Abstract
Background: Protein phosphatase 2A inhibition is one of the pre-requisites for human cell transformation. Previously, we have identified an endogenous inhibitor of PP2A, CIP2A (Cancerous Inhibitor of Protein Phosphatase 2A) in human fibrosarcoma cells (HT1080) using tandem affinity purification. CIP2A over expression has been demonstrated in almost every tumour type studied so far. However, our understanding on the mechanisms regulating CIP2A expression in human cancers, especially in sarcomas, is still emerging. Methods: Human fibrosarcoma (HT1080) cells were treated with small molecule inhibitors against the three major signalling pathways, namely p38, MEK and JNK pathways to identify the pathway regulating CIP2A expression in the sarcoma cells. This was followed by verification of the results using small interfering RNAs (siRNA) for the kinases. Results: In line with previous observations, small molecule inhibitor for MEK pathway (PD98059) decreased CIP2A mRNA and protein expression. Interestingly, small molecule inhibitor for the JNK pathway, SP600125 decreased mRNA and protein levels of CIP2A oncoprotein with negligible effect of SB203580 (p38 kinase) inhibitor on CIP2A expression in HT1080 cells. However, siRNAs specific to either JNK1 or JNK2 kinases did not result in decrease in CIP2A expression. Contrarily, two different CIP2A siRNAs, which were used as positive controls, decreased JNK2 expression in HT1080 cells. Conclusion: Although it is well established that SP600125 inhibits JNK kinases, it has also been shown to inhibit a spectra of other kinases. SP600125 inhibits CIP2A protein expression both in time and concentration dependent manner. However, depletion of both JNK1 and JNK2 kinases using specific siRNAs fails to decrease CIP2A protein expression levels, thereby indicating the need to verify the results obtained by treatment with small molecular inhibitors of kinases by independent approaches like two different target specific siRNAs. Finally, fortuitously we identify JNK2 as a CIP2A downstream target in HT1080 cells.
Highlights
It has been recently established that regardless of phenotypic variability between different cancer types, perturbation of a limited number of genetic elements is sufficient to induce transformation in different human cell types[1]
It was demonstrated that activation of RAS and telomerase (TERT), along with inactivation of the tumour suppressor proteins P53 and Retinoblastoma protein (RB) can immortalize a variety of human cell types, which can subsequently transform to a tumourigenic state in response to inhibition of protein phosphatase 2A (PP2A)[1,2]
Either of the following double-stranded oligonucleotides was transiently transfected into HT1080 cell line as Cancerous Inhibitor of Protein Phosphatase 2A (CIP2A) small interfering RNAs (siRNA): CIP2A.1, 5 ́-CUGUGGUUGUGUUUGCACUTT-3 ́, and CIP2A.2, 5 ́-ACCAUUGAUAUCCUUAGAATT-3 ́
Summary
It has been recently established that regardless of phenotypic variability between different cancer types, perturbation of a limited number of genetic elements is sufficient to induce transformation in different human cell types[1]. Cancerous Inhibitor of Protein Phosphatase 2A (CIP2A) is a recently identified oncogene, which has been demonstrated to inhibit the endogenous tumour suppressive activity of PP2A in cancer cells[6]. Methods: Human fibrosarcoma (HT1080) cells were treated with small molecule inhibitors against the three major signalling pathways, namely p38, MEK and JNK pathways to identify the pathway regulating CIP2A expression in the sarcoma cells. This was followed by verification of the results using small interfering RNAs (siRNA) for the kinases. Depletion of both JNK1 and JNK2 kinases using specific siRNAs fails to decrease CIP2A protein expression levels, thereby indicating the need to verify the results obtained by treatment with
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