This article has Open Peer Review reports available.
Characterization of STAT3 activation and expression in canine and human osteosarcoma
© Fossey et al; licensee BioMed Central Ltd. 2009
Received: 17 October 2008
Accepted: 10 March 2009
Published: 10 March 2009
Dysregulation of signal transducer and activator of transcription 3 (STAT3) has been implicated as a key participant in tumor cell survival, proliferation, and metastasis and is often correlated with a more malignant tumor phenotype. STAT3 phosphorylation has been demonstrated in a subset of human osteosarcoma (OSA) tissues and cell lines. OSA in the canine population is known to exhibit a similar clinical behavior and molecular biology when compared to its human counterpart, and is often used as a model for preclinical testing of novel therapeutics. The purpose of this study was to investigate the potential role of STAT3 in canine and human OSA, and to evaluate the biologic activity of a novel small molecule STAT3 inhibitor.
To examine STAT3 and Src expression in OSA, we performed Western blotting and RT-PCR. OSA cells were treated with either STAT3 siRNA or small molecule Src (SU6656) or STAT3 (LLL3) inhibitors and cell proliferation (CyQUANT), caspase 3/7 activity (ELISA), apoptosis (Western blotting for PARP cleavage) and/or viability (Wst-1) were determined. Additionally, STAT3 DNA binding after treatment was determined using EMSA. Expression of STAT3 targets after treatment was demonstrated with Western blotting, RT-PCR, or gel zymography.
Our data demonstrate that constitutive activation of STAT3 is present in a subset of canine OSA tumors and human and canine cell lines, but not normal canine osteoblasts. In both canine and human OSA cell lines, downregulation of STAT3 activity through inhibition of upstream Src family kinases using SU6656, inhibition of STAT3 DNA binding and transcriptional activities using LLL3, or modulation of STAT3 expression using siRNA, all resulted in decreased cell proliferation and viability, ultimately inducing caspase-3/7 mediated apoptosis in treated cells. Furthermore, inhibition of either Src or STAT3 activity downregulated the expression of survivin, VEGF, and MMP2, all known transcriptional targets of STAT3.
These data suggest that STAT3 activation contributes to the survival and proliferation of human and canine OSA cells, thereby providing a potentially promising target for therapeutic intervention. Future investigational trials of LLL3 in dogs with spontaneous OSA will help to more accurately define the role of STAT3 in the clinical setting.
Signal transducers and activators of transcription (STAT) proteins comprise a family of transcription factors that play important roles in cell survival, growth, proliferation, differentiation, apoptosis, metastasis, and angiogenesis [1–3]. Accumulating evidence suggests that constitutively activated STAT3 contributes to tumor development and progression in numerous forms of cancer including those of the breast, head and neck, prostate, skin, ovary, lung, bone, and blood [3–5]. Constitutively activated STAT3 correlates with a more malignant tumor phenotype, resistance to chemotherapeutics, and is associated with decreased survival in some cancers [6–8]. As such, STAT3 may represent a novel target for therapeutic intervention in several cancers. In support of this, a variety of inhibitors of STAT3 have been shown to inhibit tumor cell growth and induce apoptosis both in vitro and in vivo [1, 9, 10]. Interestingly, STAT3 is not required for the proliferation of normal cells, and multiple studies have demonstrated that normal cells are more tolerant of loss of STAT3 function. .
Constitutive phosphorylation of STAT3 is thought to occur via aberrant upstream signaling, as no naturally occurring activating mutations in the STAT3 gene have been identified . STAT3 is phosphorylated following stimulation of receptor tyrosine kinases by their respective growth factors (i.e, Met/HGF, Kit/SCF), binding of cytokines to their receptors (IL-6, oncostatin M), and by activation of nonreceptor tyrosine kinases such as the Src family kinases (SFKs). In particular, the SFKs are instrumental in multiple signaling pathways involved in the initiation and/or progression of numerous forms of cancer . Indeed, STAT3 was identified as a phosphorylated substrate of v-src  necessary for enabling v-src induced adhesion-independence and malignant transformation [11, 12]. STAT3 is now known to be a substrate for SFK members including Fyn and Lyn in addition to Src itself [12, 15]. Furthermore, recent studies demonstrated that SFK inhibition in various carcinoma tumor cell lines resulted in loss of STAT3 activity .
Although the contribution of STAT3 to epithelial cancers and hematologic malignancies has been described in detail, little is known about the potential role of STAT3 dysregulation in sarcomas. One study found that STAT3 activation was present in approximately fifty percent of Ewing sarcoma tissues as assessed by immunostaining . More recent work investigating the potential role of STAT3 activation in pediatric sarcomas including osteosarcoma (OSA), rhabdomyosarcoma, and Ewing sarcoma demonstrated that constitutive STAT3 phosphorylation occurs in a high percentage of these tumors . Moreover, STAT3 inhibition via a novel small molecule STAT3 inhibitor (STA-21) or a dominant negative form of STAT3 resulted in inhibition of proliferation and apoptosis of sarcoma cell lines expressing high levels of phospho-STAT3 . With respect to OSA, approximately 20% of tumors on an OSA tissue microarray were shown to express high levels of phospho-STAT3 suggesting that this dysregulation is not a consequence of adaptation to tissue culture. As such, STAT3 may represent a target for therapeutic intervention in pediatric OSA.
The ability to rapidly advance therapeutics in pediatric OSA is limited by the low incidence of this disease. However, new therapies are critical as approximately 30–40% of affected patients still die of disease, and no substantial improvement in this outcome has occurred in over ten years. To study human OSA, several animal models have been developed including a variety of transgenic and xenograft rodent models [17–19]. While studies employing these models have been informative, they do not truly recapitulate the biology of OSA that occurs spontaneously in vivo.
OSA occurs in dogs with a frequency far greater than humans (over 10,000 new cases per year in the United States), and evidence suggests that canine OSA exhibits a similar biology to its human counterpart including early metastasis and dysregulated expression of ezrin, Met, and Her2/Neu [20–22]. Additionally, recent work has found that canine and pediatric OSA possess overlapping transcriptional profiles, supporting the notion that these diseases are similar at the molecular level. Indeed, canine OSA has been used as a large animal spontaneous model of the human disease to investigate the clinical efficacy of immunotherapeutic approaches and the activity of an IGF1 inhibitor among several others [23–25].
Given the similarities of canine and pediatric OSA, we investigated the potential role of STAT3 in the canine disease. Our data demonstrate that constitutive activation of STAT3 is present in a substantial subset of canine OSA tumors and human and canine cell lines and that downregulation of STAT3 activity through inhibition of upstream Src family kinases using a small molecule inhibitor (SU6656), direct inhibition of STAT3 DNA binding and transcriptional activities using a novel small molecule inhibitor (LLL3), or modulation of STAT3 expression using siRNAs, all resulted in decreased cell proliferation and viability, ultimately inducing caspase-3 mediated apoptosis in treated cells. Furthermore, inhibition of either Src or STAT3 activity downregulated the expression of survivin, VEGF, and MMP2, all known transcriptional targets of STAT3. These data suggest that STAT3 activation contributes to the survival and proliferation of both human and canine OSA, thereby providing a potentially promising target for therapeutic intervention.
Cell Lines and Reagents
Canine OSA cell lines, OSA7, 8, 11 M, 16, 29, and 32 were provided by Dr. Jaime Modiano (University of Minnesota, Minneapolis, MN). The canine D17 OSA cell line and human OSA cell lines U2OS, SJSA and MG63 were purchased from American Type Cell Culture Collection (ATCC). The canine lines and human line SJSA were maintained in RPMI-1640 supplemented with 10% fetal bovine serum, non-essential amino acids, sodium pyruvate, penicillin, streptomycin, L-glutamine, and HEPES (4-(2-hydroxethyl)-1-piperazineethanesulfonic acid) at 35°C, supplemented with 5% CO2. The remaining human cell lines were cultured in DMEM with 10% FBS and same supplements as listed for the canine lines. Normal canine osteoblasts (Cell Applications, Inc, San Diego, CA) were cultured in canine osteoblast medium (also from Cell Applications). The Src inhibitor, SU6656 was purchased from EMD Chemicals (San Diego, CA). The novel STAT3 small molecule inhibitor LLL3 (also known as compound 1), an analog of the small molecule STAT3 inhibitor STA-21, was generously provided by P.K. Li (College of Pharmacy, The Ohio State University, Columbus, OH) [1, 26, 27]. Concentrations of LLL3 used in experiments were determined after consultation with J. Lin. Canine OSA tumor and normal muscle were obtained from patients treated at The Ohio State University College of Veterinary Medicine Teaching Hospital in compliance with established hospital policies regarding sample collection as part of the Biospecimen Repository at the Teaching Hospital. The protocol for sample collection was approved by the OSU IACUC. Fresh tissue samples were immediately flash frozen in liquid nitrogen and stored in The Ohio State University College of Veterinary Medicine Comparative Oncology Biospecimen Repository. These tumors were evaluated by board certified veterinary pathologists at The Ohio State University College of Veterinary Medicine.
To determine the effect of HGF stimulation on STAT3 and Src phosphorylation in normal canine osteoblasts or OSA cells, cells were collected, washed once with PBS, and re-suspended in 1 mL PBS with 50 ng mL-1 rhHGF for 15 minutes or left untreated. To determine the effects of culture conditions on Src and STAT3 phosphorylation, 2–5 × 106 OSA cells were serum starved for 2 hours while in suspension or adherent culture, or left in serum-containing media while in adherent conditions for 2 hours prior to collection. To determine the downstream effects of Src and STAT3 inhibition, 2 × 106 OSA cells were treated with SU6656, LLL3, or DMSO and collected after 24, 48, or 72 hours or starved for 2 hours and treated for 2 hours with DMSO or SU6656. Cells were collected then resuspended in lysis buffer consisting of 20 mM Tris-HCl pH 8.0, 137 mM NaCl, 10% glycerol, 1% IPEGAL CA-630, 10 mM ethylenediaminetetraacetic acid (EDTA), 1 mg mL-1 aprotinin, 1 mg mL-1 leupeptin, 1 mg mL-1 pepstatin A, 1 mM phenylmethylsulphonyl fluoride, 1 mM sodium orthovanadate, and 10 mM sodium fluoride (all from Sigma, St. Louis, MO) for 1 hour at 4°C. Additionally, protein lysates were prepared from frozen canine normal muscle or OSA tumor samples by pulverizing tissues in liquid nitrogen and resuspending them in this same lysis buffer with proteinase and phosphatase inhibitors. The results of Western blotting for phosphorylated STAT3 for these samples were from two separate blots. Due to limited sample availability, the Western blots from different tumor samples were batched and run on 2 different occasions and all samples could not be subsequently re-run together on one blot. Following protein quantification via the Bradford Assay, 50 to 100 ug protein was separated by SDS-PAGE, and transferred to PVDF membranes. The membranes were then incubated overnight with anti-p-STAT3 (Y705, Cell Signaling Technology, Danvers, MA), anti-p-Src (Y418, Invitrogen, Carlsbad, CA), anti-PARP (BD Biosciences, San Jose, CA), anti-VEGF (Calbiochem, Gibbstown, NJ), or anti-survivin antibody (Novus Biologicals, Littleton, CO). The membranes were incubated with appropriate horseradish peroxidase linked secondary antibodies, washed, and exposed to substrate (SuperSignal West Dura Extended Duration Substrate, Pierce, Rockford, IL). Blots were stripped, washed, and reprobed for β-actin (Santa Cruz Biotechnology, Santa Cruz, CA), total STAT3 (Cell Signaling Technology, Danvers, MA) or total Src (Cell Signaling Technology, Danvers, MA).
For the Src family kinase (SFK) RT-PCR, RNA was extracted from untreated canine OSA cells using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. For determining levels of mRNA for STAT3 downstream transcriptional targets VEGF and MMP2, human and canine OSA cells were treated with either DMSO, LLL3 (40 uM), or SU6656 (10 uM) for 72 hours and RNA extracted from cells in the same manner. To generate cDNA, 2 μg of total RNA and the M-MLV reverse transcriptase kit (Invitrogen, Carlsbad, CA) were used according to the manufacturer's instructions. Next, 1/20 of the resultant cDNA was used for each PCR reaction in a total volume of 25 μl. Primers designed and utilized for canine SFK members were as follows: (forward/reverse sequences) Src (5'-GGCCCTCTATGACTATGAG/GGTGGTGAGGCGGTGGCACAGGC-3'); Fyn (5'-GCGGCCGGAGGCCAAGGACTC/GTCGCTCAGCATCTTTTCGG-3'); Yes (5'-GATGCTTGGGAAATCCCTCG/GCAGCCCGAAGATCTCGGTG-3'); Lyn (5'-GGTAGCCTTGTACCCCTATG/CTTAATAACATCACCATGCACAGGGTC-3'); and GAPDH (5'-ACCACAGTCCATGCCATCAC/TCCACCACCCTGTTGCTGTA-3'). The annealing temperature utilized for Src and Fyn was 62°C and 58°C was used for all other SFK member PCR reactions. Primers designed and utilized for canine STAT3 transcriptional targets were as follows: (forward/reverse sequences) VEGF (5'-GTCCCAGGCTGCGCCTATGG/GTTTAACTCAAGCTGCCTCGCC-3'); MMP2 (5'-GGAGACTCTCACTTTGATGACG/GGTGAAGGGGAAGACACAGGGG-3'); and GAPDH (5'-ACCACAGTCCATGCCATCAC/TCCACCACCCTGTTGCTGTA-3'). Annealing temperatures for these reactions was 55°C. Primers designed and utilized for the human STAT3 transcriptional target VEGF and GAPDH were as follows: (forward/reverse sequences) VEGF (5'-CCTGGTGGACATCTTCCAGGAGTACCC/CTAATGCCCTGGAGCCTCCC-3') and GAPDH (5'-ACCACAGTCCATGCCATCAC/TCCACCACCCTGTTGCTGTA-3'). An annealing temperature of 60°C was used for all PCR reactions with human primers. All PCR products were run on a 2% agarose gel with ethidium bromide and visualized using the Alpha Imager system (Alpha Innotech Corp, San Leandro, CA).
STAT3 siRNA transfection
Canine STAT3 small interfering RNA (siRNA) was designed and produced using the Silencer siRNA Construction kit (Ambion, Austin, TX) according to the manufacturer's protocol. Sequences of template canine DNA were as follows: Sense STAT3 siRNA: 5'-AACTCTCTGGTCGACAGTACTCCTGTCTC-3'; Antisense STAT3 siRNA: 5'-AAAGTACTGTCGACCAGAGAGCCTGTCTC-3'; Sense negative control scrambled siRNA: 5'-AAACGTGACACGTTCGGAGAACCTGTCTC-3'; Antisense negative control scrambled siRNA: 5'-AATTCTCCGAACGTGTCACGTCCTGTCTC-3'. 7.5 × 104 OSA cells were plated and left overnight in 1% serum-containing media. The following day, the media was changed to Opti-MEM media (Invitrogen, Carlsbad, CA) and either media alone, STAT3 or negative control scrambled siRNA (50 pMol of each siRNA) with transfection agent Lipofectamine 2000 (Invitrogen, Carlsbad, CA), or Lipofectamine 2000 alone was added to cells according to manufacturer's protocol. Transfection was repeated again at 48 hours. Cells were collected and processed for Western blotting as described above to detect levels of STAT3, VEGF, survivin, and beta-actin 48 hours after initial transfection. Cell viability was determined at 0, 72, and 96 hours after initial transfection using WST-1 reagent according to the manufacturer's instructions after plating OSA cells as described above (Clontech, Mountain View, CO). Cell viability was calculated as a percentage of the control wells. Additionally, levels of apoptosis were determined at 48 hours using the SensoLyte® Homogeneous AMC Caspase- 3/7 Assay kit (Anaspec Inc, San Jose, CA) as described below after harvesting cell lysate. Images of cells 72 hours post transfection were recorded using digital photography.
Detection of Apoptosis/Caspase 3,7 Activity
1.1 × 104 OSA cells were seeded in 96-well plates overnight and incubated with media, DMSO, or increasing concentrations of LLL3 or SU6656 for 24 hours. Wells with media only were included as controls. Levels of caspase- 3/7 activity were determined using the SensoLyte® Homogeneous AMC Caspase- 3/7 Assay kit (Anaspec Inc, San Jose, CA) according to manufacturer's instructions. Briefly, caspase 3/7 substrate solution was added to all wells and incubated prior to measuring fluorescence on a SpectraMax microplate reader (Molecular Devices Corp, Sunnyvale, CA) using excitation at 354 nm and emission detection at 442 nm. Levels of caspase 3/7 activity were reported after subtraction of fluorescence levels of wells with media only.
To confirm that SU6656 and LLL3 impair STAT3 DNA binding, we used the Pierce LightShift Chemiluminescent EMSA kit (Thermo Fisher Scientific Inc, Rockford, IL) that employs a chemiluminescent detection system to detect protein:DNA interactions according to manufacturer's instructions. Briefly, nuclear protein from human and canine OSA cell lines treated 72 hours with media, DMSO, SU6656 10 uM, or LLL3 40 uM was collected using the NucBuster™ Protein Extraction kit (EMD Chemicals Inc, Gibbstown, NJ). Binding reactions using equal amounts of nuclear protein from each treatment group were incubated for 20 minutes at room temperature with DNA probes. The canine STAT3 DNA binding sequence in the promoter for survivin (sense 5'-GCCTTGCATTCCCAGAAGGCCGCGG-3') was used for binding reactions for canine OSA; the STAT3 binding sequence in the human survivin promoter (sense 5'-GAGACTCAGTTTCAAATAAATAAATAAAC-3') was used for the human OSA cell line. Both were purchased from Operon Biotechnologies (Huntsville, AL) in a biotinylated form. Reactions with biotinylated target DNA only and nuclear protein with biotinylated target DNA and excess unlabelled target DNA to compete for binding were included. STAT3 specificity was confirmed by incubation with 6 ug of anti-STAT3 antibody (Santa Cruz Biotechnology Inc, Santa Cruz, CA) to super-shift the protein-DNA complex. Following gel electrophoresis, DNA was transferred to a nylon membrane. The DNA was cross-linked and the biotin-labeled DNA detected by chemiluminescence according to manufacturer's instructions.
2 × 103 cells were seeded in 96-well plates overnight and incubated with DMSO or increasing concentrations of LLL3 or SU6656 with daily treatment. Each drug concentration was performed in four replicate wells. Media was removed at days 1, 3, and 5 and plates frozen at -80°C overnight before processing with the CyQUANT® Cell Proliferation Assay Kit (Molecular Probes, Eugene, OR) according to manufacturer's instructions. Fluorescence was measured with a SpectraMax microplate reader (Molecular Devices Corp, Sunnyvale, CA) with excitation at 485 nm and emission detection at 530 nm. Cell proliferation was calculated as a percentage of the control (DMSO treated) wells.
7.5 × 104 OSA cells were seeded in 6-well plates overnight and incubated with media only, DMSO, or increasing concentrations of SU6656 or LLL3 for 72 hours. Media was collected and processed and gel zymography performed as described previously . Images were recorded using the Alpha Imager system (Alpha Innotech Corp, San Leandro, CA) and analyzed using Image J.
All experiments were performed two to three times. Statistical analysis of the data was performed using the Student's t-test. P values of ≤ 0.05 were considered statistically significant.
STAT3 is constitutively phosphorylated in OSA tumor tissues and cell lines
STAT3 siRNA induces downregulation of STAT3 and its downstream targets survivin and VEGF with subsequent loss of viability and apoptosis of canine OSA cells
SU6656 inhibits phosphorylation of Src and STAT3 in OSA lines
Downregulation of Src or STAT3 leads to decreased STAT3 DNA binding
Inhibition of Src or STAT3 decreases cell proliferation, induces caspase-3,7 dependent apoptosis, and downregulates survivin in OSA cell lines
Inhibition of Src or STAT3 leads to loss of MMP2 and VEGF expression in OSA cells
The contribution of STAT3 to cancer has been widely confirmed by numerous studies demonstrating dysregulated activation in a variety of human tumors such as breast, lung, pancreatic, ovarian, skin, and renal carcinomas, as well as many hematopoietic tumors [3–5]. Given the prevalence of STAT3 dysregulation in cancer, its role in promoting cell cycle progression, cellular transformation, and prevention of apoptosis, and its unique position downstream of multiple activating tyrosine kinases, STAT3 represents a potential target for therapeutic intervention . While the function of STAT3 in carcinomas and hematopoietic neoplasia has been intensively studied, little is known about the potential role of STAT3 dysregulation in sarcomas. The most detailed work done to date investigating the role of STAT3 in human sarcomas revealed elevated levels of pSTAT3 in a subset of pediatric sarcomas which included osteosarcomas (OSAs)  as well as in OSA cancer stem cells . The purpose of this study was to investigate the role of STAT3 dysregulation in OSA and to explore the biologic consequences of STAT3 inhibition in OSA cell lines.
Several animal models have been developed to study human OSA including radiation-induced OSA in Sprague-Dawley rats , subcutaneous  and orthotopic  implantation of OSA cells in mice, as well as a variety of transgenic mouse models that develop OSA spontaneously. While studies employing these models have been informative, they do not truly recapitulate the biology of OSA that occurs spontaneously in vivo, particularly with respect to the development of appendicular OSA and chemotherapy-resistant metastases.
While OSA is the most common malignant bone tumor in children, it occurs with a far greater frequency in the canine population (1000 cases per year compared to greater than 10,000 cases per year in the United States). Beyond clinical similarities such as tumor location and chemotherapy resistance, canine and human OSA share similar biologic features including early metastasis, dysregulated expression of ezrin, Met, and Her2/Neu, and overlapping transcriptional profiles [20–22, 28, 33–35].
Spontaneous canine OSA has been used to evaluate novel therapeutics such as liposome encapsulated muramyl tripeptide, IGF-1R inhibitors, and more recently, rapamycin. [23, 36–39]. Together, these data support the idea that canine OSA is a relevant, spontaneous, large animal model of human OSA that closely recapitulates the biology of human OSA. As such, we chose to evaluate both human and canine OSA cell lines, as well as fresh canine OSA tumors for evidence of STAT3 dysregulation as demonstration of abnormalities in both species would lay the foundation for preclinical testing of STAT3 inhibitors in dogs with OSA.
Constitutive phosphorylation of Src and STAT3 was detected by Western blotting in all human and canine OSA cell lines tested despite alterations in culture conditions as well as in a subset (38%) of fresh frozen OSA derived from canine patients, but not in normal canine osteoblasts. This finding supports previous studies in which human OSA cell lines and 19% of tissue microarray samples were positive for elevated STAT3 phosphorylation . Indeed, all human and canine OSA cell lines tested expressed survivin, MMP2 and VEGF, downstream targets of STAT3 important in preventing apoptosis, enhancing invasion, and promoting metastasis. Increased expression of survivin has been associated with reduced survival in OSA  as well as chemotherapy and radiotherapy resistance. Expression of VEGF in OSA is also strongly predictive for pulmonary metastasis and poor prognosis . These data support the notion that constitutive activation of both Src and STAT3 is common in both canine and human OSA.
Studies have shown that inhibition of STAT3 activation through various methods such as RNA interference, double-stranded decoy oligodeoxynucleotides, and small molecule inhibitors results in decreased viability and apoptosis of a variety of human tumor cell lines, including those derived from sarcomas [1, 42–46]. Previous work has shown that downregulation of STAT3 using a dominant-negative form decreased viability of human OSA cells resulting in induction of apoptosis . Additionally, STAT3 knockdown using RNA interference in colon cancer cell lines reduced survivin expression resulting in anoikis . Transfection of canine OSA cell lines with STAT3 siRNA led to loss of STAT3, survivin, and VEGF expression with subsequent decreases in cell proliferation and induction of apoptosis through the caspase 3/7 pathway. Similar effects were seen in both canine and human OSA cell lines treated with the small molecule Src inhibitor SU6656 and the novel small molecule STAT3 inhibitor LLL3. These data are consistent with findings of previous studies of STAT3 inhibition in human carcinoma and leukemia cell lines.
Abrogation of STAT3 DNA binding in both human and canine OSA cell lines after treatment with both inhibitors, as shown by EMSA, delineates the mechanism of loss of downstream target mRNA and protein expression. Interestingly, the STAT3 small molecule inhibitor STA-21, the compound from which LLL3 was derived, was found to act in a similar manner, preventing STAT3 activity by blocking dimerization, translocation into the nucleus, and execution of its signaling pathway in breast cancer cells at roughly the same concentration . Treatment with STA-21 suppressed cell growth, reduced cell number, and induced apoptosis in human sarcomas through caspase 3, 8, and 9 dependent pathways . These results support the notion that the biologic effects manifested in OSA cell lines following either loss of total STAT3 or loss of STAT3 DNA binding are directly related to downregulation of various STAT3 transcriptional targets responsible for survival.
To correlate the effects of Src family kinase inhibition with STAT3 activation, we evaluated the effects of inhibition of Src on OSA cell lines. Previous work has shown that the small molecule SFK inhibitor SU6656 blocks the phosphorylation of Src, Fyn, Lyn, and Yes with approximately equal potencies . SU6656 has been shown to decrease activation of STAT3 and subsequent downstream targets in NIH3T3 cells resulting in cell cycle arrest and apoptosis in human lung cancer cells [30, 49]. Our data demonstrated that not only are these SFK members expressed in the canine OSA cell lines tested, but that inhibition of Src phosphorylation in both human and canine OSA cell lines with SU6656 abrogated Src signaling resulting in a concomitant decrease in STAT3 phosphorylation. These data suggest that Src contributes to activation of STAT3 in the human and canine OSA cell lines and that inhibition of Src represents another method for abrogating STAT3 activity in OSA. Indeed, small molecule SFK inhibitors have been evaluated in human OSA and Ewing's sarcoma resulting in suppression of tumor cell migration, apoptosis of tumor cell lines, and inhibition of tumor growth in xenograft models [46, 47, 50–52]. Given the apparent role of Src in STAT3 activation, it is possible that SFK inhibitors are working, in part, through modulation of STAT3 phosphorylation.
Our data demonstrate that constitutive activation of STAT3 is present in both canine and human OSA cell lines and in a subset of canine OSA fresh tumor specimens. Downregulation of STAT3 activity in OSA cell lines, either directly or through Src inhibition reduced proliferation and expression of STAT3 transcriptional targets, and induced apoptosis through caspase-3/7 activation. Data generated in both the canine and human OSA cell lines were concordant, suggesting that dysregulation of STAT3 may be common in this disease. This work serves as the foundation for future experiments with LLL3 and other inhibitors of STAT3 both in mouse models of OSA with eventual application in dogs with spontaneous OSA, as a prelude to clinical work in children.
This work was supported by a grant from the American Kennel Club Canine Health Foundation. We would like to thank The Ohio State University College of Veterinary Medicine Comparative Oncology Biospecimen Repository and the Biomedical Media Services for their assistance.
- Chen CL, Loy A, Cen L, Chan C, Hsieh FC, Cheng G, Wu B, Qualman SJ, Kunisada K, Yamauchi-Takihara K, et al: Signal transducer and activator of transcription 3 is involved in cell growth and survival of human rhabdomyosarcoma and osteosarcoma cells. BMC Cancer. 2007, 7: 111-10.1186/1471-2407-7-111.View ArticlePubMedPubMed CentralGoogle Scholar
- Germain D, Frank DA: Targeting the cytoplasmic and nuclear functions of signal transducers and activators of transcription 3 for cancer therapy. Clin Cancer Res. 2007, 13 (19): 5665-5669. 10.1158/1078-0432.CCR-06-2491.View ArticlePubMedGoogle Scholar
- Haura EB, Turkson J, Jove R: Mechanisms of disease: Insights into the emerging role of signal transducers and activators of transcription in cancer. Nat Clin Pract Oncol. 2005, 2 (6): 315-324. 10.1038/ncponc0195.View ArticlePubMedGoogle Scholar
- Aggarwal BB, Sethi G, Ahn KS, Sandur SK, Pandey MK, Kunnumakkara AB, Sung B, Ichikawa H: Targeting signal-transducer-and-activator-of-transcription-3 for prevention and therapy of cancer: modern target but ancient solution. Ann N Y Acad Sci. 2006, 1091: 151-169. 10.1196/annals.1378.063.View ArticlePubMedGoogle Scholar
- Alvarez JV, Febbo PG, Ramaswamy S, Loda M, Richardson A, Frank DA: Identification of a genetic signature of activated signal transducer and activator of transcription 3 in human tumors. Cancer Res. 2005, 65 (12): 5054-5062. 10.1158/0008-5472.CAN-04-4281.View ArticlePubMedGoogle Scholar
- Barre B, Vigneron A, Perkins N, Roninson IB, Gamelin E, Coqueret O: The STAT3 oncogene as a predictive marker of drug resistance. Trends Mol Med. 2007, 13 (1): 4-11. 10.1016/j.molmed.2006.11.001.View ArticlePubMedGoogle Scholar
- Benekli M, Xia Z, Donohue KA, Ford LA, Pixley LA, Baer MR, Baumann H, Wetzler M: Constitutive activity of signal transducer and activator of transcription 3 protein in acute myeloid leukemia blasts is associated with short disease-free survival. Blood. 2002, 99 (1): 252-257. 10.1182/blood.V99.1.252.View ArticlePubMedGoogle Scholar
- Duan Z, Foster R, Bell DA, Mahoney J, Wolak K, Vaidya A, Hampel C, Lee H, Seiden MV: Signal transducers and activators of transcription 3 pathway activation in drug-resistant ovarian cancer. Clin Cancer Res. 2006, 12 (17): 5055-5063. 10.1158/1078-0432.CCR-06-0861.View ArticlePubMedGoogle Scholar
- Turkson J, Jove R: STAT proteins: novel molecular targets for cancer drug discovery. Oncogene. 2000, 19 (56): 6613-6626. 10.1038/sj.onc.1204086.View ArticlePubMedGoogle Scholar
- Deng J, Grande F, Neamati N: Small molecule inhibitors of Stat3 signaling pathway. Curr Cancer Drug Targets. 2007, 7 (1): 91-107. 10.2174/156800907780006922.View ArticlePubMedGoogle Scholar
- Schlessinger K, Levy DE: Malignant transformation but not normal cell growth depends on signal transducer and activator of transcription 3. Cancer Res. 2005, 65 (13): 5828-5834. 10.1158/0008-5472.CAN-05-0317.View ArticlePubMedPubMed CentralGoogle Scholar
- Silva CM: Role of STATs as downstream signal transducers in Src family kinase-mediated tumorigenesis. Oncogene. 2004, 23 (48): 8017-8023. 10.1038/sj.onc.1208159.View ArticlePubMedGoogle Scholar
- Johnson FM, Saigal B, Tran H, Donato NJ: Abrogation of signal transducer and activator of transcription 3 reactivation after Src kinase inhibition results in synergistic antitumor effects. Clin Cancer Res. 2007, 13 (14): 4233-4244. 10.1158/1078-0432.CCR-06-2981.View ArticlePubMedGoogle Scholar
- Smith PD, Crompton MR: Expression of v-src in mammary epithelial cells induces transcription via STAT3. Biochem J. 1998, 331 (Pt 2): 381-385.View ArticlePubMedPubMed CentralGoogle Scholar
- Schreiner SJ, Schiavone AP, Smithgall TE: Activation of STAT3 by the Src family kinase Hck requires a functional SH3 domain. J Biol Chem. 2002, 277 (47): 45680-45687. 10.1074/jbc.M204255200.View ArticlePubMedGoogle Scholar
- Lai R, Navid F, Rodriguez-Galindo C, Liu T, Fuller CE, Ganti R, Dien J, Dalton J, Billups C, Khoury JD: STAT3 is activated in a subset of the Ewing sarcoma family of tumours. J Pathol. 2006, 208 (5): 624-632. 10.1002/path.1941.View ArticlePubMedGoogle Scholar
- Berlin O, Samid D, Donthineni-Rao R, Akeson W, Amiel D, Woods VL: Development of a novel spontaneous metastasis model of human osteosarcoma transplanted orthotopically into bone of athymic mice. Cancer Res. 1993, 53 (20): 4890-4895.PubMedGoogle Scholar
- Asai T, Ueda T, Itoh K, Yoshioka K, Aoki Y, Mori S, Yoshikawa H: Establishment and characterization of a murine osteosarcoma cell line (LM8) with high metastatic potential to the lung. Int J Cancer. 1998, 76 (3): 418-422. 10.1002/(SICI)1097-0215(19980504)76:3<418::AID-IJC21>3.0.CO;2-5.View ArticlePubMedGoogle Scholar
- Tinkey PT, Lembo TM, Evans GR, Cundiff JH, Gray KN, Price RE: Postirradiation sarcomas in Sprague-Dawley rats. Radiat Res. 1998, 149 (4): 401-404. 10.2307/3579704.View ArticlePubMedGoogle Scholar
- Flint AF, U'Ren L, Legare ME, Withrow SJ, Dernell W, Hanneman WH: Overexpression of the erbB-2 proto-oncogene in canine osteosarcoma cell lines and tumors. Vet Pathol. 2004, 41 (3): 291-296. 10.1354/vp.41-3-291.View ArticlePubMedGoogle Scholar
- Khanna C, Wan X, Bose S, Cassaday R, Olomu O, Mendoza A, Yeung C, Gorlick R, Hewitt SM, Helman LJ: The membrane-cytoskeleton linker ezrin is necessary for osteosarcoma metastasis. Nat Med. 2004, 10 (2): 182-186. 10.1038/nm982.View ArticlePubMedGoogle Scholar
- Liao AT, McMahon M, London CA: Characterization, expression and function of c-Met in canine spontaneous cancers. Veterinary and Comparative Oncology. 2005, 3 (2): 61-72. 10.1111/j.1476-5810.2005.00067.x.View ArticlePubMedGoogle Scholar
- Khanna C, Prehn J, Hayden D, Cassaday RD, Caylor J, Jacob S, Bose SM, Hong SH, Hewitt SM, Helman LJ: A randomized controlled trial of octreotide pamoate long-acting release and carboplatin versus carboplatin alone in dogs with naturally occurring osteosarcoma: evaluation of insulin-like growth factor suppression and chemotherapy. Clin Cancer Res. 2002, 8 (7): 2406-2412.PubMedGoogle Scholar
- Nardin A, Lefebvre ML, Labroquere K, Faure O, Abastado JP: Liposomal muramyl tripeptide phosphatidylethanolamine: Targeting and activating macrophages for adjuvant treatment of osteosarcoma. Curr Cancer Drug Targets. 2006, 6 (2): 123-133. 10.2174/156800906776056473.View ArticlePubMedGoogle Scholar
- Dow S, Elmslie R, Kurzman I, MacEwen G, Pericle F, Liggitt D: Phase I study of liposome-DNA complexes encoding the interleukin-2 gene in dogs with osteosarcoma lung metastases. Hum Gene Ther. 2005, 16 (8): 937-946. 10.1089/hum.2005.16.937.View ArticlePubMedGoogle Scholar
- Bhasin D, Cisek K, Pandharkar T, Regan N, Li C, Pandit B, Lin J, Li P-K: Design, synthesis, and studies of small molecule STAT3 inhibitors. Bioorg, and Med Chem Lett. 2008, 18: 391-395. 10.1016/j.bmcl.2007.10.031.View ArticleGoogle Scholar
- Song H, Wang R, Wang S, Lin J: A low-molecular-weight compound discovered through virtual database screening inhibits Stat3 function in breast cancer cells. Proc Natl Acad Sci USA. 2005, 102 (13): 4700-4705. 10.1073/pnas.0409894102.View ArticlePubMedPubMed CentralGoogle Scholar
- Liao AT, McCleese J, Kamerling S, Christensen JG, London CA: A novel small molecule Met inhibitor, PF236 exhibits biological activity against canine osteosarcoma. Vet Comp Oncol. 2376, 5: 177-196. 10.1111/j.1476-5829.2007.00137.x.View ArticleGoogle Scholar
- Lin TY, London CA: A functional comparison of canine and murine bone marrow derived cultured mast cells. Vet Immunol Immunopathol. 2006, 114 (3–4): 320-334. 10.1016/j.vetimm.2006.09.001.View ArticlePubMedGoogle Scholar
- Song L, Turkson J, Karras JG, Jove R, Haura EB: Activation of Stat3 by receptor tyrosine kinases and cytokines regulates survival in human non-small cell carcinoma cells. Oncogene. 2003, 22 (27): 4150-4165. 10.1038/sj.onc.1206479.View ArticlePubMedGoogle Scholar
- Bromberg J, Darnell JE: The role of STATs in transcriptional control and their impact on cellular function. Oncogene. 2000, 19 (21): 2468-2473. 10.1038/sj.onc.1203476.View ArticlePubMedGoogle Scholar
- Wilson H, Huelsmeyer M, Chun R, Young KM, Friedrichs K, Argyle DJ: Isolation and characterisation of cancer stem cells from canine osteosarcoma. Vet J. 2008, 175 (1): 69-75. 10.1016/j.tvjl.2007.07.025.View ArticlePubMedGoogle Scholar
- MacEwen EG, Kutzke J, Carew J, Pastor J, Schmidt JA, Tsan R, Thamm DH, Radinsky R: c-Met tyrosine kinase receptor expression and function in human and canine osteosarcoma cells. Clin Exp Metastasis. 2003, 20 (5): 421-430. 10.1023/A:1025404603315.View ArticlePubMedGoogle Scholar
- Paoloni M, Davis S, Lana S, Withrow SJ, Meltzer B, Khanna C: Comparative gene expression profiling of canine and human osteosarcoma. Proceedings of the Genes Dogs and Cancer: 4th International Canine Cancer Conference. 2006, 4: 19-Google Scholar
- Paoloni M, Khanna C: Translation of new cancer treatments from pet dogs to humans. Nat Rev Cancer. 2008, 8 (2): 147-156. 10.1038/nrc2273.View ArticlePubMedGoogle Scholar
- Kurzman ID, MacEwen EG, Rosenthal RC, Fox LE, Keller ET, Helfand SC, Vail DM, Dubielzig RR, Madewell BR, Rodriguez CO, et al: Adjuvant therapy for osteosarcoma in dogs: results of randomized clinical trials using combined liposome-encapsulated muramyl tripeptide and cisplatin. Clin Cancer Res. 1995, 1 (12): 1595-1601.PubMedGoogle Scholar
- MacEwen EG, Kurzman ID, Helfand S, Vail D, London C, Kisseberth W, Rosenthal RC, Fox LE, Keller ET, Obradovich J, et al: Current studies of liposome muramyl tripeptide (CGP 19835A lipid) therapy for metastasis in spontaneous tumors: a progress review. J Drug Target. 1994, 2 (5): 391-396. 10.3109/10611869408996814.View ArticlePubMedGoogle Scholar
- MacEwen EG, Kurzman ID, Rosenthal RC, Smith BW, Manley PA, Roush JK, Howard PE: Therapy for osteosarcoma in dogs with intravenous injection of liposome-encapsulated muramyl tripeptide. J Natl Cancer Inst. 1989, 81 (12): 935-938. 10.1093/jnci/81.12.935.View ArticlePubMedGoogle Scholar
- Moore AS, Dernell WS, Ogilvie GK, Kristal O, Elmslie R, Kitchell B, Susaneck S, Rosenthal R, Klein MK, Obradovich J, et al: Doxorubicin and BAY 12-9566 for the treatment of osteosarcoma in dogs: a randomized, double-blind, placebo-controlled study. J Vet Intern Med. 2007, 21 (4): 783-790. 10.1892/0891-6640(2007)21[783:DABFTT]2.0.CO;2.View ArticlePubMedGoogle Scholar
- Clark JC, Dass CR, Choong PF: A review of clinical and molecular prognostic factors in osteosarcoma. J Cancer Res Clin Oncol. 2008, 134 (3): 281-297. 10.1007/s00432-007-0330-x.View ArticlePubMedGoogle Scholar
- Kaya M, Wada T, Akatsuka T, Kawaguchi S, Nagoya S, Shindoh M, Higashino F, Mezawa F, Okada F, Ishii S: Vascular endothelial growth factor expression in untreated osteosarcoma is predictive of pulmonary metastasis and poor prognosis. Clin Cancer Res. 2000, 6 (2): 572-577.PubMedGoogle Scholar
- Aoki Y, Feldman GM, Tosato G: Inhibition of STAT3 signaling induces apoptosis and decreases survivin expression in primary effusion lymphoma. Blood. 2003, 101 (4): 1535-1542. 10.1182/blood-2002-07-2130.View ArticlePubMedGoogle Scholar
- Kanda N, Seno H, Konda Y, Marusawa H, Kanai M, Nakajima T, Kawashima T, Nanakin A, Sawabu T, Uenoyama Y, et al: STAT3 is constitutively activated and supports cell survival in association with survivin expression in gastric cancer cells. Oncogene. 2004, 23 (28): 4921-4929. 10.1038/sj.onc.1207606.View ArticlePubMedGoogle Scholar
- Niu G, Shain KH, Huang M, Ravi R, Bedi A, Dalton WS, Jove R, Yu H: Overexpression of a dominant-negative signal transducer and activator of transcription 3 variant in tumor cells leads to production of soluble factors that induce apoptosis and cell cycle arrest. Cancer Res. 2001, 61 (8): 3276-3280.PubMedGoogle Scholar
- Buettner R, Mora LB, Jove R: Activated STAT signaling in human tumors provides novel molecular targets for therapeutic intervention. Clin Cancer Res. 2002, 8 (4): 945-954.PubMedGoogle Scholar
- Guan H, Zhou Z, Gallick GE, Jia SF, Morales J, Sood AK, Corey SJ, Kleinerman ES: Targeting Lyn inhibits tumor growth and metastasis in Ewing's sarcoma. Mol Cancer Ther. 2008, 7 (7): 1807-1816. 10.1158/1535-7163.MCT-08-0058.View ArticlePubMedPubMed CentralGoogle Scholar
- Timeus F, Crescenzio N, Fandi A, Doria A, Foglia L, Cordero di Montezemolo L: In vitro antiproliferative and antimigratory activity of dasatinib in neuroblastoma and Ewing sarcoma cell lines. Oncol Rep. 2008, 19 (2): 353-359.PubMedGoogle Scholar
- Mendel DB, Laird AD, Smolich BD, Blake RA, Liang C, Hannah AL, Shaheen RM, Ellis LM, Weitman S, Shawver LK, et al: Development of SU5416, a selective small molecule inhibitor of VEGF receptor tyrosine kinase activity, as an anti-angiogenesis agent. Anticancer Drug Des. 2000, 15 (1): 29-41.PubMedGoogle Scholar
- Bowman T, Broome MA, Sinibaldi D, Wharton W, Pledger WJ, Sedivy JM, Irby R, Yeatman T, Courtneidge SA, Jove R: Stat3-mediated Myc expression is required for Src transformation and PDGF-induced mitogenesis. Proc Natl Acad Sci USA. 2001, 98 (13): 7319-7324. 10.1073/pnas.131568898.View ArticlePubMedPubMed CentralGoogle Scholar
- Manetti F, Santucci A, Locatelli GA, Maga G, Spreafico A, Serchi T, Orlandini M, Bernardini G, Caradonna NP, Spallarossa A, (eds), et al: Identification of a novel pyrazolo[3,4-d] pyrimidine able to inhibit cell proliferation of a human osteogenic sarcoma in vitro and in a xenograft model in mice. Journal of Medicinal Chemistry. 2007, 50 (23): 5579-5588. 10.1021/jm061449r.Google Scholar
- Shor AC, Keschman EA, Lee FY, Muro-Cacho C, Letson GD, Trent JC, Pledger WJ, Jove R: Dasatinib inhibits migration and invasion in diverse human sarcoma cell lines and induces apoptosis in bone sarcoma cells dependent on SRC kinase for survival. Cancer Res. 2007, 67 (6): 2800-2808. 10.1158/0008-5472.CAN-06-3469.View ArticlePubMedGoogle Scholar
- Spreafico A, Schenone S, Serchi T, Orlandini M, Angelucci A, Magrini D, Bernardini G, Collodel G, Di Stefano A, Tintori C, et al: Antiproliferative and proapoptotic activities of new pyrazolo[3,4-d]pyrimidine derivative Src kinase inhibitors in human osteosarcoma cells. Faseb J. 2008, 22 (5): 1560-1571. 10.1096/fj.07-9873com.View ArticlePubMedGoogle Scholar
- Liao AT, Chien MB, Shenoy N, Mendel DB, McMahon G, Cherrington JM, London CA: Inhibition of constitutively active forms of mutant kit by multitargeted indolinone tyrosine kinase inhibitors. Blood. 2002, 100 (2): 585-593. 10.1182/blood-2001-12-0350.View ArticlePubMedGoogle Scholar
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/9/81/prepub
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.