Protein inhibitor of activated STAT3 expression in lung cancer - PubMed (original) (raw)

Protein inhibitor of activated STAT3 expression in lung cancer

Amy Kluge et al. Mol Oncol. 2011 Jun.

Abstract

Protein Inhibitor of Activated Signal Transducer and Activators of Transcription 3 (PIAS3) is an endogenous inhibitor of STAT3 transcriptional activity. We have previously demonstrated the concentration-dependent negative regulatory effect of PIAS3 on STAT3 signaling and its capacity to decrease lung cancer proliferation and synergize with epidermal growth factor inhibition. We now investigate PIAS3 expression in both non-small cell lung cancer (NSCLC) cell lines and human resected NSCLC specimens. We also investigated the mechanism by which some lung cancers have significantly decreased PIAS3 expression. Expression of PIAS3 is variable in lung cancer cells lines with 2 of 3 squamous cell carcinoma (SCC) cell lines having no or little PIAS3 protein expression. Similarly, the majority of human SCCs of the lung lack PIAS3 expression by immunohistochemistry; this despite the finding that SCCs have significantly higher levels of PIAS3 mRNA compared to adenocarcinomas. High PIAS3 expression generally correlates with decreased phosphorylated STAT3 in both SCC cell lines and human specimens compatible with the negative regulatory effect of this protein on STAT3 signaling. To investigate this variable expression of PIAS3 we first performed sequencing of the PIAS3 gene that demonstrated single nucleotide polymorphisms but no mutations. Exposure of lung cancer cells to 5-azacytidine and trichostatin A results in a significant increase in PIAS3 mRNA and protein expression. However, methylation-specific PCR demonstrates a lack of CpG island methylation in the promoter region of PIAS3. Exposure of cells to an agent blocking proteosomal degradation results in a significant increase in PIAS3. Our data thus shows that SCC of the lung commonly lacks PIAS3 protein expression and that post-translational modifications may explain this finding in some cases. PIAS3 is a potential therapeutic molecule to target STAT3 pathway in lung cancer.

Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

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Figures

Figure 1

Figure 1

Western blot demonstrates variable expression of PIAS3 in NSCLC cell lines. Phosphorylated STAT3 expression and STAT3 expression were also evaluated. β‐actin was used as a protein loading control.

Figure 2

Figure 2

Immunohistochemistry for PIAS3 in resected lung cancer. Panel A demonstrates a squamous cell carcinoma with no PIAS3 staining and panel B demonstrates an adenocarcinoma with 3+ staining.

Figure 3

Figure 3

Single nucleotide polymorphisms found in A549 sense sequence, change from C/T or G/T. Verified the SNP changes by sequencing the A549 antisense and confirmed the G/A and C/A changes. These C/T and G/T SNPs were found in other sequenced cell lines, both adenocarcinomas and squamous cell carcinomas, as well as in NuLi cells, an immortalized normal lung epithelial cell line.

Figure 4

Figure 4

A‐ PIAS3 expression and β‐actin expression in H1650 cells. Cells were treated with 5AZA for 48 h followed by a 24 h treatment with TSA. B‐ Relative expression of PIAS3 standardized to β‐actin expression shows that 5AZA increased PIAS3 protein but that the combination of 5AZA and TSA increased expression about 8‐fold higher. C‐ A ∼3‐fold increase in relative PIAS3 mRNA expression in H1650 cells can be seen after exposure to 5AZA + TSA by real‐time quantitative PCR.

Figure 5

Figure 5

Localization of PIAS3 qMSP assays on Chromosome 1: Transcription Start Site (TSS) is shown as pink vertical lines, the green box represents the first exon, and the open box represents the PIAS3 promoter region. Open red boxes represent location of PIAS3 qMSP assays used for methylation profiling.

Figure 6

Figure 6

Relative mRNA expression of adenocarcinoma v. squamous cell carcinoma cell lines.

Figure 7

Figure 7

Post‐translational proteosomal degradation by MG‐132 in adenocarcinoma v. squamous cell carcinoma cell lines.

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