Evaluation of Caspase-9b and PP2Acα2 as potential biomarkers for chronic lymphocytic leukemia
© Domínguez-Berrocal et al. 2016
Received: 17 February 2016
Accepted: 26 April 2016
Published: 4 May 2016
Disruption of alternative splicing in apoptotic factors has been associated to chronic lymphocytic leukemia among other cancers and hematological malignancies. The proapoptotic proteins Caspase-9 and PP2Acα are functionally related in a direct interaction, which constitutes a promising target for cancer therapy. Both proteins present aberrant mRNA splicing variants that are antiapoptotic (Caspase-9b) and catalytically inactive (PP2Acα2), respectively.
In this work we have analyzed the relative abundance of the aberrant spliced forms Caspase-9b and PP2Acα2 in several cell lines and chronic lymphocytic leukemia patients and correlated it with several parameters of the disease. Despite 40 % of the patients presented Caspase-9b dysregulation, there was no direct association between alterations in Caspase-9b relative abundance and the parameters analyzed in medical records. More importantly, PP2Acα2 dysregulation was observed in 88 % of CLL patients and was related with advanced stages of the malignancy.
Caspase-9b dysregulation seemed to be associated with the disease, although the differences between healthy donors and CLL patients were not statistically significant. However, PP2Acα2 dysregulation was significantly different between healthy donors and CLL patients and correlated with Binet B and C stages; therefore, we propose the use of PP2Acα2 dysregulation as a potential biomarker for advanced stages of chronic lymphocytic leukemia.
KeywordsCaspase-9 Caspase-9b PP2Acα PP2Acα2 Chronic lymphocytic leukemia Alternative splicing Biomarker
Chronic lymphocytic leukemia (CLL) is the most common B-cell malignancy in Caucasian aging adults, rarely younger than 50 years old . Disruption of alternative splicing in many apoptotic factors is related to hematological malignancies and cancer, as CLL [2–6]. Abnormally expressed splicing factors in tumor cells induce the production of mRNA isoforms that are nonexistent or less abundant in normal cells, thus contributing to cancer development, tumor progression, different response to therapy and chemorefractoriness [7, 8].
Caspase-9 is a key point in the apoptotic signal transduction. The expression of its mRNA spliced variant Caspase-9b, lacking exons 3 to 6, inhibits apoptosis in a dominant-negative manner , which may establish a threshold to regulate Caspase-9 activation and prevent undesired apoptosis . Caspase-9b is dysregulated in astrocytoma  and in several subtypes of non-small-cell lung cancer (NSCLC). Moreover, its overexpression was responsible of maintaining the tumorigenic capacity of NSCLC cells and made cells resistant to erlotinib . Targeting the alternative splicing of Caspase-9 sensitized NSCLC cells to chemotherapies, increasing their efficiency and limiting their toxic side-effects .
The direct interaction between Caspase-9 and PP2Acα has been previously described . PP2A is one of the major Ser/Thr phosphatases, whose dysregulation is associated with multiple cancers among other functions . A catalytically inactive aberrant isoform of PP2A catalytic C subunit, PP2Acα2, has been reported, missing exon 5, which is close to the active site. PP2Acα2 has only been observed overexpressed in peripheral blood mononuclear cells (PBMC) under starvation conditions but the ratio PP2Acα:PP2Acα2 was reestablished once they were transferred to culture medium. When analyzed in cell lines, PP2Acα2 was present either as mRNA or as protein but at almost undetectable levels .
The aim of the present work was to study the expression ratios Caspase-9: Caspase-9b and PP2Acα:PP2Acα2 in cell lines and more importantly in healthy donors and CLL patients to evaluate their association with the disease.
Material and methods
Cell lines culture
HeLa cells were cultured in DMEM + 10 % FBS, Daudi and Jurkat cells in RPMI 1640 + 10 % FBS and SH-SY5Y in DMEM F12 + 10 % FBS, 1 % Glutamine and 1 % Hepes 1 M. All cell lines were grown at 37 °C and 5 % CO2 and underwent passage three times a week.
B cells isolation
Fresh blood from healthy donors was obtained from the Établissement français du sang. CLL samples were obtained from the Hematology Department of Saint Louis hospital (Paris). PBMC were isolated by Ficoll gradient centrifugation for 20 min at 2300 rpm, they were collected and washed twice with PBS. B cells were isolated using DynaI negative isolation kit (Invitrogen), reaching around 98 % purity.
Conventional PCR and Real Time PCR
Total RNA was extracted with TRIzol® (Life Technologies), cDNA was obtained performing RT-PCR using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) and conventional PCR was set up with Kapa HiFi DNA polymerase (Kapa Biosystems) at 95 °C-3’, 30 cycles of 98 °C-20”, 55 °C-20”, 72 °C-1’30” and a final extension of 72 °C-5’. The primers used were FWD: 5’-ATGGACGAAGCGGATCGG-3’ and REV: 5’-TTATGATGTTTTAAAGAAAAGTT-3’ for Caspase-9 and FWD 5’-GACGAGAAGGTGTTCACCAA-3’ and REV 5’-TTACAGGAAGTAGTCTGGGGTAC-3’ for PP2Acα.
Real Time PCR was performed using TaqMan PCR Mastermix in a 7500 Fast Real Time Applied Biosystems device. Caspase9 and PP2Acα probes corresponded to Hs00154261_m1 and Hs01003394_mH Life Technologies references. Caspase9b probe was previously described  and PP2Acα2 primers and probe were designed: 5’-CAAGAAGTTCCCCATGAGGGATATA-3’ (forward), 5’-CAACGATAACAATAGTTTGGAGCACT-3’ (reverse) and 5’-CGTTACTACATTCCGGTCATGGCACCA-3’ (probe). GAPDH was used as housekeeping reference gene (Hs99999905_m1, Life Technologies). Data analysis of relative expression was calculated following the 2-ΔΔC T method . Differences between healthy donors and CLL patients were statistically evaluated with Student's t test and with a Two-way ANOVA with replication with a significance level of P < 0.005.
Results and discussion
Caspase-9b and PP2Acα2 expression in cell lines
Caspase-9b and PP2Acα2 expression in healthy donors and CLL patients
A cohort of 24 healthy donors and 25 CLL patients (15 men and 10 women) was analyzed by Real Time PCR. Figure 2b summarizes Caspase-9b and PP2Acα2 expression in CLL patients. 80 % of healthy donors showed Caspase-9b normal ratio and the rest were slightly over healthy values (Fig. 2c). Unlike in NSCLC , where 36 % of the patients were moderately dysregulated and 42 % were highly dysregulated, in CLL only 40 % of the patients had some alteration in Caspase-9b expression (Fig. 2d), although the highly dysregulated also overcame the moderate percentage (28 % versus 12 %).
According to Shultz et al. criteria for Caspase-9b dysregulation, 80 % of the healthy donors that we analyzed fitted into normal values for Caspase-9b. However, PP2Acα2 was expressed in healthy donors in a PP2Acα:PP2Acα2 ratio from negligible values up to 1.27 in 80 % of the samples (Fig. 2c). Therefore, we formulated classification criteria that would fit a similar percentage of healthy donors in each category as the ones described by Schultz for Caspase-9b. We established a mRNA ratio PP2Acα:PP2Acα2 ≥ 1.33 for healthy expression, 0.67 < X < 1.33 for moderately dysregulated and ≤ 0.67 for highly dysregulated. According to this, 88 % of CLL patients analyzed had overexpression levels of PP2Acα2 (Figs. 2b and d). The efficacy of the use of PP2Acα2 as a CLL biomarker and the applicability of our criteria was validated with the calculation of several clinical parameters. The prevalence of the disease in the whole cohort was 51.02 % and the use of PP2Acα2 as a biomarker presented a sensitivity of 78.57 %, a specificity of 85.71 %, a positive predictive value of 88 % and a negative predictive value of 75 %. According to these results, the detection of a PP2Acα2 overexpression in a patient will allow to diagnose CLL in a 78.57 % of the cases. The specificity of this biomarker assures that 85.71 % of the patients without a dysregulation of PP2Acα2 don’t present the disease and only a 14.29 % would be diagnosed as false positives.
Available medical records of the CLL patients analyzed
Age at diagnosis
White blood cells
Several markers as CD38, ZAP-70 and IgVH mutation status among others have been implemented as CLL progression and diagnosis predictors not without certain controversy [22–24]; therefore we propose also the use of PP2Acα2 high dysregulation as a potential CLL biomarker associated to severe stages of the disease.
Disruption of the PP2Acα:PP2Acα2 ratio in CLL patients may also alter the equilibrium of Caspase-9/PP2Acα interaction, giving rise to aberrant Caspase-9/PP2Acα2 complexes. Interestingly, we have previously reported a bifunctional peptide termed DPT-C9h, capable of dissociating Caspase-9/PP2Acα complex . Given that Caspase-9 binding site is present in both PP2Acα and PP2Acα2, DPT-C9h may also interfere in the hypothetical Caspase-9/PP2Acα2 interaction, contributing to a possible modulation of the DPT-C9h mechanism of action and differential treatment response .
This bifunctional peptide induced apoptosis in CLL B cells without affecting healthy B cells nor the rest of peripheral blood mononuclear cells . The bifunctional peptide DPT-C9h was also capable of inducing apoptosis in several cancer cell lines and in tumor xenograft models with treatment response in a different extent, which may be related to the relative abundance of PP2Acα2 in the cell lines and patients tested . Further experiments will be performed to shed light on the role of PP2Acα2 in CLL and the hypothetical differential response to DPT-C9h depending on the level of PP2Acα2 dysregulation.
Caspase-9b has been related to disease state in astrocytoma  and to play an important role in NSCLC treatment response . Our results provide first evidences for the presence of aberrant PP2Acα/PP2Acα2 ratios in CLL advanced stage patients. Therefore, the dysregulation of the splicing variants of the association Caspase-9/PP2Acα is emerging as a valuable tool as biomarkers for prognosis in cancers that present aberrant expression of Caspase-9b or PP2Acα2 spliced variants. Further studies will need to be performed to evaluate Caspase-9/Caspase-9b and PP2Acα/PP2Acα2 ratios in other types of cancer and their possible application as biomarkers.
Ethics approval and consent to participate
The experiments with human samples included in this work were performed in accordance with the Declaration of Helsinki. No ethics committee approval was required for these experiments. Written informed consent was obtained from all patients.
chronic lymphocytic leukemia
Dulbecco’s modified Eagle medium
drug phosphatase technology
fetal bovine serum
non-small-cell lung cancer
peripheral blood mononuclear cells
reverse transcription polymerase chain reaction
This work was supported by grants from the Ministerio de Economía y Competitividad (Spain) SAF2012-31405, SAF2015-67077-R and the Generalitat Valenciana (Spain) (PROMETEO/2012/061) for sample preparation, experiment performance, data collection and analysis and manuscript writing and INSERM (France) for sample extraction, preparation and transport. We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- Rozman C, Montserrat E. Chronic lymphocytic leukemia. N Engl J Med. 1995;333:1052–7.View ArticlePubMedGoogle Scholar
- Venables JP. Unbalanced alternative splicing and its significance in cancer. BioEssays. 2006;28:378–86.View ArticlePubMedGoogle Scholar
- Makishima H, Visconte V, Sakaguchi H, Jankowska AM, Kar SA, Jerez A, Przychodzen B, Bupathi M, Guinta K, Afable MG, Sekeres MA, Padgett RA, Tiu RV, Maciejewski JP: Mutations in the spliceosome machinery, a novel and ubiquitous pathway in leukemogenesis. Blood. 2012;119:3203–10.View ArticlePubMedPubMed CentralGoogle Scholar
- Klein A, Miera O, Bauer O, Golfier S, Schriever F. Chemosensitivity of B cell chronic lymphocytic leukemia and correlated expression of proteins regulating apoptosis, cell cycle and DNA repair. Leukemia. 2000;14:40–6.View ArticlePubMedGoogle Scholar
- Ringshausen I, Schneller F, Bogner C, Hipp S, Duyster J, Peschel C, Decker T: Constitutively activated phosphatidylinositol-3 kinase (PI-3 K) is involved in the defect of apoptosis in B-CLL: Association with protein kinase Cδ. Blood. 2002;100:3741–8.View ArticlePubMedGoogle Scholar
- Packham G, Stevenson FK. Bodyguards and assassins: Bcl-2 family proteins and apoptosis control in chronic lymphocytic leukaemia. Immunology. 2005;114:441–9.View ArticlePubMedPubMed CentralGoogle Scholar
- Sampath J, Long PR, Shepard RL, Xia X, Devanarayan V, Sandusky GE, Perry WL, Dantzig AH, Williamson M, Rolfe M, Moore RE: Human SPF45, a splicing factor, has limited expression in normal tissues, is overexpressed in many tumors, and can confer a multidrug-resistant phenotype to cells. Am J Pathol. 2003;163:1781–90.
- Wang L, Lawrence MS, Wan Y, Stojanov P, Sougnez C, Stevenson K, Werner L, Sivachenko A, DeLuca DS, Zhang L, Zhang W, Vartanov AR, Fernandes SM, Goldstein NR, Folco EG, Cibulskis K, Tesar B, Sievers QL, Shefler E, Gabriel S, Hacohen N, Reed R, Meyerson M, Golub TR, Lander ES, Neuberg D, Brown JR, Getz G, Wu CJ. SF3B1 and other novel cancer genes in chronic lymphocytic leukemia. N Engl J Med. 2011;365:2497–506.
- Seol DW, Billiar TR. A caspase-9 variant missing the catalytic site is an endogenous inhibitor of apoptosis. J Biol Chem. 1999;274:2072–6.View ArticlePubMedGoogle Scholar
- Srinivasula SM, Ahmad M, Guo Y, Zhan Y, Lazebnik Y, Fernandes-Alnemri T, Alnemri ES: Identification of an endogenous dominant-negative short isoform of caspase-9 that can regulate apoptosis. Cancer Res. 1999;59:999–1002.
- Waltereit R, Weller M. The role of caspases 9 and 9-short (9S) in death ligand- and drug-induced apoptosis in human astrocytoma cells. Mol Brain Res. 2002;106:42–9.View ArticlePubMedGoogle Scholar
- Shultz JC, Goehe RW, Murudkar CS, Wijesinghe DS, Mayton EK, Massiello A, Hawkins AJ, Mukerjee P, Pinkerman RL, Park MA, Chalfant CE. SRSF1 regulates the alternative splicing of caspase 9 via a novel intronic splicing enhancer affecting the chemotherapeutic sensitivity of non-small cell lung cancer cells. Mol Cancer Res. 2011;9:889–900.View ArticlePubMedPubMed CentralGoogle Scholar
- Shultz JC, Chalfant CE. Caspase 9b: a new target for therapy in non-small-cell lung cancer. Expert Rev Anticancer Ther. 2011;11:499–502.View ArticlePubMedGoogle Scholar
- Arrouss I, Nemati F, Roncal F, Wislez M, Dorgham K, Vallerand D, Rabbe N, Karboul N, Carlotti F, Bravo J, Mazier D, Decaudin D, Rebollo A: Specific targeting of Caspase-9/PP2A interaction as potential new anti-cancer therapy. PLoS One. 2013;8:e60816.View ArticlePubMedPubMed CentralGoogle Scholar
- Lechward K, Awotunde OS, Swia̧Tek W, Muszyńska G. Protein phosphatase 2A: Variety of forms and diversity of functions. Acta Biochim Pol. 2001;48:921–33.PubMedGoogle Scholar
- Migueleti DLS, Smetana JHC, Nunes HF, Kobarg J, Zanchin NIT. Identification and characterization of an alternatively spliced isoform of the human protein phosphatase 2A catalytic subunit. J Biol Chem. 2012;287:4853–62.View ArticlePubMedPubMed CentralGoogle Scholar
- Vu NT, Park MA, Shultz JC, Goehe RW, Hoeferlin LA, Shultz MD, Smith SA, Lynch KW, Chalfant CE. HnRNP U enhances caspase-9 splicing and is modulated by AKT-dependent phosphorylation of hnRNP L. J Biol Chem. 2013;288:8575–84.View ArticlePubMedPubMed CentralGoogle Scholar
- Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2 − ΔΔCT method. Methods. 2001;25:402–8.View ArticlePubMedGoogle Scholar
- Shultz JC, Goehe RW, Wijesinghe DS, Murudkar C, Hawkins AJ, Shay JW, Minna JD, Chalfant CE:. Alternative splicing of caspase 9 is modulated by the phosphoinositide 3-kinase/Akt pathway via phosphorylation of SRp30a. Cancer Res. 2010;70:9185–96.View ArticlePubMedPubMed CentralGoogle Scholar
- Binet JL, Auquier A, Dighiero G, Chastang C, Piguet H, Goasguen J, Vaugier G, Potron G, Colona P, Oberling F, Thomas M, Tchernia G, Jacquillat C, Boivin P, Lesty C, Duault MT, Monconduit M, Belabbes S, Gremy F. A new prognostic classification of chronic lymphocytic leukemia derived from a multivariate survival analysis. Cancer. 1981;48:198–206.
- Matutes E, Owusu-Ankomah KA, Morilla RM, García Marco J, Houlihan A, Que TH, Catovsky D: The immunological profile of B-cell disorders and proposal of a scoring system for the diagnosis of CLL. Leukemia. 1994;8:1640–5.PubMedGoogle Scholar
- Rassenti LZ, Huynh L, Toy TL, Chen L, Keating MJ, Gribben JG, Neuberg DS, Flinn IW, Rai KR, Byrd JC, Kay NE, Greaves A, Weiss A, Kipps TJ. ZAP-70 compared with immunoglobulin heavy-chain gene mutation status as a predictor of disease progression in chronic lymphocytic leukemia. N Engl J Med. 2004;351:893–901.View ArticlePubMedGoogle Scholar
- Inamdar KV, Bueso-Ramos CE. Pathology of chronic lymphocytic leukemia: an update. Ann Diagn Pathol. 2007;11:363–89.View ArticlePubMedGoogle Scholar
- Matutes E, Wotherspoon A, Catovsky D. Differential diagnosis in chronic lymphocytic leukaemia. Best Pract Res Clin Haematol. 2007;20:367–84.View ArticlePubMedGoogle Scholar
- Arrouss I, Decaudin D, Choquet S, Azar N, Parizot C, Zini JM, Nemati F, Rebollo A: Cell penetrating peptides as a therapeutic strategy in chronic lymphocytic leukemia. Protein Pept Lett. 2015;22:539–46.View ArticlePubMedGoogle Scholar