-β1 over-expression.

(B) Human oral cancer

Along with others, we have been studying for some time the effects of TGF-β in a series of malignant oral keratinocyte cell lines derived from human oral squamous cell carcinomas (SCC). Interestingly, the majority of these cells retain a full or partial response to the anti-proliferative effects of TGF-β1 (Prime et al., 1994; Fahey et al., 1996; Malliri et al., 1996). Even in those lines that are not growth-inhibited by TGF-β1, a restricted transcriptional response to the ligand is retained (Paterson et al., 1995, 2002). Further, despite the fact that p53 cooperates with Smad proteins to mediate certain TGF-β signals (Cordenonsi et al., 2003), all of the human oral SCC lines that have been studied to date contain mutant p53, regardless of whether they are growth-inhibited or refractory to TGF-β1. Experiments are currently in progress to determine the exact mechanism to account for why certain cell lines fail to respond to TGF-β1, but one such mechanism is the failure to express Smad4 protein (Paterson et al., 2002). Intriguingly, over-expression of endogenous TGF-β1 in Smad4-deficient cells leads to tumor suppression in vivo (Paterson et al., 2002). Analysis of our more recent data demonstrates that the tumor suppression in this context is not due to the inhibition of primary tumor formation but, rather, occurs as a result of tumor regression, possibly by the potentiation of mitochondria-regulated apoptosis (unpublished observations).

As the TGF-β signal transduction pathways have become unraveled, genetic defects responsible for variations in TGF-β responsiveness have been examined in oral carcinomas. Structural defects of TβRI (Chen et al., 2001; Knobloch et al., 2001) and TβRII (Garrigue-Antar et al., 1995; Wang et al., 1997), leading to a complete abrogation of TGF-β signaling, have been reported, but such abnormalities have proved to be rare. The fact that most cell lines retain a ligand-induced transcriptional response suggests the occurrence of more subtle alterations in the majority of human oral cancers. During the transition from normality to malignancy and from carcinoma to metastases, TβR-II protein expression decreases significantly, suggesting that down-regulation of TβR-II, rather than gene mutations, is important in the pathogenesis of the disease (Paterson et al., 2001). To examine the functional significance of decreased TβR-II activity in oral cancer, we transfected dominant-negative TβR-II cDNA into a human malignant oral keratinocyte cell line. The results demonstrated reduction of TGF-β-induced growth inhibition but retention of ligand-induced transcriptional responses. The partial inhibition of TβR-II signaling in these circumstances not only induced a metastatic phenotype in vivo, but also was associated with an enhanced growth rate, increased migration and invasion in vitro, and a loss of tumor cell differentiation (Huntley et al., 2004).

In contrast to carcinomas from glandular tissues (Dalal et al., 1993; Friess et al., 1993; Picon et al., 1998), the level of TGF-β1 protein in oral cancer remains unchanged during the progression from normality to lymph node metastases (Paterson et al., 2001); other studies report increased TGF-β1 expression in head and neck cancers (Pasini et al., 2001). Malignant human oral keratinocytes in vitro produce less TGF-β1 than their normal counterparts, most probably due to adaptation to cell culture conditions (Fahey et al., 1996). Analysis of recent data indicates that while TGF-β1 expression is not a useful biomarker of prognosis in patients with head and neck cancer (Logullo et al., 2003), it does act in an autocrine capacity to stimulate cell motility (Hasina et al., 1999).

Loss of heterozygosity at chromosome 18q has been reported frequently in head and neck squamous cell carcinoma (van Dyke et al., 1994; Frank et al., 1997; Jones et al., 1997), suggesting that defects in Smad2 and Smad4 would be a common finding. Data to verify this proposal, however, are limited and somewhat controversial. One immunohistochemical study has shown consistent expression of Smad2, Smad3, and Smad4 protein (Muro-Cacho et al., 2001), while others have demonstrated normal expression of Smad2 and Smad3 but loss of Smad4 (Yan et al., 2000). To complicate matters further, Muro-Cacho et al.(2001) demonstrated normal expression of Smad2 but defective Smad2 phosphorylation in tissue specimens, the significance of which remains unclear. Analysis of our own data shows that Smad2 and Smad3 mRNA and protein are expressed in 14 of 14 oral carcinoma cell lines, but in two lines, Smad4 mRNA expression is markedly reduced, and Smad4 protein is undetectable, which results in loss of TGF-β-induced growth inhibition but retention of transcriptional responses (Paterson et al., 2002); transfection with wild-type Smad4 cDNA restores ligand-induced growth inhibition (unpublished observations). The mechanism of Smad4 loss in these two cell lines is currently under investigation, but it does not appear to be due to inactivating mutations in the coding sequence of the gene, and suggests the involvement of epigenetic events or promoter mutations.

Evidence is emerging to show that alterations in transcriptional regulators may also play a role in abrogating Smad signaling in head and neck cancer. A mutation in the p300 co-activator gene, for example, was recently described in a human oral carcinoma cell line, and transfection of the wild-type p300 restored TGF-β-induced Smad-dependent transcriptional activation (Suganuma et al., 2002).

We have shown that TGF-β1 induces EMT in human oral carcinoma cell lines (even in cells that are markedly growth-inhibited by the ligand), that the response is dependent on the presence of mutant Ras, and that the phenomenon involves Erk, PI3 kinase, and p38 (unpublished observations). Further, we have preliminary data to show that the AP-1 complex is involved in EMT, suggesting that the TGF-β and Ras signaling pathways converge on AP-1 to drive transdifferentiation.

The biological consequences of TGF-β1-induced EMT in oral cancer are unclear, but if it functions to promote tumor progression, investigators will have to be cognizant of the use of retinoids as a therapeutic modality. The effects of the synthetic retinoid N-(4-hydroxyphenyl)retinamide (4-HPR or Fenretinide), for example, which has been shown to be an effective chemopreventive agent in ongoing clinical trials of different cancers, including head and neck cancer (Oridate et al., 1996), appear to be mediated, albeit in part, by TGF-β (Roberson et al., 1997; Herbert et al., 1999; Borger et al., 2000; La et al., 2003), which raises the possibility that Fenretinide may have dual effects, depending on the presence of oncogenic Ras. Significantly, Ras is rarely mutated in oral cancer of Western origin, but in Asia, abnormalities leading to activated Ras occur frequently (Paterson et al., 1996). It is conceivable, therefore, that the use of Fenretinide for the treatment of pre-malignant oral lesions in India and southeast Asia could have disastrous clinical consequences. Interestingly, a Phase III chemoprevention trial involving some 47,447 smokers treated with β-carotene had to be stopped because of the increase in lung cancer and patient mortality (ATBC Cancer Prevention Study, 1994; Omenn et al., 1996); smoking-induced lung cancer is known to be associated with a high incidence of K-Ras mutations (Minamoto et al., 2000).

(VIII) Concluding Remarks

Many investigators have been discouraged by the complexity of the role of TGF-β in malignancy and have dismissed the possibility of therapeutic intervention. The development of TGF-β agonists could possibly be of value for the treatment of early-stage malignancy, while antagonists may be beneficial for the control of metastatic dissemination. The farnesyl transferase inhibitors are known to inhibit the activated Ras pathway while at the same time enhancing TβR-II expression and responsiveness to TGF-β1 (Adnane et al., 2000; Alcock et al., 2002). These agents, however, have yet to show great promise in the clinic, particularly in Phase II and Phase III trials of common cancers (Downward, 2003). A variety of different strategies has been used in the past to antagonize TGF-β activity, not only in the treatment of experimental cancer but also for the management of fibrotic disorders of the lung, kidney, heart, and skin. Experimental approaches include the use of TGF-β antibodies (Border et al., 1990), retroviral-mediated gene therapy of a dominant-negative TβR-II construct (Shah et al., 2002), decorin (Giri et al., 1997), betaglycan (Bandyopadhyay et al., 2002), and small molecule inhibitors of TβR-I or their substrates (Laping et al., 2002; Yakymovych et al., 2002). One approach which is attracting increasing attention is the use of a soluble TGF-β receptor that appears selectively to inhibit metastatic disease in the MMTV-neu transgenic mouse model in the absence of effects either on normal physiology or on tumorigenesis at the primary site (Yang et al., 2002). These findings support previous observations demonstrating the value of a soluble TGF-β receptor in the prevention of mammary tumor and pancreatic adenocarcinoma metastases (Muraoka et al., 2002; Rowland-Goldsmith et al., 2002). The use of a soluble TGF-β receptor, therefore, not only appears to be a valuable tumor suppressor, but also circumvents previous concerns that depletion of TGF-β induces auto-immune-like phenomena (Kulkarni et al., 1993). Alternatively, it may be that targeted delivery of a TGF-β antagonist to the site of the primary tumor and/or metastatic tumor deposit is another way to prevent systemic side-effects.

As noted leaders in the field have stated, “The perceived role of TGF-β in carcinogenesis has undergone more plot twists than an Agatha Christie mystery” (Roberts and Wakefield, 2003). It seems likely that TGF-β will continue to intrigue and tantalize the scientific community for some years to come.

TABLE

Examples of TGF- β Signal Transduction Defects in Cancer

Signaling Component	Defect	Cancer
NB: The frequency of specific alterations varies between the signaling components and between the tumor types.
TβR-I	Mutation	Ovarian, pancreatic, breast
	Decreased expression	Gastric, head and neck
TβR-II	Mutation	Gastric, colorectal
	Decreased expression	Breast, head and neck
Smad2	Mutation	Colorectal, lung, pancreatic
Smad3	Reduced expression	Gastric
Smad4	Deletion	Colorectal, pancreatic
	Mutation	Pancreatic
	Reduced expression	Oral
Smad7	Over-expression	Colorectal, pancreatic
Ski	Over-expression	Melanoma
p300	Mutation	Cervical, oral
NMA	Over-expression	Colorectal, hepatocellular

Figure.

The role of TGF-β in epithelial carcinogenesis. In normal epithelial cells with an intact TGF-β signaling pathway, TGF-β functions as a tumor suppressor by inhibiting cell proliferation and enhancing apoptosis, together with the regulation of replicative senescence and suppression of telomerase. During multi-step tumorigenesis, cells acquire defects in the TGF-β signaling pathway, which abrogates or diminishes the responses of the tumor cells to TGF-β, and oncogene signaling may also alter the cellular response to TGF-β. Some late-stage tumors are also known to overproduce TGF-β. Consequently, TGF-β shifts from a suppressor to a promoter of tumor progression, by increasing angiogenesis, enhancing motility/invasion, and, at times, inducing an epithelial-mesenchymal transdifferentiation.

Footnotes

Acknowledgements

The authors are most grateful to the Shirley Glasstone Hughes Memorial Fund (British Dental Association) for support of MP. We also thank J. Beddoe and L. Jones for help in preparation of the manuscript.

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