Aberrant Cytoplasmic Localization of N-CoR in Colorectal Tumors

[Cell Cycle 6:14, 1748-1752, 15 July 2007]; ©2007 Landes Bioscience

Report

Aberrant Cytoplasmic Localization of N-CoR in Colorectal Tumors Vanessa Fernández-Majada1 Jaume Pujadas2 Felip Vilardell2 Gabriel Capella2 Marty W. Mayo3 Anna Bigas1,†,* Lluis Espinosa1,†,*

Abstract

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We have previously shown that IKKs are aberrantly activated in colon cancer cells leading to SMRT phosphorylation and its release from the chromatin. We now show that IKKa phosphorylates the homologous N‑CoR corepressor in serines 2345 and 2348 creating a functional 14‑3‑3 binding domain (RKpS2348KSP). Moreover, we have analyzed the subcellular localization of N‑CoR in 43 colorectal cancer samples and we have found that aberrant cytoplasmic distribution of N‑CoR is a general trait of these tumors.

Introduction

2Laboratori

Different multiprotein complexes including several combinations of NCoRs and HDACs are responsible to specify gene silencing thus regulating cell differentiation, embryonic development and tissue and stem cell homeostasis.1‑5 �� Nuclear corepressors N‑CoR and SMRT physically interact with repression elements such as mSin3, Sharp and HDACs but also with different transcription factors such as MyoD, p65, RBPjk, etc. This latter interaction is responsible for driving the repression complexes to specific gene promoters where they modify the acetylation status and chromatin condensation.6 Phenotypic changes occurring during tumor development and progression require the activation and/or repression of specific sets of genes. In general, decreased acetylation of cell cycle regulatory genes such as p21 or p16 results in their transcriptional repression thus leading to increased cell proliferation in tumors. Therefore, HDAC inhibitors have become candidates for tumor therapy in hematologic and solid tumors.7,8 However, there are many examples where histone acetylation and specific gene activation may favor tumor progression. For example, activation of TCF/b‑catenin target genes, which are crucial for colorectal tumor development, requires the recruitment of TIP49 and the acetylation of nearby histones.9 In addition cell cycle progression mediated by E2F1 depends on the recruitment of acetyl‑transferases such as p300, pCAF and the HAT‑Tip60 complex.10 In this sense, the role of coactivator/corepressor complex recruitment in tumorigenesis depends on the specificity of their target genes. Although repressor elements are distributed mainly in the nucleus, there is increasing evidence that these proteins can translocate to the cytoplasm in response to phosphorylation events. Specific protein kinases such as PI3K, CaMK‑IV, ERK and IKKa have been reported to phosphorylate N‑CoR and SMRT in different situations.11‑15 Moreover, phosphorylation of SMRT by IKKa results in increased affinity of the corepressor for the 14‑3‑3 adaptor proteins, thus inducing its cytoplasmic export. The 14‑3‑3 family is generally regulating subcellular localization of multiple proteins including cdc25,16 FKHRL1,17 HDACs18 or NFkB.19 We have previously shown that IKKs are aberrantly activated and recruited to the chromatin in colon cancer cells correlating with phosphorylation and release of SMRT, and leading to increased activity of different Notch‑target genes. We now show that IKKa also phosphorylates the N‑CoR corepressor thus creating a functional 14‑3‑3 binding domain, and demonstrate that aberrant cytoplasmic distribution of N‑CoR is a general trait in colorectal tumors.

†These authors contributed equally to this work.

Original manuscript submitted: 03/12/07 Revised manuscript submitted: 05/08/07 Manuscript accepted: 05/10/07

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Previously published online as a Cell Cycle E-publication: http://www.landesbioscience.com/journals/cc/abstract.php?id=4429

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*Correspondence to: Anna Bigas; Institut d’Investigacio Biomèdica de Bellvitge, Gran Vía Km 2.7; Hospitalet, Barcelona 08907 Spain; Tel.: 932.607.404; Fax: 932.607.426; Email: [email protected] / Lius Espinosa; Institut d’Investigacio Biomèdica de Bellvitge, Gran Vía Km 2.7; Hospitalet, Barcelona 08907 Spain; Tel.: 932.607.404; Fax: 932.607.426; Email: [email protected]

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Charlottesville, Virginia USA

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3Department of Biochemistry and Molecular Genetics; The University of Virginia;

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de Recerca Translacional; Institut d’Investigacio Biomèdica de Bellvitge; Institut Català Oncologia; Hospitalet, Barcelona Spain

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Oncologia Molecular; Institut d’Investigacio Biomèdica de Bellvitge; Hospitalet, Barcelona Spain

Key words

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Acknowledgements

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N-CoR, IKK, 14-3-3, colorectal cancer, cytoplasmic export

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We thank Julia Inglés-Esteve for critical reading of the manuscript and helpful discussion. V.F.-M. is recipient of Generalitat de Catalunya (DURSI) Predoctoral Fellowship 2005-FI00458. This work was supported by Instituto de Salud Carlos III Grant PI041890, Fundació la Marató de TV3 Grant 051730, Fundación Mutua Madrileña, MEC Grant AGL2004-07579-04 and Fundació la Caixa Grant BM05-254-0.

Materials and Methods Plasmids. Expression vectors for myc‑14‑3‑3 have been previously described.20 GST‑N‑CoR constructs were generated by PCR and cloned in‑frame into the PGEX‑5.1 vector (Pharmacia). GST‑N‑CoR mutant was generated with the QuickChange 1748

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N-CoR Phosphorylation by IKKα

Site‑Directed Mutagenesis Kit (Stratagene). All constructs sequences were confirmed by automated sequencing. Antibodies. a-N‑CoR (sc‑C20), a-HDAC1 (sc‑7872) and a‑14‑3‑3b (K19) (sc‑629) were purchased from Santa Cruz Biotechnology. a‑IKKa (op‑133) and a-tubulin from Sigma. a‑P‑Akt (9271), a‑MEK1/2 (9122), a‑P‑ERK1/2 (9106), a‑ERK1/2 (9102) from Cell Signaling. Secondary antibodies conjugated with horseradish peroxidase (HRP) were purchased from DAKO. Cell lines and culture reagents. HEK‑293, HS27, HCT‑116, SW480 and Ls174T were cultured in DMEM 10% FBS. BAY11‑7082 was purchased from Calbiochem (7082) and used at 10 mM. Protein kinase assays. Nuclei from different cell lines were isolated and lysed for 30 min at 4˚C in 500 ml of PBS containing 0.5% Triton X‑100, 1 mM EDTA, 100 mM Na‑orthovanadate, 0.25 mM PMSF and complete protease inhibitor cocktail (Roche). After centrifugation, supernatants were precleared twice for 2 h and incubated with 1 mg of the a‑IKKa overnight at 4˚C. After incubation with Figure �� 1. Nuclear IKKa phosphorylates N‑CoR in colorectal cancer cell lines. (A) Protein sequence align‑ ProteinA‑Sepharose beads, precipitates ment of SMRT and N‑CoR. Grey box indicates the putative14‑3‑3 binding motif and underlined residues were extensively washed and assayed show the IKKa phosphorylation consensus. (B) Western blot analysis of different kinases and phosphorylated for kinase activity on GST‑N‑CoR‑wt substrates in nuclear and cytoplasmic cell extracts from the indicated cell lines. a‑tubulin is used to show the or GST‑N‑CoR‑S2345A,S2348A. absence of cytoplasmic contamination in the nuclear extracts. (C) Kinase32activity assay of nuclear extracts For immunodepletion experiments, from different cell lines on GST‑N‑CoR (AA2256‑22452) detected by P ATP incorporation. (D) Kinase activity of precipitated nuclear IKKa on GST‑N‑CoR‑wt (AA2256‑22452) and GST‑N‑CoR‑�� S2345A,S2348A 1/5 dilution of the unbound fraction (MUT)�� in �� HCT116 and �� HS27 �� as control cells. Phosphorylation was detected by 32P ATP incorporation. (from IP) was used in the kinase assay. Nuclear levels of IKKa in the different cell lines and HDAC1 as a nuclear input control are shown. (E) Kinase Immunoprecipitation assays. Cells activity on GST‑N‑CoR of the unbound fraction from nuclear extracts immunoprecipitated with nonspecific were lysed for 30 min at 4˚C in 500 ml IgG or anti‑IKKa antibody. Levels of IKKa in the unbound and bound fractions of each precipitation is shown of PBS containing 0.5% Triton X‑100, in the lower panel. 1 mM EDTA, 100 mM Na‑ orthovanadate, 0.25 mM PMSF and complete protease inhibitor cocktail (Roche). After centrifugation, Sections were hydrated and permeabilized and antigen retrieval supernatans were incubated for 3 hours at 4˚C with 2 mg of indicated was achieved by boiling in 20 mM Na Citrate pH 6.0 (2 min). antibody coupled to ProteinA‑sepharose beads. Beads were exten- HRP‑conjugated secondary antibodies were detected with the diamisively washed with the precipitation buffer and samples were assayed nobenzidine peroxidase substrate kit (Dako Cytomation). by western blot. Pull‑down assays. Pull‑down assays have been previously Results described.21 Brieftly, GST fusion proteins were purified and incubate Nuclear IKKa phosphorylates N‑CoR in a conserved 14‑3‑3 with 400 mg of cell lysates for 2 hours at 4˚C in lysis buffer. After extensive washing, pulled down proteins were analyzed by western blot. binding domain. It has been previously shown that IKKa phosHuman samples. A total of 42 colorectal carcinomas were obtained phorylates SMRT in Ser2410 thus creating a 14‑3‑3 binding site and leading to cytoplasmic translocation of SMRT.14 from the HUB‑ICO-IDIBELL tumor bank. In 31 cases paired normal (PS2410SRK) �� mucosa was available. All patients gave written consent to donate the This mechanism operates not only in response to specific stimulation tumor specimen to the biobank. The study was approved by the Ethics (such as laminin attachment) but also in cancer cells carrying activated Committee of our institution. Samples were processed for IHC. IKKa.22 By sequence analysis of the N‑CoR protein, we have identified Immunohistochemistry (IHC). For histological analysis, samples a putative IKK phosphorylation/14‑3‑3‑binding site (RKS2348KSP) were fixed in 4% formaldehyde, dehydrated and parafin‑embedded. in the homologous region of the N‑CoR protein (Fig. 1A). www.landesbioscience.com

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Figure�� 2. N‑CoR physically interacts with 14‑3‑3 proteins in an IKKa depend‑ ent manner. (A) Cell lysates from normal HS27 and colorectal tumor cell lines SW480 and Ls174T were precipitated with anti‑N‑CoR antibody or IgG as a control. The presence of 14‑3‑3 in the precipitates was determined by immunoblotting with anti‑14‑3‑3 antibody recognizing different isoforms. Inputs represents 1/10 of the total lysates. The levels of N‑CoR and 14‑3‑3 are shown. a‑tubulin is used as a loading input control. (B‑C) Pull‑down assay with GST‑N‑CoR‑wt (AA2256‑2452) or GST‑N‑CoR‑�� S2345A,S2348A (MUT)� and cell lysates from HEK‑293T cells transfected with or without myc‑14‑3‑3 protein (B), untreated or treated with the IKK inhibitor BAY11‑7082 (C). The presence of 14‑3‑3 in the precipitates was determined by western blot with an anti‑myc antibody. Coomassie staining of GST protein is shown in lower panel. Inputs represent the 1/10 of total lysates.

We previously demonstrated that colorectal cancer cells contain nuclear active IKKa. We have now characterized different colorectal cancer cell lines for the presence of kinase activities previously found to phosphorylate Nuclear Corepressors such as PI3K,13 MEK1,12 IKKa and other IKKs. We found that only IKKa, and to a lesser extent the regulatory subunit IKKg, are present in the nuclear compartment of all colorectal cancer cells but not in the nontransformed HS27 cell line (Fig. 1B). In contrast, we could not detect any increase in PI3K or MEK1/2 activities as measured by the presence of P‑Akt or P‑ERK respectively (Fig. 1B). We next investigated whether N‑CoR was a substrate for IKKa in these cells. First, by in vitro phosphorylation assay using �� GST‑N‑CoR (AA2256‑2452) as a substrate, we demonstrated that N‑CoR is consistently phosphorylated when incubated with nuclear extracts from colorectal tumor cell lines. In contrast, we did not detect any phosphorylation in the GST‑N‑CoR incubated with nuclear extracts from nontransformed HS27 cells (Fig. 1C). To further demonstrate that IKKa was responsible for N‑CoR phosphorylation, we precipitated IKKa from nuclear extracts from HCT116 and HS27 cells and determined the kinase activity of the precipitates on the GST‑N‑CoR construct. In Figure 1D we show that N‑CoR is specifically phosphorylated by the IKKa precipitates obtained from HCT116 nuclear extracts whereas immuno-depletion of IKKa eliminates the kinase activity of the unbound fraction on GST‑N‑CoR (Fig. 1E). Moreover, this phosphorylation takes place in the core of the 14‑3‑3 binding consensus, since it is greatly reduced when using a GST‑N‑CoR mutated in serines 2345 and 2348 to alanine (Fig. 1D). These results indicate that IKKa specifically phosphorylates the 14‑3‑3 binding motif of N‑CoR in colorectal cancer cells. N‑CoR physically interacts with 14‑3‑3 proteins in an IKKaphosphorylation‑dependent manner. To test the functionality of this putative 14‑3‑3 binding domain of N‑CoR, we analyzed whether this corepressor was physically interacting with 14‑3‑3 proteins. 1750

Figure�� 3. N‑CoR is localized in the cytoplasm of colorectal carcinomas. (A) IHC staining of a representative biopsy from human colorectal tissue with anti‑N‑CoR antibody. One section of each normal, hyperplastic and carcino‑ ma tissue from the same sample is represented. Nuclei were stained in blue with hematoxylin. Images were obtained at 200x and 600x. (B) Summary of 31 paired and 12 nonpaired colorectal samples analyzed indicating the subcellular distribution of N‑CoR as detected by ICH.

By immunoprecipitation experiments with a‑N‑CoR antibody, we demonstrated that endogenous 14‑3‑3 physically interacts with N‑CoR in SW480 and Ls174T colorectal cancer cells (Fig. 2A) and to a lesser extent in nontransformed HS27 cells. By pull‑down experiments, using the GST‑N‑CoR fragment (AA2256‑2452) and the mutant �� GST‑N‑CoR‑S2345A,S2348A�� , we showed that the interaction between N‑CoR and 14‑3‑3 requires the serines in the 14‑3‑3 binding motif (residues 2345‑2348) (Fig. 2B). Since these serines are targets for IKKa activity (Fig. 1D) we tested the effect of the IKKa inhibitor BAY11‑7082 on this interaction. As shown in Fig. 2C, treatment with BAY11‑7082 abrogated the interaction between N‑CoR and 14‑3‑3 comparable to the mutation Ser2345‑2348Ala. These results indicate that 14‑3‑3 physically interacts with N‑CoR and that this interaction is dependent on phosphorylation of serines 2345 and 2348 in the core of the 14‑3‑3 binding motif, which is conserved between SMRT and N‑CoR corepressors. N‑CoR is aberrrantly distributed in the cytoplasm of color‑ ectal carcinomas. Since we demonstrated that IKKa is activated in primary human colorectal tumors22 and N‑CoR binds to 14‑3‑3 in an IKKa‑phosphorylation dependent manner, we hypothesized that N‑CoR distribution may be altered in these tumors, similar to that previously observed for SMRT corepressor. To test this possibility we performed IHC with the a‑N‑CoR antibody on 43 colorectal carcinomas. Our results demonstrated that N‑CoR corepressor is excluded from the nucleus in 98% of the tumor samples (42 out of 43) (Fig. 3A and B) compared to its nuclear distribution in the adjacent normal mucosa (30 out of 31). A comparable nuclear distribution of N‑CoR was observed in 11 cases where hyperplastic crypts were identified in the normal mucosa (Fig. 3A and 3B). These results indicate that nuclear export of N‑CoR is not related to increased proliferation but it is associated with tumorigenesis.

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Discussion In this work, we have identified a specific region on N‑CoR protein that is phosphorylated by IKKa thus generating a functional 14‑3‑3 binding domain. In addition we show that N‑CoR corepressor is localized in the cytoplasm in colorectal tumors, most likely as a result of aberrant IKKa activation (see model in Fig. 4). This is similar to our previous observation with SMRT corepressor22 and may reflect the high conservation of functional elements between N‑CoR and SMRT corepressors including the regions responsible for IKKa‑mediated cytoplasmic export. In contrast, MEKK1 and PI3K specifically regulate SMRT and N‑CoR cytoplasmic export, respectively indicating the existence of other nonconserved domains.13,15 Other post‑transcriptional modifications may also regulate functional N‑CoR and SMRT specificity, since it has recently been shown that N‑CoR is sumoylated.23 N‑CoR and SMRT exert their function by recruiting repression elements to specific promoters and several transcription factors have been shown to interact with NCoRs such as Nuclear Receptors, RBPjk, p65, MyoD. Although specific functions for different NCoRs are not well characterized and both proteins are ubiquitously expressed, deletion of the N‑CoR gene in mice results in an embryonic lethal phenotype indicating that SMRT cannot rescue the lack of this gene.1 In fact, N‑CoR proteins have been more generally associated to differentiation processes, and N‑CoR mutant embryos have defects in erythroid, lymphoid and neural differentiation.1 In addition, subcellular localization of N‑CoR has directly been linked to differentiation of astrocytes, thus requiring the cytoplasmic localization of N‑CoR protein after PI3K phosphorylation for neural stem cells to undergo astrocyte differentiation.13 Nevertheless, although nothing is known about the role of N‑CoR in intestinal differentiation, we have observed that a few normal colonic cells located at the bottom of the crypts are negative for N‑CoR staining (unpublished data), suggesting that lack of nuclear N‑CoR may be associated with the undifferentiated phenotype. This observation is functionally comparable with the cytoplasmic export of N‑CoR observed in tumor tissue and may indicate the presence of specific N‑CoR‑dependent changes in gene expression associated to the progenitor‑like program. Since cytoplasmic translocation of SMRT by 14‑3‑3 is required for proteasomal degradation,14 we speculate that the absence of N‑CoR protein in few normal cells located in the bottom of the crypts and cytoplasmic distribution in tumors may be the result of the same cellular mechanism in a different context. We have observed a similar aberrant distribution of N‑CoR in 11 bladder carcinoma samples (unpublished results), in contrast to that observed in glioblastoma tumors and normal astrocytes where N‑CoR cytoplasmic translocation is associated with cell differentiation.24 This indicates that regulation of specific sets of genes (pro‑ versus anti‑differentiating genes) by N‑CoR is context‑dependent. Moreover, the fact that both N‑CoR and SMRT are exported to the cytoplasm in colorectal cancer indicates the existence of a general derepression mechanism that is operating in this particular tumor. In this sense, detection of aberrantly distributed NCoRs may be a useful tool in the diagnosis of premalignant lesions in colorectal and bladder cancers. Since histone deacetylase inhibitors are being used in the cancer therapies based on their capacity to activate tumor suppressor genes,8 it is extremely important to determine which are the targets for N‑CoR/SMRT repression in different types of cancer, whether nuclear export of corepressors is restricted to colorectal tumors and www.landesbioscience.com

Figure�� 4. Model for IKKa‑mediated transcriptional activation in colorectal carcinomas. In normal cells N‑CoR and SMRT are in the nucleus bound to their specific target genes. In colorectal carcinomas, activated IKKa phos‑ phorylates a conserved residue in both N‑CoR and SMRT thus creating a 14‑3‑3 binding motif. Once phosphorylated, 14‑3‑3 proteins are able to induce cytoplasmic translocation of Nuclear Corepressors.

how sensitive are N‑CoR/SMRT target genes to HDAC inhibitor therapies. It has been recently shown that cells with mutant APC, a common feature in colorectal cancer, are more resistant to the pro‑apoptotic effect of HDAC inhibitors such as valproic acid (VA) or suberoylanilide hydroxamic acid (SAHA).25 Moreover, there is also evidence for anti‑tumoral drugs that exert their pro‑apoptotic activity by inducing the formation of repression complexes containing N‑CoR, Sin3A and HDACs.26 Thus, a better understanding of the mechanisms regulating gene repression in specific context is required to improve cancer treatment. References 1. Jepsen K, Hermanson O, Onami TM, Gleiberman AS, Lunyak V, McEvilly RJ, Kurokawa R, Kumar V, Liu F, Seto E, Hedrick SM, Mandel G, Glass CK, Rose DW, Rosenfeld MG. Combinatorial roles of the nuclear receptor corepressor in transcription and development. Cell 2000; 102:753‑63. 2. Dressel U, Bailey PJ, Wang SC, Downes M, Evans RM, Muscat GE. A dynamic role for HDAC7 in MEF2‑mediated muscle differentiation. J Biol Chem 2001; 276:17007‑13. 3. Wu XY, Li H, Park EJ, Chen JD. SMRTe inhibits MEF2C transcriptional activation by targeting HDAC4 and 5 to nuclear domains. J Biol Chem 2001; 276:24177‑85. 4. Bernstein BE, Mikkelsen TS, Xie X, Kamal M, Huebert DJ, Cuff J, Fry B, Meissner A, Wernig M, Plath K, Jaenisch R, Wagschal A, Feil R, Schreiber SL, Lander ES. A bivalent chromatin structure marks key developmental genes in embryonic stem cells. Cell 2006; 125:315‑26. 5. Lee TI, Jenner RG, Boyer LA, Guenther MG, Levine SS, Kumar RM, Chevalier B, Johnstone SE, Cole MF, Isono K, Koseki H, Fuchikami T, Abe K, Murray HL, Zucker JP, Yuan B, Bell GW, Herbolsheimer E, Hannett NM, Sun K, Odom DT, Otte AP, Volkert TL, Bartel DP, Melton DA, Gifford DK, Jaenisch R, Young RA. Control of developmental regulators by Polycomb in human embryonic stem cells. Cell 2006; 125:301‑13. 6. Kao HY, Ordentlich P, Koyano‑Nakagawa N, Tang Z, Downes M, Kintner CR, Evans RM, Kadesch T. A histone deacetylase corepressor complex regulates the Notch signal transduction pathway. Genes Dev 1998; 12:2269‑77. 7. Villar‑Garea A, Esteller M. Histone deacetylase inhibitors: Understanding a new wave of anticancer agents. Int J Cancer 2004; 112:171‑8. 8. Bolden JE, Peart MJ, Johnstone RW. Anticancer activities of histone deacetylase inhibitors. Nat Rev Drug Discov 2006; 5:769‑84. 9. Feng Y, Lee N, Fearon ER. TIP49 regulates beta‑catenin‑mediated neoplastic transformation and T‑cell factor target gene induction via effects on chromatin remodeling. Cancer Res 2003; 63:8726‑34. 10. Taubert S, Gorrini C, Frank SR, Parisi T, Fuchs M, Chan HM, Livingston DM, Amati B. E2F‑dependent histone acetylation and recruitment of the Tip60 acetyltransferase complex to chromatin in late G1. Mol Cell Biol 2004; 24:4546‑56. 11. Jang MK, Goo YH, Sohn YC, Kim YS, Lee SK, Kang H, Cheong J, Lee JW. Ca2+/calmodulin‑dependent protein kinase IV stimulates nuclear factor‑{kappa}B transactivation via phosphorylation of the p65 subunit. J Biol Chem 2001; 276:20005‑10. 12. Hong SH, Privalsky ML. The SMRT corepressor is regulated by a MEK‑1 kinase pathway: Inhibition of corepressor function is associated with SMRT phosphorylation and nuclear export. Mol Cell Biol 2000; 20:6612‑25. 13. Hermanson O, Jepsen K, Rosenfeld MG. N‑CoR controls differentiation of neural stem cells into astrocytes. Nature 2002; 419:934‑9.

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