鈣離子扮演一個重要的信號分子參與各式各樣的訊息級聯反應來調節細胞中諸多生理活動，例如：細胞週期運轉。在癌化過程中經常觀察到鈣離子調控失衡和不受控制的細胞週期有關。鈣離子所介導的最重要事件之一是通過鈣離子依賴性相關轉錄因子之活化進行的轉錄調控。叉頭框蛋白M1是一種致癌的轉錄因子與細胞週期主要的調節因子，其過度激活和過度表達也常發生在許多惡性腫瘤中。累積的證據顯示，在不同的細胞環境中，鈣離子和叉頭框蛋白M1之間存在調節關係。然而，對鈣離子訊號傳遞和叉頭框蛋白M1調控之其中的分子機制仍未被充分了解。因此本篇研究藉由使用藥物來改變胞內鈣離子水平，探討卵巢癌細胞中鈣離子調控叉頭框蛋白M1所扮演的角色。我們發現，胞內鈣離子的螯合將叉頭框蛋白M1束縛至細胞核周圍，而上升的胞內鈣離子則促進了細胞核的叉頭框蛋白M1累積。此外，藉由降低胞內鈣離子或LY294002引起的AKT失活，導致叉頭框蛋白M1的絲氨酸磷酸化形式減少與導致其細胞核周圍的錨定。相反地，鈣離子載體誘導的鈣離子內流激活磷酸化AKT，接著將叉頭框蛋白M1在絲氨酸殘基上磷酸化並使其轉移到細胞核中。CaMKII、Rac1和PKCα訊息傳遞途徑也可能通過不同的鈣離子下游效應蛋白參與控制鈣離子介導的叉頭框蛋白M1之次細胞分布。儘管尚未確定鈣離子依賴性叉頭框蛋白M1磷酸化與次細胞分布的明確功能，但我們的發現有助於進一步了解鈣離子訊號中調控叉頭框蛋白M1的機制。

Calcium (Ca2+) acts as a pivotal signal molecule that participates in various signaling cascades, modulating numerous biological events such as cell cycle-progression. Dysregulated Ca2+ homeostasis associated with uncontrolled cell cycling is frequently observed during carcinogenesis. One of the most important Ca2+-mediated events is transcriptional regulation via activation of Ca2+-dependent transcription factors (TFs). FOXM1 (Forkhead box M1) is an oncogenic TF and a master cell-cycle regulator, and its overactivation and upregulation often occur in many malignancies. Cumulative evidence has shown that there is a regulatory relationship between Ca2+ and FOXM1 in different cellular contexts. However, the molecular mechanisms underlying Ca2+ signaling and FOXM1 regulation are still poorly understood. In this study, alterations of intracellular Ca2+ levels were achieved by using pharmacological agents in ovarian cancer cells to investigate the role of Ca2+ on FOXM1. We found that intracellular Ca2+ chelation tethered FOXM1 to the nuclear envelope, while enhanced intracellular Ca2+ promoted FOXM1 nuclear accumulation. Moreover, AKT inactivation by decreasing intracellular Ca2+ or LY294002 caused a reduction of the serine-phosphorylated form of FOXM1, resulting in its perinuclear sequestration. Conversely, ionomycin-induced Ca2+ influx activated p-AKT, which in turn phosphorylated FOXM1 on serine residues and led to its nuclear translocation. The possible involvement of the CaMKII (Ca2+/calmodulin (CaM)-dependent protein kinase II), Rac1 (Rac family small GTPase 1) and PKCα (protein kinase C alpha) pathways was also implicated in controlling Ca2+-mediated FOXM1 subcellular localization via distinct downstream Ca2+ effectors. Although the specific functions for Ca2+-dependent FOXM1 phosphorylation and subcellular distribution have not been determined yet, our findings provide further insight into the mechanisms of Ca2+ signaling in FOXM1 regulation.

Agassandian M., Chen B.B., Pulijala R., Kaercher L., Glasser J.R., Mallampalli R.K. (2012) Calcium-calmodulin kinase I cooperatively regulates nucleocytoplasmic shuttling of CCTα by accessing a nuclear export signal. Mol. Biol. Cell 23:2755–2769.
Aggarwal A., Prinz-Wohlgenannt M., Tennakoon S., Höbaus J., Boudot C., Mentaverri R., Brown E.M., Baumgartner-Parzer S., Kállay E. (2015) The calcium-sensing receptor: a promising target for prevention of colorectal cancer. Biochim. Biophys. Acta 1853:2158–2167.
Alvarez-Fernández M., Halim V.A., Aprelia M., Mohammed S., Medema R.H. (2011) Protein phosphatase 2A (B55α) prevents premature activation of forkhead transcription factor FoxM1 by antagonizing cyclin A/cyclin-dependent kinase-mediated phosphorylation. J. Biol. Chem. 286:33029‒33036.
Anders L., Ke N., Hydbring P., Choi Y.J., Widlund H.R., Chick J.M., Zhai H., Vidal M., Gygi S.P., Braun P., Sicinski P. (2011) A systemic screen for CDK4/6 substrates links FOXM1 phosphorylation to senescence suppression in cancer cells. Cancer Cell 20:620‒634.
Bagur R., Hajnóczky G. (2017) Intracellular Ca2+ sensing: role in calcium homeostasis and signaling. Mol. Cell 66:780–788.
Berchtold C.M., Wu Z.H., Huang T.T., Miyamoto S. (2007) Calcium-dependent regulation of NEMO nuclear export in response to genotoxic stimuli. Mol. Cell. Biol. 27:497–509.
Berchtold M.W., Villalobo A. (2014) The many faces of calmodulin in cell proliferation, programmed cell death, autophagy, and cancer. Biochim. Biophys. Acta 1843:398–435.
Berridge M.J., Bootman M.D., Lipp P. (1998) Calcium - a life and death signal. Nature 395:645–648.
Berridge M.J., Bootman M.D., Roderick H.L. (2003) Calcium signaling: dynamics, homeostasis and remodelling. Nat. Rev. Mol. Cell Biol. 4:517–529.
Berridge M.J. (2004) Calcium signal transduction and cellular control mechanisms. Biochim. Biophys. Acta 1742:3–7.
Bertoli C., Skotheim J., de Bruin R. (2013) Control of cell cycle transcription during G1 and S phases. Nat. Rev. Mol. Cell Biol. 14:518–528.
Bhagavathula N., Hanosh A.W., Nerusu K.C., Appelman H., Chakrabarty S., Varani J. (2007) Regulation of E-cadherin and β-catenin by Ca2+ in colon carcinoma is dependent on calcium-sensing receptor expression and function. Int. J. Cancer 121:1455–1462.
Boubriak I.I., Malhas A.N., Drozdz M.M., Pytowski L., Vaux D.J. (2017) Stress-induced release of Oct-1 from the nuclear envelope is mediated by JNK phosphorylation of lamin B1. PLoS One 12:e0177990.
Brazil D.P., Yang Z.Z., Hemmings B.A. (2004) Advances in protein kinase B signalling: AKTion on multiple fronts. Trends Biochem. Sci. 29:233‒242.
Brunet J.P., Cotte-Laffitte J., Linxe C., Quero A.M., Géniteau-Legendre M., Servin A. (2000) Rotavirus infection induces an increase in intracellular calcium concentration in human intestinal epithelial cells: role in microvillar actin alteration. J. Virol. 74:2323–2332.
Chen Y.F., Chen Y.T., Chiu W.T., Shen M.R. (2013) Remodeling of calcium signaling in tumor progression. J. Biomed. Sci. 20:23.
Chen Y.J., Dominguez-Brauer C., Wang Z., Asara J.M., Costa R.H., Tyner A.L., Lau L.F., Raychaudhuri P. (2009) A conserved phosphorylation site within the forkhead domain of FoxM1B is required for its activation by cyclin-CDK1. J. Biol. Chem. 284:30695–30707.
Chen Y.W., Chen Y.F., Chen Y.T, Chiu W.T., Shen M.R. (2016) The STIM1-Orai1 pathway of store-operated Ca2+ entry controls the checkpoint in cell cycle G1/S transition. Sci. Rep. 6:22142.
Clevers H. (2006) Wnt/beta-catenin signaling in development and disease. Cell 127:469–480.
Cohn O., Feldman M., Weil L., Kublanovsky M., Levy D. (2016) Chromatin associated SETD3 negatively regulates VEGF expression. Sci. Rep. 6:37115.
Dai C., Hang Y., Shostak A., Poffenberger G., Hart N., Prasad N., Phillips N., Levy S.E., Greiner D.L., Shultz L.D., Bottino R., Kim S.K., Powers A.C. (2017) Age-dependent human β cell proliferation induced by glucagon-like peptide 1 and calcineurin signaling. J. Clin. Invest. 127:3835–3844.
Deb T.B., Coticchia C.M., Dickson R.B. (2004) Calmodulin-mediated activation of Akt regulates survival of c-Myc-overexpressing mouse mammary carcinoma cells. J. Biol. Chem. 279:38903–38911.
Dewenter M., von der Lieth A., Katus H.A., Backs J. (2017) Calcium signaling and transcriptional regulation in cardiomyocytes. Circ. Res. 121:1000‒1020.
Dunn K.W., Kamocka M.M., McDonald J.H. (2011) A practical guide to evaluating colocalization in biological microscopy. Am. J. Physiol. Cell Physiol. 300:C723–C742.
Feske S. (2007) Calcium signaling in lymphocyte activation and disease. Nat. Rev. Immunol. 7:690–702.
Fisher W.G., Yang P.C., Medikonduri R.K., Jafri M.S. (2006) NFAT and NFкB activation in T lymphocytes: a model of differential activation of gene expression. Ann. Biomed. Eng. 34:1712–1728.
FitzHarris G., Larman M., Richards C., Carroll J. (2005) An increase in [Ca2+]i is sufficient but not necessary for driving mitosis in early mouse embryos. J. Cell Sci. 118:4563–4575.
Flaherty P., Radhakrishnan M.L., Dinh T., Rebres R.A., Roach T.I., Jordan M.I., Arkin A.P. (2008) A dual receptor crosstalk model of G-protein-coupled signal transduction. PLoS Comput. Biol. 4:e1000185.
Gocher A.M., Azabdaftari G., Euscher L.M., Dai S., Karacosta L.G., Franke T.F., Edelman A.M. (2017) Akt activation by Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2) in ovarian cancer cells. J. Biol. Chem. 292:14188–14204.
González J.M., Navarro-Puche A., Casar B., Crespo P., Andrés V. (2008) Fast regulation of AP-1 activity through interaction of lamin A/C, ERK1/2, and c-Fos at the nuclear envelope. J. Cell Biol. 183:653–666.
Görlich D., Mattaj I.W. (1996) Nucleocytoplasmic transport. Science 271:1513–1518.
Heessen S., Fornerod M. (2007) The inner nuclear envelope as a transcription factor resting place. EMBO Rep. 8:914–919.
Ho C., Wang C., Mattu S., Destefanis G., Ladu S., Delogu S., Armbruster J., Fan L., Lee S.A., Jiang L., Dombrowski F., Evert M., Chen X., Calvisi D.F. (2012) AKT and N-Ras co-activation in the mouse liver promotes rapid carcinogenesis via mTORC1, FOXM1/SKP2, and c-Myc pathways. Hepatology 55:833‒845.
Hutchins J.R., Dikovskaya D., Clarke P.R. (2013) Regulation of Cdc2/cyclin B activation in Xenopus egg extracts via inhibitory phosphorylation of Cdc25C phosphatase by Ca2+/calmodulin-dependent protein kinase II. Mol. Biol. Cell 14:4003–4014.
Huynh K.M., Soh J.W., Dash R., Sarkar D., Fisher P.B., Kang D. (2011) FOXM1 expression mediates growth suppression during terminal differentiation of HO-1 human metastatic melanoma cells. J. Cell. Physiol. 226:194–204.
Ivanova H., Kerkhofs M., La Rovere R.M., Bultynck G. (2017) Endoplasmic reticulum-mitochondrial Ca2+ fluxes underlying cancer cell survival. Front. Oncol. 7:70.
Ivorra C., Kubicek M., González J.M., Sanz-González S.M., Alvarez-Barrientos A., O'Connor J.E., Burke B., Andrés V. (2006) A mechanism of AP-1 suppression through interaction of c-Fos with lamin A/C. Genes Dev. 20:307–320.
Kaur G., Ly-Huynh J.D., Jans D.A. (2014) Intracellular calcium levels can regulate importin-dependent nuclear import. Biochem. Biophys. Res. Commun. 450:812–817.
Koo C.Y., Muir K.W., Lam E.W. (2012) FOXM1: from cancer initiation to progression and treatment. Biochim. Biophys. Acta 1819:28–37.
Korver W., Roose J., Clevers H. (1997) The winged-helix transcription factor Trident is expressed in cycling cells. Nucleic Acids Res. 25:1715–1719.
Kuo I.Y., Ehrlich B.E. (2015) Signaling in muscle contraction. Cold Spring Harb. Perspect. Biol. 7: a006023.
Lammerding J., Fong L.G., Ji J.Y., Reue K., Stewart C.L., Young S.G., Lee R.T. (2006) Lamins A and C but not lamin B1 regulate nuclear mechanics. J. Biol. Chem. 281:25768–25780.
Laoukili J., Alvarez M., Meijer L.A., Stahl M., Mohammed S., Kleij L., Heck A.J., Medema R.H. (2008a) Activation of FoxM1 during G2 requires cyclin A/Cdk-dependent relief of autorepression by the FoxM1 N-terminal domain. Mol. Cell Biol. 28:3076‒3087.
Laoukili J., Alvarez-Fernandez M., Stahl M., Medema R.H. (2008b) FOXM1 is degraded at mitotic exit in a Cdh1-dependent manner. Cell Cycle 7:2720‒2726.
Leung T.W., Lin S.S., Tsang A.C., Tong C.S., Ching J.C., Leung W.Y., Gimlich R., Wong G.G., Yao K.M. (2001) Over-expression of FOXM1 stimulates cyclin B1 expression. FEBS Lett. 507:59–66.
Li M., Yang J., Zhou W., Ren Y., Wang X., Chen H., Zhang J., Chen J., Sun Y., Cui L., Liu X., Wang L., Wu C. (2017) Activation of an AKT/FOXM1/STMN1 pathway drives resistance to tyrosine kinase inhibitors in lung cancer. Br. J. Cancer 117:974–983.
Li N., Jiang P., Du W., Wu Z., Li C., Qiao M., Yang X., Wu M. (2011) Siva1 suppresses epithelial-mesenchymal transition and metastasis of tumor cells by inhibiting stathmin and stabilizing microtubules. Proc. Natl. Acad. Sci. U.S.A. 108:12851–12856.
Li Y., Zhang T., Zhang Y., Zhao X., Wang W. (2018) Targeting the FOXM1-regulated long noncoding RNA TUG1 in osteosarcoma. Cancer Sci. 109:3093–3104.
Liao G.B., Li X.Z., Zeng S., Liu C., Yang S.M., Yang L., Hu C.J., Bai J.Y. (2018) Regulation of the master regulator FOXM1 in cancer. Cell Commun. Signal. 16:57.
Liu L., Wu N., Wang Y., Zhang X., Xia B., Tang J., Cai J., Zhao Z., Liao Q., Wang J. (2019) TRPM7 promotes the epithelial-mesenchymal transition in ovarian cancer through the calcium-related PI3K/AKT oncogenic signaling. J. Exp. Clin. Cancer Res. 38:106.
Lv C., Zhao G., Sun X., Wang P., Xie N., Luo J., Tong T. (2016) Acetylation of FOXM1 is essential for its transactivation and tumor growth stimulation. Oncotarget 7:60366–60382.
Ma R.Y., Tong T.H., Cheung A.M., Tsang A.C., Leung W.Y., Yao K.M. (2005) Raf/MEK/MAPK signaling stimulates the nuclear translocation and transactivating activity of FOXM1c. J. Cell Sci. 118:795‒806.
Major M.L., Lepe R., Costa R.H. (2004) Forkhead box M1B transcriptional activity requires binding of Cdk-cyclin complexes for phosphorylation-dependent recruitment of p300/CBP coactivators. Mol. Cell Biol. 24:2649‒2661.
Monteith G.R., Davis F.M., Roberts-Thomson S.J. (2012) Calcium channels and pumps in cancer: changes and consequences. J. Biol. Chem. 287:31666‒31673.
Moser B., Hochreiter B., Herbst R., Schmid J.A. (2017) Fluorescence colocalization microscopy analysis can be improved by combining object-recognition with pixel-intensity-correlation. Biotechnol. J. 12:1600332.
Myatt S.S., Kongsema M., Man C.W., Kelly D.J., Gomes A.R., Khongkow P., Karunarathna U., Zona S., Langer J.K., Dunsby C.W., Coombes R.C., French P.M., Brosens J.J., Lam E.W. (2014) SUMOylation inhibits FOXM1 activity and delays mitotic transition. Oncogene 33:4316–4329.
Nandi D., Cheema P.S., Jaiswal N., Nag A. (2018) FoxM1: repurposing an oncogene as a biomarker. Semin. Cancer Biol. 52:74–84.
Naraghi M., Neher E. (1997) Linearized buffered Ca2+ diffusion in microdomains and its implications for calculation of [Ca2+] at the mouth of a calcium channel. J. Neurosci. 17:6961–6973.
Nicholson-Fish J.C., Cousin M.A., Smillie K.J. (2016) Phosphatidylinositol 3-kinase couples localized calcium influx to activation of Akt in central nerve terminals. Neurochem. Res. 41:534–543.
Ogura S., Yoshida Y., Kurahashi T., Egawa M., Furuta K., Kiso S., Kamada Y., Hikita H., Eguchi H., Ogita H., Doki Y., Mori M., Tatsumi T., Takehara T. (2018) Targeting the mevalonate pathway is a novel therapeutic approach to inhibit oncogenic FoxM1 transcription factor in human hepatocellular carcinoma . Oncotarget 9:21022–21035.
Oheim M., van’t Hoff M., Feltz A., Zamaleeva A., Mallet J.M., Collot M. (2014) New red-fluorescent calcium indicators for optogenetics, photoactivation and multi-color imaging. Biochim. Biophys. Acta 1843: 2284‒2306.
Parfitt A.M., Kleerekofer M. (1980) The divalent ion homeostasis system: physiology and metabolism of calcium, phosphorus, magnesium, and bone. In: Maxwell M.H., Kleeman C.R., editors. Clinical Disorders of Fluid and Electrolyte Metabolism. McGraw-Hill, New York. 269–398.
Park H.J., Wang Z., Costa R.H., Tyner A., Lau L.F., Raychaudhuri P. (2008) An N-terminal inhibitory domain modulates activity of FoxM1 during cell cycle. Oncogene 27:1696–1704.
Patel R., Holt M., Philipova R., Moss S., Schulman H., Hidaka H., Whitaker M. (1999) Calcium/calmodulin-dependent phosphorylation and activation of human Cdc25-C at the G2/M phase transition in HeLa cells. J. Biol. Chem. 274:7958–7968.
Prat J. (2012) New insights into ovarian cancer pathology. Ann. Oncol. 23:111–117.
Price L.S., Langeslag M., ten Klooster J.P., Hordijk P.L., Jalink K., Collard J.G. (2003) Calcium signaling regulates translocation and activation of Rac. J. Biol. Chem. 278:39413–39421.
Roderick H.L., Cook S.J. (2008) Ca2+ signalling checkpoints in cancer: remodelling Ca2+ for cancer cell proliferation and survival. Nat. Rev. Cancer 8:361–375.
Russa A.D., Maesawa C., Satoh Y. (2009) Spontaneous [Ca2+]i oscillations in G1/S phase-synchronized cells. J. Electron Microsc. 58:321–329.
Schajnovitz A., Itkin T., D′Uva G., Kalinkovich A., Golan K., Ludin A., Cohen D., Shulman Z., Avigdor A., Nagler A., Kollet O., Seger R., Lapidot T. (2011) CXCL12 secretion by bone marrow stromal cells is dependent on cell contact and mediated by connexin-43 and connexin-45 gap juctions. Nat. Immunol. 12:391‒398.
Schimmel J., Eifler K., Sigurethsson J.O., Cuijpers S.A.G., Hendriks I.A., Verlaan-de Vries M., Kelstrup C.D., Francavilla C., Medema R.H., Olsen J.V., Vertegaal A.C.O. (2014) Uncovering SUMOylation dynamics during cell-cycle progression reveals FoxM1 as a key mitotic SUMO target protein. Mol. Cell 53:1053‒1066.
Schwarz D.S., Blower M.D. (2016) The endoplasmic reticulum: structure, function and response to cellular signaling. Cell. Mol. Life Sci. 73:79‒94.
Serebryannyy L., Misteli T. (2018) Protein sequestration at the nuclear periphery as a potential regulatory mechanism in premature aging. J. Cell Biol. 217:21‒37.
Shutes A., Onesto C., Picard V., Leblond B., Schweighoffer F., Der C.J. (2007) Specificity and mechanism of action of EHT1864, a novel small molecule inhibitor of Rac family small GTPases. J. Biol. Chem. 282:35666–35678.
Smith M.K., Colbran R.J., Brickey D.A., Soderling T.R. (1992) Functional determinants in the autoinhibitory domain of calcium/calmodulin-dependent protein kinase II. Role of His282 and multiple basic residues. J. Biol. Chem. 267:1761–1768.
Swulius M.T., Waxham M.N. (2008) Ca2+/calmodulin-dependent protein kinases. Cell. Mol. Life Sci. 65:2637–2657.
Wagstaff K.M., Jans D.A. (2009) Importins and beyond: non-conventional nuclear transport mechanisms. Traffic 10:1188–1198.
Wang J.L., Lin Y.W., Chen H.M., Kong X., Xiong H., Shen N., Hong J., Fang J.Y. (2011) Calcium prevents tumorigenesis in a mouse model of colorectal cancer. PLoS One 6:e22566.
Weeraratna A.T., Jiang Y., Hostetter G., Rosenblatt K., Duray P., Bittner M., Trent J.M. (2002) Wnt5a signaling directly affects cell motility and invasion of metastatic melanoma. Cancer Cell 1:279–288.
Wong M.H., Samal A.B., Lee M., Vlach J., Novikov N., Niedziela-Majka A., Feng J.Y., Koltun D.O., Brendza K.M., Kwon H.J., Schultz B.E., Sakowicz R., Saad J.S., Papalia G.A. (2019) The KN-93 moleculce inhibits calcium/calmodulin -dependent protein kinase II (CaMKII) activity by binding to Ca2+/CaM. J. Mol. Biol. 431:1440‒1459.
Xu N., Luo K.Q., Chang D.C. (2003) Ca2+ signal blockers can inhibit M/A transition in mammalian cells by interfering with the spindle checkpoint. Biochem. Biophys. Res. Commun. 306:737–745.
Yao K.M., Sha M., Lu Z., Wong G.G. (1997) Molecular analysis of a novel winged helix protein, WIN. Expression pattern, DNA binding property, and alternative splicing within the DNA binding domain. J. Biol. Chem. 272:19827‒19836.
Zhang N., Wei P., Gong A., Chiu W.T., Lee H.T., Colman H., Huang H., Xue J., Liu M., Wang Y., Sawaya R., Xie K., Yung W.K., Medema R.H., He X., Huang S. (2011) FoxM1 promotes β-catenin nuclear localization and controls Wnt target-gene expression and glioma tumorigenesis. Cancer Cell 20:427–442.
Zhang X., Lv Q.L., Huang Y.T., Zhang L.H., Zhou H.H. (2017) Akt/FoxM1 signaling pathway-mediated upregulation of MYBL2 promotes progression of human glioma. J. Exp. Clin. Cancer Res. 36:105.
Zhang X., Zhang L., Du Y., Zheng H., Zhang P., Sun Y., Wang Y., Chen J., Ding P., Wang N., Yang C., Huang T., Yao X., Qiao Q., Gu H., Cai G., Cai S., Zhou X., Hu W. (2017) A novel FOXM1 isoform, FOXM1D, promotes epithelial-mesenchymal transition and metastasis through ROCKs activation in colorectal cancer. Oncogene 36:807–819.