Exploringstathmincontrolofcellsurvival ... ·...

DOI: 10.19185/matters.201602000021 Matters (ISSN: 2297-8240) | 1

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DisciplinesCell Biology

KeywordsSignal TransductionMicrotubulesApoptosis

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Submitted Jan 12, 2016 Published Feb 22, 2016

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Exploring stathmin control of cell survivalthrough negative feedback of a JNK-dependentpathwayJessica Leung, Melissa Plooster, Lynne CassimerisBiological Sciences, Lehigh University;

AbstractPrevious studies demonstrated that stathmin, a microtubule destabilizing protein, pro-motes cell survival and/or prevents apoptosis, although the underlyingmolecular mech-anism is unknown. Based on a recent work [1], we are testing the hypothesis thatstathmin normally functions to keep the levels of active c-Jun N-terminal kinases (JNK)low. In this model, stathmin, phosphorylated by JNK, functions in a negative feed-back loop to inhibit the JNK pathway and limit JNK activation. This model predictsthat active JNK will rise in the absence of stathmin; prolonged activation of JNK wouldthen trigger apoptosis. As a first test of whether stathmin regulates JNK activity tocontrol cell survival, we found that treatment with either JNK-IN-8, a JNK inhibitor,or depletion of JNK1,2, prevented cell death in stathmin-depleted HeLa cells. Using alocalization-dependent biosensor [2], we found that active JNK levels were higher instathmin-depleted cells. Expression of a stathmin phosphomimic restored active JNKlevel and prevented apoptosis. These data support a model where phosphorylated stath-min, acting independently of the microtubule cytoskeleton, prevents JNK hyperactiva-tion to promote cell survival.

ObjectiveTo address whether stathmin depletion activates apoptosis via loss of a negative feed-back loop and hyperactivation of JNK, we asked whether treatment with a JNK inhibitor,JNK depletion or expression of a stathmin phosphomimic, abrogates apoptosis normallyobserved in stathmin-depleted cells.

IntroductionStathmin/Oncoprotein 18 has been linked to numerous human cancers, where increasedstathmin expression is highly correlated with cancer stage progression and chemo-resistance [3] [4]. Previous studies have demonstrated that stathmin depletion is suffi-cient to slow proliferation and increase cell death in cancer cells [5] [6] [7] [3], althoughthe mechanism responsible has not been thoroughly described. It is thought that stath-min acts via microtubule destabilization to control cell fate, where stathmin depletionresults in greater microtubule stability, a phenotype superficially similar to Taxol treat-ment [3]. Stathmin’s microtubule-destabilizing function is inactivated by phosphoryla-tion of up to four serines, where several kinases target one or more sites [8] [9]. Recentwork suggested, but did not test, a potential microtubule-independent function for phos-phorylated stathmin, where stathmin is both a target of JNK and, in its phosphorylatedform, an upstream inhibitor of this kinase [1]. In this way, phosphorylated stathminfunctions as part of a negative feedback loop to prevent hyperactivation of JNK. Impor-tantly, prolonged JNK activation results in apoptosis [10] [11] .

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Figure LegendFigure a. Loss of JNK activity abrogates cell death in stathmin-depleted HeLa cells.Cells were treated with a JNK inhibitor, JNK-IN-8 (20 nM final concentration), or DMSOcontrol beginning approximately 4 h post-siRNA transfection and maintained until cellviability measurements by Trypan Blue exclusion at 48 h post-transfection. Treatmentwith JNK inhibitor in control cells transfected with non-targeting (NT) siRNA causedminimal cell death compared to the NT siRNA treatment alone. Administering the JNKinhibitor to stathmin (STMN)-depleted HeLa cells significantly reduced the percentageof dead cells. Data shown from three independent experiments, >100 cells per conditionper experiment (*p ≤0.05; **p ≤0.01).Figure b. HeLa cells were depleted of stathmin and/or JNK1,2 by siRNA transfection.The antibody to JNK recognizes both JNK 1 and 2. GAPDH was used as a loading con-trol.Figure c. Cell viability was assessed in cells depleted of stathmin and/or JNK1,2 by theCellTox Green continuous cytotoxicity assay at 24, 48, and 72 h post-transfection. Lossof JNK activity by siRNA knockdown caused minimal cell death. Double knockdownof stathmin and JNK was sufficient to minimize cell death up to 72 h post-transfection.Data shown from three independent experiments, >100 cells per condition per experi-ment. Color coding for each condition is given below the x axis. (*p ≤0.05; **p ≤0.01;***p ≤0.001)Figure d. JNK activity is elevated in stathmin-depleted cells. Control and stathmin-depleted HeLa cells were transfected with JNK KTR-mRuby2 (JNK activity biosensor) 4h post-siRNA transfection. Cells were fixed at time points following transfection andimaged for quantification of JNK activity. Representative images of mRuby2-taggedJNK KTR localizations are shown. Cell outlines are marked by dashed bluelines. Thecytoplasmic to nuclear (C/N) ratios of JNK KTR intensity are shown and measured asdescribed in Methods. Time, in hours, post-transfection is given over the images. Scalebar = 10 µm.Figure e. Integrated C/N intensity ratios of JNK KTR localizations in cells fixed attime points 24-72 h post NT- or stathmin-siRNAtransfections. The greater C/N ratioin stathmin-depleted cells represents nuclear export of the biosensor, indicating greaterJNK activity. Grey boxes represent non-targeting (NT) siRNA; blue boxes represent



stathmin (STMN)-depleted cells. Data shown from three independent experiments, >50cells per condition per experiment (**p ≤0.01)Figure f. Phase contrast and fluorescence (mRuby2-JNK KTR) images of a cell just be-fore apoptosis and a neighboring cell that survives. The corresponding C/N JNK KTRintensity ratios are given below each image. Scale bar = 10 µm.Figure g. JNK activity was quantified by calculating the intensity of the JNK KTR re-porter in nucleus and cytoplasm for individual cells inthe 30 min prior to cell death.Comparison of JNK activity in cells approaching death versus surviving neighboringcells indicates that active JNK levels are significantly increased in stathmin-depletedcells in the 30 min prior to cell death. Blue boxes represent cells prior to death at thetimes given; green boxes represent neighboring, surviving cells. Data shown from threeindependent experiments, >100 cells per condition per experiment (****p ≤0.0001)Figure h. Expression of FLAG-tagged stathmin or phosphorylation site mutants in HeLacells depleted of endogenous stathmin (by siRNA targeted to the 5’ UTR of stathmin).Tubulin is shown as a loading control.Figure i. Expression of a stathmin phosphomimic abrogates cell death. Cell viabilitywas assessed by the CellTox Green assay in cells depleted of endogenous stathmin andexpressing the FLAG-tagged constructs at 24, 48, and 72 h post-transfection. Expres-sion of either FLAG-tagged wildtype stathmin or stathmin Tetra-E mutant (phospho-mimic) reduced cell death up to 72 h post-transfection. In contrast, expression of thenonphosphorylatable stathmin Tetra-A mutant, which maintains the ability to regulatemicrotubule stability, fails to prevent cell death in HeLa cells depleted of the endogenousprotein. Color coding is shown in the upper right. Data shown from three independentexperiments, >100 cells per condition per experiment (*p ≤0.05; **p ≤0.01; ***p ≤0.001)Figure j. Expression of a stathmin phosphomimic restores JNK activity to basal levels.Analysis of JNK activity by localization of mRuby2-JNK KTR in cells depleted of endoge-nous stathmin and expressing either stathmin or phosphorylation site mutants. Expres-sion of the FLAG-tagged stathmin or the phosphomimic (Tetra-E) in cells depleted ofendogenous stathmin significantly reduced cytoplasmic to nuclear localization ratiosof the JNK KTR, indicative of reduced active JNK compared to stathmin-depleted cellsalone. Color coding for the boxes is given below the x axis. Data shown from threeindependent experiments, >50 cells per condition per experiment (*p ≤0.05; **p ≤0.01;***p ≤0.001; ****p ≤0.0001).

Results & DiscussionStathmin depletion increases apoptosis in p53-deficient cancer cells [5] [6]; however,the mechanism by which stathmin regulates cell survival remains unresolved. We hy-pothesized that stathmin promotes cell survival independently of its regulation of themicrotubule cytoskeleton by maintaining low levels of JNK activity. In the absence ofstathmin and loss of a negative feedback loop, active JNK levels should rise and eventu-ally activate apoptosis. HeLa cells are a convenient model system to test this hypothesissince they are easily transfected, and previous studies demonstrated that stathmin de-pletion induces apoptosis in these cells [7] [6] [12]. Using a specific JNK inhibitor orsiRNA knockdown to abolish JNK activity, we addressed whether JNK is necessary forinduction of cell death in stathmin-depleted cells. Treatment with the small-moleculeJNK inhibitor, JNK-IN-8, restored cell viability to control levels in stathmin-depletedcells (Figure a). Depletion of JNK 1,2 (Figure b) did not impact cell survival, while co-depletion of stathmin and JNK 1,2 abrogated the cell death observed in cells depleted ofstathmin alone (Figure c). These results demonstrate that JNK activity is required forstathmin-depletion-induced cell death.A model where stathmin normally functions in a negative feedback loop to limit JNKactivation predicts that basal levels of JNK activity will rise in the absence of stathmin.We were unable to detect increased phosphorylated (active) JNK in stathmin-depletedcells by western blots. It is possible that increased active JNK occurs asynchronouslyin a population of cells, much like the asynchrony in the onset of cell death [12], and,therefore, is undetectable by western blots from cell populations. In order to assess JNKactivity in individual cells over time, we used a fluorescently tagged JNK kinase translo-cation reporter (KTR), a localization-based biosensor of JNK kinase activity [2]. We con-



firmed that the JNK KTR localizes appropriately in our system and is translocated fromthe nucleus to the cytoplasm when cells are treated with a small-molecule JNK activator(Supplemental Figure S1). Applying the sensor to our experiments, we found that ac-tive JNK levels were elevated for at least 72 h post-transfection in stathmin-depleted cellscompared to cells treatedwith non-targeting siRNA, although the increased JNK activitywas modest (Figure d,e). We further investigated the subset of stathmin-depleted cellsthat undergo apoptosis by long-term, live cell imaging. Stathmin-depleted cells express-ing the mRuby2-tagged JNK KTR were observed at 24-72 h post-transfection, and JNKactivity was quantified on a single cell basis for the 30-min interval prior to cell death.This analysis revealed that JNK activity is significantly elevated in stathmin-depletedcells in the 30 min prior to apoptosis when compared to the neighboring surviving cells(Figure f,g). These data support a role for elevated JNK in stathmin depletion-inducedapoptosis, but it is not yet known if JNK activity accumulates over time in all stathmin-depleted cells or if a subset of stathmin-depleted cells is more likely to hyperactivateJNK and undergo apoptosis.To explore whether JNK hyperactivation occurs due to loss of phosphorylated stath-min, possibly acting to inhibit JNK activation, or whether stathmin depletion functionsby stabilizing the microtubule cytoskeleton, we expressed FLAG-tagged stathmin orphospho-mutant constructs in cells depleted of the endogenous protein (Figure h). Ex-pression of FLAG-tagged stathmin abrogates cell death in these cells. A phosphomimic,with all four serine residues mutated to glutamic acid, also abrogates cell death in theabsence of endogenous stathmin (Figure i). This Tetra-E mutant has little microtubuledestabilizing activity compared to the unphosphorylated protein [9] [13]. In contrast, anonphosphorylatable Tetra-A stathmin mutant, with serines mutated to alanine, retainsits ability to destabilize microtubules [9] [13] [14] and fails to prevent cell death after de-pletion of endogenous stathmin (Figure i). These results support the idea that stathminacts independently of the microtubule cytoskeleton in order to mediate cell viability.Additionally, expression of JNK KTR reveals that expression of FLAG-tagged stathminor the Tetra-E phosphomimic restores JNK activity to levels comparable to control cells(Figure j; Supplemental Figure S2). In comparison, JNK activity remains elevated in cellsexpressing the Tetra-A mutant. These data support the hypothesis that phosphorylatedstathmin maintains low JNK activity, which is necessary to prevent cell death/promotecell survival.Earlier studies demonstrated that JNK-dependent phosphorylation of stathmin protectsagainst stress-induced apoptosis [15], supporting our current hypothesis that stathmin,phosphorylated by active JNK, acts in a negative feedback loop to inhibit the JNK ac-tivation pathway. However, the target inhibited by phosphorylated stathmin remainsunresolved. Any number of kinases upstream of JNK and its activator MKK-4 in theMAPK cascade may be the critical component [1], though another hypothesis suggestsJNK activation is sustained by the inhibition of MAP kinase phosphatases [16]. To date,these potential feedback mechanisms for stathmin depletion-stimulated apoptosis re-main unexplored; however, our results support the idea that stathmin functions inde-pendently of the microtubule cytoskeleton to mediate cell survival, likely acting in anegative feedback loop to maintain basal JNK activity.Stathmin has been shown to bind to two tubulin dimers [9], and this has been typicallydescribed as stathmin functioning to sequester tubulins and limit their polymerization.Given that we have uncovered a non-microtubule based function for stathmin, it is in-teresting to speculate that tubulin sequesters stathmin, and not the other way around.By sequestering stathmin, tubulin could prevent its phosphorylation and subsequentinhibition of the JNK activation pathway.The data presented here provide initial tests of a stathmin-JNK negative feedback loopand demonstrate the potential significance of such a feedback loop in controlling cellsurvival/apoptosis decisions. But much remains unknown, including where in the JNKactivation pathway phosphorylated stathmin acts as an inhibitor. We have not uncov-ered the link between JNK hyperactivation and apoptosis under our experimental con-ditions. Initial experiments focused on the E3 ubiquitin ligase, ITCH, which when acti-vated by long-term JNK activation degrades the Caspase 8 inhibitor, c-FLIP [17] [18]. Todate we have not detected a role for ITCH in activation of apoptosis. ITCH depletion did

not abrogate cell death in stathmin-depleted cells, indicating that ITCH is not sufficientto activate apoptosis after stathmin depletion.An unsolved question is why so many cancers overexpress stathmin. Indeed, increasedstathmin expression has been correlated with cancer progression [19], and at least inhepatocellular cancer, patient survival is predicted equally well by either stathmin over-expression or by p53 mutations [20]. It is possible that those cancer cells overexpressingstathmin are selected because the high level of stathmin prevents JNK hyperactivationand subsequent apoptosis.

Additional Information

Methods and Supplementary MaterialPlease see https://sciencematters.io/articles/201602000021.Supported by NIH GM100381.The authors are indebted to undergraduates Kyle Peters and Arianna Caruso for per-forming the ITCH experiments.

Ethics StatementNot applicable.

Citations

[1] Enpeng Zhao et al. “Stathmin Mediates Hepatocyte Resistance toDeath from Oxidative Stress by down Regulating JNK”. In: PLoSONE 9.10 (Oct. 2014), e109750. doi:10.1371/journal.pone.0109750. url: http://dx.doi.org/10.1371/journal.pone.0109750.

[2] Sergi Regot et al. “High-Sensitivity Measurements of MultipleKinase Activities in Live Single Cells”. In: Cell 157.7 (June 2014),pp. 1724–1734. doi: 10.1016/j.cell.2014.04.039.url: http://dx.doi.org/10.1016/j.cell.2014.04.039.

[3] Barbara Belletti and Gustavo Baldassarre. “Stathmin: a proteinwith many tasks. New biomarker and potential target in cancer”.In: Expert Opinion on Therapeutic Targets 15.11 (Nov. 2011),pp. 1249–1266. doi: 10.1517/14728222.2011.620951.url: http://dx.doi.org/10.1517/14728222.2011.620951.

[4] Wei Nie et al. “Overexpression of stathmin 1 is a poor prognosticbiomarker in non-small cell lung cancer”. In: Lab Invest 95.1 (Nov.2014), pp. 56–64. doi: 10.1038/labinvest.2014.124.url: http://dx.doi.org/10.1038/labinvest.2014.124.

[5] E Alli, J-M Yang, and W N Hait. “Silencing of stathmin inducestumor-suppressor function in breast cancer cell lines harboringmutant p53”. In: Oncogene 26.7 (Aug. 2006), pp. 1003–1012. doi:10.1038/sj.onc.1209864. url:http://dx.doi.org/10.1038/sj.onc.1209864.

[6] Bruce K. Carney and Lynne Cassimeris. “Stathmin/oncoprotein18, a microtubule regulatory protein, is required for survival ofboth normal and cancer cell lines lacking the tumor suppressor,p53”. In: Cancer Biology and Therapy 9.9 (May 2010), pp. 699–709.doi: 10.4161/cbt.9.9.11430. url:http://dx.doi.org/10.4161/cbt.9.9.11430.

[7] Hui-Zhong Zhang et al. “Silencing stathmin gene expression bysurvivin promoter-driven siRNA vector to reverse malignantphenotype of tumor cells”. In: Cancer Biology and Therapy 5.11(Nov. 2006), pp. 1457–1461. doi:10.4161/cbt.5.11.3272. url:http://dx.doi.org/10.4161/cbt.5.11.3272.

[8] Lynne Cassimeris. “The oncoprotein 18/stathmin family ofmicrotubule destabilizers”. In: Current Opinion in Cell Biology14.1 (Feb. 2002), pp. 18–24. doi:10.1016/s0955-0674(01)00289-7. url:http://dx.doi.org/10.1016/s0955-0674(01)00289-7.

[9] Michel O. Steinmetz. “Structure and thermodynamics of thetubulin–stathmin interaction”. In: Journal of Structural Biology158.2 (May 2007), pp. 137–147. doi:10.1016/j.jsb.2006.07.018. url: http://dx.doi.org/10.1016/j.jsb.2006.07.018.

[10] Anning Lin. “Activation of the JNK signaling pathway: Breakingthe brake on apoptosis”. In: Bioessays 25.1 (Dec. 2002), pp. 17–24.doi: 10.1002/bies.10204. url:http://dx.doi.org/10.1002/bies.10204.

[11] Jing LIU and Anning LIN. “Role of JNK activation in apoptosis: Adouble-edged sword”. In: Cell Res 15.1 (Jan. 2005), pp. 36–42. doi:10.1038/sj.cr.7290262. url:http://dx.doi.org/10.1038/sj.cr.7290262.

[12] Victoria C Silva et al. “A delay prior to mitotic entry triggerscaspase 8-dependent cell death in p53-deficient Hela andHCT-116 cells”. In: Cell Cycle 14.7 (Jan. 2015), pp. 1070–1081. doi:10.1080/15384101.2015.1007781. url: http://dx.doi.org/10.1080/15384101.2015.1007781.

[13] P. Holmfeldt et al. “The Catastrophe-promoting Activity ofEctopic Op18/Stathmin Is Required for Disruption of MitoticSpindles But Not Interphase Microtubules”. In: Molecular Biologyof the Cell 12.1 (Jan. 2001), pp. 73–83. doi:10.1091/mbc.12.1.73. url:http://dx.doi.org/10.1091/mbc.12.1.73.

[14] T. Wittmann, G. M. Bokoch, and C. M. Waterman-Storer.“Regulation of Microtubule Destabilizing Activity ofOp18/Stathmin Downstream of Rac1”. In: Journal of BiologicalChemistry 279.7 (Nov. 2003), pp. 6196–6203. doi:10.1074/jbc.m307261200. url:http://dx.doi.org/10.1074/jbc.m307261200.

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[15] D. C. H. Ng et al. “c-Jun N-terminal Kinase Phosphorylation ofStathmin Confers Protection against Cellular Stress”. In: Journalof Biological Chemistry 285.37 (July 2010), pp. 29001–29013. doi:10.1074/jbc.m110.128454. url:http://dx.doi.org/10.1074/jbc.M110.128454.

[16] Hideaki Kamata et al. “Reactive Oxygen Species PromoteTNFu�-Induced Death and Sustained JNK Activation by InhibitingMAP Kinase Phosphatases”. In: Cell 120.5 (Mar. 2005),pp. 649–661. doi: 10.1016/j.cell.2004.12.041. url:http://dx.doi.org/10.1016/j.cell.2004.12.041.

[17] Lufen Chang et al. “The E3 Ubiquitin Ligase Itch Couples JNKActivation to TNFu�-induced Cell Death by Inducing c-FLIPLTurnover”. In: Cell 124.3 (Feb. 2006), pp. 601–613. doi:10.1016/j.cell.2006.01.021. url: http://dx.doi.org/10.1016/j.cell.2006.01.021.

[18] M. Gao. “Jun Turnover Is Controlled Through JNK-DependentPhosphorylation of the E3 Ligase Itch”. In: Science 306.5694 (Oct.2004), pp. 271–275. doi: 10.1126/science.1099414. url:http://dx.doi.org/10.1126/science.1099414.

[19] G. Chen. “Overexpression of Oncoprotein 18 Correlates withPoor Differentiation in Lung Adenocarcinomas”. In: Molecularand Cellular Proteomics 2.2 (Feb. 2003), pp. 107–116. doi:10.1074/mcp.m200055-mcp200. url: http://dx.doi.org/10.1074/mcp.M200055-MCP200.

[20] R-H Yuan et al. “Stathmin overexpression cooperates withp53mutation andosteopontin overexpression, and is associated withtumour progression, early recurrence, and poor prognosis inhepatocellular carcinoma”. In: J. Pathol. 209.4 (2006), pp. 549–558.doi: 10.1002/path.2011. url:http://dx.doi.org/10.1002/path.2011.

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