Sgk1 Enhances RANBP1 Transcript Levels and Decreases Taxol Sensitivity in RKO Colon Carcinoma Cells

SHORT COMMUNICATION Sgk1 enhances RANBP1 transcript levels and decreases taxol sensitivity in RKO colon carcinoma cells

R Amato1,8, D Scumaci2,8, L D’Antona1, R Iuliano2,3, M Menniti1, M Di Sanzo2, MC Faniello2, E Colao3, P Malatesta3, A Zingone4, V Agosti2, FS Costanzo2, AM Mileo5, MG Paggi5, F Lang6, G Cuda2, P Lavia7 and N Perrotti1,3

The serum- and glucocorticoid-regulated kinase (Sgk1) is essential for hormonal regulation of epithelial sodium channel-mediated sodium transport and is involved in the transduction of growth factor-dependent cell survival and proliferation signals. Growing evidence now points to Sgk1 as a key element in the development and/or progression of human cancer. To gain insight into the mechanisms through which Sgk1 regulates cell proliferation, we adopted a proteomic approach to identify up- or downregulated proteins after Sgk1-speciﬁc RNA silencing. Among several proteins, the abundance of which was found to be up- or downregulated upon Sgk1 silencing, we focused our attention of RAN-binding protein 1 (RANBP1), a major effector of the GTPase RAN. We report that Sgk1-dependent regulation of RANBP1 has functional consequences on both mitotic microtubule activity and taxol sensitivity of cancer cells.

Oncogene (2013) 32, 4572–4578; doi:10.1038/onc.2012.470; published online 29 October 2012 Keywords: Sgk1; RANBP1; taxol sensitivity; mitotic microtubule stabilization

INTRODUCTION and Ca2 þ channels, Na þ /H þ exchangers, amino-acid transporters The serum- and glucocorticoid-inducible kinase (Sgk1) was and glucose transporters, upregulation of nuclear factors NFkBand originally described as a key enzyme with serine/threonine b-catenin, as well as downregulation of the transcription factors 3 specificity acting in the hormonal regulation of sodium absorption Foxo3a/FKHRL1. Indeed, Sgk1 deficiency in a knock-out mouse by the amiloride-sensitive epithelial sodium channel. The gene model has recently been demonstrated to confer resistance to 14 coding for Sgk1 is a serum and glucocorticoid-sensitive gene, later chemical carcinogenesis induction of colonic tumors in vivo and found to be similarly regulated by other hormones, osmotic cell to counteract the appearance of intestinal tumors in adenomatous 15 shrinkage and other triggers of cell stress.1–3 The kinase protein polyposis coli (APC) deficiency. product is activated by oxidative stress, insulin or growth factors To gain more insight into pathways affected by Sgk1 activity in colon through PDK1-dependent mechanisms Kobayashi et al.4,5 The cancer cells, in this work we have adopted a proteomic approach hydrophobic motif (HOURS-motif) mTOR kinase phosphorylates to identify proteins that are either upregulated or downregulated Sgk1 at S422;6 PDK1 then binds phospho-S422 in the Sgk1 in the human colon carcinoma cell line RKO, a well-studied model 16,17 HOURS-motif to achieve subsequent phosphorylation at T256 and of human colon carcinoma, with and without specific silencing full activation of the kinase.5 Initially, Sgk1 was demonstrated to for Sgk1, by means of Sgk1-specific short hairpin (sh) RNAs. participate in the regulation of the renal epithelial sodium Using two-dimensional electrophoresis (2DE) gel electrophoresis channel.7–9 Since then, a wide variety of channels, transporters followed by Nanoscale liquid chromatography coupled with tandem and further functions of Sgk1 have been identified.2 mass spectrometry (LC-MS/MS) analysis we discovered that Sgk1 Sgk1-sensitive functions include the inhibition of apoptosis.3 silencing is associated with significant modifications in the abundance Specifically, Sgk1 transmits insulin and IGF1-dependent survival of a distinct group of proteins. Among the best identified protein signals.10,11 Sgk1 also contributes to IL2-dependent antiapoptotic spots, RAN-binding protein 1 (RANBP1) showed significant down- signals in kidney cancer cells.12 In addition, Sgk1 directly regulation in Sgk1-silenced cells. RANBP1 protein abundance is known phosphorylates MDM2, which directs p53 to ubiquitylation and to affect many steps of mitotic progression, including the dynamic 15,18–22 23,24 proteosomal degradation, thus affecting the half-life of p53 and, in activity of microtubules. RAN is a major regulator of mitosis turn, cell cycle, differentiation and survival.13 Taken together these and was recently identified as a potential therapeutic target in cancers 25 data indicate that Sgk1 is potentially implicated in human carcino- expressing higher Ras/MEK/ERK and PI3K/Akt/mTORC1 activities. genesis, a role that is further supported by the involvement of Sgk1 itself has been suggested to have a role in microtubule Sgk1 in other pathways that include activation of K þ channels organization by phosphorylating the protein product of the N-myc

1Department of Human Health, University Magna Graecia at Catanzaro, Campus S Venuta, Localita` Germaneto Viale Europa, Catanzaro, Italy; 2Department of Experimental and Clinical Medicine, University Magna Graecia at Catanzaro, Campus S Venuta, Localita` Germaneto Viale Europa, Catanzaro, Italy; 3Unit of Medical Genetics and Pathology University Hospital, Policlinico Mater Domini, Catanzaro, Italy; 4Multiple Myeloma Section, Metabolism Branch, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD, USA; 5Department for the Development of Therapeutic Programs, National Cancer Institute ‘Regina Elena’, Rome, Italy; 6Department of Physiology, University of Tuebingen, Tu¨bingen, Germany and 7IBPM Institute of Molecular Biology and Pathology, CNR National Research Council, c/o Sapienza University of Rome, Rome, Italy. Correspondence: Dr P Lavia, IBPM Institute of Molecular Biology and Pathology, CNR National Research Council, c/o Sapienza University of Rome, 00185 Rome, Italy or Professor N Perrotti, Scienze della Salute, UniversitA˜ Magna Graecia di Catanzaro Viale Europa, localitA˜ Germaneto, Catanzaro, CZ 88100, Italy. E-mail: [email protected] or [email protected] 8These authors contributed equally to this work. Received 17 January 2012; revised 1 August 2012; accepted 21 August 2012; published online 29 October 2012 Sgk1, RANBP1 and taxol sensitivity R Amato et al 4573 downstream regulated gene.26 We therefore elected to focus on molecular function and cell localization is reported in Sgk1-dependent regulation of RANBP1 abundance. We report Supplementary File 5. Eighteen protein spots varied by over that Sgk1 regulates RANBP1 at the gene transcription level and two-fold. As shown in Table 1, nine proteins were consistently that this has functional consequences on microtubule stability and upregulated and nine were downregulated in ShSgk1 RKO cells, on the cell sensitivity to taxol. compared with their Shscrl control counterpart. Among the spots consistently downregulated in ShSgk1 RKO cells, spot 6 was positively identified as the RAN-specific GTPase-activating protein RESULTS AND DISCUSSION RANBP1 (Figures 1a and b), with an identification score 4400, 8 Differential 2DE protein profiling of RKO colon carcinoma cells unique peptides, defined by convention, with a score X30 proficient or defective for the Sgk1 kinase (Supplementary File 6), and a 67% coverage of the full-length Here, we have examined the protein profile of colon carcinoma- protein sequence. The effects of Sgk1 silencing on RANBP1 derived RKO cell lines in which the Sgk1 kinase was either abundance were confirmed by mono-dimensional SDS– inactivated by using viral-driven Sgk1-specific ShRNAs (named polyacrylamide gel electrophoresis followed by western blot in thereafter ShSgk1-Rko cells), or expressed at wild-type level RKO cells (Figure1c) as well as in other cell lines such as EpH4 and (interfered with scrambled RNA, indicated thereafter as scrambled Hela cells (data not shown). Shscrl-RKO cells). Sgk1-directed ShRNAs abolished or drastically reduced Sgk1 expression (Supplementary File 1); the ShSgk1 and the Shscrl RKO cells have been extensively characterized Sgk1 modulates transcription of the RANBP1-coding gene in previous work. Sgk1 downregulation had significant conse- We first focused on the search for possible mechanisms of quences in cell cycle progression, yielding a decrease in the Sgk1-dependent regulation of RANBP1. Sgk1 was unable to percentage of G2/M phase cells and an increase in the percentage phosphorylate GST–RANBP1 and no molecular interaction was of S phase cells in ShSgk1 cells, compared with Sgk1-proficient demonstrated between Sgk1 and RANBP1 in standard protein– 13,28,29 cultures (Supplementary File 2), in agreement with previous data protein interaction assays, including co-immunoprecipitation obtained using dominant negative technology in Hela cells.13 and GST pull-down assays (data not shown). In direct support We comparatively examined the protein expression profiles of of this idea, adenovirus-driven Sgk1 overexpression yielded an both Sgk1 and scrambled sh RKO cells in 2DE assays. The increased abundance of RANBP1 protein in RKO cells (Figure 2a), experiments were performed in triplicate, ensuring reproducibility proving that fluctuations in Sgk1 expression can affect the levels of of spot detection among each gel by 97% in ShSgk1 and 98% in RanBP1. Shscrl RKO cells, respectively. A pair of representative 2DE maps is The reduced RANBP1 abundance in Sgk1-silenced cells shown in Supplementary File 3. suggests, therefore, that Sgk1 has roles in either RANBP1 protein stabilization or in upregulating transcription of the RANBP1 gene. Quantitative reverse transcriptase–PCR assays demonstrated that Identification of differentially expressed proteins RANBP1 mRNA levels significantly decreased in Sgk1-silenced RKO After automatic spot detection, background subtraction and cells (Figure 2b), strongly suggesting a role of Sgk1 in regulation of volume normalization, we detected 800±9.4 protein spots in RANBP1 gene transcription. ShSgk1 and 830±6.1 protein spots in Shscrl RKO cells, respec- To elucidate these mechanisms, we transfected RKO cells with tively. Only reproducibly detected spots were subjected to the murine wild-type RANBP1 promoter. The sequence and statistical analysis. A list of up- or downregulated proteins in functional organization of the RANBP1 promoter are characterized ShSgk1 RKO cells is provided as Supplementary File 4, and their in depth, and critical functional elements are identified, including classification according to their involvement in cellular processes, a TATA-less initiator element (arrowed in Figure 2c) as well as Sp1-

Table 1. List of the proteins consistently up- or downregulated in ShSgk1 RKO cells, compared with their Shscrl control counterpart

Spot number Protein name Fold changes of proteins upregulated Fold changes of in ShRNA 458 Sgk1 RKO cells proteins downregulated in ShRNA 458 Sgk1 RKO cells

Spot 17 Flavin reductase (EC 1.5.1.30) Detected only in ShRNA 458 Sgk1 RKO cells Spot 19 3-Hydroxyacyl-CoA dehydrogenase type-2 Detected only in ShRNA 458 Sgk1 RKO cells Spot 20 3-Hydroxyacyl-CoA dehydrogenase type-2 Detected only in ShRNA 458 Sgk1 RKO cells Spot 34 Matrin-3 Detected only in ShRNA 458 Sgk1 RKO cells Spot 5 Proteasome subunit alpha type 3 Detected only in ShRNA 458 Sgk1 RKO cells Spot 12 Alpha-2-HS-glycoprotein precursor (Fetuin-A) 2.6 Spot 16 Phosphatidylethanolamine-binding protein 1 2.4 Spot 17 b Peroxiredoxin-1 (EC 1.11.1.15) 2.0 Spot 23 Proliferation-associated protein 2G4 2.0 Spot 30 HNRNPH1 Heterogeneous nuclear À 2.0 ribonucleoprotein H Spot 6 Ran-speciﬁc GTPase-activating protein À 2.0 Spot 8b Elongation factor 1-delta À 2.0 Spot 11b Nucleosome assembly protein 1-like 1 À 2.3 Spot 10 Calumenin precursor (Crocalbin) À 2.8 Spot 1 14-3-3 Protein sigma (Stratiﬁn) À 3.3 Spot7 Proteasome activator complex subunit 1 À 3.6 Spot 7b Prohibitin À 3.8 Spot 3 Peroxiredoxin-2 À 5.3 Abbreviation: ShRNA, short hairpin RNA.

& 2013 Macmillan Publishers Limited Oncogene (2013) 4572 – 4578 Sgk1, RANBP1 and taxol sensitivity R Amato et al 4574

Figure 1. Sgk1-speciﬁc sh RNA silencing downregulates RANBP1 protein levels in RKO cells. (a) Blow-up of RANBP1 spot in 2DE gel from ShSgk1-Rko cells (top left) and Shscrl RKO cells (top right) from 2DE gels shown in Supplementary File 1. RanBP1 abundance clearly decreases in ShSgk1Rko cells (left) compared with ShScrl-Rko cells (right). In the same panels, the protein appearing just above RanBP1 only in ShSgk1Rko cells (left) (spot 5 in Table 1) was identiﬁed as proteasome subunit alpha type 3, a potentially insteresting Sgk1 target unrelated to RanBP1. Actin was used as a loading control. Materials and methods for 2DE polyacrylamide gel electrophoresis, Nanoscale LC-MS/MS analysis and data analysis are detailed in Supplementary File 3.27 Methods for the establishment of ShSgk1 RKO cell lines are detailed in Supplementary File 1. (b) Signal intensity values (mean±s.d.) from RANBP1 densitometric scanning from three independent 2DE gel experiments in ShSgk1-Rko and Sh scrlRKO cells. (c) Western blot of proteins from ShSgk1-Rko and Shscrl-RKO cells. Cell extracts (40–50 mg aliquots) were loaded on SDS–polyacrylamide gel electrophoresis for immunoblotting using Sgk1 antibody (rabbit polyclonal, Upstate, Lake Placid, NY, USA), RANBP1 antibody (goat polyclonal, Santa Cruz, Santa Cruz, CA, USA), and beta tubulin antibody (rabbit polyclonal).

Oncogene (2013) 4572 – 4578 & 2013 Macmillan Publishers Limited Sgk1, RANBP1 and taxol sensitivity R Amato et al 4575 and E2F-binding sites (see Figure 2c for details). The E2F- and These assays demonstrate that Sp1 is a genuine target of Sgk1 and Sp1-binding sites are highly conserved in the murine and human that Sgk1 phosphorylates serine 59 in the transactivation domain promoters. We assessed the sensitivity of the RANBP1 promoter, of Sp1, a substrate shared with PKC-alpha and Erk1 kinases.35 either in the wild-type form (pTS-A), or mutagenized in transcription factor-binding sites, to Sgk1 levels in chloramphenicol Downregulating Sgk1 abundance mimicks RANBP1 silencing in acetyl transferase (CAT) reporter assays. We found that adenovirally RKO cells and modulates the cell response to taxol driven Sgk1 overexpression increased RANBP1 promoter activity RANBP1 protein abundance affects multiple aspects of mitosis, (Figure 2d). When RANBP1 mutant promoters were transfected in including the dynamic activity of the spindle microtubules. Sgk1-overexpressing cells, we found that a mutant version lacking RANBP1 inactivation by either antibody microinjection18 or by the E2F-binding element (pmE construct) retained the ability RNA interference21,22 induced microtubule hyper-stabilization, to be upregulated by Sgk1, whereas mutational inactivation of apoptosis during mitosis—reminiscent of the effects of the the Sp1-binding element (pmS construct) reduced drastically the microtubule-stabilizing drug taxol—and increased cell death in promoter responsiveness to Sgk1-dependent activation (Figure 2d). response to taxol in several transformed cell lines.36 Given that These data indicate that Sgk1 upregulates RANPB1 transcription Sgk1 acts as a positive regulator of RANBP1 expression, and that the Sp1 transcription factor has a role in Sgk1-dependent downregulating Sgk1 levels should have similar functional RANBP1 promoter activation. consequences to those caused by direct RANBP1 silencing. If so, downregulation of Sgk1 should enhance the sensitivity of cells to Sgk1 phopshorylates Sp1 Taxol. SP1 was traditionally thought of as a housekeeping factor. In a first set of experiments, we examined the ability of taxol to Growing evidence, however, indicates that phosphorylation, arrest mitotic progression. This can be measured by assessing the acetylation, sumoylation, ubiquitylation and glycosylation are fraction of RKO cells, either silenced or overexpressing Sgk1, that among the post-translational modifications that can modulate arrest in prometaphase after taxol treatment (representative the trans-activating potential and stability of Sp1.34 Given the examples of prometaphase arrest are shown n Figure 3a). Taxol implication of Sp1 in Sgk1-dependent modulation of RANBP1 administration (10 nM) for 16 h yielded a significant increase in transcription, we wondered whether Sgk1 modifies Sp1 and hence prometaphase-blocked cells in ShSgk1 compared with control its transactivation potential. In vitro phosphorylation assays Shscrl RKO cultures; furthermore, at identical taxol concentrations, showed indeed that active Sgk1 was able to phosphorylate adenovirally driven Sgk1 overexpression significantly decreased GST-purified Sp1 (Figure 2e, lane 4). Sgk1 also phosphorylates this fraction (percent variations are quantified in the graph in GST-delta 1 Sp1 (residues 1–110), containing the N-terminal Figure 3a); thus, Sgk1 expression affects taxol-induced mitotic transactivation domain (Figure 2e lower panel), but is unable to arrest in cancer cells in culture, most likely via modulation of phosphorylate a S59A delta 1Sp1 mutant (Figure 2f and RanBP1 expression, although the prosurvival function of Sgk1, Supplementary File 7 for an additional independent experiment). when overexpressed, might also contribute to this effect.

Figure 2. Sgk1 regulates RANBP1 gene expression. (a) Western blot of proteins from RKO cells infected with adenovirus overexpressing Myc Sgk1 (AdSgk1-Rko cells) and control (Psi5-Rko cells) infected with control adenovirus. RANBP1 and Myc-Sgk1 were detected using goat polyclonal and rabbit polyclonal anti-Myc (both from Santa Cruz). Beta tubulin, detected by rabbit polyclonal, was used as a loading control. Adenovirus expressing wild-type Myc-tagged Sgk1 was prepared as described.30 Briefly to make an E1-substituted virus, Psi5 adenovirus was used as a donor virus to supply the viral backbone. The Psi5 packaging site was flanked by directly repeated loxP sites. 293/CRE 8 cells were used to produce recombinant virus particles in the presence of the shuttle plasmid with a single loxP site containing the Myc-Sgk1 sequence, sublconed from the original pciNeoSgk1.5 Cesium chloride gradient-purified viruses were used at 1:250 dilution in cell culture medium. Sgk1 expression was detectable 24 h after infection for at least 72 h. Cells infected with adenovirus driving Sgk1 expression are named AdSgk1-Rko cells, control cells infected with Psi5 adenovirus are named Psi5-Rko cells. (b) Quantitative PCR (q-PCR) quantification of RANBP1 mRNA levels in ShSgk1- and ShScrl-Rko cells. cDNA was synthesized from 1 mg total RNA using SuperScript III RNase HOURS-reverse transcriptase (Invitrogen, San Giuliano Milanese, Italy) and 2.5 mM random hexamers (Invitrogen). cDNA aliquots were amplified in an Applied Biosystems 7500 thermal cycler (Applied Biosystems, Branchburg, NJ, USA) using SYBR Green PCR master mix (Applied Biosystems) in the presence of RANBP1-specific primers (fw: 50-AGA AAG CAG GAT CAG GCA AA-30, rev: 50-AGC TTT TCC GCC ACT TTT TC-30) for 40 cycles (95 1C 10 min 1 cycle; 95 1C10s,551C 60 s). RANBP1 q-PCR products were normalized to the housekeeping Hypoxanthine phophoribosyl-transferase (HPRT) gene. Results, expressed as the mean±s.e.m. of quadruplicates samples, were evaluated by t-test. (c) Map of the murine wild-type RANBP1 promoter (pTS-A) with relevant transcription factor-binding sites. Basal transcription requires a proximal promoter region encompassing the major transcription start sites (arrowed) and harboring a bona fide Sp1-binding site (Sp1.2), an initiator element termed Htf9 footprinted element and a target site for E2F-4/p107 factors. G1/S upregulation of RanBP1 transcription is controlled by upstream elements, including binding sites for E2F-1/pRb (E2F-b site) and Sp1 (Sp1.3 site) factors. The position of mutagenized sites is indicated: pmE, mutant promoter in the E2F-b element; pmS, mutant promoter in the Sp1.3 element (details in Di Fiore et al.31). (d) CAT activity driven by pTS-A (wild-type), pmE (E2F-mutagenized) and pmS (Sp1-mutagenized) promoter constructs in AdSgk1 vs Psi5RKO cells. Twenty-four hours after infection AdSgk1- and AdSgk1-Rko cells were transfected with 10 mg of RANBP1 promoter reporter vectors and 1 mg of co-transfected beta-gal reporter for control. Twenty-four hours after transfection cells were lysed. The lysate was cleared by centrifugation and assayed for CAT activity as 32,33 described, in the presence of 1 mCi of [14C]chloramphenicol (50 mCi/mmol; New England Nuclear Corp.) and 20 ml of 4 mM acetyl coenzyme A. After autoradiography, the spots corresponding to the separated acetylated chloramphenicol forms were cut out and counted. Counts in the acetylated chloramphenicol spots recovered from AdSgk1-Rko cell extracts are expressed relative to the counts recorded in the acetylated chloramphenicol spot recovered from Psi5-Rko cell extracts (taken as 100). Results, expressed as the mean±s.e.m. of three independent experiments, were evaluated by t-test. (e) In vitro phosphorylation assays of GST, GST-purified Sp1 or GST-delta 1S59 Sp1 (kindly provided by Dr Kyoung Lim, Ajou University School of Medicine, Suwon, Korea) in the absence or presence of Sgk1 as indicated. The gel on the right shows the separation of bacterially produced GST-Sp1 and GST-delta1 Sp1 in pGEX3T3. Dialyzed GST, GST-SP1, GST-SP1-S59 delta1 were used as substrates in an in vitro kinase assay. Phosphorylation was detected by 32P incorporation into the GST fusion protein in the presence of [g-32P]ATP (0.02 mCi per sample). Samples were incubated in kinase buffer (200 mM MgCl2, 100 mM Tris-HCl pH 7.5, 2 mM DTT, 100 mM ATP) at room temperature for 30 min on a rotating wheel. The reaction was blocked by adding 50 ml SDS sample buffer. Samples were resolved by SDS–polyacrylamide gel electrophoresis, and phosphoproteins were detected by autoradiography. (f) In vitro phosphorylation assays of GST-delta 1 S59 Sp1 and GST-delta 1 S59A Sp1. Phosphorylation was performed as described above. The GST-delta1 S59A mutant was produced by means of QuikChange Site-Directed Mutagenesis Kit following the instructions of the manufacturer.

& 2013 Macmillan Publishers Limited Oncogene (2013) 4572 – 4578 Sgk1, RANBP1 and taxol sensitivity R Amato et al 4576

Oncogene (2013) 4572 – 4578 & 2013 Macmillan Publishers Limited Sgk1, RANBP1 and taxol sensitivity R Amato et al 4577 The ability of Sgk1 to modulate the extent of taxol-induced promoter elements.31 Disrupting cell cycle-regulated RANBP1 trans- mitotic arrest is expected to also affect the apoptotic response to cription displays the most significant consequences in mitosis: taxol. To address this question, Sgk1-defective and -proficient indeed, RANBP1 overexpression yields multipolar spindles with cultures were subjected to short-term (4 h) exposure to a higher fragmented centrosomes,19 whereas downregulation induces micro- 21,22,36 taxol concentration (100 nM). Cells in early apoptotic stages were tubule stabilization and impairs the spindle dynamic activity. revealed by their binding to Annexin V in fluorescence-activated Loss of control of these mechanisms, associated with RANBP1- cell sorting analysis. This treatment induced low levels of deregulated expression, ultimately affects the accuracy of chromo- apoptosis in ShScrl RKO control samples, whereas apoptosis some segregation and can contribute to cancer development. significantly increased in ShSgk1 cell lines (Figure 3b). These data This work shows that Sgk1 regulates RANBP1 gene transcription are consistent with results previously obtained by direct RANBP1 in RKO colon carcinoma cells. Experiments with site-specific downregulation.36 The increase in cell death was reversed by RANBP1 promoter mutants identify the SP1 site as a pivotal transfection of shSgk1 cells with a vector encoding RFP-RanBP1. element in Sgk1-dependent RANBP1 transcriptional regulation. We This strongly suggests that the effects of Sgk1 silencing on the cell concomitantly show that Sgk1 phosphorylates Sp1 in serine 59 in sensitivity to taxol are mediated, at least in part, via RanBP1 the N-terminal activation domain, reported to be essential for Sp1- downregulation (Figure 3b). We also evaluated the effects of long- dependent regulation of gene expression. Interestingly, it has term (16 h) exposure to taxol (10 nM) on apoptosis, assessed by recently been suggested that Sp transcription factors, particularly measuring the appearance of sub-G1 cells by propidium iodine Sp1, act in concert with the PI3K-dependent pathway to regulate staining (late apoptosis). Detailed cell cycle features of taxol- the alterations in cell metabolism caused by malignant transfor- treated hScrl and ShSgk1 RKO cultures are shown in mation.40 The results presented here indicate that Sgk1 silencing Supplementary File 8. Taxol treatment significantly increased the enhances taxol-induced apoptosis compared with wild-type cells, induction of sub-G1 cells both in ShScrl and ShSgk1 RKO cultures, similar to direct RANBP1 inactivation; moreover, an ectopically though more effectively in Sgk1-silenced cultures (Figure 3c). expressed RANBP1-encoding transgene counteracted the effects Interestingly, RANBP1 expression from a GFP-tagged expression of taxol in Sgk1 downregulated cells; thus, at least these effects of construct in ShSgk1 cells almost completely prevented taxol- Sgk1 are largely mediated by RANBP1 in RKO cells. Although induced apoptosis (Figure 3c), while having a negligible effect in reduced Sp1ser59 phosphorylation will likely affect a broad set of ShScrl cultures that express native RANBP1 levels. These results are genes, hence directly or indirectly affecting the expression of fully consistent with those obtained using Annexin V and confirm several proteins (as also indicated by our proteomics profiling the importance of RANBP1 downregulation in sensitizing colon of Sgk1-downregulated RKO cells), the present results identify a cancer cells to cell death induction by taxol. regulatory axis that includes Sgk1, Sp1 and RANBP1, which affects RANBP1 is a major effector of the GTPase RAN: it cooperates the response of colon cancer cells to taxol. Mitotic regulatory with RANGAP1 (RAN GTPase-activating protein) in regulating GTP genes modulate the response of cancer cells to microtubule- hydrolysis on RAN; in addition, it binds RANGTP and modulates its targeting drugs, particularly taxanes, which are widely employed association with or dissociation from effectors of the family of in cancer chemotherapy.41 An important implication of the nuclear transport receptors (for example, importin beta and present findings is that cancers overexpressing SGK1 might be exportin/CRM1).37,38 Through this dual activity, RANBP1 modulates more resistant to taxane-dependent induction of cell death due to RAN-dependent signals in all downstream-regulated processes, their elevated RANBP1 expression level. Sgk1 emerges, therefore, that is interphase nuclear import and export, mitotic spindle as a crucial factor in carcinogenesis and drug resistance. As Sgk1 is organization and nuclear reformation after mitosis.23,24,39 itself regulated at both the transcriptional and post-translational Among RAN network members, RANBP1 has the unique property level,2 the functional relation between Sgk1 and RANBP1 may also of being transcriptionally regulated during the cell cycle. RANBP1 contribute to the link between metabolism, chronic stress42 and transcriptional control is ensured via E2F- and Sp1-regulated cancer.

Figure 3. Taxol-induced apoptosis in RKO cells with or without Sgk1 expression. (a) Representative immunoflourescent images of 4’,6-diamidino-2-phenylindole/pericentrin (green)/beta tubulin (red) M-arrested cells in taxol (10 nmol/16 h). The graph shows the induction of M arrest in taxol (10 nmol)-treated RKO cells with either downregulated (ShSgk1) or overexpresed (Ad-Sgk1) RANBP1 relative to controls (Scr-RKO cells and Psi5 RKO cells, taken as 100). Cells were grown on coverslips in 6-well culture dishes in Dulbecco’s modified eagle medium (Invitrogen). When roughly 50% confluent, they were serum-starved overnight, then supplemented with 10% fetal bovine serum and later treated with 10 nMol taxol for 16 h. Cells were finally fixed in 3.7% formaldehyde for 20 min, permeabilized in 0.5% Triton X-100 for 1 min and washed with phosphate-buffered saline (PBS) (pH 7.4). Samples were incubated with pericentrin rabbit antibody (Babco, 1:500 dilution) and beta tubulin mouse antibody (Life Span Biosciences, 1:500 dilution) for 2 h at room temperature in PBS (pH 7.4) containing bovine serum albumin (1 mg/ml) and Tween-20 (0.2%), then washed in PBS and incubated with FITC-conjugated goat anti-rabbit Ig to detect pericentrin, TRITC-conjugated goat anti-mouse Ig to detect beta tubulin and 4’,6-diamidino-2-phenylindole (0.05% mgmlÀ 1). Samples were visualized under a Leica TC SP2 microscope (Leica, Wetzlar, Germany) with a Â 63 objective and processed with Leica confocal software. Digital Zoom is indicated in the scale bar. Mitotic arrest was calculated by visually counting prometaphase-arrested cells among the total of the cells in a microscopic field (50 microscopic fields were considered for each condition, corresponding to the following cell numbers: ShScrl: 4248 cells; ShSgk1: 1474 cells; AdPsi: 423 cells; AdSgk1: 831 cells). The data from either ShSgk1-silenced or AdSgk1-infected cultures (calulated as mean±s.d.) are expressed as the percent variation relative to the appropriate control, that is ShScrl and AdPsi5 RKO cultures, respectively. Differences were statististically evaluated using the t-test. (b) Early apoptosis after short taxol exposure (100 nmol, 4 h). Representative fluorescence-activated cell sorting panels show the Annexin V reactivity of cells in untreated and taxol-treated ShScr and ShSgk1 RKO cells alone or transfected with RFP-RANBP1 plasmid. Early apoptosis was quantified by fluorescence-activated cell sorting analysis of FITC annexin- stained cell populations. Histograms represent the percentage of early apoptotic cells in ShScr and ShSgk1 RKO cultures in the absence or presence of Taxol. Note that exogenous RFP-RANBP1 expression in ShSgk1 RKO cells significantly decreases the early apoptosis induction by taxol compared with that observed in Sgk1-silenced cells. FITC-Annexin V was sorted through channel FL1-A and RFP RANBP1 through FL2-A channels. Results are expressed as the mean±s.d. of 10 points from two independent experiments and statistically evaluated using the t-test. (c) Late apoptosis after taxol exposure (10 nmol, 16 h). Examplifying panels of propidium iodide-stained cell populations from untreated and taxol-treated ShScr and Sh-Sgk1 RKO cultures, alone or transfected with GFP-RANBP1 (demonstrated by the green fluorescence of the transfected cells). The hisotgrams in the graph below represent the percentage of late apoptotic (sub-G1) cells in ShScr and ShSgk1 RKO cultures, in the absence or presence of taxol. Expression of exogenous GFP-RANBP1 drastically reduced the percentage of late apoptotic cells induced in ShSgk1 RKO cultures in response to taxol. Results are expressed as the mean±s.d. of six points from two independent experiments and statistically evaluated using the t-test.

& 2013 Macmillan Publishers Limited Oncogene (2013) 4572 – 4578 Sgk1, RANBP1 and taxol sensitivity R Amato et al 4578 CONFLICT OF INTEREST 19 Di Fiore B, Ciciarello M, Mangiacasale R, Palena A, Tassin AM, Cundari E, Lavia P. The authors declare no conflict of interest. Mammalian RanBP1 regulates centrosome cohesion during mitosis. J Cell Sci 2003; 116: 3399–3411. 20 Peloponese Jr JM, Haller K, Miyazato A, Jeang KT. Abnormal centrosome amplifi- ACKNOWLEDGEMENTS cation in cells through the targeting of Ran-binding protein-1 by the human T cell leukemia virus type-1 Tax oncoprotein. Proc Natl Acad Sci USA 2005; 102: 18974–9. This work was supported in part by the Italian Association for Cancer Research 21 Li HY, Ng WP, Wong CH, Iglesias PA, Zheng Y. Coordination of chromosome (AIRC grant IG10164 to PL). RA was supported in part by INAIL, CZ. alignment and mitotic progression by the chromosome-based Ran signal. 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Oncogene (2013) 4572 – 4578 & 2013 Macmillan Publishers Limited