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Abstract

Recent efforts on developing more direct and effective targets for cancer therapy have revolved around a family of transcription factors known as STATs (signal transducers and activators of transcription). STAT proteins are latent cytoplasmic transcription factors that become activated in response to extracellular signaling proteins. STAT proteins have been convincingly reported to possess oncogenic properties in a plethora of human cancers, including oral and oropharyngeal cancer. Signal transduction pathways mediated by these oncogenic transcription factors and their regulation in oral cancer are the focus of this review.

Introduction

Squamous cell carcinoma (SCC) of the head and neck, including the oral cavity and oropharynx, is the sixth most common cancer worldwide (Vokes et al., 1993). Despite recent advances in surgery, radiation, and chemotherapy, the prognosis for head and neck cancer remains dismal, especially if malignant tumors are not diagnosed during the early stages of the disease. Several cellular and molecular events that underlie the occurrence and progression of head and neck SCC, however, are gradually unfolding, including oncogenic alterations and de-regulations in cell death (Todd et al., 1997; Williams, 2000; Nagler et al., 2002; Loro et al., 2003). In this regard, p53 mutations (Maestro et al., 1992; Ahomadegbe et al., 1995) as well as overexpression of cyclin D1 (Jares et al., 1994; Callender et al., 1994; Gimenez-Conti et al., 1996; Quon et al., 2001) and epidermal growth factor receptor (EGFR) (Ozanne et al., 1986; Maxwell et al., 1989) are common alterations. Moreover, overproduction of EGFR and its ligand, transforming growth factor-α (TGF-α), has been described as an early event in head and neck carcinogenesis (Grandis and Tweardy, 1993; Grandis et al., 1996). Elucidation of such mechanisms responsible for critical tumorigenic abnormalities that influence tumor behavior in head and neck cancer are valuable in the diagnosis and development of more effective treatments.

The STAT (signal transducer and activator of transcription) family of proteins includes 7 members (Stat-1, 2, 3, 4, 5A, 5B, and 6), encoded by distinct genes in mammalian cells (Darnell, 1997). The STAT family members are latent cytoplasmic transcription factors that become activated in response to extracellular signaling proteins, including growth factors, cytokines, hormones, and peptides (Darnell et al., 1994; Darnell, 1997; Bromberg and Darnell, 2000). The transmission of signals involves activation of various tyrosine kinases that phosphorylate STAT proteins. Activated STAT proteins transmit signals by translocating into the nucleus and driving transcription of specific genes through interactions with DNA elements in the promoters of target genes (Darnell, 1997; Bromberg and Darnell, 2000).

The STAT family of transcription factors plays a critical role in regulating physiological responses to stimulation by cytokines and growth factors: STAT knockout mice phenotypically present defects similar to those observed in knockouts of cytokines and their receptors (Darnell et al., 1994; Zhong et al., 1994; Schindler and Darnell, 1995; Darnell, 1997; Takeda and Akira, 2000; Levy and Darnell, 2002). In contrast, persistent STAT activation, in particular Stat3 and Stat5, has been convincingly implicated in oncogenesis (Bowman et al., 2000; Bromberg and Darnell, 2000; Bromberg, 2001, 2002; Levy and Darnell, 2002). This review provides a synopsis of recent advances that elucidate the role of STAT proteins in oral cancer, highlighting the important implications of these molecules in oral carcinogenesis, growth, and survival, and possible applications for cancer therapeutics.

STAT Signaling: Activation and Outcome

Activation of STAT molecules involves their tyrosine phosphorylation by several tyrosine kinases. These kinases are activated as a result of interactions between cytokines or growth factors and their respective receptors, inducing receptor dimerization or oligomerization. Cytokine receptors, devoid of intrinsic kinase activity, require the mediation of Janus kinases (JAKs) to induce STAT receptor docking and activation. Upon binding of a cytokine to its corresponding receptor, associated JAKs undergo auto-phosphorylation and subsequently phosphorylate tyrosine residues within the cytoplasmic tail of receptors. These receptors recruit and serve as docking sites for STAT monomers through their phosphotyrosine residues and SH2 domain, respectively (Darnell, 1997; Bromberg and Darnell, 2000; Levy and Darnell, 2002). In contrast, receptor tyrosine kinases such as EGFR possess intrinsic tyrosine kinase activity and facilitate recruitment and subsequent direct activation of STAT proteins (Darnell, 1997; Bromberg and Darnell, 2000; Levy and Darnell, 2002). In addition to cytokine and growth factor receptors, non-receptor tyrosine kinases such as Src and Abl can, either directly or through association with JAKs, induce STAT phosphorylation (Chaturvedi et al., 1997; Olayioye et al., 1999; Wang et al., 2000; Garcia et al., 2001; Schreiner et al., 2002).

Regardless of the mode of activation, phosphorylated STAT proteins form immediate dimer complexes through reciprocal SH2 phosphotyrosine interactions. STAT dimers undergo an immediate disengagement from the receptor or kinase and subsequently translocate to the nucleus. Once in the nucleus, STAT dimers bind to DNA and induce transcription of specific target genes (Darnell, 1997; Levy and Darnell, 2002). Interestingly, different STAT complexes interact with different co-activators and induce distinct genes based on their binding affinities for promoter response elements (Seidel et al., 1995; Ehret et al., 2001).

Structural Elements of STAT Molecules

The STAT molecule is structurally characterized by the presence of distinct N-terminal, C-terminal, and DNA binding regions, which are essential for activation and function (Fig. 1).

(a) N-terminal region

The N-terminal region consists of an oligomerization domain, which stabilizes the association of two adjacent DNA-bound STAT dimers (Vinkemeier et al., 1996). The N-terminal region is also involved in nuclear translocation and STAT de-activation, thus exerting transcriptional control (Shuai et al., 1996b; Strehlow and Schindler, 1998).

(b) DNA-binding region

The DNA-binding domain of STATs is an essential element for function as well as regulation. The centrally positioned DNA-binding domain spans the region separating the C- and N-terminal domains and enables STAT to interact with specific response elements within the promoter sequence of target genes (Darnell, 1997). Moreover, a linker domain of unknown function, spanning conserved residues between the DNA-binding and SH2 domains, may be implicated in DNA-binding stability and transcriptosome assembly (Yang et al., 2002).

(c) C-terminal region

Within the C-terminal region resides a well-conserved Src homology 2 (SH2) domain. This structural motif is critical for STAT interaction with specific phosphotyrosine residues of other proteins (Gupta et al., 1996). This interaction facilitates tyrosine phosphorylation of a STAT molecule at a tyrosine residue (~ 701) by upstream kinases, followed by dimerization through reciprocal SH2 phosphotyrosine binding with another STAT molecule. Preceded by the SH2 domain, the transactivation domain (TAD) lies within the extreme C-terminal region of the STAT molecule and contributes to functional specificity and transcriptional activation of target genes (Kim and Baumann, 1997; Moriggl et al., 1997; Hoey and Schindler, 1998; Leonard and O’Shea, 1998). The latter function is mediated through interaction of the C-terminal TAD of STAT proteins with other transcriptional co-activators and mediators such as p300/CBP, c-Jun, and histone acetyltransferases (HATs) (Levy and Darnell, 2002). In some STATs, particularly Stat1 and Stat3, a critical serine residue 727 (Ser727), within TAD, enhances the DNA-binding affinity and transcriptional activity (Wen et al., 1995; Bromberg et al., 1998; Turkson et al., 1999).

Negative Regulation of STAT Signaling

Several classes of negative regulators of STAT proteins have been discovered.

(a) Cytoplasmic inhibitors of STATs

Suppressors of cytokine signaling (SOCS) proteins are transcriptional targets of STATs and function in a classic negative feedback loop to inhibit further STAT activation by interacting with either JAK catalytic or receptor sites (Starr and Hilton, 1998, 1999; Kile and Alexander, 2001; Krebs and Hilton, 2001; Levy and Darnell, 2002). Cytoplamic tyrosine phosphatases, including SH2-containing protein tyrosine phosphatase-1 (SHP-1) and SHP-2, also inhibit STATs by dephosphorylating upstream receptors and associated JAK kinases (David et al., 1995; Starr and Hilton, 1999; You et al., 1999; Aoki and Matsuda, 2000; Kile et al., 2001; Qu, 2002).

(b) Nuclear inhibitors of STATs

The protein inhibitors of activated STATs (PIAS) are nuclear inhibitors of activated STAT proteins that function to repress DNA binding and dimer-dependent transcription (Chung et al., 1997a; Shuai, 2000). At least two members, PIAS1 and PIAS3, have been shown specifically to inhibit Stat1 and Stat3, respectively (Chung et al., 1997a; Shuai, 2000). However, these proteins are also involved in STAT independent processes, and their specificity to STATs remains controversial. In addition to PIAS, naturally occurring truncated STATs, lacking the transactivation domain TAD, can act as non-functional STATs and obstruct DNA binding of functional STAT proteins (Fig. 1) (Wen et al., 1995; Caldenhoven et al., 1996). Finally, nuclear tyrosine phosphatases that function to dephosphorylate active nuclear STATs have also been recently identified (Shuai, 2000; ten Hoeve et al., 2002).

Regulation and Crosstalk in STAT Signaling

(a) Interactions with MAPKs

Other signal transduction pathways within the cell have been shown to regulate STAT proteins, through indirect kinase-mediated cross-interactions. Among these kinases, mitogen-activated protein kinases (MAPKs) have been implicated in the regulation of STAT proteins through crosstalk signaling (Decker and Kovarik, 2000). ERK-dependent repression of Src- or Jak2-mediated Stat3 signaling involves inhibition of tyrosine phosphorylation, DNA binding, and transcriptional activity (Jain et al., 1998). In this regard, ERK2 has been shown to interact physically with Stat3, involving Ser727 of the Stat3 molecule (Jain et al., 1998). Similar to Stat3, it has been shown that Stat5a contains a putative ERK phosphorylation site and also physically interacts with ERK1/2 (Pircher et al., 1999). Moreover, the inhibitory effect of ERK signaling on IL-6-mediated Stat3 activation has been shown to involve repression of the upstream Jak1 and Jak2 (Sengupta et al., 1998). In addition to ERKs, various inflammatory and stress agents are able to inhibit STAT signaling via a p38 MAPK-mediated mechanism, possibly involving repression of Jak1 (Ahmed and Ivashkiv, 2000) or induction of SOCS3 expression (Bode et al., 1999, 2001). Moreover, activation of the JNK family of MAPK by various stresses or by its upstream kinase MMK7 has also been shown to repress EGF-mediated Stat3 activation (Lim and Cao, 1999).

(b) Crosstalk with other cellular proteins

In addition to crosstalk with MAPKs, STAT proteins are also subject to regulation by other cellular proteins, including protein kinase Cδ (PKCδ), glucocorticoids, and protein kinase R (PKR) (Decker and Kovarik, 2000). Moreover, the inhibition of Stat3 by a novel inhibitor GRIM-19 (gene associated with retinoid-IFN-induced mortality 19) (Lufei et al., 2003; Zhang et al., 2003) has been documented. The interaction between Grim-19 and Stat3 has also been shown to involve the Stat3 Ser727 residue (Zhang et al., 2003).

(c) Serine phosphorylation and its potential significance

Serine phosphorylation of STAT proteins has been suggested to play an important role in STAT function (Decker and Kovarik, 2000). In general, STAT serine (in addition to tyrosine) phosphorylation occurs in response to growth factor and cytokine stimulation (Wen et al., 1995; Wen and Darnell, 1997). This activation is mediated by either MAPK or PKCδ signaling in a pathway-dependent manner (Decker and Kovarik, 2000). However, the function of STAT serine phosphorylation in the regulation of STATs remains elusive, in that serine phosphorylation enhances maximum transcriptional activity of STAT proteins (especially Stat1 and Stat3) (Wen et al., 1995; Darnell, 1997; Wen and Darnell, 1997), while causing decreases in STAT tyrosine phosphorylation (Chung et al., 1997b). With respect to the former, overexpression of a phosphoserine mutant of Stat3 was shown to abolish cell transformation by v-Src (Bromberg et al., 1998). The precise role of serine phosphorylation of STAT proteins is further complicated by the findings that MAPKs, which can exert negative regulation on STAT signaling, also induce serine phosphorylation of STAT proteins (Decker and Kovarik, 2000). In light of the fact that repression of Stat3 tyrosine phosphorylation by MEK/ERK signaling can occur independent of Ser727 phosphorylation (Sengupta et al., 1998), the exact role and functional significance of STAT serine phosphorylation necessitates further elucidation.

STATs and Tumorigenesis

(a) Oncogenic role of STATs

Several lines of evidence support a significant role of STAT molecules, especially Stat3 and Stat5, in carcinogenesis. The ability of STAT molecules to act as oncogenes was established through experiments with a constitutively active mutant of Stat3 (Stat3C), which was capable of inducing malignant transformation in fibroblasts (Bromberg et al., 1999). In addition, dominant-negative STAT mutants induce apoptosis and growth arrest in cancer cells (Grandis et al., 1998; Garcia et al., 2001), suggesting a fundamental role for constitutively active STAT molecules in malignancy. However, it should be noted that not all STATs promote survival. In fact, there is compelling evidence that Stat1 induces cell-cycle arrest and promotes apoptosis through activation of the Cdk inhibitor, p21 (Chin et al., 1996; Sahni et al., 1999) and caspase 1 (Chin et al., 1997). Some investigators have proposed that Stat1 may function as a tumor suppressor, in that Stat1-/- mice potentiate tumor development (Kaplan et al., 1998; Bromberg, 2001). In contrast, over-activation of Stat3 and Stat5 signaling induces specific target genes which prevent apoptosis and stimulate cell proliferation (Bowman et al., 2000; Bromberg, 2001, 2002).

(b) Aberrations in STAT regulators

With the exception of Stat5 chromosomal translocation in a subset of acute promyelocytic-like leukemias (Ahmed and Ivashkiv, 2000), mutations in STAT proteins that result in aberrant activation have not been reported in cancer. Thus, STAT oncogenic signaling has been linked to aberrations in STAT cellular regulators, including genetic alterations in activators and repressors of STAT signaling. Indeed, several viral as well as cellular oncogenes, including various oncogenic tyrosine kinases, induce transformation selectively by activating STAT proteins (Migone et al., 1995; Yu et al., 1995; Garcia et al., 1997; Lund et al., 1997; Bromberg et al., 1998; Zong et al., 1998; Wen et al., 1999; Bowman et al., 2000; Bromberg, 2001, 2002). The JAK family of kinases has been shown to drive the neoplastic transformation by oncogenic STAT proteins (Schwaller et al., 1998; Bowman et al., 2000). In addition, oncogenic transformation by Src has been consistently linked to STATs (Yu et al., 1995; Cao et al., 1996; Chaturvedi et al., 1997; Garcia et al., 1997; Bromberg et al., 1998; Bowman et al., 2000), and transformation by the Src oncoprotein has been shown to require Stat3 activation (Bromberg et al., 1998; Turkson et al., 1998; Zhang et al., 2000). Interestingly, Jak1 was shown to be required for maximal Stat3 activation by Src kinases in mammalian cells (Zhang et al., 2000). Moreover, v-Abl and BCR-Abl oncoproteins lead to STAT constitutive activation through JAK-dependent and -independent mechanisms (Shuai et al., 1996a; Danial et al., 1998; Danial and Rothman, 2000).

Genetic alterations in negative regulators of STAT proteins have also been reported in several cancer models. Interestingly, SOCS1 gene hypermethylation has been reported in several types of cancer, including hepatocellular carcinomas (Yoshikawa et al., 2001), multiple myeloma (Galm et al., 2003), hepatoblastoma (Nagai et al., 2003), leukemia (Chen et al., 2003), and pancreatic ductal neoplasms (Fukushima et al., 2003), suggesting an important role for SOCS1 in tumor progression. The methylation-induced silencing of SOCS1, leading to constitutive Stat3 activation in hepatocellular carcinoma cell lines, provides a possible mechanism for aberrant activation of STATs in these cells (Yoshikawa et al., 2001). Finally, PIAS3 expression is also repressed in anaplastic large cell lymphoma, possibly contributing to constitutive Stat3 levels (Zhang et al., 2002).

STAT Molecules in Tumor Growth and Survival

(a) Cell death regulation

Regulation of apoptosis by modulating cell death regulatory proteins is an integral function of oncogenic STAT signaling (Bowman et al., 2000; Bromberg, 2001, 2002). Stat3 and Stat5 especially possess potent anti-apoptotic properties. Constitutive Stat3 signaling has been shown to be essential for the survival of myeloma cells (Catlett-Falcone et al., 1999). Moreover, v-Src-mediated constitutive activation of Stat3 up-regulates bcl-X gene expression in transfected NIH3T3 cells (Catlett-Falcone et al., 1999). Stat3 regulates transcription of the Bcl-X gene, most likely through multiple Stat3 binding elements found within the Bcl-X promoter (Seidel et al., 1995). In head and neck SCC, Stat3 antisense gene therapy leads to decreased Bcl-X_L protein expression in xenograft models, revealing Stat3-mediated Bcl-X_L anti-apoptotic functions in vivo (Grandis et al., 2000a; Song and Grandis, 2000). Stat5 activation also maintains cell survival by inducing Bcl-X_L expression (Lord et al., 2000), since Stat5a and Stat5b double-knockouts result in abrogation of Bcl-X_L expression (Dumon et al., 1999; Silva et al., 1999; Socolovsky et al., 1999). In addition to Bcl-X_L induction, up-regulation of the apoptosis inhibitor Mcl-1 expression has also been shown to mediate the anti-apoptotic actions of Stat3 and Stat5 (Epling-Burnette et al., 2001; Huang et al., 2002).

(b) Cell-cycle regulation

In addition to an anti-apoptotic response, oncogenic STAT properties also involve regulation of cell-cycle control genes (Bowman et al., 2000; Bromberg, 2001, 2002). Several cell-cycle regulatory genes—including c-Myc, cyclin D1/D2, p21, and p27—have been linked to the proliferative actions of either Stat3 or Stat5 (Bowman et al., 2000; Bromberg, 2001, 2002). In transformed fibroblasts, abrogation of Stat3 signaling by the dominant-negative Stat3β protein inhibits v-Src-induced and platelet-derived growth-factor-mediated malignant transformation through repression of c-Myc expression (Bowman et al., 2001). Similarly, v-Src transformed fibroblasts exhibit up-regulation of p21, cyclin D1, and cyclin E in a Stat3-dependent manner (Sinibaldi et al., 2000). Importantly, overexpression of cyclin D1 in head and neck SCC has been linked to aberrant activation of Stat3 (Masuda et al., 2002). Expression of cyclin D2 (Martino et al., 2001) and c-Myc (Lord et al., 2000) has also been linked to Stat5-mediated cell-cycle control (Huang et al., 2002).

STAT Proteins in Specific Cancer Types

STAT constitutive activation has been detected in a plethora of primary tumors and tumor cell lines (Bowman et al., 2000; Bromberg, 2001, 2002), including breast cancer (Watson and Miller, 1995; Garcia et al., 1997; Sartor et al., 1997; Bromberg, 2000), head and neck cancer (Grandis et al., 2000a; Song and Grandis, 2000), non-small-cell lung cancer (Fernandes et al., 1999), prostate cancer (Ni et al., 2000, 2002; Dhir et al., 2002; Mora et al., 2002), melanoma (Florenes et al., 1999; Kirkwood et al., 1999), leukemia (Gouilleux-Gruart et al., 1996; Shuai et al., 1996a; Schuringa et al., 2000; Benekli et al., 2002, 2003), and EBV-transformed cells (Weber-Nordt et al., 1996). In breast cancer, constitutive activation of Stat1 and Stat3 has been identified in cell lines (Garcia et al., 1997, 2001) and primary tumor nuclear extracts (Garcia et al., 2001), but not in mammary epithelial cells (Garcia et al., 2001) or healthy breast tissues (Garcia et al., 2001). This activation has been linked to EGFR and Src overexpression and overactivation (Shuai et al., 1996a; Garcia et al., 2001). In non-small-cell lung cancer, constitutive Stat3 activation has been linked to an autocrine ErbB-1/TGF-alpha loop mediated by ErbB-2 (HER2/neu) receptor tyrosine kinase (Fernandes et al., 1999) and to tumor survival (Song et al., 2003). Activation of Stat3 in prostate cancer (Dhir et al., 2002; Ni et al., 2002) has been shown to potentiate malignant phenotypes and induce cell growth in prostate cancer cells (Ni et al., 2000).

STAT in Head and Neck Cancer

In head and neck SCC, there is compelling evidence that STAT constitutive activation is linked to cancer development and growth (Grandis et al., 2000a; Song and Grandis, 2000; Kijima et al., 2002).

(a ) In vitro studies

In squamous epithelial cells, there is evidence that overactivation of EGFR and its ligand (TGF-α) results in constitutive activation of both Stat1 and Stat3 (Song and Grandis, 2000). Accordingly, SIF-A (Stat3 homodimer) and SIF-B (Stat3/Stat1 heterodimer) are the predominant protein-DNA complexes formed in response to treatment of head and neck SCC cell lines with TGF-α (Grandis et al., 2000a). The resulting activation of Stat1 and Stat3 molecules, however, is associated with different signaling events: Antisense targeting of Stat3, but not Stat1, caused cell growth inhibition in vitro, revealing the critical role of Stat3 in head and neck SCC growth (Grandis et al., 1998). Moreover, transfection of dominant-negative constructs of Stat3 or antisense oligonucleotide targeting of the Stat3 translational start site resulted in significant growth inhibition in vitro (Grandis et al., 1998, 2000a). Interestingly, similar to Stat3, constitutive activation of Stat5b, but not Stat5a, also contributes to head and neck SCC growth (Leong et al., 2002).

(b) In vivo studies

Stat3 antisense gene therapy delivered to head and neck SCC xenograft models also leads to increased tumor apoptosis in vivo (Grandis et al., 2000a; Song and Grandis, 2000). In tumors and adjacent normal mucosa from head and neck SCC patients, Stat3 protein expression, tyrosine phosphorylation, and DNA-binding activity are constitutively amplified compared with levels in the normal mucosa from healthy individuals (Grandis et al., 2000a). Interestingly, tissue distribution of active Stat3 in normal mucosa differs significantly between healthy individuals and patients with head and neck cancer. While phosphorylated Stat3 is expressed in only basal epithelial layers of normal mucosa from healthy individuals, phosphorylated Stat3 is detected throughout the entire epithelium in normal mucosa from head and neck cancer patients, implicating a role for Stat3 activation in early carcinogenesis (Grandis et al., 2000a). In addition, Stat1 and Stat3 expression are inversely correlated with respect to tumor differentiation in head and neck SCC. While Stat1 is detected to a higher degree in well-differentiated tumors, Stat3 is up-regulated in poorly differentiated tumors (Arany et al., 2003).

Regulators of Oncogenic STAT Signaling in Head and Neck Cancer

Deregulation of various upstream pathways involving overexpression or overactivity of specific cytokines, growth factors and their respective receptors has been implicated in the constitutive activation of STAT proteins (Bowman et al., 2000; Bromberg, 2001, 2002).

(a) TGF-α and EGFR

In head and neck cancer, there is significant evidence suggesting that aberrant TGF-α/EGFR signaling leads to constitutive activation of Stat3 (Grandis et al., 1998, 2000a; Song and Grandis, 2000; Leong et al., 2002). EGFR has been shown to interact directly with Stat-1, -3, -5 in A431 cells (Olayioye et al., 1999). Moreover, targeting of TGF-α and EGFR with either TGF-α neutralizing antibodies or EGFR-specific tyrosine kinase inhibitors abrogates Stat3 activation in vitro (Grandis et al., 1998). Furthermore, repression of Stat3 constitutive activity, with an antisense gene therapy approach targeting the EGFR gene, has been documented in vivo (Grandis et al., 2000b; Song and Grandis, 2000). Similarly, Stat3 targeting indicates the critical role of Stat3 in TGF-α/EGFR-mediated growth in head and neck SCC cells (Grandis et al., 1998, 2000b).

(b) Src kinases

Recent evidence has unveiled the role of Src kinases in EGFR-mediated STAT activation (Xi et al., 2003). Activation of Src kinases correlates with constitutive activation of Stat3 and Stat5 in head and neck SCC. In addition, the direct interaction among EGFR, Src, and STAT proteins suggests that Src kinases contribute to oncogenic STAT signaling by facilitating STAT interactions with EGFR (Xi et al., 2003). The importance of Src kinases in constitutive STAT activation is not exclusive to head and neck cancer, in that recent reports have indicated an essential role of Src in transformed cells and breast cancer cells (Bowman et al., 2001; Garcia et al., 2001).

(c) Cytokines and JAKs

Despite the strong association between aberrant TGF-α/EGFR signaling and activation of STAT proteins in head and neck cancer, the requirement of EGFR stimulation for mediating constitutive Stat3 activation remains unclear. The ability of constitutively active Stat3 to serve as a growth-promoting signal independent of EGFR signaling supports the existence of other pathways contributing to oncogenic Stat3 signaling in head and neck cancer (Kijima et al., 2002). Consistent with this view, autocrine/paracrine stimulation of IL-6-mediated gp130/JAK signaling has been linked to EGFR-independent constitutive activation of Stat3 in head and neck SCC cells (Sriuranpong et al., 2003). Head and neck SCC cells express high levels of several pro-inflammatory and pro-angiogenic cytokines, including IL-1α, IL-6, IL-8, granulocyte-macrophage colony-stimulating factor, and VEGF in vitro and in vivo, which may also play a possible role in autocrine and/or paracrine signaling leading to oncogenic STAT activation (Chen et al., 1998, 1999; Ondrey et al., 1999). Importantly, targeting the gp130 receptor or inhibiting JAK kinases abrogates constitutive Stat3 activation and inhibits cell growth in head and neck cancer (Sriuranpong et al., 2003). However, the role of JAK kinases in oncogenic STAT signaling remains controversial, since JAK kinases were shown not to be required for STAT activation and cell growth in head and neck SCC (Xi et al., 2003). Such discrepancies may be the result of differences in levels of JAK kinase inhibition.

(d) Other regulators

In addition to signaling effects by cytokines and growth factors, ligand-independent activation of Stat3 linked to inhibition of cyclin-dependent kinase-2 has also been described, implying that cell-cycle inhibitors may contribute to the regulation of persistent Stat3 activation in head and neck SCC (Steinman et al., 2003). Finally, aberrations in negative regulators of STAT proteins may also result in constitutive STAT activation. In this regard, we recently observed that SOCS1 expression may be subject to down-regulation in oral SCC cells. Constitutive activation of STAT proteins in head and neck cancer most likely involves multiple upstream regulatory elements (depicted in Fig. 2), which may differ among different cell lines, resulting in variations in tumor phenotypes with respect to constitutive activation of STAT proteins.

Targeting of STATs

STAT latent transcription factors, along with several other transcription factors, are critical for mediating deregulation of oncogenic pathways in cancer (Darnell, 2002). The central role of STAT proteins (in particular, Stat3 and Stat5) in malignant transformation and cancer progression presents STATs as a potentially effective target for cancer therapy. In light of that, several strategies for the design of specific inhibitors that hamper STAT signaling have been developed (Turkson and Jove, 2000; Buettner et al., 2002).

(a) Inhibition of upstream kinases

Because tyrosine phosphorylation is critical for STAT activation, kinase inhibitors have become the focus of intensive investigation. Inhibition of specific upstream tyrosine kinases, including JAKs, Src, and EGFR, has been effective in inhibiting oncogenic STAT signaling and growth in myeloma, breast, and prostate cancer cells (Fry et al., 1994; Catlett-Falcone et al., 1999; Ni et al., 2000; Turkson and Jove, 2000; Garcia et al., 2001; Buettner et al., 2002; Mora et al., 2002). Recent findings indicate that a selective JAK inhibitor, AG490, represses Stat3 activation and cell growth in head and neck SCC (Sriuranpong et al., 2003). Similarly, our recent findings suggest that targeting of JAK kinase in oral SCC effectively reduces cell growth and inhibits Stat3 signaling. In addition to JAK inhibition, inhibitors of Src kinases also abrogate Stat3 and Stat5 activation and induce cell growth inhibition in head and neck SCC (Xi et al., 2003). Finally, targeting of EGFR has a potent growth-inhibitory effect in head and neck cancer (Ford and Grandis, 2003). Consistent with this view, EGFR antisense therapy inhibits constitutive Stat3 signaling and induces cell growth inhibition (Grandis et al., 2000b). Interestingly, delivery of phosphopeptides, spanning critical tyrosine residues within the EGFR C-terminal domain, to head and neck SCC cells disrupts EGFR-mediated Stat3 DNA-binding activity and cell growth (Shao et al., 2003). Similarly, EGFR binding peptide aptamers inhibit EGFR-mediated Stat3 activation in A431 and breast cancer cells (Buerger et al., 2003).

(b) Direct targeting

Direct targeting of STAT proteins provides the most specific therapeutic approach. Targeting of Stat3, through transfection of dominant-negative constructs or application of antisense oligonucleotide treatment, results in growth arrest and apoptosis in head and neck SCC (Grandis et al., 1998), breast cancer (Li and Shaw, 2002), prostate cancer (Mora et al., 2002), non-small-cell carcinoma (Song et al., 2003), and leukemia cells (Nakajima et al., 1996; Epling-Burnette et al., 2001). Furthermore, Stat3 antisense gene therapy in head and neck xenograft models leads to increased tumor apoptosis in vivo (Grandis et al., 2000a; Song and Grandis, 2000; Kijima et al., 2002). Importantly, a decoy oligonucleotide corresponding to the Stat3 response element induces cell growth inhibition in head and neck SCC without affecting the proliferation of normal oral keratinocytes (Leong et al., 2003), highlighting the dependence of head and neck SCC cells on persistent Stat3 activity. Finally, the development of short peptides that target Stat3 dimerization and inhibit DNA-binding suppresses gene regulation and transformation mediated by oncogenic Stat3 in vivo (Turkson et al., 2001).

(c) Alternative strategies

Alternatively, a combination of retinoic acid (RA) and IFN signaling suggests a potentially useful strategy in the treatment of cancer including head and neck cancer (Toma et al., 1994; Lingen et al., 1998; Gonçalves et al., 2001). Importantly, the up-regulation of Grim-19, shown to be critical for IFN/RA-induced cell death, leads to inhibition of Stat3 proteins in breast cancer cells (Zhang et al., 2003). In view of the fact that targeting of Stat3 severely hinders tumor survival in head and neck cancer, we have recently reported that a non-steroidal anti-inflammatory drug, sulindac, abrogates Stat3 signaling in oral cancer cells (Nikitakis et al., 2002a). Similarly, our findings suggest that cyclopentenone prostaglandins, especially 15-deoxy-Δ^12,14-PGJ₂, also target Stat3 signaling and induce apoptosis in oral cancer cells (Nikitakis et al., 2002b). Elucidation of mechanisms of STAT inhibition by novel inhibitors may help us identify other critical regulators of STAT proteins in head and neck cancer.

Conclusions and Perspectives

Compelling evidence has accumulated revealing a critical role of STAT proteins in carcinogenesis. Although constitutive activation of STAT proteins is not the only contributor to transformation and cancer progression, its crucial role has been clearly demonstrated in head and neck cancer. The mechanisms responsible for aberrant STAT activation in head and neck cancer remain uncertain and need further exploration. Moreover, knowledge of the cross-interaction of STAT molecules with other critical cellular proteins involved in growth regulation and survival may better serve to explain head and neck carcinogenesis. Importantly, the association between transcriptional co-activators and co-repressors with STAT molecules in regulating transcription of target genes will prove valuable in our understanding of the molecular basis of head and neck cancer.

Malignancies in which STAT activation is linked to tumor progression and survival are preferentially susceptible to STAT-targeted therapy. The observed dependence of such malignancies, but not normal cells, on aberrant STAT activation for growth and survival has wide implications for cancer therapeutics (Turkson and Jove, 2000; Buettner et al., 2002). That STAT activation is transient in normal physiological conditions may also help explain the enhanced sensitivity to cell death for cancer cells compared with normal cells. However, the consequence of targeting STAT molecules in therapeutics requires more vigorous investigation, especially given the importance of STAT proteins in normal cellular processes. More challenging is to elucidate selective inhibition of transcriptional regulation for STAT-regulated genes related to tumor growth and survival. Elucidation of mechanisms that further define the dependence of head and neck cancer cells on STAT proteins will also provide important insights for the development of more effective therapeutic strategies in head and neck cancer.

Figure 1.
Functional domains of STAT proteins. (A) Schematic diagram of a normal STAT molecule and (B) STAT variant harboring a truncation in the C-terminal transactivation domain. N-terminal (NH₂), Coiled Coil interactive domain (Coiled Coil), DNA-binding domain (DNABD), Linker domain (Linker), Src-Homology 2 domain (SH2), transactivation domain (TAD), phosphotyrosine (pY) and phosphoserine (pS) residues, and C-terminal (COOH) are depicted.

Figure 2.
Model of persistent Stat3 activation and possible modes of regulation in oral squamous carcinoma cells. Mechanisms involving aberrant Stat3 activation may include overexpression of receptors, kinases, and ligands, and de-regulation of positive or negative regulators. Overexpression and overactivation of EGFR and its ligand (TGF-α) result in aberrant activation of Stat3, possibly involving constitutive activity of Src kinases. Production of IL-6 and overactivation of gp130 and associated JAK kinases can lead to persistent Stat3 activity. Constitutive Stat3 signaling leads to activation of downstream genes, including anti-apoptotic protein Bcl-X_L and cell cycle regulator Cyclin D1. Possible negative regulators of activated Stat3 are also depicted, including Stat3 inducible suppressors of cytokine signaling (SOCS) and protein inhibitors of activated Stat3 (PIAS3). Activation of MAPKs (ERK, JNK, and p38) by MAPK kinases (MEK1/2, MEK4/7, MEK3/6) may also regulate constitutive Stat3 activity through cross-interactions.

Footnotes

Acknowledgements

This work was supported by grants from PHS/NIH DE13118, DE12606, and DE014935.

References

Ahmed ST, Ivashkiv LB (2000). Inhibition of IL-6 and IL-10 signaling and Stat activation by inflammatory and stress pathways. J Immunol 165:5227–5237.

Ahomadegbe JC, Barrois M, Fogel S, Le Bihan ML, Douc-Rasy S, Duvillard P, et al. (1995). High incidence of p53 alterations (mutation, deletion, overexpression) in head and neck primary tumors and metastases; absence of correlation with clinical outcome. Frequent protein overexpression in normal epithelium and in early non-invasive lesions. Oncogene 10:1217–1227.

Aoki N, Matsuda T (2000). A cytosolic protein-tyrosine phosphatase PTP1B specifically dephosphorylates and deactivates prolactin-activated STAT5a and STAT5b. J Biol Chem 275:39718–39726.

Arany I, Chen SH, Megyesi JK, Adler-Storthz K, Chen Z, Rajaraman S, et al. (2003). Differentiation-dependent expression of signal transducers and activators of transcription (STATs) might modify responses to growth factors in the cancers of the head and neck. Cancer Lett 199:83–89.

Benekli M, Xia Z, Donohue KA, Ford LA, Pixley LA, Baer MR, et al. (2002). Constitutive activity of signal transducer and activator of transcription 3 protein in acute myeloid leukemia blasts is associated with short disease-free survival. Blood 99:252–257.

Benekli M, Baer MR, Baumann H, Wetzler M (2003). Signal transducer and activator of transcription proteins in leukemias. Blood 101:2940–2954.

Bode JG, Nimmesgern A, Schmitz J, Schaper F, Schmitt M, Frisch W, et al. (1999). LPS and TNFalpha induce SOCS3 mRNA and inhibit IL-6-induced activation of STAT3 in macrophages. FEBS Lett 463:365–370.

Bode JG, Ludwig S, Freitas CA, Schaper F, Ruhl M, Melmed S, et al. (2001). The MKK6/p38 mitogen-activated protein kinase pathway is capable of inducing SOCS3 gene expression and inhibits IL-6-induced transcription. Biol Chem 382:1447–1453.

Bowman T, Garcia R, Turkson J, Jove R (2000). STATs in oncogenesis. Oncogene 19:2474–2488.

10.

Bowman T, Broome MA, Sinibaldi D, Wharton W, Pledger WJ, Sedivy JM, et al. (2001). Stat3-mediated Myc expression is required for Src transformation and PDGF-induced mitogenesis. Proc Natl Acad Sci USA 98:7319–7324.

11.

Bromberg J (2000). Signal transducers and activators of transcription as regulators of growth, apoptosis and breast development. Breast Cancer Res 2:86–90.

12.

Bromberg JF (2001). Activation of STAT proteins and growth control. Bioessays 23:161–169.

13.

Bromberg J (2002). Stat proteins and oncogenesis. J Clin Invest 109:1139–1142.

14.

Bromberg J, Darnell JE Jr (2000). The role of STATs in transcriptional control and their impact on cellular function. Oncogene 19:2468–2473.

15.

Bromberg JF, Horvath CM, Besser D, Lathem WW, Darnell JE Jr (1998). Stat3 activation is required for cellular transformation by v-src. Mol Cell Biol 18:2553–2558.

16.

Bromberg JF, Wrzeszczynska MH, Devgan G, Zhao Y, Pestell RG, Albanese C et al. (1999). Stat3 as an oncogene. Cell 98:295–303.

17.

Buerger C, Nagel-Wolfrum K, Kunz C, Wittig I, Butz K, Hoppe-Seyler F, et al. (2003). Sequence-specific peptide aptamers, interacting with the intracellular domain of the epidermal growth factor receptor, interfere with Stat3 activation and inhibit the growth of tumor cells. J Biol Chem 278:37610–37621.

18.

Buettner R, Mora LB, Jove R (2002). Activated STAT signaling in human tumors provides novel molecular targets for therapeutic intervention. Clin Cancer Res 8:945–954.

19.

Caldenhoven E, van Dijk TB, Solari R, Armstrong J, Raaijmakers JA, Lammers JW, et al. (1996). STAT3beta, a splice variant of transcription factor STAT3, is a dominant negative regulator of transcription. J Biol Chem 271:13221–13227.

20.

Callender T, el Naggar AK, Lee MS, Frankenthaler R, Luna MA, Batsakis JG (1994). PRAD-1 (CCND1)/cyclin D1 oncogene amplification in primary head and neck squamous cell carcinoma. Cancer 74:152–158.

21.

Cao X, Tay A, Guy GR, Tan YH (1996). Activation and association of Stat3 with Src in v-Src-transformed cell lines. Mol Cell Biol 16:1595–1603.

22.

Catlett-Falcone R, Landowski TH, Oshiro MM, Turkson J, Levitzki A, Savino R, et al. (1999). Constitutive activation of Stat3 signaling confers resistance to apoptosis in human U266 myeloma cells. Immunity 10:105–115.

23.

Chaturvedi P, Sharma S, Reddy EP (1997). Abrogation of interleukin-3 dependence of myeloid cells by the v-src oncogene requires SH2 and SH3 domains which specify activation of STATs. Mol Cell Biol 17:3295–3304.

24.

Chen CY, Tsay W, Tang JL, Shen HL, Lin SW, Huang SY, et al. (2003). SOCS1 methylation in patients with newly diagnosed acute myeloid leukemia. Genes Chromosomes Cancer 37:300–305.

25.

Chen Z, Colon I, Ortiz N, Callister M, Dong G, Pegram MY et al. (1998). Effects of interleukin-1alpha, interleukin-1 receptor antagonist, and neutralizing antibody on proinflammatory cytokine expression by human squamous cell carcinoma lines. Cancer Res 58:3668–3676.

26.

Chen Z, Malhotra PS, Thomas GR, Ondrey FG, Duffey DC, Smith CW et al. (1999). Expression of proinflammatory and proangiogenic cytokines in patients with head and neck cancer. Clin Cancer Res 5:1369–1379.

27.

Chin YE, Kitagawa M, Su WC, You ZH, Iwamoto Y, Fu XY (1996). Cell growth arrest and induction of cyclin-dependent kinase inhibitor p21 WAF1/CIP1 mediated by STAT1. Science 272:719–722.

28.

Chin YE, Kitagawa M, Kuida K, Flavell RA, Fu XY (1997). Activation of the STAT signaling pathway can cause expression of caspase 1 and apoptosis. Mol Cell Biol 17:5328–5337.

29.

Chung CD, Liao J, Liu B, Rao X, Jay P, Berta P, et al. (1997a). Specific inhibition of Stat3 signal transduction by PIAS3. Science 278:1803–1805.

30.

Chung J, Uchida E, Grammer TC, Blenis J (1997b). STAT3 serine phosphorylation by ERK-dependent and -independent pathways negatively modulates its tyrosine phosphorylation. Mol Cell Biol 17:6508–6516.

31.

Danial NN, Rothman P (2000). JAK-STAT signaling activated by Abl oncogenes. Oncogene 19:2523–2531.

32.

Danial NN, Losman JA, Lu T, Yip N, Krishnan K, Krolewski J, et al. (1998). Direct interaction of Jak1 and v-Abl is required for v-Abl-induced activation of STATs and proliferation. Mol Cell Biol 18:6795–6804.

33.

Darnell JE Jr (1997). STATs and gene regulation. Science 277:1630–1635.

34.

Darnell JE Jr (2002). Transcription factors as targets for cancer therapy. Nat Rev Cancer 2:740–749.

35.

Darnell JE Jr, Kerr IM, Stark GR (1994). Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins. Science 264:1415–1421.

36.

David M, Chen HE, Goelz S, Larner AC, Neel BG (1995). Differential regulation of the alpha/beta interferon-stimulated Jak/Stat pathway by the SH2 domain-containing tyrosine phosphatase SHPTP1. Mol Cell Biol 15:7050–7058.

37.

Decker T, Kovarik P (2000). Serine phosphorylation of STATs. Oncogene 19:2628–2637.

38.

Dhir R, Ni Z, Lou W, DeMiguel F, Grandis JR, Gao AC (2002). Stat3 activation in prostatic carcinomas. Prostate 51:241–246.

39.

Dumon S, Santos SC, Debierre-Grockiego F, Gouilleux-Gruart V, Cocault L, Boucheron C, et al. (1999). IL-3 dependent regulation of Bcl-xL gene expression by STAT5 in a bone marrow derived cell line. Oncogene 18:4191–4199.

40.

Ehret GB, Reichenbach P, Schindler U, Horvath CM, Fritz S, Nabholz M, et al. (2001). DNA binding specificity of different STAT proteins. Comparison of in vitro specificity with natural target sites. J Biol Chem 276:6675–6688.

41.

Epling-Burnette PK, Liu JH, Catlett-Falcone R, Turkson J, Oshiro M, Kothapalli R, et al. (2001). Inhibition of STAT3 signaling leads to apoptosis of leukemic large granular lymphocytes and decreased Mcl-1 expression. J Clin Invest 107:351–362.

42.

Fernandes A, Hamburger AW, Gerwin BI (1999). ErbB-2 kinase is required for constitutive stat 3 activation in malignant human lung epithelial cells. Int J Cancer 83:564–570.

43.

Florenes VA, Lu C, Bhattacharya N, Rak J, Sheehan C, Slingerland JM, et al. (1999). Interleukin-6 dependent induction of the cyclin dependent kinase inhibitor p21WAF1/CIP1 is lost during progression of human malignant melanoma. Oncogene 18:1023–1032.

44.

Ford AC, Grandis JR (2003). Targeting epidermal growth factor receptor in head and neck cancer. Head Neck 25:67–73.

45.

Fry DW, Kraker AJ, McMichael A, Ambroso LA, Nelson JM, Leopold WR, et al. (1994). A specific inhibitor of the epidermal growth factor receptor tyrosine kinase. Science 265:1093–1095.

46.

Fukushima N, Sato N, Sahin F, Su GH, Hruban RH, Goggins M (2003). Aberrant methylation of suppressor of cytokine signalling-1 (SOCS-1) gene in pancreatic ductal neoplasms. Br J Cancer 89:338–343.

47.

Galm O, Yoshikawa H, Esteller M, Osieka R, Herman JG (2003). SOCS-1, a negative regulator of cytokine signaling, is frequently silenced by methylation in multiple myeloma. Blood 101:2784–2788.

48.

Garcia R, Yu CL, Hudnall A, Catlett R, Nelson KL, Smithgall T, et al. (1997). Constitutive activation of Stat3 in fibroblasts transformed by diverse oncoproteins and in breast carcinoma cells. Cell Growth Differ 8:1267–1276.

49.

Garcia R, Bowman TL, Niu G, Yu H, Minton S, Muro-Cacho CA, et al. (2001). Constitutive activation of Stat3 by the Src and JAK tyrosine kinases participates in growth regulation of human breast carcinoma cells. Oncogene 20:2499–2513.

50.

Gimenez-Conti IB, Collet AM, Lanfranchi H, Itoiz ME, Luna M, Xu HJ, et al. (1996). p53, Rb, and cyclin D1 expression in human oral verrucous carcinomas. Cancer 78:17–23.

51.

Gonçalves A, Camerlo J, Bun H, Gravis G, Genre D, Bertucci F, et al. (2001). Phase II study of a combination of cisplatin, all-trans-retinoic acid and interferon-alpha in squamous cell carcinoma: clinical results and pharmacokinetics. Anticancer Res 21:1431–1437.

52.

Gouilleux-Gruart V, Gouilleux F, Desaint C, Claisse JF, Capiod JC, Delobel J, et al. (1996). STAT-related transcription factors are constitutively activated in peripheral blood cells from acute leukemia patients. Blood 87:1692–1697.

53.

Grandis JR, Tweardy DJ (1993). Elevated levels of transforming growth factor alpha and epidermal growth factor receptor messenger RNA are early markers of carcinogenesis in head and neck cancer. Cancer Res 53:3579–3584.

54.

Grandis JR, Melhem MF, Barnes EL, Tweardy DJ (1996). Quantitative immunohistochemical analysis of transforming growth factor-alpha and epidermal growth factor receptor in patients with squamous cell carcinoma of the head and neck. Cancer 78:1284–1292.

55.

Grandis JR, Drenning SD, Chakraborty A, Zhou MY, Zeng Q, Pitt AS, et al. (1998). Requirement of Stat3 but not Stat1 activation for epidermal growth factor receptor-mediated cell growth In vitro. J Clin Invest 102:1385–1392.

56.

Grandis JR, Drenning SD, Zeng Q, Watkins SC, Melhem MF, Endo S, et al. (2000a). Constitutive activation of Stat3 signaling abrogates apoptosis in squamous cell carcinogenesis in vivo. Proc Natl Acad Sci USA 97:4227–4232.

57.

Grandis JR, Zeng Q, Drenning SD (2000b). Epidermal growth factor receptor-mediated stat3 signaling blocks apoptosis in head and neck cancer. Laryngoscope 110:868–874.

58.

Gupta S, Yan H, Wong LH, Ralph S, Krolewski J, Schindler C (1996). The SH2 domains of Stat1 and Stat2 mediate multiple interactions in the transduction of IFN-alpha signals. EMBO J 15:1075–1084.

59.

Hoey T, Schindler U (1998). STAT structure and function in signaling. Curr Opin Genet Dev 8:582–587.

60.

Huang M, Dorsey JF, Epling-Burnette PK, Nimmanapalli R, Landowski TH, Mora LB, et al. (2002). Inhibition of Bcr-Abl kinase activity by PD180970 blocks constitutive activation of Stat5 and growth of CML cells. Oncogene 21:8804–8816.

61.

Jain N, Zhang T, Fong SL, Lim CP, Cao X (1998). Repression of Stat3 activity by activation of mitogen-activated protein kinase (MAPK). Oncogene 17:3157–3167.

62.

Jares P, Fernandez PL, Campo E, Nadal A, Bosch F, Aiza G, et al. (1994). PRAD-1/cyclin D1 gene amplification correlates with messenger RNA overexpression and tumor progression in human laryngeal carcinomas. Cancer Res 54:4813–4817.

63.

Kaplan DH, Shankaran V, Dighe AS, Stockert E, Aguet M, Old LJ, et al. (1998). Demonstration of an interferon gamma-dependent tumor surveillance system in immunocompetent mice. Proc Natl Acad Sci USA 95:7556–7561.

64.

Kijima T, Niwa H, Steinman RA, Drenning SD, Gooding WE, Wentzel AL, et al. (2002). STAT3 activation abrogates growth factor dependence and contributes to head and neck squamous cell carcinoma tumor growth in vivo. Cell Growth Differ 13:355–362.

65.

Kile BT, Alexander WS (2001). The suppressors of cytokine signalling (SOCS). Cell Mol Life Sci 58:1627–1635.

66.

Kile BT, Nicola NA, Alexander WS (2001). Negative regulators of cytokine signaling. Int J Hematol 73:292–298.

67.

Kim H, Baumann H (1997). The carboxyl-terminal region of STAT3 controls gene induction by the mouse haptoglobin promoter. J Biol Chem 272:14571–14579.

68.

Kirkwood JM, Farkas DL, Chakraborty A, Dyer KF, Tweardy DJ, Abernethy JL, et al. (1999). Systemic interferon-alpha (IFN-alpha) treatment leads to Stat3 inactivation in melanoma precursor lesions. Mol Med 5:11–20.

69.

Krebs DL, Hilton DJ (2001). SOCS proteins: negative regulators of cytokine signaling. Stem Cells 19:378–387.

70.

Leonard WJ, O’Shea JJ (1998). Jaks and STATs: biological implications. Annu Rev Immunol 16:293–322.

71.

Leong PL, Xi S, Drenning SD, Dyer KF, Wentzel AL, Lerner EC, et al. (2002). Differential function of STAT5 isoforms in head and neck cancer growth control. Oncogene 21:2846–2853.

72.

Leong PL, Andrews GA, Johnson DE, Dyer KF, Xi S, Mai JC, et al. (2003). Targeted inhibition of Stat3 with a decoy oligonucleotide abrogates head and neck cancer cell growth. Proc Natl Acad Sci USA 100:4138–4143.

73.

Levy DE, Darnell JE Jr (2002). Stats: transcriptional control and biological impact. Nat Rev Mol Cell Biol 3:651–662.

74.

Li L, Shaw PE (2002). Autocrine-mediated activation of STAT3 correlates with cell proliferation in breast carcinoma lines. J Biol Chem 277:17397–17405.

75.

Lim CP, Cao X (1999). Serine phosphorylation and negative regulation of Stat3 by JNK. J Biol Chem 274:31055–31061.

76.

Lingen MW, Polverini PJ, Bouck NP (1998). Retinoic acid and interferon alpha act synergistically as antiangiogenic and antitumor agents against human head and neck squamous cell carcinoma. Cancer Res 58:5551–5558.

77.

Lord JD, McIntosh BC, Greenberg PD, Nelson BH (2000). The IL-2 receptor promotes lymphocyte proliferation and induction of the c-myc, bcl-2, and bcl-x genes through the trans-activation domain of Stat5. J Immunol 164:2533–2541.

78.

Loro LL, Vintermyr OK, Johannessen AC (2003). Cell death regulation in oral squamous cell carcinoma: methodological considerations and clinical significance. J Oral Pathol Med 32:125–138.

79.

Lufei C, Ma J, Huang G, Zhang T, Novotny-Diermayr V, Ong CT, et al. (2003). GRIM-19, a death-regulatory gene product, suppresses Stat3 activity via functional interaction. EMBO J 22:1325–1335.

80.

Lund TC, Garcia R, Medveczky MM, Jove R, Medveczky PG (1997). Activation of STAT transcription factors by herpesvirus Saimiri Tip-484 requires p56lck. J Virol 71:6677–6682.

81.

Maestro R, Dolcetti R, Gasparotto D, Doglioni C, Pelucchi S, Barzan L, et al. (1992). High frequency of p53 gene alterations associated with protein overexpression in human squamous cell carcinoma of the larynx. Oncogene 7:1159–1166.

82.

Martino A, Holmes JH, Lord JD, Moon JJ, Nelson BH (2001). Stat5 and Sp1 regulate transcription of the cyclin D2 gene in response to IL-2. J Immunol 166:1723–1729.

83.

Masuda M, Suzui M, Yasumatu R, Nakashima T, Kuratomi Y, Azuma K, et al. (2002). Constitutive activation of signal transducers and activators of transcription 3 correlates with cyclin D1 overexpression and may provide a novel prognostic marker in head and neck squamous cell carcinoma. Cancer Res 62:3351–3355.

84.

Maxwell SA, Sacks PG, Gutterman JU, Gallick GE (1989). Epidermal growth factor receptor protein-tyrosine kinase activity in human cell lines established from squamous carcinomas of the head and neck. Cancer Res 49:1130–1137.

85.

Migone TS, Lin JX, Cereseto A, Mulloy JC, O’Shea JJ, Franchini G, et al. (1995). Constitutively activated Jak-STAT pathway in T cells transformed with HTLV-I. Science 269:79–81.

86.

Mora LB, Buettner R, Seigne J, Diaz J, Ahmad N, Garcia R, et al. (2002). Constitutive activation of Stat3 in human prostate tumors and cell lines: direct inhibition of Stat3 signaling induces apoptosis of prostate cancer cells. Cancer Res 62:6659–6666.

87.

Moriggl R, Berchtold S, Friedrich K, Standke GJ, Kammer W, Heim M, et al. (1997). Comparison of the transactivation domains of Stat5 and Stat6 in lymphoid cells and mammary epithelial cells. Mol Cell Biol 17:3663–3678.

88.

Nagai H, Naka T, Terada Y, Komazaki T, Yabe A, Jin E, et al. (2003). Hypermethylation associated with inactivation of the SOCS-1 gene, a JAK/STAT inhibitor, in human hepatoblastomas. J Hum Genet 48:65–69.

89.

Nagler RM, Kerner H, Laufer D, Ben Eliezer S, Minkov I, Ben Itzhak O (2002). Squamous cell carcinoma of the tongue: the prevalence and prognostic roles of p53, Bcl-2, c-erbB-2 and apoptotic rate as related to clinical and pathological characteristics in a retrospective study. Cancer Lett 186:137–150.

90.

Nakajima K, Yamanaka Y, Nakae K, Kojima H, Ichiba M, Kiuchi N, et al. (1996). A central role for Stat3 in IL-6-induced regulation of growth and differentiation in M1 leukemia cells. EMBO J 15:3651–3658.

91.

Ni Z, Lou W, Leman ES, Gao AC (2000). Inhibition of constitutively activated Stat3 signaling pathway suppresses growth of prostate cancer cells. Cancer Res 60:1225–1228.

92.

Ni Z, Lou W, Lee SO, Dhir R, DeMiguel F, Grandis JR, et al. (2002). Selective activation of members of the signal transducers and activators of transcription family in prostate carcinoma. J Urol 167:1859–1862.

93.

Nikitakis NG, Hamburger AW, Sauk JJ (2002a). The nonsteroidal anti-inflammatory drug sulindac causes down-regulation of signal transducer and activator of transcription 3 in human oral squamous cell carcinoma cells. Cancer Res 62:1004–1007.

94.

Nikitakis NG, Siavash H, Hebert C, Reynolds MA, Hamburger AW, Sauk JJ (2002b).15-PGJ2, but not thiazolidinediones, inhibits cell growth, induces apoptosis, and causes downregulation of Stat3 in human oral SCCa cells. Br J Cancer 87:1396–1403.

95.

Olayioye MA, Beuvink I, Horsch K, Daly JM, Hynes NE (1999). ErbB receptor-induced activation of stat transcription factors is mediated by Src tyrosine kinases. J Biol Chem 274:17209–17218.

96.

Ondrey FG, Dong G, Sunwoo J, Chen Z, Wolf JS, Crowl-Bancroft CV, et al. (1999). Constitutive activation of transcription factors NF-(kappa)B, AP-1, and NF-IL6 in human head and neck squamous cell carcinoma cell lines that express pro-inflammatory and pro-angiogenic cytokines. Mol Carcinogen 26:119–129.

97.

Ozanne B, Richards CS, Hendler F, Burns D, Gusterson B (1986). Over-expression of the EGF receptor is a hallmark of squamous cell carcinomas. J Pathol 149:9–14.

98.

Pircher TJ, Petersen H, Gustafsson JA, Haldosen LA (1999). Extracellular signal-regulated kinase (ERK) interacts with signal transducer and activator of transcription (STAT) 5a. Mol Endocrinol 13:555–565.

99.

Qu CK (2002). Role of the SHP-2 tyrosine phosphatase in cytokine-induced signaling and cellular response. Biochim Biophys Acta 1592:297–301.

100.

Quon H, Liu FF, Cummings BJ (2001). Potential molecular prognostic markers in head and neck squamous cell carcinomas. Head Neck 23:147–159.

101.

Sahni M, Ambrosetti DC, Mansukhani A, Gertner R, Levy D, Basilico C (1999). FGF signaling inhibits chondrocyte proliferation and regulates bone development through the STAT-1 pathway. Genes Dev 13:1361–1366.

102.

Sartor CI, Dziubinski ML, Yu CL, Jove R, Ethier SP (1997). Role of epidermal growth factor receptor and STAT-3 activation in autonomous proliferation of SUM-102PT human breast cancer cells. Cancer Res 57:978–987.

103.

Schindler C, Darnell JE Jr (1995). Transcriptional responses to polypeptide ligands: the JAK-STAT pathway. Annu Rev Biochem 64:621–651.

104.

Schreiner SJ, Schiavone AP, Smithgall TE (2002). Activation of STAT3 by the Src family kinase Hck requires a functional SH3 domain. J Biol Chem 277:45680–45687.

105.

Schuringa JJ, Wierenga AT, Kruijer W, Vellenga E (2000). Constitutive Stat3, Tyr705, and Ser727 phosphorylation in acute myeloid leukemia cells caused by the autocrine secretion of interleukin-6. Blood 95:3765–3770.

106.

Schwaller J, Frantsve J, Aster J, Williams IR, Tomasson MH, Ross TS, et al. (1998). Transformation of hematopoietic cell lines to growth-factor independence and induction of a fatal myelo- and lymphoproliferative disease in mice by retrovirally transduced TEL/JAK2 fusion genes. EMBO J 17:5321–5333.

107.

Seidel HM, Milocco LH, Lamb P, Darnell JE Jr, Stein RB, Rosen J (1995). Spacing of palindromic half sites as a determinant of selective STAT (signal transducers and activators of transcription) DNA binding and transcriptional activity. Proc Natl Acad Sci USA 92:3041–3045.

108.

Sengupta TK, Talbot ES, Scherle PA, Ivashkiv LB (1998). Rapid inhibition of interleukin-6 signaling and Stat3 activation mediated by mitogen-activated protein kinases. Proc Natl Acad Sci USA 95:11107–11112.

109.

Shao H, Cheng HY, Cook RG, Tweardy DJ (2003). Identification and characterization of signal transducer and activator of transcription 3 recruitment sites within the epidermal growth factor receptor. Cancer Res 63:3923–3930.

110.

Shuai K (2000). Modulation of STAT signaling by STAT-interacting proteins. Oncogene 19:2638–2644.

111.

Shuai K, Halpern J, ten Hoeve J, Rao X, Sawyers CL (1996a). Constitutive activation of STAT5 by the BCR-ABL oncogene in chronic myelogenous leukemia. Oncogene 13:247–254.

112.

Shuai K, Liao J, Song MM (1996b). Enhancement of antiproliferative activity of gamma interferon by the specific inhibition of tyrosine dephosphorylation of Stat1. Mol Cell Biol 16:4932–4941.

113.

Silva M, Benito A, Sanz C, Prosper F, Ekhterae D, Nunez G, et al. (1999). Erythropoietin can induce the expression of bcl-x(L) through Stat5 in erythropoietin-dependent progenitor cell lines. J Biol Chem 274:22165–22169.

114.

Sinibaldi D, Wharton W, Turkson J, Bowman T, Pledger WJ, Jove R (2000). Induction of p21WAF1/CIP1 and cyclin D1 expression by the Src oncoprotein in mouse fibroblasts: role of activated STAT3 signaling. Oncogene 19:5419–5427.

115.

Socolovsky M, Fallon AE, Wang S, Brugnara C, Lodish HF (1999). Fetal anemia and apoptosis of red cell progenitors in Stat5a-/-5b-/- mice: a direct role for Stat5 in Bcl-X(L) induction. Cell 98:181–191.

116.

Song JI, Grandis JR (2000). STAT signaling in head and neck cancer. Oncogene 19:2489–2495.

117.

Song L, Turkson J, Karras JG, Jove R, Haura EB (2003). Activation of Stat3 by receptor tyrosine kinases and cytokines regulates survival in human non-small cell carcinoma cells. Oncogene 22:4150–4165.

118.

Sriuranpong V, Park JI, Amornphimoltham P, Patel V, Nelkin BD, Gutkind JS (2003). Epidermal growth factor receptor-independent constitutive activation of STAT3 in head and neck squamous cell carcinoma is mediated by the autocrine/paracrine stimulation of the interleukin 6/gp130 cytokine system. Cancer Res 63:2948–2956.

119.

Starr R, Hilton DJ (1998). SOCS: suppressors of cytokine signalling. Int J Biochem Cell Biol 30:1081–1085.

120.

Starr R, Hilton DJ (1999). Negative regulation of the JAK/STAT pathway. Bioessays 21:47–52.

121.

Steinman RA, Wentzel A, Lu Y, Stehle C, Grandis JR (2003). Activation of Stat3 by cell confluence reveals negative regulation of Stat3 by cdk2. Oncogene 22:3608–3615.

122.

Strehlow I, Schindler C (1998). Amino-terminal signal transducer and activator of transcription (STAT) domains regulate nuclear translocation and STAT deactivation. J Biol Chem 273:28049–28056.

123.

Takeda K, Akira S (2000). STAT family of transcription factors in cytokine-mediated biological responses. Cytokine Growth Factor Rev 11:199–207.

124.

ten Hoeve J, de Jesus Ibarra-Sanchez M, Fu Y, Zhu W, Tremblay M, David M, et al. (2002). Identification of a nuclear Stat1 protein tyrosine phosphatase. Mol Cell Biol 22:5662–5668.

125.

Todd R, Donoff RB, Wong DT (1997). The molecular biology of oral carcinogenesis: toward a tumor progression model. J Oral Maxillofac Surg 55:613–623.

126.

Toma S, Monteghirfo S, Tasso P, Nicolo G, Spadini N, Palumbo R, et al. (1994). Antiproliferative and synergistic effect of interferon alpha-2a, retinoids and their association in established human cancer cell lines. Cancer Lett 82:209–216.

127.

Turkson J, Jove R (2000). STAT proteins: novel molecular targets for cancer drug discovery. Oncogene 19:6613–6626.

128.

Turkson J, Bowman T, Garcia R, Caldenhoven E, De Groot RP, Jove R (1998). Stat3 activation by Src induces specific gene regulation and is required for cell transformation. Mol Cell Biol 18:2545–2552.

129.

Turkson J, Bowman T, Adnane J, Zhang Y, Djeu JY, Sekharam M, et al. (1999). Requirement for Ras/Rac1-mediated p38 and c-Jun N-terminal kinase signaling in Stat3 transcriptional activity induced by the Src oncoprotein. Mol Cell Biol 19:7519–7528.

130.

Turkson J, Ryan D, Kim JS, Zhang Y, Chen Z, Haura E, et al. (2001). Phosphotyrosyl peptides block Stat3-mediated DNA binding activity, gene regulation, and cell transformation. J Biol Chem 276:45443–45455.

131.

Vinkemeier U, Cohen SL, Moarefi I, Chait BT, Kuriyan J, Darnell JE Jr (1996). DNA binding of in vitro activated Stat1 alpha, Stat1 beta and truncated Stat1: interaction between NH2-terminal domains stabilizes binding of two dimers to tandem DNA sites. EMBO J 15:5616–5626.

132.

Vokes EE, Weichselbaum RR, Lippman SM, Hong WK (1993). Head and neck cancer. N Engl J Med 328:184–194.

133.

Wang YZ, Wharton W, Garcia R, Kraker A, Jove R, Pledger WJ (2000). Activation of Stat3 preassembled with platelet-derived growth factor beta receptors requires Src kinase activity. Oncogene 19:2075–2085.

134.

Watson CJ, Miller WR (1995). Elevated levels of members of the STAT family of transcription factors in breast carcinoma nuclear extracts. Br J Cancer 71:840–844.

135.

Weber-Nordt RM, Egen C, Wehinger J, Ludwig W, Gouilleux-Gruart V, Mertelsmann R, et al. (1996). Constitutive activation of STAT proteins in primary lymphoid and myeloid leukemia cells and in Epstein-Barr virus (EBV)-related lymphoma cell lines. Blood 88:809–816.

136.

Wen X, Lin HH, Shih HM, Kung HJ, Ann DK (1999). Kinase activation of the non-receptor tyrosine kinase Etk/BMX alone is sufficient to transactivate STAT-mediated gene expression in salivary and lung epithelial cells. J Biol Chem 274:38204–38210.

137.

Wen Z, Darnell JE Jr (1997). Mapping of Stat3 serine phosphorylation to a single residue (727) and evidence that serine phosphorylation has no influence on DNA binding of Stat1 and Stat3. Nucleic Acids Res 25:2062–2067.

138.

Wen Z, Zhong Z, Darnell JE Jr (1995). Maximal activation of transcription by Stat1 and Stat3 requires both tyrosine and serine phosphorylation. Cell 82:241–250.

139.

Williams HK (2000). Molecular pathogenesis of oral squamous carcinoma. Mol Pathol 53:165–172.

140.

Xi S, Zhang Q, Dyer KF, Lerner EC, Smithgall TE, Gooding WE, et al. (2003). Src kinases mediate STAT growth pathways in squamous cell carcinoma of the head and neck. J Biol Chem 278:31574–31583.

141.

Yang E, Henriksen MA, Schaefer O, Zakharova N, Darnell JE Jr (2002). Dissociation time from DNA determines transcriptional function in a STAT1 linker mutant. J Biol Chem 277:13455–13462.

142.

Yoshikawa H, Matsubara K, Qian GS, Jackson P, Groopman JD, Manning JE, et al. (2001). SOCS-1, a negative regulator of the JAK/STAT pathway, is silenced by methylation in human hepatocellular carcinoma and shows growth-suppression activity. Nat Genet 28:29–35.

143.

You M, Yu DH, Feng GS (1999). Shp-2 tyrosine phosphatase functions as a negative regulator of the interferon-stimulated Jak/STAT pathway. Mol Cell Biol 19:2416–2424.

144.

Yu CL, Meyer DJ, Campbell GS, Larner AC, Carter-Su C, Schwartz J, et al. (1995). Enhanced DNA-binding activity of a Stat3-related protein in cells transformed by the Src oncoprotein. Science 269:81–83.

145.

Zhang J, Yang J, Roy SK, Tininini S, Hu J, Bromberg JF, et al. (2003). The cell death regulator GRIM-19 is an inhibitor of signal transducer and activator of transcription 3. Proc Natl Acad Sci USA 100:9342–9347.

146.

Zhang Q, Raghunath PN, Xue L, Majewski M, Carpentieri DF, Odum N, et al. (2002). Multilevel dysregulation of STAT3 activation in anaplastic lymphoma kinase-positive T/null-cell lymphoma. J Immunol 168:466–474.

147.

Zhang Y, Turkson J, Carter-Su C, Smithgall T, Levitzki A, Kraker A, et al. (2000). Activation of Stat3 in v-Src-transformed fibroblasts requires cooperation of Jak1 kinase activity. J Biol Chem 275:24935–24944.

148.

Zhong Z, Wen Z, Darnell JE Jr (1994). Stat3: a STAT family member activated by tyrosine phosphorylation in response to epidermal growth factor and interleukin-6. Science 264:95–98.

149.

Zong CS, Zeng L, Jiang Y, Sadowski HB, Wang LH (1998). Stat3 plays an important role in oncogenic Ros- and insulin-like growth factor I receptor-induced anchorage-independent growth. J Biol Chem 273:28065–28072.