KLF6 Degradation After Apoptotic DNA Damage

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FEBS Letters 580 (2006) 6981–6986

KLF6 degradation after apoptotic DNA damage

Michaela S. Bancka,*, Simon W. Beavend,1, Goutham Narlaa,b, Martin J. Walshc, Scott L. Friedmand, Andreas S. Beutlera a Department of Medicine, Division of Hematology/Oncology, P.O. Box 1079, Mount Sinai School of Medicine, One Gustave Levy Place, Room 24-42A, New York, NY 10029, United States b Department of Human Genetics, Mount Sinai School of Medicine, New York, NY 10029, United States c Department of Pediatrics, Mount Sinai School of Medicine, New York, NY 10029, United States d Division of Liver Diseases, Mount Sinai School of Medicine, New York, New York 10029, United States

Received 27 July 2006; revised 24 October 2006; accepted 25 October 2006

Available online 13 November 2006

Edited by Varda Rotter

about the molecular roles or fate of KLF6 in neoplastic cells Abstract Kru¨ppel-like factor 6 (KLF6) is a cancer gene (www.sanger.ac.uk/genetics/CGP/Census/). Here, we demon- or in the response to anti-neoplastic therapies. strate that KLF6 protein is rapidly degraded when apoptosis is We found it notable to observe that KLF6 protein decreased induced via the intrinsic pathway by cisplatin, adriamycin, or in all of several types of cancer cell lines in response to DNA UVB irradiation in multiple cell lines (HCT116, SW40, HepG2, damage induced by cisplatin. The decrease in KLF6 protein PC3-M, Skov3, NIH-3T3, 293T, GM09706, and MEF, IMR- occurred only when the degree of DNA damage was extensive 90). KLF6 degradation occurred in the presence or absence of enough to induce apoptosis. It occurred whenever apoptosis p53, was associated with ubiquitination, mediated by the protea- was induced through the intrinsic pathway (i.e. DNA damage) some (half-life 16 min, unstimulated), and independent of casp- but not when induced through the extrinsic pathway (i.e. death ases and calpain. KLF6 was unchanged by apoptosis via the receptors). KLF6 was found to be ubiquitinated and to be extrinsic/death-receptor pathway. Deregulation of KLF6 stabil- degraded by the proteasome. ity may alter its tumor suppressor function and/or the response of tumors to chemotherapeutics. Regulation of KLFs at the level of gene expression has been 2006 Federation of European Biochemical Societies. Published well documented [12]. More recently, regulation at the protein by Elsevier B.V. All rights reserved. level has been reported for KLF1, KLF2, KLF5 and KLF4 [13–16]. The latter study used serum starvation as a means Keywords: Kru¨ppel-like factor; KLF6; DNA-damage; of inducing growth arrest and demonstrated that under these Apoptosis; Chemosensitivity; Proteasomal degradation; conditions KLF4 was degraded by the proteasome. Here, we Ubiquitin; Cancer cells; Carcinogenesis report similar ﬁndings for KLF6 in the setting of a cellular perturbation with relevance for cancer biology, namely chemotherapy/DNA damage induced apoptosis.

1. Introduction 2. Materials and methods Kru¨ppel-like factor 6 (KLF6) is a member of the Kru¨ppel- like family of C2H2 zinc finger transcription factors and has 2.1. Cell lines and materials HepG2 cells, IMR-90 and HCT116 cells were obtained from the been linked to carcinogenesis by a high rate of somatic muta- American Tissue Culture Collection (ATCC). Dulbecco’s Modified tions in malignancies of the prostate, colon, stomach, liver, Eagle’s Medium (DMEM), McCoy’s 5A medium, antibiotics (penicil- lung, neck, pituitary, and nervous system [1–6]. Decreased lin, streptomycin 100 mg/ml) and L-glutamine (30 mg/ml) were ob- expression of KLF6 has been associated with poor prognosis tained from Cellgro. Fetal bovine serum (FBS) was from Hyclone. The polyclonal antibody anti-KLF6 (R-173) and anti p21 (C-19) in adenocarcinoma of the lung [7] and in prostate cancer [8]. were obtained from Santa Cruz Biotechnology. Monoclonal antibody We recently reported that a germline single nucleotide poly- recognizing the HA epitope was obtained from Santa Cruz. Secondary morphism (SNP) in the KLF6 gene increases the risk for pros- antibodies were from Amersham. The proteasome inhibitors ALLN, tate cancer in men with a family history of this disease, thereby MG132 and lactacystine were obtained from Calbiochem. The caspase linking KLF6 to an inheritable cancer syndrome [9]. inhibitor ZVAD-fmk was purchased from Alexis Biochemicals (San Diego, CA). The calpain inhibitor calpeptin was from Sigma. Lipofect- We have previously demonstrated that KLF6 contributes to amine 2000 was obtained from Invitrogen. cell cycle regulation via transcriptional activation of the p21 promoter, as well as direct sequestration of cyclin D1 protein 2.2. Western blot analysis and chemiluminescence through competition with cdk4. It also inhibits transcription Whole cell protein extracts were obtained by lysing cells in lysis buf- of c-jun [2,10,11]. All of these activities induce G1 arrest and fer (50 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.1% a decrease in cell proliferation. However, little else is known NP40, 10% glycerol, 20 mM b-glycerolphosphate, 0.1 mM sodium vanadate, 1 mM NaF, 1 mM PMSF and 1 protease inhibitor tablet/ 10 mL buffer). For Western blot, 30–50 lg of total cell extracts were *Corresponding author. Fax: +1 212 241 4096. resolved on a 10% SDS–PAGE. Following transfer onto nitrocellulose E-mail address: [email protected] (M.S. Banck). membranes, proteins of interest were detected by incubating with primary antibodies and processed by the standard ECL detection method 1Present address: Division of Digestive Disease, UCLA School of under conditions recommended by the manufacturer (Amersham). Medicine, Los Angeles, CA 90095, United States. Nitrocellulose membranes were then subjected to chemiluminescence

0014-5793/$32.00 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2006.10.077 6982 M.S. Banck et al. / FEBS Letters 580 (2006) 6981–6986 analysis using a Fuji LAS-3000 Luminescent Image Analyzer. The expressed by transfected cDNA [10], suggesting that it is emitted light units were recorded by a digital camera, and quantitated. derived from one transcript and represents two states of posttranslational modification. Exposure of HCT116 cells to 2.3. Real-time quantitative PCR cisplatin 5–20 lM for 24 h led to arrest of the cell cycle in S Total cellular RNA was extracted using RNeasy Mini kit (Qiagen). The RNA was treated with DNase (Qiagen). A total of 1 lg of RNA and G2 phase and induction of p21 but not to apoptosis as was reverse transcribed per reaction using first strand complementary shown by absence of PARP cleavage (Fig. 1, lanes 2 and 3). DNA synthesis with random primers (Promega). QRT-PCR was per- Exposure to cisplatin to higher concentrations of 60–120 lM formed using the following PCR primers on an ABI PRISM led to apoptosis as shown by PARP cleavage and an increase 7900HT Sequence Detection System (Applied Biosystems): KLF6 For- of the number of cells in the sub-G1 fraction of the DNA con- ward: 50-CGG ACG CAC ACA GGA GAA AA-30 and KLF6 Re- verse: 50-CGG TGT GCT TTC GGA AGT G-30. All experiments tent profile (lanes 4 and 5). These observations of cell cycle ar- were done in triplicates and normalized to actin. Fluorescent signals rest and apoptosis of HCT116 cells in the presence of the were analyzed during each of 20 cycles consisting of denaturation concentrations of cisplatin used in this experiment is in accor- (95 C, 15 s), annealing (56 C, 15 s), and extension (72 C, 40 s). dance with observations made by others [17]. The low molecular component of the KLF6 double band disappeared at 2.4. Cell cycle analysis by flow cytometry 60 lM and no KLF6 was detected at 120 lM, i.e. at apoptotic For flow cytometry analysis, cells were washed twice in PBS, trypsin- ized with 1 ml of trypsin-EDTA, centrifuged, and washed in PBS once cisplatin doses. We next investigated the cause of the decrease more. Next, cells were resuspended in 250 llof1· PBS and 750 llof in KLF6 protein by addressing whether the decline was due to 100% ethanol, which was added drop wise while cells were gently vor- protein degradation or decrease in mRNA. HCT116 cells over- texed. To allow for proper fixation, cells were kept on ice for at least expressing KLF6 were exposed to 120 lM cisplatin. As shown 30 min, then centrifuged at 1000 rpm for 10 min at 4 C, washed twice in PBS, and resuspended in 1 ml of PBS and RNaseA treated for in Fig. 2, we observed a decrease in KLF6 protein while 30 min in a 37 C water bath. Finally, cells were incubated with propi- mRNA levels remained stable suggesting active degradation dium iodide and scanned by flow cytometry using a Becton-Dickinson of KLF6 protein upon exposure to cisplatin. Excalibur. A total of 10000 cells were analyzed per experiment. 3.2. Cisplatin-induced KLF6 protein degradation is observed in all cancer cell lines tested and with a variety of forms of 3. Results and discussion DNA damage We were interested to investigate whether KLF6 degrada- 3.1. Cisplatin induces degradation of KLF6 protein but only in tion is universally observed in response to apoptotic doses of apoptotic doses DNA damage. Therefore, we tested different cell lines, includ- As in all human cancer cell lines, KLF6 is detected in ing p53 deficient HCT116 cells with different genotoxic agents HCT116 cells as a double-band (Fig. 1A, lane 1). Both compo- to induce apoptosis. Table 1 summarizes all cell lines tested at nents of the double-band are intensified when KLF6 is over- apoptotic doses of different forms of genotoxic stress, includ- ing drugs used in chemotherapy for cancer as well as ultravio- let light (UVB). We observed degradation of KLF6 in all cell lines once treatment was administered in doses that induced apoptosis. Thus, although an earlier report noted that KLF6 protein disappeared after UVB and theorized that this could facilitate cell-cycle reentry after DNA repair by releasing a brake on c-jun [11] we found that doses of DNA damaging, chemotherapeutic agents and UVB that lead to degradation invariably lead to cell death while low doses that allow reentry into the cell cycle are associated with an increase in KLF6 protein (Fig. 1 and Table 1).

Fig. 1. Dose dependent bimodal response of KLF6 to DNA damage. HCT116 cells were exposed to cisplatin and harvested 24 h later. (A) 50 lg of protein from total cell extracts were run on a 10% SDS–PAGE gel and submitted to Western blot analysis for KLF6, PARP and p21. Actin served as the protein loading control. KLF6 protein levels increase with two low doses of cisplatin and decrease with two higher Fig. 2. KLF6 protein levels decrease due to accelerated protein doses. PARP cleavage occurred only with the high cisplatin doses degradation. HCT116 cells were treated with 120 lM cisplatin and indicating that KLF6 induction occurred with sub-apoptotic doses of submitted to Western Blot analysis (upper panel) and real time PCR cisplatin and decreased only with high, apoptotic doses. (B) Cell cycle (lower panel). KLF6 protein disappeared but its mRNA remained proﬁle of cells from the same culture dish after staining with propidium stable after exposure of the cells to extensive, apoptotic DNA damage iodide. as indicated by PARP cleavage. M.S. Banck et al. / FEBS Letters 580 (2006) 6981–6986 6983

Table 1 Degradation of KLF6 () observed in multiple cell lines during apoptosis induction via the intrinsic/DNA damage pathway but not via the extrinsic/ death receptor pathway ( () = stable KLF protein levels) Cell lines Apoptotic agents Figures Status Species Origin/cell type Name Genetics Intrinsic pathway Extrinsic (DNA damage) pathway (death receptors) Cisplatin UVB Other TNF-a Trail Malignant Human Colon cancer HCT116 p53+/+ ()() Figs. 1 and 4 p53/ ()() Fig. 3 SW40 c-abl / p53mutant Fig. 3 Hepatoblastoma HepG2 Fig. 3 and 7 Prostate cancer PC3-M Not shown Ovarian cancer Skov3 Not shown Transformed Mouse Fibroblasts NIH3T3 Not shown Human Embryonic kidney 293T Not shown Primary Human Fibroblasts IMR-90 Diploid Fig. 3 GM09706 ATM/ Not shown Mouse Embryonic ﬁbroblasts MEF Diploid Not shown

Of note, the IC50 for cisplatin is different among human tors such as TNF-receptors, CD95, and TRAIL-receptors is cancer cell lines, even among those of colonic origin [17,18]. mediated via the extrinsic pathway. We tested whether the Therefore, while some cell lines are more sensitive to cisplatin decrease in KLF6 protein was a feature of apoptosis in general than others (e.g. IMR-90 diploid fibroblasts require less cis- or whether it would be limited to apoptosis triggered by DNA platin to undergo apoptosis within 24 h than HepG2 hepato- damage. HCT116 cells are sensitive to the death receptor blastoma cells), KLF6 degradation invariably occurs at ligands TRAIL and TNFa [19]. We therefore exposed concentrations that ultimately lead to apoptosis. The rate of HCT116 cells to TNFa or TRAIL in the presence of cyclohex- the decline in KLF6 protein, a composite of protein degrada- imide (CHX). For this experiment, TRAIL was used in combi- tion but stable levels of mRNA as shown in Figs. 2 and 3. nation with CHX because TRAIL can trigger a protective pathway in via the activation of NFj-B, whose targets include 3.3. KLF6 degradation is specific for stimulation by the intrinsic genes encoding intracellular inhibitors of apoptosis [20].As pathway and does not occur when apoptosis is induced by can be seen in Fig. 4, KLF6 protein levels remained stable TNF-a or TRAIL upon treatment of HCT116 cells with either ligand at doses Apoptosis can be induced via two principal pathways. Ultra- that induced a strong apoptotic response. CHX alone did violet irradiation and chemotherapeutic agents induce DNA not induce apoptosis (data not shown). Thus, loss of KLF6 damage and induce apoptosis via the intrinsic apoptotic path- protein occurs in the setting of apoptotic DNA damage and way. Apoptosis that is triggered by stimulation of death recep- not with death receptor induced cell death.

Fig. 3. KLF6 protein degradation after cisplatin occurs in all cell lines tested. (A) Cells were harvested 24 h after treatment with increasing doses of cisplatin and submitted to Western Blot analysis. (B) Rate of KLF6 degradation. 6984 M.S. Banck et al. / FEBS Letters 580 (2006) 6981–6986

determined the stability of KLF6 in the presence of the proteasome inhibitor ALLN. Fig. 5 shows that KLF6 degradation in normally proliferating cells was completely inhibited in the presence of ALLN. We next examined whether KLF6 under- goes ubiquitination. As seen in Fig. 6, a high molecular weight smear, characteristic of ubiquitinated proteins is seen in 293T cells co-transfected with Flag-tagged KLF6 and HA-tagged ubiquitin. We conclude that KLF6 has a very short half-life and that degradation occurs through the proteasome in normally proliferating cells. To test whether KLF6 degradation after DNA damage also occurs via the proteasome we treated cells with an apoptotic dose of cisplatin in the presence of the proteasome inhibitor lactacystine. As shown in Fig. 7, KLF6 levels were diminished Fig. 4. KLF6 level is stable upon stimulation the extrinsic apoptotic pathway. KLF6 levels remain stable when the extrinsic pathway is within 4 h and no KLF6 was detected after 18 h in cells treated stimulated by TRAIL or TNFa. HCT116 human colon carcinoma with cisplatin alone. However, cisplatin-mediated degradation cells were treated with 50 ng/ml of TRAIL in the presence of 5 lg/ml of was completely inhibited in the presence of lactacystine. Simi- CHX (A) or 100 ng/ml of TNFa (B). PARP cleavage is shown to lar results were obtained using two other proteasome inhibi- conﬁrm apoptotic cell death. tors, ALLN and MG132 (data not shown) but not with the pan-caspase inhibitor Z-VAD-FMK or the calpain inhibitor 3.4. KLF6 has a short half-life and is degraded by the proteasome To assess the stability of KLF6 in untreated cells we inhibited new protein synthesis with CHX. As shown in Fig. 5, 50% of KLF6 had disappeared after 16 min. The majority of proteins in mammalian cells are degraded by the 26S proteasome [21]. In this pathway, the protein is marked for recogni- tion by the proteasome and subsequent degradation by covalent linkage to multiple molecules of ubiquitin. To deter- mine whether KLF6 is degraded by this mechanism we ﬁrst

Fig. 6. KLF6 is ubiquitinated in vivo. 293T cells were transiently transfected with pcDNA-KLF6 and hemagglutinin-tagged ubiquitin (pHA-Ub) and cultured for 2 h with the proteasome inhibitor ALLN or vehicle alone (DMSO). Equal amounts of protein were immuno- precipitated with anti-HA-antibody and Western blotted with anti KLF6 or anti-Flag antibody.

Fig. 5. KLF6 half-life is increased by proteasome inhibition. (A) 293T cells were transiently transfected with pcDNA3-Flag-KLF6 treated with 100 lg/ml CHX to inhibit protein synthesis and submitted to Western Blot analysis. (B) The quantity of KLF6 protein after CHX Fig. 7. KLF6 is degraded by the proteasome upon DNA damage. treatment was determined by chemiluminescence and is shown relative HepG2 cells were treated with different inhibitors of protein degrada- to the light emission of untreated cells. Quantification was performed tion 15 minutes prior to exposure to cisplatin (60 lM). (A) 40 lMof for the combined lower and upper band. T1/2 was estimated to be Lactacystine prevented KLF6 degradation upon exposure to cisplatin. 16 min. (C) Proteasome inhibition by ALLN 50 mM added 2 h prior to (B) Degradation of KLF6 proceeded unaltered in the presences the beginning of the experiment prevented KLF6 degradation Z-VAD-FMK (100 ng/ml), a pan-caspase inhibitor and prevented increasing the relative abundance of the upper component of the PARP cleavage as shown. (C) There was no effect by the calpain KLF6 double band. inhibitor, calpeptin (40 lM). M.S. Banck et al. / FEBS Letters 580 (2006) 6981–6986 6985 calpeptin. We conclude that KLF6 is rapidly and actively Eng, S.L., Arthur, M.J., Martignetti, J.A. and Friedman, S.L. degraded by the proteasome after lethal DNA damage. (2004) Kruppel-like factor 6 (KLF6) is a tumor-suppressor gene Protein degradation through the ubiquitin/proteasome path- frequently inactivated in colorectal cancer. Gastroenterology 126, 1090–1103. way is a key regulatory process in the control of apoptosis, cell [3] Kremer-Tal, S., Reeves, H.L., Narla, G., Thung, S.N., Schwartz, cycle control and DNA repair [22]. Degradation of proteins M., Difeo, A., Katz, A., Bruix, J., Bioulac-Sage, P., Martignetti, that either promote DNA repair or are anti-apoptotic/pro-sur- J.A. and Friedman, S.L. (2004) Frequent inactivation of the vival may precede cell death facilitating apoptosis when DNA tumor suppressor Kruppel-like factor 6 (KLF6) in hepatocellular carcinoma. Hepatology 40, 1047–1052. damage is too extensive to allow successful repair [23].An [4] Cho, Y.G., Kim, C.J., Park, C.H., Yang, Y.M., Kim, S.Y., Nam, example of a DNA repair-related protein that is degraded in S.W., Lee, S.H., Yoo, N.J., Lee, J.Y. and Park, W.S. (2005) this situation is PARP [24]. Two examples of anti-apoptotic Genetic alterations of the KLF6 gene in gastric cancer. Oncogene proteins that are degraded following DNA damage are c-FLIP 24, 4588–4590. and Bcl2. Degradation of c-FLIP may contribute to sensitiza- [5] Li, D., Yea, S., Li, S., Chen, Z., Narla, G., Banck, M., Laborda, J., Tan, S., Friedman, J.M., Friedman, S.L. and Walsh, M.J. tion of cells towards apoptosis mediated by p53 [25]. In all of (2005) KLF6 promotes preadipocyte differentiation through these examples degradation is a specific regulatory event and histone deacetylase 3 (HDAC3)-dependent repression of Dlk1. not the consequence of unspecific processes. In the present J. Biol. Chem. 280, 26941–26952. study, we showed KLF6 levels are controlled by proteasomal [6] Wang, S., Chen, X., Zhang, W. and Qiu, F. (2004) KLF6mRNA expression in primary hepatocellular carcinoma. J. Huazhong degradation linked to DNA damage. Low levels of DNA dam- Univ. Sci. Technol. Med. 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