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Regulation of Cancerous inhibitor of PP2A (CIP2A) by small molecule inhibitor for c-Jun NH2-Terminal Kinases (JNKs), SP600125, in Human Fibrosarcoma (HT1080) cells

[version 2; peer review: 2 approved with reservations]
PUBLISHED 11 Dec 2013
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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.

Keywords

Fibrosarcoma, HT1080, SP600125, CIP2A, JNK2, siRNA, PP2A

Revised Amendments from Version 1

The manuscript has now been revised based on the comments received from the respectable reviewer. New data showing additional controls, along with the specific explanation to clarify the siRNA based specificity issues, has been now added. Notably, an additional small siRNA screen involving kinases previously demonstrated to be sensitive to SP600125 has been carried out, and resulted in identification of CHK1 as the kinase positively regulating CIP2A expression in HT1080 cells. This is in accordance to recent observations seen in gastric, prostate, breast and cervical cancer cells (Khanna et al., Cancer Research 2013). Additionally, the requested p-values and text have been modified to accommodate and explain the new findings.

See the author's detailed response to the review by Kristopher Clark

Introduction

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 types1. Experimentally, 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. Various independent studies have shown that inhibition of PP2A activity is a pre-requisite for human cell transformation1,35. Therefore, understanding the mechanisms by which PP2A is inhibited in cancer cells is vital for developing new anti-cancer therapies.

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 cells6. Several layers of evidence, both from us and others, have shown CIP2A to be required for malignant cell growth and in vivo tumour formation6. In addition, the prognostic role of CIP2A has been demonstrated in several human tumours6. Moreover, since CIP2A overexpression has been observed at a high frequency in most human cancers studied so far6,7, identification of mechanisms regulating its expression in human cancers becomes important to address.

Although several transcription factors like MYC8, ETS19, E2F110 and ATF211 have been identified as positive regulators of CIP2A in various carcinomas, factors influencing CIP2A expression in non-hematopoietic mesenchymal cells or sarcomas, are yet to be discovered. Notably, CIP2A amplification has been observed in soft-tissue sarcomas12. In addition, since CIP2A was identified using HT1080 (human fibrosarcoma cell line) cell extracts13 this cell line was selected to dissect the mechanisms for high CIP2A expression in sarcomas. Since p38, ERK and JNK signalling pathways are commonly perturbed in cancers, we assessed the role of these pathways in CIP2A expression in HT1080 cells. To this end, respective small molecule kinase inhibitors, namely SB203580 (p38 pathway inhibitor), PD98059 (MEK pathway inhibitor) and SP600125 (JNK pathway inhibitor) were used to inhibit signalling through these pathways in HT1080 cells.

Material and methods

Chemicals

SP600125 was purchased from Calbiochem (Cat No. - 420119, Merck-Millipore CAS 129-56-6, San Diego, CA) and stocked as a 20 mM solution in DMSO. PD98059 was purchased from Calbiochem (Cat No. - 513000, Merck-Millipore, San Diego, CA) and stocked as 40 mM stock in DMSO. SB203580 was purchased from Calbiochem (Cat No. - 559389, Merck-Millipore, San Diego, CA) and stocked as 20 mM.

RNAi

The siRNAs to inhibit CIP2A expression were obtained from Eurofins MWG operon (Ebersberg, Germany). Either of the following double-stranded oligonucleotides was transiently transfected into HT1080 cell line as CIP2A siRNAs: CIP2A.1, 5´-CUGUGGUUGUGUUUGCACUTT-3´, and CIP2A.2, 5´-ACCAUUGAUAUCCUUAGAATT-3´. As a control, a scrambled siRNA with the sequence 5´-UAACAAUGAGAGCACGGCTT-3´ was used instead. HP-validated siRNAs for human JNK1 and JNK2 were purchased from Qiagen Sciences (Germantown, MD). Either of the following oligonucleotides were transiently transfected into HT1080 at 30%–50% confluency in a six-well plate were transfected with the siRNA in antibiotic free medium using RNAiMAX Reagent (Invitrogen, Carlsbad, CA), according to the manufacturer’s instructions.

Immunoblotting

Proteins were extracted in hot Laemmli sample buffer and subjected to immunoblot analysis. Thirty micrograms of total protein extracts was separated by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (Bio-Rad Laboratories, Helsinki, Finland) and transferred to nitrocellulose membranes (Thermo Scientific Pierce Protein Biology Products, Rockford, USA). Membranes were blocked with 5% nonfat milk in Tris-buffered saline (TBS; 20 mM Trizma Base and 150 mM NaCl dissolved in distilled water and adjusted with HCl to pH 7.5) containing 0.1%-NP40 (Igepal Ca-630; Sigma-Aldrich)8. Nitrocellulose membranes (Thermo Scientific Pierce Protein Biology Products, Rockford, USA) were incubated with antibodies to JNK1 (Cat. No. sc-1648: 1:500 dilution, Santa Cruz Biotechnology, Santa Cruz, CA), JNK2 (Cat. No. sc-827:1:500 dilution, Santa Cruz Biotechnology, Santa Cruz, CA) in 5% milk in TBS-NP40 (Igepal Ca-630; Sigma-Aldrich) at 4°C overnight, with a 1:5000 dilution of the rabbit polyclonal anti-CIP2A antibody8 at 4°C overnight, or with a 1:1000 dilution of goat polyclonal anti-β-Actin antibody (Cat. No. sc-47778, Santa Cruz Biotechnology) at room temperature for 1 hour.

Cell culture

HT1080 cells originally were obtained from ATCC and were cultured in DMEM (Gibco) supplemented with 10% (v/v) fetal calf serum (FCS), 2 mM glutamine, 100 units/ml penicillin and 100 µg/ml streptomycin (Bio-Whittaker Europe, Verviers, Belgium).

mRNA analysis

Total mRNA was extracted from cells using the RNeasy kit (Qiagen, Valencia, CA) and converted to cDNA by using the M-MLV Reverse Transcriptase, RNase H Minus, Point Mutant cDNA synthesis kit (Promega Corporation, Madison, WI). cDNAs were subjected to quantitative real-time polymerase chain reaction (PCR) by using the Light Cycler (Roche Diagnostics, Mannheim, Germany) and SYBR Green PCR Master Mix kit (Roche Diagnostics). Primer sequences (Sigma-Proligo, St Louis, MO) used for PCR of CIP2A were as follows: CIP2A forward, 5´-CTGGTGAGATAATCAGCAATTT-3´ and CIP2A reverse, 5´-CGAAACATTCATCAGACTTTTCA-3´. Transcript levels were normalized to levels of TATA-binding protein (TBP) or β-Actin expression, which were determined by PCR of the same samples using the following primers: TBP forward, 5´-GAATATAATCCCAAGCGGTTTG-3´, and TBP reverse, 5´-ACTTCACATCACAGCTCCCC-3´; Actin forward, 5´-CGAGCACAGAGCCTCGCCTTTGC-3´, and Actin reverse: 5´-CATAGGAATCCTTCTGACCCATG-3´.

Bioinformatics

Cancer cell line encyclopaedia (http://www.broadinstitute.org/software/cprg/?q=node/11) was used to get the expression levels of JNK1 and JNK2 in HT1080 cells14. This is a resource which provides analysis and visualization of DNA copy number, mRNA expression, mutation data and more, for 1000 cancer cell lines.

Statistics

Student T-test was used obtain the statistical significance value using Graph Pad software.

Results

SP600125 inhibits CIP2A mRNA and protein expression in a time and concentration dependent manner in HT1080 cells

To determine the oncogenic signalling pathways that maybe involved in regulating CIP2A expression in human fibrosarcoma, HT1080 cells were treated with small molecule inhibitors for the p38 (SB203580; 20 µM), JNK (SP600125; 10 µM) and ERK (PD98058; 20 µM) signalling pathways. As previously observed in gastric cancer cells9, PD98058 reduced CIP2A mRNA and protein expression in HT1080 cells (Figure 1A and B). Importantly, while SB203580 showed negligible effect, SP600125 potently inhibited CIP2A mRNA and protein expression in HT1080 (Figure 1A and B) cells. Furthermore, inhibition of CIP2A protein expression by SP600125 was observed to be both time and concentration dependent (Figure 1C and D).

f7dd5dc2-bb91-48a8-b0fa-8b1f829b43d9_figure1.gif

Figure 1. SP600125 positively regulates CIP2A expression in HT1080 cells.

A. qRT-PCR showing the effect of small molecule inhibitors against the p38 (SB203580), MEK-ERK (PD98059) and JNK (SP600125) pathways on CIP2A mRNA expression (12 h timepoint; Shown is mean values + S.D., or representative results from three independent experiments. Student T-test was used obtain the statistical significance value) B. Western blot showing the effect of small molecule inhibitors against the p38 (SB203580), MEK-ERK (PD98059) and JNK (SP600125) pathways on CIP2A protein expression (24 h timepoint). Shown is the representative picture of two independent experiments. C and D. Effect of small molecule inhibitors against JNK (SP600125) pathway on CIP2A protein expression in concentration (C) and time (D) dependent manner. Shown is the representative picture of two independent experiments.

Neither JNK1 nor JNK2 regulate CIP2A protein expression in HT1080

SP600125-mediated inhibition of CIP2A expression suggested the involvement of the c-Jun N-Terminal Kinases (JNKs) in regulation of CIP2A expression in HT1080 cells. Since the JNK3 isoform is well known to be neural-specific15, specific validated siRNAs against JNK1 and JNK2 isoforms were transfected into HT1080 cells and CIP2A expression estimated. Although, both JNK1 and JNK2 siRNAs reduced their target proteins (JNK1 and JNK2 respectively), there was no change in CIP2A protein expression with either of them individually (Figure 2A,B and Supplementary Figure 1A) or in combination (Figure 2C). Since the JNK2 isoform is expressed more than 10-fold higher than the JNK1 isoform in the HT108014 (Figure 2D) cell line we transfected two validated and specific siRNAs against JNK2. In addition, we also transfected the HT1080 cells with two different CIP2A siRNA as positive controls. In line with our previous observation (Figure 2B and C), the two different siRNAs for JNK2 knocked out JNK2 expression (Figure 2E), while CIP2A expression remained unaltered. Surprisingly, two different CIP2A siRNA, which worked well as positive controls, efficiently decreased JNK2 expression (Figure 2E). SP600125 has been demonstrated to inhibit various kinases like CDK2, DYRK1 and AMPK even more effectively than JNK kinase itself16 (Supplementary Table 1). Interestingly, recently CHK1 kinase (also inhibited by SP600125; Supplementary Table 1) was shown to inhibit CIP2A levels both in vitro and in vivo6,17,18. Therefore, we transfected HT1080 cells with 2 different siRNAs against CHK1 (Kinase almost as sensitive to SP600125 as JNK) and with siRNAs against CDK2 and DYRK1 (two other kinases that are more sensitive to SP600125 than JNK). As shown in Supplementary Figure 1B, two different siRNAs against CHK1 decreased CIP2A expression n HT1080 cells.

f7dd5dc2-bb91-48a8-b0fa-8b1f829b43d9_figure2.gif

Figure 2. JNK1 and JNK2 do not regulate CIP2A expression in HT1080 cells.

A, B, and C. Western blots showing the effect of JNK1 (A), JNK2 (B) and combination of both JNK1 and JNK2 (C) siRNAs on CIP2A protein expression, 72 h post-transfection. Shown is a representative result from two independent experiments. D. mRNA expression of JNK1 and JNK2 in HT1080 cell line from the cancer cell encyclopaedia study. E. Western blot showing the effect of two different siRNAs specific for JNK2 and CIP2A proteins and their protein expression levels 72 h post-transfection. The numbers below the blot are the quantified values for CIP2A and JNK2 protein levels normalized to Actin protein levels, relative to the levels in Scrambled (control) transfected cells. Shown is a representative result of two independent experiments.

Discussion

Even though small molecule inhibitors are an emerging therapeutic option against cancers, the specificity issues limit their potential to be used in clinics. They have been extensively used to study various cell signalling pathways. In particular SP600125 has been used to study the effect of C-Jun N-Terminal Kinases (JNKs) in various processes1921. Our results suggest that even though we were able to see decrease in CIP2A expression in HT1080 cells on treatment with SP600125 (Figure 1), at doses used previously to inhibit c-Jun NH2-terminal kinase (JNKs) activity22,23, we were not able to validate the findings using two different siRNAs specific to the JNK2 kinase (Figure 2E). Interestingly, a decade ago a previous study emphatically demonstrated the effect of SP600125 on different kinases16. The study revealed that SP600125, although a JNK inhibitor, could inhibit the activity of several other kinases like CDK2, DYRK1 and CHK116. Notably, we here identify CHK1 as the potential SP600125 sensitive kinase that positively regulates CIP2A expression in HT1080 cells (Supplementary Figure 1B).

Surprisingly, two different CIP2A siRNAs used as positive controls, decreased JNK2 expression levels in HT1080. This has also been observed in a separate study in an epithelial origin cell line, HeLa24. Interestingly, JNK2 has been shown to regulate CIP2A expression via ATF2 transcription factor in mouse embryo fibroblasts (MEFs)11 in the pre-transformed stage. Since we observe the vice versa in fully transformed HT1080 cells, it can suggest that there may be a molecular switch between JNK2 and CIP2A which may have a possible role in the RAS-transformation of mesenchymal cells. Nevertheless, the functional consequence of CIP2A-mediated JNK2 expression in mesenchymal cells would require further exploration.

Altogether, our study highlights the need for the validation of results obtained by small molecule treatments with independent approaches like two or more target specific siRNAs, shRNAs or use of inducible systems like RNAi or Tamoxifen/Tetracycline-induced overexpression systems8.

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Khanna A. Regulation of Cancerous inhibitor of PP2A (CIP2A) by small molecule inhibitor for c-Jun NH2-Terminal Kinases (JNKs), SP600125, in Human Fibrosarcoma (HT1080) cells [version 2; peer review: 2 approved with reservations]. F1000Research 2013, 2:174 (https://doi.org/10.12688/f1000research.2-174.v2)
NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article.
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ApprovedThe paper is scientifically sound in its current form and only minor, if any, improvements are suggested
Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.
Not approvedFundamental flaws in the paper seriously undermine the findings and conclusions
Version 2
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PUBLISHED 11 Dec 2013
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Reviewer Report 26 Aug 2014
Tony Hunter, Salk Institute Cancer Center, Salk Institute for Biological Studies, La Jolla, CA, USA 
Approved with Reservations
VIEWS 28
Here the author showed that the expression of CIP2A (Cancerous Inhibitor of PP2A) in HT080 human fibrosarcoma cells is inhibited by treatment with the PD98059 MEK inhibitor, as previously reported, and also more strongly by the SP600125 JNK MAPK inhibitor, ... Continue reading
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Hunter T. Reviewer Report For: Regulation of Cancerous inhibitor of PP2A (CIP2A) by small molecule inhibitor for c-Jun NH2-Terminal Kinases (JNKs), SP600125, in Human Fibrosarcoma (HT1080) cells [version 2; peer review: 2 approved with reservations]. F1000Research 2013, 2:174 (https://doi.org/10.5256/f1000research.3258.r4566)
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
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Reviewer Report 07 Feb 2014
Kristopher Clark, MRC Protein Phosphorylation Unit, University of Dundee, Dundee, UK 
Approved with Reservations
VIEWS 41
In the revised version, Dr Khanna has re-emphasized the important drawbacks of using non-specific kinase inhibitors such as SP600125 to support a role for the c-Jun N-terminal Kinases in cells. The new data help demonstrate that the author could deplete ... Continue reading
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Clark K. Reviewer Report For: Regulation of Cancerous inhibitor of PP2A (CIP2A) by small molecule inhibitor for c-Jun NH2-Terminal Kinases (JNKs), SP600125, in Human Fibrosarcoma (HT1080) cells [version 2; peer review: 2 approved with reservations]. F1000Research 2013, 2:174 (https://doi.org/10.5256/f1000research.3258.r2719)
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
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Reviewer Report 20 Aug 2013
Kristopher Clark, MRC Protein Phosphorylation Unit, University of Dundee, Dundee, UK 
Approved with Reservations
VIEWS 41
Inhibition of the protein phosphatase PP2A contributes to the acquisition of a cancerous phenotype in cells. One mechanism by which the inhibition of PP2A can be achieved is through the up-regulation of endogenous inhibitors of PP2A such as CIP2A. However, ... Continue reading
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Clark K. Reviewer Report For: Regulation of Cancerous inhibitor of PP2A (CIP2A) by small molecule inhibitor for c-Jun NH2-Terminal Kinases (JNKs), SP600125, in Human Fibrosarcoma (HT1080) cells [version 2; peer review: 2 approved with reservations]. F1000Research 2013, 2:174 (https://doi.org/10.5256/f1000research.2042.r1558)
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
  • Author Response 11 Dec 2013
    Anchit Khanna, Adult Cancer Program, Lowy Cancer Research Centre and Prince of Wales Hospital, UNSW Medicine, University of New South Wales, Sydney, Australia
    11 Dec 2013
    Author Response
    • ''The data as presented does not confirm or refute a role for the JNKs in regulating CIP2A expression in HT1080 cells, as alternative explanations for the data remain possible. For
    ... Continue reading
COMMENTS ON THIS REPORT
  • Author Response 11 Dec 2013
    Anchit Khanna, Adult Cancer Program, Lowy Cancer Research Centre and Prince of Wales Hospital, UNSW Medicine, University of New South Wales, Sydney, Australia
    11 Dec 2013
    Author Response
    • ''The data as presented does not confirm or refute a role for the JNKs in regulating CIP2A expression in HT1080 cells, as alternative explanations for the data remain possible. For
    ... Continue reading

Comments on this article Comments (0)

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Alongside their report, reviewers assign a status to the article:
Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested
Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.
Not approved - fundamental flaws in the paper seriously undermine the findings and conclusions
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