- Letter to the Editor
- Open Access
p27KIP1 and PTEN cooperate in myeloproliferative neoplasm tumor suppression in mice
© Shao et al 2016
Received: 26 April 2016
Accepted: 10 June 2016
Published: 30 June 2016
PTEN acts as a phosphatase for PIP3 and negatively regulates the PI3K/AKT pathway, and p27KIP1 is a cyclin-dependent kinase inhibitor that regulates the G1 to S-phase transition by binding to and regulating the activity of cyclin-dependent kinases. Genetic alterations of PTEN or CDKN1B (p27KIP1) are common in hematological malignancies. To better understand how mutations in these two genes might cooperate in leukemogenesis, we inactivated both genes in the hematological compartment in mice. Here, we show that the combined inactivation of Pten and Cdkn1b results in a more severe myeloproliferative neoplasm phenotype associated with lower hemoglobin, enlarged spleen and liver, and shorter lifespan compared to inactivation of Pten alone. More severe anemia and increased myeloid infiltration and destruction of the spleen contributed to the earlier death of these mice, and elevated p-AKT, cyclin D1, and cyclin D3 might contribute to the development of this phenotype. In conclusion, PTEN and p27KIP1 cooperate in tumor suppression in the hematological compartment.
PTEN (phosphatase and tension homolog deleted on chromosome 10) is a tumor suppressor gene located on chromosome 10q23 and is one of the most commonly mutated or deleted genes in human cancers, including acute lymphoblastic leukemia, juvenile myelomonocytic leukemia, and non-Hodgkin’s lymphoma [1, 2]. PTEN acts as a phosphatase for phosphatidylinositol-3,4,5-trisphosphate (PIP3) and negatively regulates the phosphatidylinositol 3-kinase (PI3K)/AKT pathway . The CDKN1B gene encodes p27KIP1, which belongs to the Cip/Kip family of cyclin-dependent kinase inhibitors. p27KIP1 is a key regulator of the G1 to S-phase transition by inhibiting cyclinD1/CDK4 and cyclinE/CDK2 complexes . Deletions and other cytogenetic aberrations involving CDKN1B have been reported in a variety of leukemias [5–7]. In addition, CDKN1B expression can be a useful prognostic molecular marker for acute myeloid leukemia, where low CDKN1B expression is associated with high proliferation and, therefore, with a favorable response to chemotherapy . Inactivation of the tumor-suppressor gene PTEN and lack of CDKN1B expression have been detected in some kinds of cancer, including most advanced prostate cancers and lymphomas [8, 9]. It has been shown that the combined loss of PTEN and p27KIP1 is associated with tumor cell proliferation and increased risk of recurrent disease in localized prostate cancer . Loss of PTEN expression is more frequent in anaplastic large-cell lymphoma, which strongly correlates with the loss of CDKN1B expression .
Targeted disruption of the murine Cdkn1b gene causes a gene dose-dependent increase in animal size without other gross morphologic abnormalities , and deletion of Pten in the hematopoietic compartment in mice promotes excessive proliferation of leukemogenic stem cells resulting in the development of myeloproliferative neoplasm (MPN) followed by acute leukemia . In mice, concomitant inactivation of Pten and Cdkn1b accelerates spontaneous neoplastic transformation of prostate cancer . In order to better understand the relation and clinical relevance of these two genes in the pathogenesis of hematological malignancies, we used Cre recombinase to simultaneously inactivate Pten and Cdkn1b in the hematopoietic compartment.
Results and discussion
We performed Western blot analysis to determine the knock-out efficiency and the consequences of inactivating PTEN and p27KIP1 on downstream molecules. Deficiency of PTEN or p27KIP1 was observed in the respective knock-out mice (Fig. 2g). It has been shown that PTEN activity leads to the induction of p27KIP1, which in turn can negatively regulate the transition through the cell cycle . However, the association between PTEN and p27KIP1 might be different in different kinds of tissues. A lack of convincing correlation between PTEN and p27KIP1 has been reported for ovarian carcinomas, indicating the possible existence of p27KIP1-independent pathways downstream of PTEN . In our study, we found that expression of p27KIP1 was reduced in the splenocytes of PM mice. PCM mice had higher phosphorylated AKT compared to PM, CM, and Ctrl mice (Fig. 2g), and cyclin D1 and cyclin D3 expression levels were elevated in PCM and PM mice. However, the levels of phosphorylated ERK1/2 were similar in all groups of mice. Previous studies showed the synergistic activity of PI3K/mTOR and JAK2 signaling pathway in the myeloproliferative neoplasms [17, 18], therefore it will be interesting to study the JAK2 activity in PTEN and p27KIP1 knockout mice model.
In conclusion, our results show that PTEN deficiency can promote tumor progression by a decrease in p27KIP1 levels in the hematological compartment and that PTEN and p27KIP1 have a cooperative role in leukemia suppression. In addition, our results show that elevated phosphorylated AKT, cyclin D1, and cyclin D3 might play an important role in the progression of the severe MPN phenotype.
Mice with conditional Pten fl/fl alleles (designated P) with a mixed genomic background of 129S4/SvJae and C57BL/6J were bred with Cdkn1b fl/fl mice (designated C) to generate PC mice. PC mice were bred with mice harboring the interferon (IFN)-inducible Mx1-Cre transgene (designated M) to generate PCM (Pten fl/fl Cdkn1b fl/fl Mx1-Cre), PM (Pten fl/fl Mx1-Cre), and CM (Cdkn1b fl/fl Mx1-Cre) mice. Mice without Mx1-Cre were used as healthy controls (designated Ctrl).
The mice were housed under controlled environmental conditions with free access to water and food. Illumination was on between 0600 and 1800 h. All mice were monitored daily. Groups of 4-week-old mice were injected with 400 µg polyinosinic-polycytidylic acid (pI–pC; Sigma, St Louis, MO). Blood was taken weekly and analyzed with a hematology analyzer KX-21 (Sysmex Europe, Norderstedt, Hamburg, Germany). Three weeks after injection, groups of mice were sacrificed and their tissues were harvested for further analysis. Mice were euthanized by cervical dislocation after carbon dioxide inhalation. In addition, groups of mice were kept for a survival study. If mice had ruffled fur and become listless or lost more than 10 % of their body weight, they were euthanized. All experimental protocols were approved by the regional ethical committee of the University of Gothenburg, Sweden.
Genotyping was performed by PCR amplification of genomic DNA extracted from mouse tails. The Pten fl allele was detected with forward primer 5′-CAAGCACTCTGCGAACTGAG-3′ and reverse primer 5′-AAGTTTTTGAAGGCAAGATGC-3′, yielding a 328-bp fragment from the Pten fl allele and a 156-bp fragment from the Pten + allele. The P27 fl allele was detected with forward primer 5′-TAGGGGAAATGGATAGTAGATGTTAGGACC-3′ and reverse primer 5′-GGTATAATATGGAAAGTGACTCTAATGGCC-3′, yielding a 400-bp fragment from the P27 fl allele and a 370-bp fragment from the P27 + allele. The Mx1-Cre transgene was detected with forward primer 5′-GCGGTCTGGCAGTAAAAACTATC-3′ (oIMR 1084) and reverse primer 5′-GTGAAACAGCATTGCTGTCACTT-3′ (oIMR 1085) to yield a 100 bp fragment.
Fluorescence-activated cell sorting, colony assays, and histology
Splenocytes and bone marrow cells were incubated with antibodies against Gr1 (PE-Cy7/RB6-8C5), CD11b (V450/M1/70), c-kit (PE/2B8), Sca1 (PE-Cy7/D7), Lin- (FITC) and CD45 (V500/30-F11) and analyzed with FACS Diva software (BD Biosciences, San Jose, CA, USA). For colony assays, splenocytes (1 × 105) and bone marrow cells (2 × 104) harvested from experimental mice were seeded in duplicate wells in methylcellulose medium (MethoCult M3434; StemCell Technologies, Vancouver, BC, Canada). Six days later, the numbers of colonies were scored. For bone marrow cells, on the 7th day the cultured cells were washed, collected, and replated. Histology was performed as described [19, 20].
Tissue pieces (50–100 mg) were lysed in ice-cold buffer (50 mM Tris–HCl, 120 mM NaCl, 5 mM MgCl2, 1 % Triton X-100, 0.1 % sodium dodecyl sulfate, 1 % NP-40, 20 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 2 mM orthovanadate, and the Complete Mini protease inhibitor cocktail). Lysates were homogenized, and centrifuged at 20,000g for 20 min, and equal amounts of total protein of the supernatant were size-fractionated on 10–15 % sodium dodecyl sulfate polyacrylamide gels. The proteins were transferred onto nitrocellulose membranes and incubated with antibodies against phosphorylated ERK1/2 (9106), total ERK (9102), phosphorylated AKT (9271), PTEN (9559), p27KIP1 (2552; Cell Signaling, Danvers, MA), Cyclin D1(sc-718), Cyclin D3 (sc-182), and Beta-actin (sc-47778; Santa Cruz Biotechnology, Inc., Dallas, TX). Protein bands were visualized with a horseradish peroxidase-conjugated secondary antibody (170-5046 and 170-5047; Bio-Rad Laboratories, Inc., Hercules, California) and the Enhanced Chemiluminescence Kit (Amersham, Little Chalfont, Buckinghamshire, United Kingdom). Band density was measured by Quantity One software (Bio-Rad Laboratories, Inc. USA).
Data are plotted as the mean ± SEM. Differences in the concentrations and percentages of white blood cells, the colony-forming ability of hematopoietic cells, and the proliferation of cells in culture were determined with Student’s t test. Differences in mouse survival were assessed by the Mann–Whitney U test.
phosphatase and tensin homolog
cyclin-dependent kinase inhibitor 1B
protein kinase B
Ptenfl/fl Cdkn1bfl/fl Mx1-Cre
lineage-negative (lin−), Sca-1+, c-Kit+
extracellular signal–regulated kinase
mechanistic target of rapamycin
Janus kinase 2
standard error of the mean
JS designed and carried out experimental work and wrote the manuscript. VML designed the work and wrote the manuscript. SL carried out experimental work and analysis of data. KB carried out the acquisition and analysis of data. SYW, KL analysed the data and revised the manuscript. All authors read and approved the final manuscript.
We thank Matthew Fero from Cancer Research Center, Seattle, USA for kindly providing the mouse models.
The authors declare that they have no competing interests.
Availability of data and materials
Consent for publication
Ethics approval and consent to participate
All animal experimental protocols were approved by the regional ethical committee of the University of Gothenburg, Sweden.
This study was supported by Stiftelsen Assar Gabrielssons Fond (FB14-18), internal university funding from Gothenburg University and Sahlgrenska University Hospital (to V.M.L).
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.
- Liu YL, Castleberry RP, Emanuel PD. PTEN deficiency is a common defect in juvenile myelomonocytic leukemia. Leuk Res. 2009;33:671–7.View ArticlePubMedGoogle Scholar
- Gutierrez A, Sanda T, Grebliunaite R, Carracedo A, Salmena L, Ahn Y, et al. High frequency of PTEN, PI3K and AKT abnormalities in T-cell acute lymphoblastic leukemia. Blood. 2009;114:647–50.View ArticlePubMedPubMed CentralGoogle Scholar
- Rossi DJ, Weissman IL. Pten, tumorigenesis, and stem cell self-renewal. Cell. 2006;125:229–31.View ArticlePubMedGoogle Scholar
- Chu IM, Hengst L, Slingerland JM. The Cdk inhibitor p27 in human cancer: prognostic potential and relevance to anticancer therapy. Nat Rev Cancer. 2008;8:253–67.View ArticlePubMedGoogle Scholar
- Komuro H, Valentine MB, Rubnitz JE, Saito M, Raimondi SC, Carroll AJ, et al. p27KIP1 deletions in childhood acute lymphoblastic leukemia. Neoplasia. 1999;1:253–61.View ArticlePubMedPubMed CentralGoogle Scholar
- Haferlach C, Bacher U, Kohlmann A, Schindela S, Alpermann T, Kern W, et al. CDKN1B, encoding the cyclin-dependent kinase inhibitor 1B (p27), is located in the minimally deleted region of 12p abnormalities in myeloid malignancies and its low expression is a favorable prognostic marker in acute myeloid leukemia. Haematologica. 2011;96:829–36.View ArticlePubMedPubMed CentralGoogle Scholar
- Dietrich S, Hullein J, Lee SC, Hutter B, Gonzalez D, Jayne S, et al. Recurrent CDKN1B (p27) mutations in hairy cell leukemia. Blood. 2015;126:1005–8.View ArticlePubMedGoogle Scholar
- Di Cristofano A, De Acetis M, Koff A, Cordon-Cardo C, Pandolfi PP. Pten and p27KIP1 cooperate in prostate cancer tumor suppression in the mouse. Nat Genet. 2001;27:222–4.View ArticlePubMedGoogle Scholar
- Uner AH, Saglam A, Han U, Hayran M, Sungur A, Ruacan S. PTEN and p27 expression in mature T-cell and NK-cell neoplasms. Leuk Lymphoma. 2005;46:1463–70.View ArticlePubMedGoogle Scholar
- Halvorsen OJ, Haukaas SA, Akslen LA. Combined loss of PTEN and p27 expression is associated with tumor cell proliferation by Ki-67 and increased risk of recurrent disease in localized prostate cancer. Clin Cancer Res. 2003;9:1474–9.PubMedGoogle Scholar
- Fero ML, Rivkin M, Tasch M, Porter P, Carow CE, Firpo E, et al. A syndrome of multiorgan hyperplasia with features of gigantism, tumorigenesis, and female sterility in p27(Kip1)-deficient mice. Cell. 1996;85:733–44.View ArticlePubMedGoogle Scholar
- Guo W, Lasky JL, Chang CJ, Mosessian S, Lewis X, Xiao Y, et al. Multi-genetic events collaboratively contribute to Pten-null leukaemia stem-cell formation. Nature. 2008;453:529–33.View ArticlePubMedPubMed CentralGoogle Scholar
- Wey S, Luo B, Tseng CC, Ni M, Zhou H, Fu Y, et al. Inducible knockout of GRP78/BiP in the hematopoietic system suppresses Pten-null leukemogenesis and AKT oncogenic signaling. Blood. 2012;119:817–25.View ArticlePubMedPubMed CentralGoogle Scholar
- Kogan SC, Ward JM, Anver MR, Berman JJ, Brayton C, Cardiff RD, et al. Bethesda proposals for classification of nonlymphoid hematopoietic neoplasms in mice. Blood. 2002;100:238–45.View ArticlePubMedGoogle Scholar
- Sun H, Lesche R, Li DM, Liliental J, Zhang H, Gao J, et al. PTEN modulates cell cycle progression and cell survival by regulating phosphatidylinositol 3,4,5,-trisphosphate and Akt/protein kinase B signaling pathway. Proc Natl Acad Sci USA. 1999;96:6199–204.View ArticlePubMedPubMed CentralGoogle Scholar
- Kurose K, Zhou XP, Araki T, Cannistra SA, Maher ER, Eng C. Frequent loss of PTEN expression is linked to elevated phosphorylated Akt levels, but not associated with p27 and cyclin D1 expression, in primary epithelial ovarian carcinomas. Am J Pathol. 2001;158:2097–106.View ArticlePubMedPubMed CentralGoogle Scholar
- Bartalucci N, Tozzi L, Bogani C, Martinelli S, Rotunno G, Villeval JL, et al. Co-targeting the PI3K/mTOR and JAK2 signalling pathways produces synergistic activity against myeloproliferative neoplasms. J Cell Mol Med. 2013;17:1385–96.View ArticlePubMedPubMed CentralGoogle Scholar
- Nagai Y, Kawahara M, Sugino N, Shimazu Y, Hishizawa M, Yamashita K, et al. A case of minor BCR-ABL1 positive acute lymphoblastic leukemia following essential thrombocythemia and originating from a clone distinct from that harboring the JAK2-V617F mutation. Exp Hematol Oncol. 2014;3:6.View ArticlePubMedPubMed CentralGoogle Scholar
- Reddy P, Liu L, Adhikari D, Jagarlamudi K, Rajareddy S, Shen Y, et al. Oocyte-specific deletion of Pten causes premature activation of the primordial follicle pool. Science. 2008;319:611–3.View ArticlePubMedGoogle Scholar
- Rajareddy S, Reddy P, Du C, Liu L, Jagarlamudi K, Tang W, et al. p27kip1 (cyclin-dependent kinase inhibitor 1B) controls ovarian development by suppressing follicle endowment and activation and promoting follicle atresia in mice. Mol Endocrinol. 2007;21:2189–202.View ArticlePubMedGoogle Scholar