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Characterization of STAT3 expression, signaling and inhibition in feline oral squamous cell carcinoma.

Brown ME, Bear MD, Rosol TJ, Premanandan C, Kisseberth WC, London CA - BMC Vet. Res. (2015)

Bottom Line: Exposure of SCCF2 cells to LLL12 resulted in decreased expression of pSTAT3 and total STAT3, apoptosis as assessed by caspase 3/7 activation, inhibition of colony formation and reduced expression of the STAT3 transcriptional target survivin.The OSCC cell lines with low basal STAT3 phosphorylation did not exhibit these effects, suggesting that STAT3 inhibition was responsible for the observed results.However, given the up-regulation of several STAT3 transcriptional targets following treatment, further investigation of STAT3 and its related signaling pathways in OSCC is warranted.

View Article: PubMed Central - PubMed

Affiliation: Department of Veterinary Clinical Sciences, College of Veterinary Medicine, The Ohio State University, Columbus, OH, USA.

ABSTRACT

Background: Signal transducer and activator of transcription 3 (STAT3) plays a critical role in tumor development by regulating signaling pathways involved in cell proliferation, survival, metastasis and angiogenesis. STAT3 is activated in many cancers, including head and neck squamous cell carcinoma (HNSCC) in people. Feline oral squamous cell carcinoma (OSCC) is similar to advanced or recurrent HNSCC as it is poorly responsive to traditional therapies and carries a poor long-term prognosis. The purpose of this study was to characterize expression and activation of STAT3 in feline OSCC cell lines and tumor samples and to investigate the biologic activity of a novel, allosteric STAT3 inhibitor, LLL12, in feline OSCC cell lines.

Results: We evaluated 3 feline OSCC cell lines and one of these (SCCF2) exhibited high levels of constitutive STAT3 phosphorylation and high sensitivity to LLL12 treatment. Exposure of SCCF2 cells to LLL12 resulted in decreased expression of pSTAT3 and total STAT3, apoptosis as assessed by caspase 3/7 activation, inhibition of colony formation and reduced expression of the STAT3 transcriptional target survivin. In contrast, the STAT3 transcriptional targets VEGF and MCL-1 increased after LLL12 treatment. This was, in part, likely due to LLL12 mediated upregulation of HIF-1α, which is known to drive VEGF and MCL-1 expression. The OSCC cell lines with low basal STAT3 phosphorylation did not exhibit these effects, suggesting that STAT3 inhibition was responsible for the observed results. Lastly, immunohistochemistry for pSTAT3 was performed using a feline OSCC tissue microarray, demonstrating expression in 48 % of samples tested.

Conclusions: These data demonstrate that LLL12 has biologic activity against a feline OSCC cell line expressing pSTAT3 and that STAT3 represents a target for therapeutic intervention in this disease. However, given the up-regulation of several STAT3 transcriptional targets following treatment, further investigation of STAT3 and its related signaling pathways in OSCC is warranted.

No MeSH data available.


Related in: MedlinePlus

Effects of LLL12 treatment on pSTAT3 and STAT3 expression in feline OSCC cell lines. a Feline OSCC cell lines were treated with DMSO or 0.2 μM LLL12 for 12 h prior to collection. Protein lysates were generated, separated by SDS-PAGE and Western blotting for pSTAT3, STAT3 and β-actin was performed. b SCCF2 cells were treatment with 0.2 μM LLL12 alone, 10 μM MG132 alone or MG132 in combination with LLL12 for 12 h. c Feline OSCC cell lines were treated with DMSO or 0.2 μM LLL12 for 12 h. RNA was collected and quantitative RT-PCR for STAT3 was performed
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Fig3: Effects of LLL12 treatment on pSTAT3 and STAT3 expression in feline OSCC cell lines. a Feline OSCC cell lines were treated with DMSO or 0.2 μM LLL12 for 12 h prior to collection. Protein lysates were generated, separated by SDS-PAGE and Western blotting for pSTAT3, STAT3 and β-actin was performed. b SCCF2 cells were treatment with 0.2 μM LLL12 alone, 10 μM MG132 alone or MG132 in combination with LLL12 for 12 h. c Feline OSCC cell lines were treated with DMSO or 0.2 μM LLL12 for 12 h. RNA was collected and quantitative RT-PCR for STAT3 was performed

Mentions: To assess the direct effects of LLL12 on pSTAT3, two OSCC cell lines with high (SCCF2) and low (SCCF3) basal STAT3 activation were chosen for further analysis. Cells were treated with DMSO or 0.2 μM LLL12 for 12 h and Western blotting for pSTAT3 and STAT3 was performed. Western blot analyses revealed that pSTAT3 and, to a lesser degree, total STAT3, were both downregulated in SCCF2 following treatment with LLL12 (Fig. 3a). In contrast, no change in pSTAT3 or STAT3 expression was observed in the SCCF3 cell line.Fig. 3


Characterization of STAT3 expression, signaling and inhibition in feline oral squamous cell carcinoma.

Brown ME, Bear MD, Rosol TJ, Premanandan C, Kisseberth WC, London CA - BMC Vet. Res. (2015)

Effects of LLL12 treatment on pSTAT3 and STAT3 expression in feline OSCC cell lines. a Feline OSCC cell lines were treated with DMSO or 0.2 μM LLL12 for 12 h prior to collection. Protein lysates were generated, separated by SDS-PAGE and Western blotting for pSTAT3, STAT3 and β-actin was performed. b SCCF2 cells were treatment with 0.2 μM LLL12 alone, 10 μM MG132 alone or MG132 in combination with LLL12 for 12 h. c Feline OSCC cell lines were treated with DMSO or 0.2 μM LLL12 for 12 h. RNA was collected and quantitative RT-PCR for STAT3 was performed
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4536595&req=5

Fig3: Effects of LLL12 treatment on pSTAT3 and STAT3 expression in feline OSCC cell lines. a Feline OSCC cell lines were treated with DMSO or 0.2 μM LLL12 for 12 h prior to collection. Protein lysates were generated, separated by SDS-PAGE and Western blotting for pSTAT3, STAT3 and β-actin was performed. b SCCF2 cells were treatment with 0.2 μM LLL12 alone, 10 μM MG132 alone or MG132 in combination with LLL12 for 12 h. c Feline OSCC cell lines were treated with DMSO or 0.2 μM LLL12 for 12 h. RNA was collected and quantitative RT-PCR for STAT3 was performed
Mentions: To assess the direct effects of LLL12 on pSTAT3, two OSCC cell lines with high (SCCF2) and low (SCCF3) basal STAT3 activation were chosen for further analysis. Cells were treated with DMSO or 0.2 μM LLL12 for 12 h and Western blotting for pSTAT3 and STAT3 was performed. Western blot analyses revealed that pSTAT3 and, to a lesser degree, total STAT3, were both downregulated in SCCF2 following treatment with LLL12 (Fig. 3a). In contrast, no change in pSTAT3 or STAT3 expression was observed in the SCCF3 cell line.Fig. 3

Bottom Line: Exposure of SCCF2 cells to LLL12 resulted in decreased expression of pSTAT3 and total STAT3, apoptosis as assessed by caspase 3/7 activation, inhibition of colony formation and reduced expression of the STAT3 transcriptional target survivin.The OSCC cell lines with low basal STAT3 phosphorylation did not exhibit these effects, suggesting that STAT3 inhibition was responsible for the observed results.However, given the up-regulation of several STAT3 transcriptional targets following treatment, further investigation of STAT3 and its related signaling pathways in OSCC is warranted.

View Article: PubMed Central - PubMed

Affiliation: Department of Veterinary Clinical Sciences, College of Veterinary Medicine, The Ohio State University, Columbus, OH, USA.

ABSTRACT

Background: Signal transducer and activator of transcription 3 (STAT3) plays a critical role in tumor development by regulating signaling pathways involved in cell proliferation, survival, metastasis and angiogenesis. STAT3 is activated in many cancers, including head and neck squamous cell carcinoma (HNSCC) in people. Feline oral squamous cell carcinoma (OSCC) is similar to advanced or recurrent HNSCC as it is poorly responsive to traditional therapies and carries a poor long-term prognosis. The purpose of this study was to characterize expression and activation of STAT3 in feline OSCC cell lines and tumor samples and to investigate the biologic activity of a novel, allosteric STAT3 inhibitor, LLL12, in feline OSCC cell lines.

Results: We evaluated 3 feline OSCC cell lines and one of these (SCCF2) exhibited high levels of constitutive STAT3 phosphorylation and high sensitivity to LLL12 treatment. Exposure of SCCF2 cells to LLL12 resulted in decreased expression of pSTAT3 and total STAT3, apoptosis as assessed by caspase 3/7 activation, inhibition of colony formation and reduced expression of the STAT3 transcriptional target survivin. In contrast, the STAT3 transcriptional targets VEGF and MCL-1 increased after LLL12 treatment. This was, in part, likely due to LLL12 mediated upregulation of HIF-1α, which is known to drive VEGF and MCL-1 expression. The OSCC cell lines with low basal STAT3 phosphorylation did not exhibit these effects, suggesting that STAT3 inhibition was responsible for the observed results. Lastly, immunohistochemistry for pSTAT3 was performed using a feline OSCC tissue microarray, demonstrating expression in 48 % of samples tested.

Conclusions: These data demonstrate that LLL12 has biologic activity against a feline OSCC cell line expressing pSTAT3 and that STAT3 represents a target for therapeutic intervention in this disease. However, given the up-regulation of several STAT3 transcriptional targets following treatment, further investigation of STAT3 and its related signaling pathways in OSCC is warranted.

No MeSH data available.


Related in: MedlinePlus