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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 SCF FBXW7 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 Ca 2+ /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 Ca 2+ channel blockers resulted in downregulation of both E2F1 protein and the expression of E2F1 target genes, whereas treatment with the Ca 2+ 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., Competing Interests: Competing interests statement:The authors declare no competing interest.

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., (© 2024. The Author(s).)

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 Ca 2+ -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., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interests with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

c-Myc, a transcription factor, induces cell proliferation and is often aberrantly or highly expressed in cancers. However, molecular mechanisms underlying this aberrantly high expression remain unclear. Here, we found that intracellular Ca 2+ concentration regulates c-Myc oncoprotein stability. We identified that calcineurin, a Ca 2+ -dependent protein phosphatase, is a positive regulator of c-Myc expression. Calcineurin depletion suppresses c-Myc targeted gene expression and c-Myc degradation. Calcineurin directly dephosphorylates Thr 58 and Ser 62 in c-Myc, which inhibit binding to the ubiquitin ligase Fbxw7. Mutations within the autoinhibitory domain of calcineurin, most frequently observed in cancer, may increase phosphatase activity, increasing c-Myc transcriptional activity in turn. Notably, calcineurin inhibition with FK506 decreased c-Myc expression with enhanced Thr 58 and Ser 62 phosphorylation in a mouse xenograft model. Thus, calcineurin can stabilize c-Myc, promoting tumor progression. Therefore, we propose that Ca 2+ signaling dysfunction affects cancer-cell proliferation via increased c-Myc stability and that calcineurin inhibition could be a new therapeutic target of c-Myc-overexpressing cancers., (© 2023. Springer Nature Limited.)

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 Ca 2+ /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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

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 Ser 294 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 Ser 294 promotes its binding to E6AP and consequent degradation. Calcineurin was also found to be required for the phosphorylation of ER-α at Ser 118 by mechanistic target of rapamycin complex 1 and the consequent activation of ER-α in response to β-estradiol treatment. Our study thus indicates that calcineurin controls both the stability and activity of ER-α by regulating its phosphorylation at Ser 294 and Ser 118 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., Competing Interests: The authors declare no competing interest.

The Calcineurin/NFAT (nuclear factor of activated T cells) pathway plays an essential role in the tumorigenic and metastatic properties in breast cancer. The molecular mechanism of the antiproliferative effect of calcineurin inhibition, however, is poorly understood. We found that calcineurin inhibition delayed cell cycle progression at G1/S, and promoted cyclin D1 degradation by inhibiting dephosphorylation at T286. Importantly, overexpression of cyclin D1 partially rescued delayed G1/S progression, thereby revealing cyclin D1 as a key factor downstream of calcineurin inhibition. Cyclin D1 upregulation is observed in human invasive breast cancers, and our findings indicate that dysregulation of T286 phosphorylation could play a role in this phenomenon. We therefore propose that targeting site specific phosphorylation of cyclin D1 could be a potential strategy for clinical intervention of invasive breast cancer.