Characterization of STAT3 activation and expression in canine and human osteosarcoma.
BACKGROUND
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Fossey
Stacey L
SL
Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University, Columbus, OH, USA. fossey.1@osu.edu
Liao
Albert T
AT
McCleese
Jennifer K
JK
Bear
Misty D
MD
Lin
Jiayuh
J
Li
Pui-Kai
PK
Kisseberth
William C
WC
London
Cheryl A
CA
D000818:Animals; D000880:Anthraquinones / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D053148:Caspase 3 / Q000378:metabolism; D053179:Caspase 7 / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D004285:Dogs; D024202:Electrophoretic Mobility Shift Assay; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D051034:Inhibitor of Apoptosis Proteins; D020778:Matrix Metalloproteinase 2 / Q000235:genetics / Q000378:metabolism; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism; D012516:Osteosarcoma / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D050796:STAT3 Transcription Factor / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D013449:Sulfonamides / Q000494:pharmacology; D000077022:Survivin; D014162:Transfection; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism
D000880:Anthraquinones; C107489:BIRC5 protein, human; D007211:Indoles; D051034:Inhibitor of Apoptosis Proteins; C534708:LLL-3 compound; D008869:Microtubule-Associated Proteins; D034741:RNA, Small Interfering; D050796:STAT3 Transcription Factor; C416927:SU 6656; D013449:Sulfonamides; D000077022:Survivin; D042461:Vascular Endothelial Growth Factor A; D016392:Proto-Oncogene Proteins pp60(c-src); D053148:Caspase 3; D053179:Caspase 7; D020778:Matrix Metalloproteinase 2