Abstract
BackgroundDysregulation 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.MethodsTo 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.ResultsOur 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.ConclusionThese 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.
Highlights
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
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 small interfering RNA (siRNA), all resulted in decreased cell proliferation and viability, inducing caspase-3 mediated apoptosis in treated cells
STAT3 is constitutively phosphorylated in OSA tumor tissues and cell lines STAT3 is known to be activated by various upstream receptor and nonreceptor tyrosine kinases including the Src family kinases (SFKs) and Met [12,30]
Summary
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. 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. 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 [1,2,3]. Activated STAT3 correlates with a more malignant tumor phenotype, resistance to chemotherapeutics, and is associated with decreased survival in some cancers [6,7,8]. 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. [11]
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