Calcineurin-mediated dephosphorylation stabilizes E2F1 protein by suppressing binding of the FBXW7 ubiquitin ligase subunit.

The transcription factor E2F1 serves as a regulator of the cell cycle and promotes cell proliferation. It is highly expressed in cancer tissues and contributes to their malignant transformation. Degradation by the ubiquitin-proteasome system may help to prevent such overexpression of E2F1 and thereby to suppress carcinogenesis. A detailed understanding of the mechanisms underlying E2F1 degradation may therefore inform the development of new cancer treatments. We here identified SCFFBXW7 as a ubiquitin ligase for E2F1 by comprehensive analysis. We found that phosphorylation of E2F1 at serine-403 promotes its binding to FBXW7 (F-box/WD repeat-containing protein 7) followed by its ubiquitination and degradation. Furthermore, calcineurin, a Ca2+/calmodulin-dependent serine-threonine phosphatase, was shown to stabilize E2F1 by mediating its dephosphorylation at serine-403 and thereby preventing FBXW7 binding. Treatment of cells with Ca2+ channel blockers resulted in downregulation of both E2F1 protein and the expression of E2F1 target genes, whereas treatment with the Ca2+ ionophore ionomycin induced upregulation of E2F1. Finally, the calcineurin inhibitor FK506 attenuated xenograft tumor growth in mice in association with downregulation of E2F1 in the tumor tissue. Impairment of the balance between the opposing actions of FBXW7 and calcineurin in the regulation of E2F1 abundance may therefore play an important role in carcinogenesis.

FOXO1 promotes cancer cell growth through MDM2-mediated p53 degradation.

FOXO1 is a transcription factor and potential tumor suppressor that is negatively regulated downstream of PI3K-PKB/AKT signaling. Paradoxically, FOXO also promotes tumor growth, but the detailed mechanisms behind this role of FOXO are not fully understood. In this study, we revealed a molecular cascade by which the Thr24 residue of FOXO1 is phosphorylated by AKT and is dephosphorylated by calcineurin, which is a Ca2+-dependent protein phosphatase. Curiously, single nucleotide somatic mutations of FOXO1 in cancer occur frequently at and near Thr24. Using a calcineurin inhibitor and shRNA directed against calcineurin, we revealed that calcineurin-mediated dephosphorylation of Thr24 regulates FOXO1 protein stability. We also found that FOXO1 binds to the promoter region of MDM2 and activates transcription, which in turn promotes MDM2-mediated ubiquitination and degradation of p53. FOXO3a and FOXO4 are shown to control p53 activity; however, the significance of FOXO1 in p53 regulation remains largely unknown. Supporting this notion, FOXO1 depletion increased p53 and p21 protein levels in association with the inhibition of cell proliferation. Taken together, these results indicate that FOXO1 is stabilized by calcineurin-mediated dephosphorylation and that FOXO1 supports cancer cell proliferation by promoting MDM2 transcription and subsequent p53 degradation.

FKBP52 and FKBP51 differentially regulate the stability of estrogen receptor in breast cancer.

Estrogen receptor α (ERα) is a transcription factor that induces cell proliferation and exhibits increased expression in a large subset of breast cancers. The molecular mechanisms underlying the up-regulation of ERα activity, however, remain poorly understood. We identified FK506-binding protein 52 (FKBP52) as a factor associated with poor prognosis of individuals with ERα-positive breast cancer. We found that FKBP52 interacts with breast cancer susceptibility gene 1 and stabilizes ERα, and is essential for breast cancer cell proliferation. FKBP52 depletion resulted in decreased ERα expression and proliferation in breast cancer cell lines, including MCF7-derived fulvestrant resistance (MFR) cells, suggesting that inhibiting FKBP52 may provide a therapeutic effect for endocrine therapy­resistant breast cancer. In contrast, FKBP51, a closely related molecule to FKBP52, reduced the stability of ERα. Consistent with these findings, FKBP51 was more abundantly expressed in normal tissues than in cancer cells, suggesting that these FKBPs may function in the opposite direction. Collectively, our study shows that FKBP52 and FKBP51 regulate ERα stability in a reciprocal manner and reveals a regulatory mechanism by which the expression of ERα is controlled.

Estrogen receptor α revised: Expression, structure, function, and stability.

Estrogen receptor α (ERα) is a ligand-dependent transcription factor that regulates the expression of estrogen-responsive genes. Approximately 70% of patients with breast cancer are ERα positive. Estrogen stimulates cancer cell proliferation and contributes to tumor progression. Endocrine therapies, which suppress the ERα signaling pathway, significantly improve the prognosis of patients with breast cancer. However, the development of de novo or acquired endocrine therapy resistance remains a barrier to breast cancer treatment. Therefore, understanding the regulatory mechanisms of ERα is essential to overcome the resistance to treatment. This review focuses on the regulation of ERα expression, including copy number variation, epigenetic regulation, transcriptional regulation, and stability, as well as functions from the point of view post-translational modifications.

Calcineurin regulates the stability and activity of estrogen receptor α.

Estrogen receptor α (ER-α) mediates estrogen-dependent cancer progression and is expressed in most breast cancer cells. However, the molecular mechanisms underlying the regulation of the cellular abundance and activity of ER-α remain unclear. We here show that the protein phosphatase calcineurin regulates both ER-α stability and activity in human breast cancer cells. Calcineurin depletion or inhibition down-regulated the abundance of ER-α by promoting its polyubiquitination and degradation. Calcineurin inhibition also promoted the binding of ER-α to the E3 ubiquitin ligase E6AP, and calcineurin mediated the dephosphorylation of ER-α at Ser294 in vitro. Moreover, the ER-α (S294A) mutant was more stable and activated the expression of ER-α target genes to a greater extent compared with the wild-type protein, whereas the extents of its interaction with E6AP and polyubiquitination were attenuated. These results suggest that the phosphorylation of ER-α at Ser294 promotes its binding to E6AP and consequent degradation. Calcineurin was also found to be required for the phosphorylation of ER-α at Ser118 by mechanistic target of rapamycin complex 1 and the consequent activation of ER-α in response to ß-estradiol treatment. Our study thus indicates that calcineurin controls both the stability and activity of ER-α by regulating its phosphorylation at Ser294 and Ser118 Finally, the expression of the calcineurin A-α gene (PPP3CA) was associated with poor prognosis in ER-α-positive breast cancer patients treated with tamoxifen or other endocrine therapeutic agents. Calcineurin is thus a promising target for the development of therapies for ER-α-positive breast cancer.

Impact of FKBP52 on cell proliferation and hormone-dependent cancers.

FK506 binding protein 52 (FKBP52) (gene name FKBP4) is a 52 kDa protein that belongs to the FKBP family; it binds to the immunosuppressant FK506 and has proline isomerase activity. In addition to its FK domain-containing peptidylprolyl isomerase activity, FKBP52 also acts as a cochaperone through the tetratricopeptide repeat domain that mediates binding to heat shock protein 90. Previous studies have reported that FKBP52 is associated with hormone-dependent, stress-related, and neurodegenerative diseases, revealing its diverse functions. In particular, the effects of FKBP52 on cancer have attracted considerable attention. FKBP52 promotes the growth of hormone-dependent cancers by activating steroid hormone receptors. Recent studies have shown that the expression of FKBP52 is increased not only in steroid hormone-dependent cancer cells but also in colorectal, lung, and liver cancers, revealing its diverse functions that contribute to cancer growth. This review summarizes reports related to hormone-dependent cancer and cell proliferation in terms of the structure of FKBP52 and its function on interacting molecules.

Dephosphorylation of the EGFR protein by calcineurin at serine 1046/1047 enhances its stability.

The epidermal growth factor receptor (EGFR) is highly expressed or abnormally activated in several types of cancers, such as lung and colorectal cancers. Inhibitors that suppress the tyrosine kinase activity of EGFR have been used in the treatment of lung cancer. However, resistance to these inhibitors has become an issue in cancer treatment, and the development of new therapies that inhibit EGFR is desired. We found that calcineurin, a Ca2+/calmodulin-activated serine/threonine phosphatase, is a novel regulator of EGFR. Inhibition of calcineurin by FK506 treatment or calcineurin depletion promoted EGFR degradation in cancer cells. In addition, we found that calcineurin dephosphorylates EGFR at serine (S)1046/1047, which in turn stabilizes EGFR. Furthermore, in human colon cancer cells transplanted into mice, the inhibition of calcineurin by FK506 decreased EGFR expression. These results indicate that calcineurin stabilizes EGFR by dephosphorylating S1046/1047 and promotes tumor growth. These findings suggest that calcineurin may be a new therapeutic target for cancers with high EGFR expression or activation.

Chk1 is a histone H3 threonine 11 kinase that regulates DNA damage-induced transcriptional repression.

DNA damage results in activation or suppression of transcription of a large number of genes. Transcriptional activation has been well characterized in the context of sequence-specific DNA-bound activators, whereas mechanisms of transcriptional suppression are largely unexplored. We show here that DNA damage rapidly reduces histone H3 Threonine 11 (T11) phosphorylation. This correlates with repression of genes, including cyclin B1 and cdk1. H3-T11 phosphorylation occurs throughout the cell cycle and is Chk1 dependent in vivo. Following DNA damage, Chk1 undergoes rapid chromatin dissociation, concomitant with reduced H3-T11 phosphorylation. Furthermore, we find that loss of H3-T11 phosphorylation correlates with reduced binding of the histone acetyltransferase GCN5 at cyclin B1 and cdk1 promoters and reduced H3-K9 acetylation. We propose a mechanism for Chk1 as a histone kinase, responsible for DNA-damage-induced transcriptional repression by loss of histone acetylation.

UV-induced activation of ATR is mediated by UHRF2.

UHRF1 (Ubiquitin-like with PHD and ring finger domains 1) regulates DNA methylation and histone modifications and plays a key role in cell proliferation and the DNA damage response. However, the function of UHRF2, a paralog of UHRF1, in the DNA damage response remains largely unknown. Here, we show that UHRF2 is essential for maintaining cell viability after UV irradiation, as well as for the proliferation of cancer cells. UHRF2 was found to physically interact with ATR in a DNA damage-dependent manner through UHRF2's TTD domain. In addition, phosphorylation of threonine at position 1989, which is required for UV-induced activation of ATR, was impaired in cells depleted of UHRF2, suggesting that UHRF2 is essential in ATR activation. In conclusion, these results suggest a new regulatory mechanism of ATR activation mediated by UHRF2.

Necessary and sufficient role for a mitosis skip in senescence induction.

Senescence is a state of permanent growth arrest and is a pivotal part of the antitumorigenic barrier in vivo. Although the tumor suppressor activities of p53 and pRb family proteins are essential for the induction of senescence, molecular mechanisms by which these proteins induce senescence are still not clear. Using time-lapse live-cell imaging, we demonstrate here that normal human diploid fibroblasts (HDFs) exposed to various senescence-inducing stimuli undergo a mitosis skip before entry into permanent cell-cycle arrest. This mitosis skip is mediated by both p53-dependent premature activation of APC/C(Cdh1) and pRb family protein-dependent transcriptional suppression of mitotic regulators. Importantly, mitotic skipping is necessary and sufficient for senescence induction. p16 is only required for maintenance of senescence. Analysis of human nevi also suggested the role of mitosis skip in in vivo senescence. Our findings provide decisive evidence for the molecular basis underlying the induction and maintenance of cellular senescence.

Decoding the Phosphatase Code: Regulation of Cell Proliferation by Calcineurin.

Calcineurin, a calcium-dependent serine/threonine phosphatase, integrates the alterations in intracellular calcium levels into downstream signaling pathways by regulating the phosphorylation states of several targets. Intracellular Ca2+ is essential for normal cellular physiology and cell cycle progression at certain critical stages of the cell cycle. Recently, it was reported that calcineurin is activated in a variety of cancers. Given that abnormalities in calcineurin signaling can lead to malignant growth and cancer, the calcineurin signaling pathway could be a potential target for cancer treatment. For example, NFAT, a typical substrate of calcineurin, activates the genes that promote cell proliferation. Furthermore, cyclin D1 and estrogen receptors are dephosphorylated and stabilized by calcineurin, leading to cell proliferation. In this review, we focus on the cell proliferative functions and regulatory mechanisms of calcineurin and summarize the various substrates of calcineurin. We also describe recent advances regarding dysregulation of the calcineurin activity in cancer cells. We hope that this review will provide new insights into the potential role of calcineurin in cancer development.

PP1 regulatory subunit NIPP1 regulates transcription of E2F1 target genes following DNA damage.

DNA damage induces transcriptional repression of E2F1 target genes and a reduction in histone H3-Thr11 phosphorylation (H3-pThr11 ) at E2F1 target gene promoters. Dephosphorylation of H3-pThr11 is partly mediated by Chk1 kinase and protein phosphatase 1γ (PP1γ) phosphatase. Here, we isolated NIPP1 as a regulator of PP1γ-mediated H3-pThr11 by surveying nearly 200 PP1 interactor proteins. We found that NIPP1 inhibits PP1γ-mediated dephosphorylation of H3-pThr11 both in vivo and in vitro. By generating NIPP1-depleted cells, we showed that NIPP1 is required for cell proliferation and the expression of E2F1 target genes. Upon DNA damage, activated protein kinase A (PKA) phosphorylated the NIPP1-Ser199 residue, adjacent to the PP1 binding motif (RVxF), and triggered the dissociation of NIPP1 from PP1γ, leading to the activation of PP1γ. Furthermore, the inhibition of PKA activity led to the activation of E2F target genes. Statistical analysis confirmed that the expression of NIPP1 was positively correlated with E2F target genes. Taken together, these findings demonstrate that the PP1 regulatory subunit NIPP1 modulates E2F1 target genes by linking PKA and PP1γ during DNA damage.

Essential role of Tip60-dependent recruitment of ribonucleotide reductase at DNA damage sites in DNA repair during G1 phase.

A balanced deoxyribonucleotide (dNTP) supply is essential for DNA repair. Here, we found that ribonucleotide reductase (RNR) subunits RRM1 and RRM2 accumulated very rapidly at damage sites. RRM1 bound physically to Tip60. Chromatin immunoprecipitation analyses of cells with an I-SceI cassette revealed that RRM1 bound to a damage site in a Tip60-dependent manner. Active RRM1 mutants lacking Tip60 binding failed to rescue an impaired DNA repair in RRM1-depleted G1-phase cells. Inhibition of RNR recruitment by an RRM1 C-terminal fragment sensitized cells to DNA damage. We propose that Tip60-dependent recruitment of RNR plays an essential role in dNTP supply for DNA repair.

Loss of maintenance DNA methylation results in abnormal DNA origin firing during DNA replication.

The mammalian maintenance methyltransferase DNMT1 [DNA (cytosine-5-)-methyltransferase 1] mediates the inheritance of the DNA methylation pattern during replication. Previous studies have shown that depletion of DNMT1 causes a severe growth defect and apoptosis in differentiated cells. However, the detailed mechanisms behind this phenomenon remain poorly understood. Here we show that conditional ablation of Dnmt1 in murine embryonic fibroblasts (MEFs) resulted in an aberrant DNA replication program showing an accumulation of late-S phase replication and causing severely defective growth. Furthermore, we found that the catalytic activity and replication focus targeting sequence of DNMT1 are required for a proper DNA replication program. Taken together, our findings suggest that the maintenance of DNA methylation by DNMT1 plays a critical role in proper regulation of DNA replication in mammalian cells.

Mammal-specific H2A variant, H2ABbd, is involved in apoptotic induction via activation of NF-κB signaling pathway.

Histone variants play specific roles in maintenance and regulation of chromatin structures. H2ABbd, an H2A variant, possesses a highly divergent structure compared with canonical H2A and is highly expressed in postmeiotic germ cells, but its functions in the regulation of gene expression are largely unknown. In the present study, we investigated the cellular phenotype associated with enforced H2ABbd expression. Among H2A variants, H2ABbd specifically caused growth defect in human cells and induced apoptosis. H2ABbd expression resulted in degradation of inhibitor of κB-α and translocation of NF-κB into nuclei, indicating the activation of NF-κB. Intriguingly, NF-κB activity was essential for H2ABbd-induced apoptosis. H2ABbd overexpression resulted in DNA damage after release from G1/S, progressed through the S phase slowly, and induced apoptosis. Furthermore, gene expression microarray analysis revealed that expression of H2ABbd activates groups of genes involved in apoptosis and postmeiotic germ cell development, suggesting that H2ABbd might influence transcription. Taken together, our data suggest that H2ABbd may contribute to specific chromatin structures and promote NF-κB activation, which could in turn induce apoptosis in mammalian cells.

Physical interaction between MPP8 and PRC1 complex and its implication for regulation of spermatogenesis.

Epigenetic modifications such as DNA methylation and histone H3 lysine 27 methylation (H3K27me) are repressive marks that silence gene expression. The M phase phosphoprotein (MPP8) associates with proteins involved in both DNA methylation and histone modifications, and therefore, is a potential candidate to mediate crosstalk between repressive epigenetic pathways. Here, by performing immunohistochemical analyses we demonstrate that MPP8 is expressed in the rodent testis, especially in spermatocytes, suggesting a role in spermatogenesis. Interestingly, we found that MPP8 physically interacts with PRC1 (Polycomb Repressive Complex 1) components which are known to possess essential function in testis development by modulating monoubiquitination of Histone H2A (uH2A) and trimethylation of Histone H3 Lysine 27 (H3K27me3) residues. Knockdown analysis of MPP8 in HeLa cells resulted in derepression of a set of genes that are normally expressed in spermatogonia, spermatids and mature sperm, thereby indicating a role for this molecule in silencing testis-related genes in somatic cells. In addition, depletion of MPP8 in murine ES cells specifically induced expression of genes involved in mesoderm differentiation, such as Cdx2 and Brachyury even in the presence of LIF, which implicated that MPP8 might be required to repress differentiation associated genes during early development. Taken together, our results indicate that MPP8 could have a role for silencing genes that are associated with differentiation of the testis and the mesoderm by interacting with epigenetic repressors modules such as the PRC1 complex.

Cooperative functions of Chk1 and Chk2 reduce tumour susceptibility in vivo.

Although the linkage of Chk1 and Chk2 to important cancer signalling suggests that these kinases have functions as tumour suppressors, neither Chk1+/- nor Chk2-/- mice show a predisposition to cancer under unperturbed conditions. We show here that Chk1+/-Chk2-/- and Chk1+/-Chk2+/- mice have a progressive cancer-prone phenotype. Deletion of a single Chk1 allele compromises G2/M checkpoint function that is not further affected by Chk2 depletion, whereas Chk1 and Chk2 cooperatively affect G1/S and intra-S phase checkpoints. Either or both of the kinases are required for DNA repair depending on the type of DNA damage. Mouse embryonic fibroblasts from the double-mutant mice showed a higher level of p53 with spontaneous DNA damage under unperturbed conditions, but failed to phosphorylate p53 at S23 and further induce p53 expression upon additional DNA damage. Neither Chk1 nor Chk2 is apparently essential for p53- or Rb-dependent oncogene-induced senescence. Our results suggest that the double Chk mutation leads to a high level of spontaneous DNA damage, but fails to eliminate cells with damaged DNA, which may ultimately increase cancer susceptibility independently of senescence.

Dephosphorylation of NFAT by Calcineurin inhibits Skp2-mediated degradation.

The transcription factor NFAT plays key roles in multiple biological activities, such as immune responses, tissue development and malignant transformation. NFAT is dephosphorylated by calcineurin, which is activated by intracellular calcium levels, and translocated into the nucleus, resulting in transcriptional activation. Calcineurin dephosphorylates various target proteins and regulates their functions. However, the regulation of NFAT degradation is largely unknown, and it is unclear whether calcineurin contributes to the stability of NFAT. We investigated the effect of calcineurin inhibition on NFAT protein stability and found that the dephosphorylation of NFAT by calcineurin promotes the NFAT stabilization, whereas calcineurin mutant that is defective in phosphatase activity was unable to stabilize NFAT. Increased intracellular calcium ion concentration, which is essential for calcineurin activation, also induced NFAT stability. In addition, we identified S-phase kinase associated protein 2 (Skp2), an F-box protein of the SCF ubiquitin ligase complex, as a factor mediating degradation of NFAT when calcineurin was depleted. In summary, these findings revealed that the dephosphorylation of NFAT by calcineurin protects NFAT from degradation by Skp2 and promotes its protein stability.

NFAT activation by FKBP52 promotes cancer cell proliferation by suppressing p53.

FK506-binding protein 52 (FKBP52) is a member of the FKBP family of proline isomerases. FKBP52 is up-regulated in various cancers and functions as a positive regulator of steroid hormone receptors. Depletion of FKBP52 is known to inhibit cell proliferation; however, the detailed mechanism remains poorly understood. In this study, we found that FKBP52 depletion decreased MDM2 transcription, leading to stabilization of p53, and suppressed cell proliferation. We identified NFATc1 and NFATc3 as transcription factors that regulate MDM2 We also found that FKBP52 associated with NFATc3 and facilitated its nuclear translocation. In addition, calcineurin, a well-known Ca2+ phosphatase essential for activation of NFAT, plays a role in MDM2 transcription. Supporting this notion, MDM2 expression was found to be regulated by intracellular Ca2+ Taken together, these findings reveal a new role of FKBP52 in promoting cell proliferation via the NFAT-MDM2-p53 axis, and indicate that inhibition of FKBP52 could be a new therapeutic tool to activate p53 and inhibit cell proliferation.

Calcineurin/NFATc1 pathway represses cellular cytotoxicity by modulating histone H3 expression.

Excess amounts of histones in the cell induce mitotic chromosome loss and genomic instability, and are therefore detrimental to cell survival. In yeast, excess histones are degraded by the proteasome mediated via the DNA damage response factor Rad53. Histone expression, therefore, is tightly regulated at the protein level. Our understanding of the transcriptional regulation of histone genes is far from complete. In this study, we found that calcineurin inhibitor treatment increased histone protein levels, and that the transcription factor NFATc1 (nuclear factor of activated T cells 1) repressed histone transcription and acts downstream of the calcineurin. We further revealed that NFATc1 binds to the promoter regions of many histone genes and that histone transcription is downregulated in a manner dependent on intracellular calcium levels. Indeed, overexpression of histone H3 markedly inhibited cell proliferation. Taken together, these findings suggest that NFATc1 prevents the detrimental effects of histone H3 accumulation by inhibiting expression of histone at the transcriptional level.