Transcription factor C/EBP-β induces tumor-suppressor phosphatase PHLPP2 through repression of the miR-17–92 cluster in differentiating AML cells

Abstract

PHLPP2, a member of the PH-domain leucine-rich repeat protein phosphatase (PHLPP) family, which targets oncogenic kinases, has been actively investigated as a tumor suppressor in solid tumors. Little is known, however, regarding its regulation in hematological malignancies. We observed that PHLPP2 protein expression, but not its mRNA, was suppressed in late differentiation stage acute myeloid leukemia (AML) subtypes. MicroRNAs (miR or miRNAs) from the miR-17-92 cluster, oncomir-1, were shown to inhibit PHLPP2 expression and these miRNAs were highly expressed in AML cells that lacked PHLPP2 protein.Studies showed that miR-17-92 cluster regulation was, surprisingly, independent of transcription factors c-MYC and E2F in these cells; instead all-trans-retinoic acid (ATRA), a drug used for terminally differentiating AML subtypes, markedly suppressed miR-17-92 expression and increased PHLPP2 protein levels and phosphatase activity. Finally, we demonstrate that the effect of ATRA on miR-17-92 expression is mediated through its target, transcription factor C/EBPβ, which interacts with the intronic promoter of the miR-17-92 gene to inhibit transactivation of the cluster. These studies reveal a novel mechanism for upregulation of the phosphatase activity of PHLPP2 through C/EBPβ-mediated repression of the miR-17-92 cluster in terminally differentiating myeloid cells. Phosphatases are pivotal components of intracellular signaling cascades, controlling essential processes like metabolism, apoptosis, proliferation and differentiation. 1 The PH-domain leucine-rich repeat protein phosphatase (PHLPP) family serine-threonine phosphatases are emerging as central factors in cell survival and death regulation. The PHLPP2 is a member of this family. 2,3 To date four PHLPP substrates, Akt, PKC, Mst1 and S6K1, have been identified, all kinases involved in cell survival or apoptosis. 4-7 Dephosphorylation inactivates Akt, S6K1 and PKC, and subsequently cell survival signaling, whereas dephosphorylation of Mst1 activates its apoptotic function.PHLPP protein expression and activity is suppressed through various mechanisms in cancers. Although most studies on PHLPP2 have focused on genetic alterations in solid tumors, 8 PHLPP2 protein expression in small cell lung and colorectal cancers is also restricted through translational suppression 9 and post-transcriptional control by microRNAs (miR or miRNAs), miR-205 and miR-224. 10,11 The PHLPP2 3' untranslated region (3'UTR) harbors complementary binding sites for a subset of miRNAs belonging to the miRNA-17-92 cluster. 12 This cluster comprises six miRNAs on chromosome 13, transcribed as a single polycistronic unit. 13,14 Also known as oncomir-1, the cluster enhances cell proliferation or inhibits apoptosis by suppressing targets such as Bim, E2F1 and PTEN and is markedly overexpressed in human cancers. [15][16][17][18][19][20][21] Reduced levels of PHLPP2 in chemoresistant miR-17-92 overexpressing mantle cell lymphomas had suggested that the phosphatase could be a target of oncomir-1. 12 Kno...

PHLPP2 as a novel metastatic and prognostic biomarker in non‐small cell lung cancer patients

“…Researchers have made efforts to identify novel and important metastatic and prognostic biomarkers to guide research and provide therapeutic targets . Another interesting finding was that we found that low PHLPP2 expression levels were associated with positive lymph node metastasis.…”

“…PHLPP2 regulates several cellular pathways in cancer . Notably, direct dephosphorylation by PHLPP2 inactivates pro‐oncogenic AGC kinases such as Akt, S6K, and PKC and activates pro‐apoptotic kinases such as Mst1 . In addition, PHLPP2 can regulate the epigenome by suppressing histone acetylation and phosphorylation to reduce the gene expression of epidermal growth factor receptor (EGFR), an oncogenic driver of tumor progression .…”

Clustered miRNAs and their role in biological functions and diseases

“…miRNA clusters have their own promoters and are often driven by RNA polymerase II (Pol II) in a cell‐, tissue‐ or disease‐specific manner. Deregulated TFs can contribute to alterations in miRNA cluster expression and this is likely to be a widespread phenomenon (Yan et al, ). For example, regulation of chrX cluster (miR‐508/513a‐2, miR‐513b/513c) by forkhead box O1 (FOXO1) (Singhal et al, ).…”

“…For example, myc proto‐oncogene (MYC) activates miR‐17/92 cluster by binding to its promoter and influences the expression of several target genes [SIN3 transcription regulator family member B ( SIN3B ), HMG‐box transcription factor 1 ( HBP1 ), and BTG anti‐proliferation factor 1 ( BTG1 )] (Bo et al, ). Downregulation of miR‐17/92 by CCAAT/enhancer binding protein beta (C/EBPβ) and of miR‐144/451 by runt related transcription factor 1 (RUNX1)/ETO fusion protein is reported in differentiating acute myeloid leukemia (AML) cells and megakaryopoiesis, respectively (Kohrs et al, ; Yan et al, ). Single TFs can regulate multiple miRNA clusters and vice versa .…”

MicroRNA: an important regulator in acute myeloid leukemia

“…Concerning that microRNAs have functions of inducing cell differentiation, we have a good reason to propose that ectopic microRNAs, such as mir‐223, may mediate abnormal differentiation and lead to leukemogenesis (Emmrich et al, ). Moreover, researchers discussed the relationship among mir‐223, nuclear factor I‐A (NFI‐A) and CCAAT/enhancer binding proteinα (C/EBPα) (Yan et al, ), they previously observed that over‐expression of mir‐223 in acute promyelocytic leukaemia (APL) cell line promoted cell differentiation and mir‐223 was up‐regulated in cell lines treated with all‐trans retinoic acid (ATRA) (Palma et al, ). Mir‐233 was also found to induce NFI‐A silencing by inhibiting the NFI‐A promoter region, which has been previously proved have important effect on granulopoiesis.…”