Constitutive Activation of Stat5b Contributes to Carcinogenesis in Vivo

[CANCER RESEARCH 63, 6763–6771, October 15, 2003] Constitutive Activation of Stat5b Contributes to Carcinogenesis in Vivo

Sichuan Xi, Qing Zhang, William E. Gooding, Thomas E. Smithgall, and Jennifer Rubin Grandis1 Departments of Otolaryngology [S. X., J. R. G.], Pharmacology [Q. Z., J. R. G.], and Molecular Genetics and Biochemistry [T. E. S.], University of Pittsburgh School of Medicine, and Department of Biostatistics, University of Pittsburgh Cancer Institute [W. E. G.], Pittsburgh, Pennsylvania 15213

ABSTRACT EGF. Like Stat3, Stat5 has been shown to regulate proliferation and inhibition of apoptosis in cancer cells. A constitutively active Stat5 The development of more effective prevention and treatment strategies mutant induced properties characteristic of transformed cells (8). for solid tumors is limited by an incomplete understanding of the critical However, previous studies have generally not distinguished Stat5a growth pathways that are activated in carcinogenesis. Signal transducers and activators of transcription (STAT) proteins have been linked to and Stat5b in carcinogenesis, and there are no reports of Stat5 acti- transformation and tumor progression. Studies to date have not eluci- vation in epithelial tumor specimens. Stat5a and Stat5b are derived dated clear and distinct roles for Stat5 genes (Stat5a and Stat5b) in human from distinct, yet closely linked genes on chromosome 11 and exhibit epithelial cancers. We analyzed the role of Stat5a/b isoforms in squamous 93% identity at the amino acid level. Stat5a/b demonstrate similar cell carcinoma of the head and neck using expression and activation patterns of expression, and are activated by the same cytokines and studies in human tissues and in a xenograft model after selective targeting. growth factors. The association of Stat5 with transformation and In a xenograft model, blockade of Stat5b, but not Stat5a, using antisense tumor progression suggests that Stat5 may play a role in human oligonucleotides resulted in tumor growth inhibition and abrogation of carcinogenesis. Stat5 target genes in vivo. Blockade of the epidermal growth factor Early genetic changes that contribute to carcinogenesis can be receptor resulted in partial abrogation of Stat5 activation, thus linking detected in the histologically normal mucosa in SCCHN patients. This epidermal growth factor receptor to Stat5 in vivo. In tissues from 33 individuals with head and neck cancer, Stat5 activation levels were cor- “condemned mucosa” is subjected to field cancerization by carcino- related with progression to a malignant phenotype, where increased ex- genic agents (e.g., tobacco and alcohol), predisposing SCCHN pa- pression and phosphorylation of Stat5b were detected consistently in tients to the development of multiple primary tumors (9, 10). Over- tumors compared with their epithelial counterparts. Thus, constitutive expression of the EGFR and its autocrine ligand, TGF-␣, has been activation of Stat5b contributes to squamous cell tumorigenesis and may detected in transformed squamous epithelium, adjacent histologically serve as a therapeutic target. normal epithelium from SCCHN patients, as well as in premalignant dysplastic lesions, compared with levels in control mucosa from patients without cancer, suggesting that this pathway is activated early INTRODUCTION in SCCHN carcinogenesis (11–13). Dysregulation of TGF-␣/EGFR Cumulative evidence supports a role for activation of STATs2 in appears to be primarily a result of transcriptional activation and not oncogenesis as reflected by elevated STAT-DNA binding activity in gene amplication or prolongation of mRNA half-life (14). The detec- a variety of primary tumor specimens and cell lines (1, 2). Several tion of increased expression of EGFR and activation of Stat3 in this potential mechanisms of STAT activation have been implicated in “at risk” mucosa from head and neck cancer patients implicates human cancer cells including activation of upstream receptor tyrosine EGFR-mediated STAT activation as an early event in SCCHN carci- kinases, such as the EGFR, as well as nonreceptor kinases. Autocrine nogenesis (15). stimulation of EGFR results in receptor dimerization, phosphoryla- The vast majority of cancers that arise in the mucosa of the upper tion, and recruitment of STATs to tyrosine residues in the cytoplasmic aerodigestive tract (Ͼ90%) are squamous cell carcinomas. The de- domain. Interaction between STAT protein src-homology 2 domains velopment of SCCHN has been linked to carcinogen exposure, gen- and the activated EGFR leads to STAT phosphorylation, dimerization, erally tobacco and alcohol, as well as to genetic alterations in the and nuclear translocation. In the nucleus, STAT dimers bind to target affected tissues. Early genetic changes that contribute to SCCHN gene promoters and regulate gene expression (3–6). Seven STAT carcinogenesis can be detected in the histologically normal-appearing genes have been identified: Stat1, -2, -3, -4, -5a, -5b, and -6. Consti- mucosa in the area of “field cancerization.” Such a broad mucosal tutive activation of STATs 1, 3, and 5 has been demonstrated in a diathesis in these patients is supported by the high frequency of variety of diverse human tumor cell lines. In general, STATs 3 and 5 multiple primary tumors. Patients who survive the initial SCCHN are involved in the development and progression of cancers, whereas tumor will most likely succumb to a second primary tumor of the Stat1 demonstrates a tumor suppressor function (2). aerodigestive tract. Identification of the critical signaling pathways To date, Stat5 activation has been demonstrated primarily in he- will facilitate the design of novel prevention and treatment strategies. matopoietic malignancies where Stat5 activation is associated with The present study was undertaken to determine the role of Stat5 specific genetic abnormalities, such as the Bcr-Abl fusion protein in activation in SCCHN tumorigenesis and test the hypothesis that Stat5 chronic myelogenous leukemia (7). A variety of cytokines and growth isoforms could serve as therapeutic targets. factors have been reported to stimulate Stat5 activation, including MATERIALS AND METHODS Received 5/29/03; revised 7/17/03; accepted 7/23/03. The costs of publication of this article were defrayed in part by the payment of page Tissues and Cells. Samples of squamous cell carcinoma and normal mu- charges. This article must therefore be hereby marked advertisement in accordance with cosa distant from the tumor (generally, several centimeters away) were ob- 18 U.S.C. Section 1734 solely to indicate this fact. tained from 33 subjects undergoing primary surgical resection for head and 1 To whom requests for reprints should be addressed, at The Eye and Ear Institute, neck cancer at the University of Pittsburgh Medical Center from 1998 to 2001 Suite 500, 200 Lothrop Street, Pittsburgh, PA 15213. Phone: (412) 647-5280; Fax: (412) 647-0108; E-mail: [email protected]. (Table 1). Samples of normal oropharyngeal mucosa were obtained from ten 2 The abbreviations used are: STAT, signal transducers and activators of transcription; gender and age-matched (Ϯ5 years) control subjects without cancer undergo- EGFR, epidermal growth factor receptor; EGF, epidermal growth factor; SCCHN, squa- ing nononcological surgical procedures, such as uvulopalatopharyngoplasty for mous cell carcinoma of the head and neck; TGF, transforming growth factor; EMSA, obstructive sleep apnea syndrome. Tissues were collected under the auspices of electrophoretic mobility shift assay; TBST, Tris-buffered saline [10 mmol/liter Tris-HCL (pH 7.5) and 150 mmol/liter NaCl] with 0.5% Tween 20; PCNA, proliferating cell nuclear an Institutional Review Board-approved protocol with informed consent ob- antigen. tained from all of the subjects. For the xenograft studies, we used the cell lines 6763

Table 1 Clinicopathologic characteristics of 33 head and neck cancer patients control on that gel as described by us previously for Stat3 activation determi- evaluated prospectively for Stat5 activation/expression nations (15). Gender Immunoblotting and Immunoprecipitation. Whole cell extracts were Male 25 (76%) mixed with 2ϫ SDS sample buffer [125 mmol/liter Tris-HCL (pH 6.8), 4% Female 8 (24%) SDS, 20% glycerol, and 10% 2 mercaptoethanol] at 1:1 ratio and were heated Age Mean, 64 years; Median, 63 years; range, 4786 years for 5 min at 100°C. Proteins (50 ␮g/lane) were separated by 12.5% SDS- Tumor site PAGE and transferred onto a nitrocellulose membrane (MSI, Westboro, MA). Oral cavity 16 (49%) Prestained molecular weight markers (Life Technologies, Inc., Gaithersburg, Oropharynx 5 (15%) Larynx 12 (36%) MD) were included in each gel. Membranes were blocked for 30 min in TBST T stage and 5% BSA. After blocking, membranes were incubated with a primary 1–2 11 (33%) antibody, rabbit antihuman Stat5a or Stat5b polyclonal antibodies (Transduc- 3–4 18 (55%) tion Labs, Lexington, KY), or rabbit antihuman Cyclin D1 polyclonal antibody Recurrence 4 (12%) N stage or mouse antihuman Bcl-xL monoclonal antibody (Santa Cruz Biotechnology), 0–1 27 (82%) in TBST and 1% BSA. After washing the membranes three times with TBST 2 6 (18%) (5 min each), they were incubated with horseradish peroxidase-conjugated Tumor differentiation secondary antibody in TBST and 1% BSA for 30 min. Subsequently, mem- Well 8 (24%) Well-moderate 3 (9%) branes were washed three times with TBST and developed using the enhanced Moderate 17 (52%) chemiluminescence detection system (Amersham Life Sciences Inc., Arlington Moderate-poor 1 (3%) Heights, IL). Stat5 activation was determined by immunoblotting with a Poor 4 (12%) phosphospecific antibody that cannot distinguish Stat5a from Stat5b (Upstate Biotechnology). Stat5a or Stat5b phosphorylation was determined by immunoprecipitation with antiphosphotyrosine monoclonal antibody (PY20; Trans- OSC-19 (16) or 1483 (17), which are well-characterized SCCHN cell lines that duction Laboratories, Inc.), followed by immunoblotting with anti- Stat5a or can form tumors in athymic nude mice. In culture, they were maintained in Stat5b (Upstate Biotechnology, Inc., Lake Placid, NY). Interaction of Stat5a or supplemented DMEM as described previously (16). Sf-9 cells (Invitrogen) and Stat5b with EGFR was determined by immunoprecipitation with anti-EGFR Sf-9 cells transfected with Stat5a or Stat5b were cultured in Grace’s complete (Santa Cruz Biotechnology), or Stat5a or Stat5b antibodies, followed by insect cell medium containing 10% fetal bovine serum and 50 ␮g/ml gentam- immunoblotting with anti-Stat5a or Stat5b antisera. icin as described previously (18). Animal Studies. Female athymic nude mice ␯/␯ (4– 6 weeks old; EMSA and Quantitation. Nuclear extracts were prepared and EMSAs 20 Ϯ 2 g; Harlan-Sprague Dawley) were implanted with 1 ϫ 106 cells were performed on 4% native polyacrylamide gels as described (19, 20). (OSC-19 or 1483) into the right and left flank with a 26-gauge needle/1-ml Complementary duplex oligonucleotides were synthesized based on the pub- tuberculin syringe. Ten days later, when the tumor nodules were established sequences of STAT DNA-binding elements with the addition of GGGG lished (ϳ2 ϫ 2 mm in diameter), the tumor implanted on one flank was at the 5Ј termini to allow radiolabeling as described previously (␤-casein treated with Stat5b antisense oligonucleotides, and the tumor on the con- promoter: 5Ј-AGATTTCTAGGAATTCAAATC-3Ј; 21). For supershift exper- tralateral flank was treated with Stat5b sense oligonucleotides, Stat5a iments, extracts were preincubated with Stat5a polyclonal antibody (L-20; antisense oligonucleotides, or no treatment. Antisense oligonucleotides Santa Cruz Biotechnology, Santa Cruz, CA) or Stat5b polyclonal antibody were injected on days 11–15, 18–22, and 25–29. There were 5–7 mice in (N-20; Santa Cruz Biotechnology). Stat5 activation levels were determined by each treatment group. For the EGFR antisense gene therapy studies, the running a positive control SCCHN cell line on each gel. Gel shift bands were tumor on one flank was treated with intratumoral injection of EGFR quantitated by densitometry and the Molecular Analyst software (AlphaDigi- antisense DNA (25 ␮g three times a week). The tumor on the contralateral doc 1000; Alpho-Innotech) with normalization of each valve to the positive flank was treated with the same dose of EGFR sense DNA as described

Fig. 1. Constitutive Stat5 activation in normal mucosa and tumors from SCCHN patients. A, nuclear extracts (20 ␮g) were prepared from representative tumors (T1–3) and normal mucosa samples (N1–3) from SCCHN patients, as well as normal mucosa biopsies from patients without cancer (C1–3). EMSA was performed with radiolabeled ␤-casein duplex oligonucleotide (5Ј-AGATTTCTAGGAATTCAAATC-3Ј). Immunoblotting was performed with phosphospecific Stat5 antisera or Stat5 antisera followed by actin as control for loading. B, cumulative results of Stat5 activation levels in 10 control and 33 pairs of SCCHN tissues showing increased constitutive Stat5 activation in tumors compared with normal mucosa from SCCHN patients (P ϭ 0.000135). In addition, Stat5 activation is elevated in tumors compared with normal mucosa from control patients (P ϭ 0.000523) and in normal mucosa from SCCHN patients compared with normal mucosa from patients without cancer (P ϭ 0.017). C, EMSA was performed using extracts of a representative SCCHN tumor and corresponding normal mucosa compared with control mucosa from a patient without cancer. Extracts were preincubated with antibody to Stat5a or Stat5b or no antibody as indicated. The supershift complexes are indicated on the right.

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Fig. 2. Stat5b expression and activation in SCCHN and control tissues. A, immunoblotting for total Stat5b protein using extracts (50 ␮g) from representative SCCHN tumors (T1–3), corresponding normal mucosa from SCCHN patients (N1–3), and control mucosa from patients without cancer (C1–3). B, cumulative results of Stat5b expression in tissues from 33 SCCHN patients and 10 control patients without cancer. Stat5b expression is elevated in the tumors compared with levels in corresponding normal mucosa from SCCHN patients (P ϭ 0.000446). In addition, Stat5b expression is elevated in tumors compared with levels in normal mucosa from patients without cancer (P ϭ 0.000357), and in normal mucosa from SCCHN patients compared with levels in normal mucosa from noncancer patients (P ϭ 0.013). C, representative coimmunoprecipitation showing phosphorylated Stat5b expression levels in representative tumors (T1–3), normal mucosa from SCCHN patients (N1–3), and control mucosa from patients without cancer (C1–3). D, cumulative results of expression of phosphorylated Stat5b in these tissues. Extracts were precipitated with antiphosphotyrosine PY20 and im- munoblotted with anti-Stat5b antisera

previously. Phosphorothioated 21-mer oligodeoxynucleotides were synthe- Academy of Sciences (1996)], and the Association for Assessment and sized on an Applied Biosystem 394 synthesizer by ␤-cyanothylphysphora- Accreditation of Laboratory Animal Care International. midite chemistry to minimize degradation of the oligonucleotides by en- Statistics. Comparisons of Stat5 expression, phosphorylation, and activa- dogenous nucleases. The antisense oligonucleotides were directed against tion levels among tissue types were conducted using a two-sample t test. the translation start site (AUG codon) and surrounding nucleotides of the Tumor growth experiments compared the tumor volume in one flank to the human Stat5a or Stat5b genes. The Stat5a antisense oligonucleotide se- paired tumor volume in the opposite flank with the signed rank test. quence was 5Ј TGA ACG GCC ATG GCG GGC TGG 3Ј and the corre- Ј sponding sense oligonucleotide sequence was 5 CCA GCC CGC CAT RESULTS GGC GCT TCA 3Ј. The Stat5b antisense oligonucleotide sequence was 5Ј CCA CAC AGC CAT GTT TAC CCG 3Ј and the corresponding sense Increased Stat5 Activation in SCCHN Carcinogenesis. We re- oligonucleotide sequence was 5Ј CGG GTA AAC ATG GCT GTG TGG 3Ј. ported previously that Stat3 is activated in both tumor and corre- ␮ Intratumoral injection of antisense oligonucleotides (7.92 nM or 50 g) in sponding normal mucosa from SCCHN patients compared with a volume of 50 ␮l was delivered five times per week for a total of 15 normal mucosa from SCCHN patients compared with levels in treatments. Tumor volumes were measured in conjunction with each treatment and calculated as length ϫ (width)2/2. Mice were sacrificed after the control mucosa from individuals without cancer (15). To determine last treatment, and tumors were harvested for analysis. Experiments were Stat5 activation in SCCHN and control tissues, nuclear extracts repeated twice to ensure reproducibility. Animal care was in strict compli- were prepared from 33 SCCHN and control tissues and analyzed ance with institutional guidelines established by the University of Pitts- for Stat5 activation by EMSA using the ␤-casein response element burgh, the Guide for the Care and Use of Laboratory Animals [National (see Table 1 for patient characteristics). Repeat EMSA analysis 6765

Fig. 3. Stat5a expression and activation in SCCHN and control tissues. A, immunoblotting for total Stat5a protein using extracts (50 ␮g) from representative SCCHN tumors (T1–3), corresponding normal mucosa from SCCHN patients (N1–3), and control mucosa from patients without cancer (C1–3). B, cumulative results of Stat5a expression in tissues from 33 SCCHN patients and 10 control patients without cancer. Stat5a expression is similar in the tumors compared with levels in corresponding normal mucosa from SCCHN patients (P ϭ 0.6215). In addition, Stat5a expression is similar in tumors compared with levels in normal mucosa from patients without cancer (P ϭ 0.7457), and in normal mucosa from SCCHN patients compared with levels in normal mucosa from noncancer patients (P ϭ 0.4032). C, representative coimmunoprecipitation showing phosphorylated Stat5a expression levels in representative tumors (T1–3), normal mucosa from SCCHN patients (N1–3), and control mucosa from patients without cancer (C1–3). D, cumulative results of expression of phosphorylated Stat5a in these samples. Extracts were precipitated with antiphosphotyrosine PY20 and im- munoblotted with anti-Stat5a antisera.

was performed on a subset of samples, and the variability was Stat5 isoform. These experiments demonstrated a 2.4-fold increase found to be Ͻ10% (data not shown). DNA binding of Stat5 was in Stat5b expression in both SCCHN tumors compared with control found to be 3-fold higher in tumors and 1.8-fold higher in normal mucosa from individuals without cancer (P ϭ 0.000357), as well mucosa from cancer subjects (harvested several centimeters away as a 1.6-fold increase of Stat5b expression levels in normal mucosa from the tumor) compared with normal mucosa from noncancer from SCCHN patients compared with control mucosa from unaf- subjects (P ϭ 0.000523 and P ϭ 0.017, respectively). In addition, fected individuals (P ϭ 0.013). In addition, Stat5b expression was the level of Stat5 activation in the tumors was 1.7-fold higher in the elevated 1.5-fold in SCCHN tumors compared with levels in nor- SCCHN tumors compared with levels in the corresponding normal mal mucosa from the same subjects (P ϭ 0.000446). Because mucosa from the same SCCHN patients (P ϭ 0.000135). Because phosphospecific antisera that distinguish Stat5a from Stat5b are it is not possible to distinguish Stat5a from Stat5b activation on gel not available, to determine Stat5a/b phosphorylation levels, coim- shift assay, we performed supershift analysis to confirm that the munoprecipitation was performed on a subset of samples where constitutive Stat5 complexes contained both Stat5a and Stat5b there was sufficient clinical material for analysis. As shown in Fig. isoforms. Immunoblotting with a phosphospecific Stat5 antibody 2, Stat5b phosphorylation was only detected in SCCHN tumors and was performed to confirm the increased Stat5 activation levels in was not found in the corresponding normal mucosa from SCCHN SCCHN tissues detected by gel shift (Fig. 1). patients or control mucosa from subjects without cancer. In con- Elevated Expression and Phosphorylation of Stat5b, but not trast, expression levels of Stat5a and phosphorylated Stat5a in Stat5a, in SCCHN Carcinogenesis. To determine the relative con- SCCHN tumors was comparable with the levels detected in normal tribution of Stat5a and Stat5b to SCCHN carcinogenesis, we per- mucosa from SCCHN subjects, as well as in normal mucosa from formed immunoblotting studies using antisera specific for each patients without cancer (Fig. 3). 6766

Fig. 4. TGF-␣/EGFR are linked to constitutive Stat5 activation in vivo. A, EMSA was performed with radiolabeled ␤-casein duplex oligonucleotide using extracts from representative SCCHN xenografts (1483 cell line) treated with an EGFR antisense gene therapy construct (Antisense) compared with tumors treated with a control EGFR sense construct (Sense). The position of the Stat5 band is indicated on the right. A representative extract was preincubated with antibody to Stat5a or Stat5b or no antibody as indicated. The supershift complexes are indicated on the right. Immunoblot- ting with phosphospecific Stat5 antisera was performed using the same extracts. B, interaction of EGFR with Stat5a/b. SCCHN cells (1483 cell line) with or without TGF-␣ stimulation (10 ng/ml) were lysed and the proteins were immunoprecipitated with anti-EGFR antibody. The immunoprecipitated proteins were subjected to SDS-PAGE analysis using a gradient gel and analyzed by immunoblotting with anti-Stat5a or anti-Stat5b antisera. Sf-9 insect cells engineered to express Stat5a or Stat5b were used as controls to verify that the interaction in the SCCHN cells was not because of antibody cross- reactivity.

Stat5 Activation Is Linked to EGFR Stimulation in Vivo. (23). To determine the link between EGFR and Stat5 in vivo, xe- SCCHN cell lines and tissues are characterized by overexpression of nografts were established in nude mice using a well-characterized EGFR where EGFR levels correlate with survival (11, 22). Stat5b SCCHN cell line, as described previously (24). In this model, the activation by EGF was reported to require overexpression of EGFR EGFR antisense (or control sense) construct was generated by cloning 6767

a relatively short sequence (40 bp) corresponding with the ATG start site of the human EGFR gene, into a U6 expression construct. Ten days later, when the tumor nodules were established (ϳ2 ϫ 2mmin diameter), the tumor implanted on one flank was treated with EGFR antisense DNA and the tumor on the contralateral flank with EGFR sense DNA. Tumors were harvested after the last treatment, and Stat5 activation levels were determined by EMSA. As shown in Fig. 4A, tumors treated with EGFR antisense DNA demonstrated decreased Stat5 activation levels (as determined by EMSA or immunoblotting) compared with tumors treated with EGFR sense DNA, thus linking Stat5 activation, in part, to EGFR stimulation in vivo. EGFR has been reported to associate with STATs in cultured epithelial cells (25, 26). In such a model, direct interaction between STAT protein SH2 domains and the activated receptor leads to STAT phosphorylation followed by dimerization of the STAT protein and translocation to the nucleus. In the nucleus, STATs bind to DNA- response elements in promoters, thus regulating growth factor/cytokine-dependent gene expression. To determine whether EGFR formed a complex with Stat5a/b in SCCHN cells, we performed coimmunoprecipitation assays and found that EGFR can interact with either Stat5a or Stat5b (Fig. 4B). Therefore, EGFR-mediated Stat5 activation may occur, in part, via direct interaction between the tyrosine kinase domain of the receptor and SH2 domain of Stat5a/b. Targeting Stat5b, but not Stat5a, Inhibits Tumor Growth and Target Gene Expression in Vivo. We reported recently that blocking Stat5b, but not Stat5a, using either dominant-negative or antisense strategies inhibited the growth of SCCHN cells in vitro (27). The detection of elevated Stat5b phosphorylation exclusively in SCCHN tumor specimens suggests that Stat5b may play a critical role in the progression of head and neck cancer. To determine the consequences of down-modulating Stat5b in vivo, we treated established SCCHN xenografts with antisense oligonucleotides targeting the translation start site of Stat5b as described previously (27). Controls included Stat5b oligonucleotides in the sense ori- entation, antisense oligonucleotides targeting the translation start site of Stat5a, or no treatment. Three groups of mice were ran- domly selected to receive 25 ␮g of Stat5b antisense oligonucleotides injected into each established tumor five times a week. Injections were administered on days 11–15, 18–22, and 25–29 after tumor implantation. As shown in Fig. 5, tumor volumes were consistently lower in the tumors that received Stat5b antisense oligonucleotides compared with tumors treated with corresponding Stat5b sense oligonucleotides or Stat5a antisense oligonucleotides. Similar results were obtained using two other SCCHN xenograft models (data not shown). After the last treatment, mice were sacrificed and tumors harvested for analysis. Western blotting of tumor lysates revealed decreased expression of Stat5a or Stat5b in the respective antisense-treated tumors demonstrating down- modulation of the specific Stat5 isoform being targeted (Fig. 6A). The specificity of the treatments was additionally demonstrated by immunoblotting, which showed that Stat5b antisense treatment did not decrease Stat5a expression, nor did Stat5a antisense treatment block expression of Stat5b protein. STATs, including Stat5, have been shown to exert their effects by modulation of gene expression. Stat5 response elements have been identified in several genes,

istration of Stat5b antisense oligonucleotides (f; A–C) Stat5b sense oligonucleotides (Œ; A), Stat5a antisense oligonucleotides (F; B), or no oligonucleotides (ࡗ; C), 10 days after tumor cell implantation. Statistical analysis (exact two-tailed signed rank test) was Fig. 5. Antisense targeting of Stat5b inhibits SCCHN growth in vivo. Three groups of performed comparing tumor volumes in the Stat5b antisense-treated flank with the tumor mice were inoculated with a well-characterized SCCHN xenograft cell line (OSC-19; Ref. volume of the opposite flank. Significant differences were observed at all time points on 16). After the formation of established tumor nodules, mice received intratumoral admin days 15–30 (P Ͻ 0.0001). 6768

Fig. 6. Expression of Stat5a/b and target genes in treated tumors. A, Stat5a or Stat5b immunoblotting demonstrating down-modulation of Stat5a or Stat5b protein expression levels in the SCCHN xenografts after treatment with the respective antisense (versus sense) oligonucleotides. In contrast, treatment with Stat5a antisense oligonucleotides did not abrogate Stat5b expression, and treatment with Stat5b antisense oligonucleotides did not decrease Stat5a expression. B, Bcl-xL and Cyclin D1 immunoblotting of SCCHN xenografts treated with Stat5b antisense (or sense) oligonucleotides demonstrating decreased target gene expression. ␤-Actin expression was performed as a control for loading. In contrast, treatment with Stat5a antisense oligonucleotides did not decrease protein expression levels of Cyclin D1 or Bcl-xL. The exper- iment was repeated with similar results obtained.

including bcl-xL and cyclin D1 (28). Analysis of the tumor lysates growth in inhibition after Stat5b targeting, xenografts were stained for demonstrated that expression of both Bcl-xL and Cyclin D1 were DNA fragmentation and proliferation indicies. Targeting Stat5b, but decreased in Stat5b antisense-treated tumors compared with levels in not Stat5a, resulted in decreased tumor cell proliferation as deter- tumors treated with Stat5b sense DNA. In contrast, treatment with mined by staining and blotting for PCNA (Fig. 7; data not shown). In Stat5a antisense oligonucleotides did not abrogate Cyclin D1 or contrast, there was no evidence of apoptosis modulation after target-

BcL-xL expression (Fig. 6B). To investigate the mechanism of tumor ing of either Stat5 isoform (data not shown). 6769

target gene expression in vivo, whereas targeting Stat5a had no effect on tumor growth or gene expression. In hematopoietic malignancies, Stat5 activation has been linked to transformation mediated by fusion genes including NPM/ALK, TEL/ JAK2, and TEL/ABL (31, 32). However, the role of Stat5 activation in epithelial tumor formation and progression has been largely unex- plored. Nonspecific epithelial cell defects in the prostates of Stat5a- deficient mice have been described (33). Using a murine breast cancer model, it was reported recently that loss of Stat5a by genetic manip- ulation delayed mammary cancer progression (34). Stat5 has been implicated in the estrogen-regulated control of T47D cells (35). Un- like Stat3, which has been shown to transform mammalian fibroblasts when constitutively activated (36), Stat5 activation alone has not been reported to be an essential event in malignant transformation. Src kinase has been shown to phosphorylate and activate either Stat5a or Stat5b, although only Stat5b was translocated to the nucleus after phosphorylation (37). Using NIH-3T3 cells, Stat5b also accelerated v-Src-induced tumorigenicity, cell motility, and cell growth (38). These studies suggest that Stat5b activation may potentiate the malignant phenotype, which is primarily induced by other transforming events. Fig. 7. Antisense targeting of Stat5b inhibits SCCHN proliferation in vivo. SCCHN The mechanism of increased Stat5 activation in human cancers has xenografts (OSC-19) were treated with Stat5b antisense (or sense) oligonucleotides or Stat5a antisense (or sense) oligonucleotides followed by immunoblotting. A, PCNA not been completely defined, and may depend on the specific cell type immunostaining or immunoblotting demonstrating decreased proliferation in Stat5b an- and activating stimuli in the tumor microenvironment. Src has been tisense (or sense) treated tumors. B, analysis of Stat5a antisense (or sense) treated tumors shown to contribute to Stat5 activation in a vulvar squamous cell demonstrated no modulation of PCNA expression. carcinoma cell line (25). We report here EGFR-Stat5 interactions in SCCHN cells, and we have detected Src-Stat5 complexes (either Stat5a or Stat5b) in SCCHN cell lines (data not shown). Activation of DISCUSSION Stat5b by EGF has been reported to require EGFR overexpression (23). Our previous studies provide compelling evidence that increased Increased expression of EGFR is characteristic of most epithelial expression of EGFR in SCCHN tumors is associated with decreased malignancies, including SCCHN, where autocrine or paracrine acti- survival, where antitumor effects are demonstrated with EGFR tar- vation of EGFR is thought to contribute to tumor progression (re- geting strategies in preclinical models (22, 24). However, in the viewed in Ref. 39). In the present study, we have demonstrated that clinical setting, only modest antitumor effects have generally been targeting EGFR in a SCCHN xenograft model, using an antisense observed, and only when EGFR targeting agents are combined with gene therapy approach, abrogated constitutive Stat5 activation, thus standard cytotoxic approaches such as chemotherapy or radiation. We linking EGFR to Stat5 in vivo. Additional investigation showed that reported recently that constitutive STAT activation is accompanied by increased expression and phosphorylation of Stat5b was associated resistance to stimulation or blockade of the EGFR axis in SCCHN with SCCHN tumorigenesis where targeting Stat5b decreased tumor cells (22). Therefore, targeting a downstream signaling pathway that progression in vivo. The antitumor effects of Stat5b blockade can be regulates growth may prove efficacious, alone, or in combination with explained, in part, by down-regulation of target genes that control EGFR blockade. growth and apoptosis. Specifically, antisense targeting of Stat5b re- We demonstrate here that expression and activation of Stat5b, sulted in decreased expression of Cyclin D1 and Bcl-x . Previous downstream of EGFR, contribute to SCCHN carcinogenesis in vivo L studies have demonstrated that the antiapoptotic gene encoding Bcl-x and may serve as a therapeutic target in cancers that demonstrate L is a downstream target of both Stat3 and Stat5 (40, 41). Similarly, constitutive Stat5 activation. Many reports have not distinguished Cyclin D1/D2 are critical cell cycle control genes that have been activation of Stat5a from Stat5b, and such a failure to determine the reported to be a target of Stat5 (42). The present study suggests that role of each Stat5 gene may obscure key differences. Studies of blockade of Stat5b in vivo leads to tumor growth inhibition primarily knockout mice have contributed to our understanding of the physio- as a result of decreased proliferation, and not increased apoptosis. logical roles of these closely linked STAT proteins. Stat5a-deficient Because both STATs 3 and 5 are activated in SCCHN, and appear to mice exhibited defective mammary gland development and lactogen- contribute to growth regulation, additional studies to elucidate mech- esis (29). In contrast, studies of Stat5b-deficient mice indicated that anisms of STAT activation are required. These cumulative results Stat5b mediates an essential function in growth hormone actions (30). implicate decreased Stat5 activation as a potential antitumor mecha- Although previous reports have suggested potentially distinct roles for nism of EGFR blocking approaches, and suggest that specific abro- Stat5 isoforms, this study provides clear evidence that Stat5b, but not gation of Stat5b may play a role in molecular targeting strategies for Stat5a, contributes to tumor progression in a human epithelial cancer. cancer therapy. Whereas levels of Stat5a expression and phosphorylation were similar in tumor tissue and paired normal mucosa from head and neck cancer subjects, as well as control mucosa from subjects without cancer, REFERENCES Stat5b expression and phosphorylation was elevated consistently in SCCHN tumors but not in their epithelial counterparts. Additional 1. Ihle, J. N., and Kerr, I. M. Jaks and Stats in signaling by the cytokine receptor superfamily. 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