Expression of Kinase Suppressor of Ras1 ... - Cancer Research

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Expression of Kinase Suppressor of Ras1 Enhances Cisplatin-Induced Extracellular Signal–Regulated Kinase Activation and Cisplatin Sensitivity Min Kim, Ying Yan, Robert L. Kortum, Scott M. Stoeger, Magdalene K. Sgagias, Kwangmoon Lee, Robert E. Lewis, and Kenneth H. Cowan Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, Nebraska

Abstract Kinase suppressor of Ras1 (KSR1) interacts with several mitogen-activated protein (MAP) kinase pathway components, including Raf, MAP/extracellular signal–regulated kinase (ERK) kinase (MEK), and ERK, and acts as a positive regulator of the Ras signaling cascade. Previous studies have shown that exposure of cells to the anticancer agent cisplatin (cisdiamminedichloroplatinum, CDDP) is associated with changes in multiple signal transduction pathways, including c-Jun-NH2kinase, ERK, and p38 pathways. Moreover, ERK activation has been linked to changes in cell survival following CDDP treatment. In this report, we have examined the effects of KSR1 expression on the sensitivity of cells to CDDP-induced apoptosis. Loss of KSR1 expression in mouse embryo fibroblasts (MEFs) derived from KSR1 knockout mice (KSR/ MEF) is associated with decreased CDDP-induced ERK activation and increased resistance to CDDP-induced apoptosis compared with wild-type MEFs (KSR+/+ MEF). Furthermore, transduction of KSR/ MEFs and MCF-7 breast cancer cells with wild-type KSR1 resulted in enhanced ERK activation following CDDP exposure and increased sensitivity to CDDP. In addition, inhibition of ERK activation by exposing MEFs to the MEK1/2-specific inhibitors PD98059 and U0126 protected both KSR+/+ and KSR/ MEFs cells from CDDP-induced apoptosis. These results indicate that KSR1-mediated regulation of ERK activity represents a novel determinant of CDDP sensitivity of cancer cells. (Cancer Res 2005; 65(10): 3986-92)

Introduction Cisplatin (cis-diamminedichloroplatinum, CDDP) is an effective anticancer agent that is useful in the treatment of a wide variety of human malignant diseases, including ovarian cancer, lung cancer, and head and neck cancer. Following exposure of cells to CDDP, the drug reacts with DNA to form intrastrand and interstrand cross-links (1). Cellular response to CDDP exposure includes activation of multiple signal transduction pathways (2, 3) and enhancement of DNA repair processes. Whereas CDDP-induced apoptosis in human cancer cells, including HeLa cervical cancer cells and A172 glioma cells, is associated with activation of several signaling pathways, including extracellular signal–regulated kinase (ERK), c-Jun-NH2-kinase (JNK), and p38, only inhibition of ERK

Note: M. Kim and Y. Yan contributed equally to this work. Requests for reprints: Kenneth H. Cowan, Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, 986805 Nebraska Medical Center, Omaha, NE 68198-6805. Phone: 402-559-4238; Fax: 402-559-4651; E-mail: [email protected]. I2005 American Association for Cancer Research.

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activation is associated with change in cellular sensitivity to CDDPinduced cell death (4, 5). Similar CDDP-induced effects are observed in B104 rat neuroblastoma cells and TKPTS mouse proximal tubule cells. In these models, CDDP-induced apoptosis is also associated with ERK activation and treatment of these cells with mitogen-activated protein (MAP)/ERK kinase (MEK)–specific inhibitors PD98059 and U0126 results in enhanced cell survival following CDDP exposure (6, 7). Cui et al. (8) also reported that exposure of A2780 human ovarian cancer cells to CDDP is associated with ERK activation. However, in these cells, inhibition of CDDP-induced ERK resulted in increased sensitivity to CDDP (8). Thus, whereas CDDP treatment induces ERK activation in several human cancer cell lines (HeLa, A172, and A2780), the effects of ERK inhibition on cell survival or cell death are apparently cell line specific. Previous studies have identified kinase suppressor of Ras1 (KSR1) as a positive regulator of the Raf/MEK/ERK kinase cascade (9). However, the precise role of KSR1 in regulating cell growth is not clear. Whereas KSR1 gene deletion studies have indicated that KSR1 gene expression is not essential for normal growth and development (10), mouse embryo fibroblasts (MEFs) in which the KSR1 gene has been deleted show diminished ERK activation following exposure of cells to growth factors or following the expression of activated Ras (10). In this report, we examined the influence of KSR1 expression on the response of cells to CDDP exposure as well as the role of this gene in CDDP-mediated cytotoxicity and CDDP-induced apoptosis. We found that MEFs deficient in KSR1 expression are resistant to CDDP-induced cytotoxicity and showed decreased ERK activation and apoptosis following CDDP exposure. Additional studies indicated that restoration of KSR1 expression in MEFs containing homozygous deletion of KSR1 gene resulted in enhanced CDDPinduced ERK activation and increased sensitivity to CDDP. These studies indicate that change in KSR1 expression can alter the cellular response to CDDP treatment.

Materials and Methods Generation of immortalized mouse embryo fibroblasts. Nonimmortalized MEFs were generated from 13.5-day KSR/ and KSR+/+ embryos as previously described (10). Cells were maintained in culture under a 3T9 protocol (11) until immortalized populations of cells emerged. To alter KSR1 expression in cells, KSR/ MEFs and MCF-7 cells were infected with a recombinant bicistronic retrovirus encoding both KSR1 and green fluorescent protein (GFP). For control, cells were also infected with vector expressing GFP only. Transduced cells were sorted by flow cytometry. Cells were excited at 458 nm and separated at 510/20 nm, with baseline fluorescence of uninfected cells having a mean intensity of 6 (range 0-15). Sorted cells were assessed for purity by fluorescence-activated cell sorting (FACS) analysis. Pools of cells were collected and placed in culture and

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KSR1 Enhances Cisplatin Sensitivity

assessed for KSR1 expression level by Western blot analysis using an antiKSR1 antibody (BD Biosciences, San Jose, CA). Cytotoxicity assays. Cell viability was assessed by microscopic examination of trypan blue–stained cells or by 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay as described previously (12). For the MTT assay, cells (1,500/well) were plated in 96-well plates and incubated at 37jC overnight. Following exposure to increasing concentrations of CDDP for 5 days at 37jC, 100 Ag MTT in PBS was added to each well and the cells were incubated for 4 hours at 37jC. Following centrifugation of plates at 500 g for 10 minutes, media was removed and 120 AL DMSO added to each well and incubated for additional 1 hour at room temperature with gentle shaking using an orbital shaker. The A 570 was determined using ELx 808 Ultra Microplate Reader (Bio-Tek Instrument, Winooski, VT). Cytotoxicity was expressed as the percentage of A 570 of treated cells relative to that of untreated cells. Western blot analysis and antibodies. Cells were plated in 15-cm dishes and incubated at 37jC until 70% confluent. CDDP at indicated doses was added onto the cells for an incubation time of 24 hours or indicated otherwise. The treated cells were then harvested, and whole cell lysates prepared and subjected to Western blot analysis as described previously (13). Antibodies used for Western blot analysis include anti-KSR1 (BD Biosciences), anti-phosphorylated-ERK (E-4, Santa Cruz Biotechnology, Santa Cruz, CA), anti-ERK (C-14, Santa Cruz Biotechnology), anti– poly(ADP-ribose) polymerase (PARP, C-2-10, Biomol Research Laboratories, Plymouth Meeting, PA), and anti-actin (I-19, Santa Cruz Biotechnology). Assays for apoptosis. For detection of apoptosis by DNA staining with 4V,6-diamidino-2-phenylindole (DAPI), treated cells were harvested with trypsin-EDTA and washed with PBS. Cells (50,000) were resuspended in 150 AL PBS containing 15% bovine serum albumin and loaded onto the bottom of a cytofunnel mounted with a microscope slide. The cells were fixed onto the slide by spinning at 500 rpm for 5 minutes in a cytocentrifuge.

Slides were air dried for 30 minutes and washed with PBS. DAPI (2.5 Ag/mL in PBS) was applied for 30 minutes at room temperature. After washing with PBS, samples were analyzed using a fluorescence microscope. For detection of apoptosis by the terminal deoxynucleotidyl transferase– mediated nick end labeling (TUNEL) method, cells were collected, washed twice with PBS, and TUNEL assay was done using MEBSTAIN Apoptosis kit (Medical and Biological Laboratories Co., Ltd., Nagoya, Japan) according to the manufacturer’s directions. FACS analyses were done on a FACSCalibur instrument (Becton Dickinson, Mansfield, MA).

Results Deletion of KSR1 alters sensitivity to cisplatin. Previous studies have shown that CDDP treatment of cells induces activation of several signal transduction pathways including ERK (4). Moreover, inhibition of ERK signaling influences overall cell survival following CDDP treatment (4). Because KSR1 has been shown to facilitate the activation of ERK following exposure of cells to growth factors, we examined the effect of KSR1 expression in determining cellular sensitivity to CDDP. For these studies, wildtype (KSR+/+) and null (KSR/) MEFs were obtained from 13.5day-old embryos as described in Materials and Methods. Although KSR1 has been shown to influence the activation of signal transduction pathways in response to growth factors, previous studies revealed no change in the in vitro growth rates of KSR+/+ and KSR/ MEFs (10). To examine the effects of KSR1 expression on CDDP sensitivity, KSR+/+ and KSR/ MEFs were exposed to increasing concentrations of CDDP for 5 days and cell survival was determined by

Figure 1. Cell viability and apoptosis in CDDP-treated wild-type and KSR/ MEFs. A, KSR+/+ and KSR/ MEFs were seeded into 96-well plates and treated with different doses of CDDP for 5 days. Cell viability was assessed using the MTT colorimetric assay as described in Materials and Methods. B and C, KSR+/+ and KSR/ MEFs were treated with CDDP at the indicated doses for 24 hours. The resulting cells were harvested and analyzed for apoptosis by DAPI staining as described in Materials and Methods. B, an aliquot of each sample (50,000 cells) was stained and examined by fluorescence microscopy. C, the percentage of apoptosis is shown as the mean (columns ) F SD (bars ) of quadruplicate samples. D, flow cytometric TUNEL analysis for apoptosis. KSR/ and KSR+/+ MEFs (2 106) were treated with 100 Amol/L CDDP (MEF / + CDDP and MEF +/+ + CDDP ) or, as a control, DMSO (MEF / and MEF +/+), for 24 hours. The resulting cells were analyzed for apoptosis using TUNEL assay as described in Materials and Methods.


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Figure 2. Introduction of KSR1 into KSR/ MEFs increased CDDP-induced cytotoxicity. A, KSR/ MEFs were infected with a recombinant bicistronic retrovirus encoding both KSR1 and GFP or GFP alone. Cells expressing GFP were isolated as described in Materials and Methods. KSR1 levels in KSR1/GFP-transduced (KSR1/GFP1 and KSR1/GFP2 ) or GFP-transduced (GFP ) KSR/ MEFs were measured by Western blot analysis. For a comparison, measurements of KSR1 levels in KSR/ and KSR+/+ MEFs were also included (KSR/ and KSR+/+). B, KSR1/GFP-transduced MEFs, KSR1/GFP1 (o) and KSR1/GFP2 (.), and GFP-transduced MEFs (4) were incubated with CDDP at indicated doses for 5 days and cell viability assessed using MTT assay.

MTT assay. As shown in Fig. 1A, KSR/ MEFs were 5-fold more resistant to CDDP treatment compared with KSR+/+ MEFs. The increased resistance of KSR/ MEFs to CDDP was confirmed by examining cell death following drug exposure using DAPI staining. As shown in Fig. 1B, microscopic examination revealed more condensed and fragmented nuclei in KSR+/+ MEFs relative to KSR/ MEFs following exposure to 100 Amol/L CDDP for 24 hours, indicating increased CDDP-induced apoptosis in KSR+/+ MEFs (Fig. 1B). Shown in Fig. 1C are the quantitative results of DAPI staining in KSR+/+ MEFs versus KSR/ MEFs following exposure to increasing concentrations of CDDP for 24 hours. The results in Fig. 1C indicate that KSR+/+ MEFs are f3-fold more sensitive to CDDP-induced apoptosis relative to KSR/ MEFs. The induction of apoptosis in MEFs by CDDP treatment was confirmed using TUNEL assay. In this assay, induction of apoptosis is represented by an increase in DNA fragments that are labeled with biotin-dUTP and detected by binding to avidin-FITC. As results shown in Fig. 1D, treatment of MEFs with CDDP resulted in an increase in mean fluorescence intensity (rightward shift) when compared with untreated samples. Consistent with the results obtained from DAPI staining analysis, KSR+/+ MEFs treated with CDDP showed higher intensity of mean fluorescence relative to KSR / MEFs treated with the same concentration of CDDP (MEF +/+ + CDDP versus MEF / + CDDP). Ectopic KSR1 expression increases cisplatin sensitivity in KSR/ cells. To confirm that the loss of KSR1 was responsible for this change in CDDP sensitivity, a KSR1 expression vector was used to reintroduce KSR1 gene expression in KSR/ MEFs. In this study, a bicistronic retroviral vector expressing both KSR1 and GFP (KSR1/GFP) and a control vector expressing GFP only (GFP) were

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used as described in Materials and Methods. Following transduction of KSR/ MEFs with each vector, GFP-expressing cells were isolated and KSR1 levels were measured by Western blot analyses. Two cell populations expressing both GFP and KSR1 were obtained and CDDP-induced cytotoxicity in these cells was examined using MTT assay. As shown in Fig. 2A, both KSR1/GFP-transduced KSR/ MEF cell populations (KSR1/GFP1 and KSR1/GFP2) expressed levels of KSR1 comparable with that found in KSR+/+ MEFs. In contrast, KSR/ MEFs transduced with the control GFP vector (GFP) had undetectable levels of KSR1 expression. MTT assay was used to assess the effect of KSR1 expression on CDDP-induced cytotoxicity in KSR/ MEFs. As shown in Fig. 2B, expression of KSR1 in KSR/ MEFs increased the sensitivity of both KSR1/GFP1 and KSR1/GFP2 cells to CDDP 4- to 5-fold relative to control GFP-transduced KSR/ MEFs. The IC50 for CDDP were similar in the both KSR1/GFP-transduced MEFs (6 Amol/L for KSR1/GFP1 and 7 Amol/L for KSR1/GFP2). These studies indicate that introduction of KSR1 into KSR/ MEFs enhances CDDP cytotoxic effect. KSR1 enhances cisplatin-stimulated apoptosis and extracellular signal–regulated kinase activation. DAPI staining following exposure to increasing doses of CDDP was also examined to evaluate changes in CDDP-induced apoptosis. As shown in Fig. 3A, CDDP treatment resulted in a dose-dependent increase in apoptosis in both KSR1/GFP-transduced and control (GFP)transduced MEFs. However, KSR1/GFP-transduced KSR/ MEFs (KSR1/GFP1) showed significant higher CDDP sensitivity compared with control GFP-transduced KSR/ MEFs (GFP). Exposure of control GFP-transduced KSR/ cells to 100 Amol/L CDDP for 24 hours induced apoptosis in only 30% cells. In contrast, KSR1/ GFP-transduced KSR/ MEFs (KSR1/GFP1) showed 90% apoptosis following exposure to 100 Amol/L CDDP. Previous studies have indicated that loss of KSR1 expression reduced epidermal growth factor (EGF) and phorbol 12-myristate 13-acetate–induced ERK activation in KSR/ MEFs and that the reintroduction of KSR1 gene into KSR/ MEFs restored their response to growth factor–induced activation of ERK (10). Because exposure of cells to CDDP has also been shown to activate ERK (4–8), we next examined the effect of the loss of KSR1 expression on the activation of ERK following exposure to increasing concentrations of CDDP in both GFP and KSR1/GFP gene–transduced MEFs. As shown in Fig. 3B, total ERK2 protein levels were similar in both control (GFP)-transduced and KSR1/ GFP-transduced KSR/ MEFs. However, exposure to 33 and 100 Amol/L CDDP for 24 hours induced greater ERK phosphorylation in KSR1/GFP-transduced MEFs compared with control GFP– transduced KSR/ MEFs. Thus, KSR1 gene expression is associated with increased CDDP-induced ERK activation in MEFs. We also examined the time course of ERK phosphorylation following CDDP exposure of cells expressing ectopic KSR1. As shown in Fig. 3C, phosphorylation of ERK was detected within 12 hours following exposure of KSR1/GFP-transduced KSR/ MEFs to 100 Amol/L CDDP, whereas control (GFP)-transduced KSR/ MEFs had little detectable ERK activation until 24 hours following exposure to 100 Amol/L CDDP (data not shown). Furthermore, as shown in Fig. 3C, CDDP-induced ERK activation begins at 12 hours following exposure to 100 Amol/L CDDP, which is concomitant with the development of cleavage of PARP precursor (Fig. 3C, P-ERK and PARP). Because apoptosis involves activation of proteolytic enzymes resulting in cleavage of several intracellular proteins including PARP (14), the detection of cleavage

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greater ERK phosphorylation in KSR+/+ MEFs compared with KSR/ MEFs. These results confirm that KSR1 expression enhances CDDP-induced ERK activation and results in enhanced cytotoxic effects of CDDP. MEK inhibitors block cisplatin-induced apoptosis. Two specific inhibitors of MEK1/2 (PD98059 and U0126) were used to evaluate the role of ERK activation in CDDP-induced apoptosis in KSR1/GFP- and control GFP–transduced KSR/ MEFs. As shown in Fig. 4A, Western blot analysis indicated exposure of control GFPtransduced MEFs to 100 Amol/L CDDP for 24 hours resulted in

Figure 3. Introduction of KSR1 into KSR/ MEFs increased CDDP-induced apoptosis and enhanced CDDP-induced ERK phosphorylation. A, KSR/ MEFs transduced with KSR1/GFP vector (solid columns ) or GFP control vector (open columns ) were incubated with CDDP at the indicated doses for 24 hours and analyzed for apoptosis by DAPI staining. The percentage of apoptosis is shown as the mean (columns ) F SD (bars ) of quadruplicate samples. B, the cells described above were treated with CDDP at the indicated concentrations for 24 hours. The treated cells were analyzed for levels of phosphorylated-ERK (P-ERK ), ERK 2 (ERK2 ), and actin (Actin) by Western blot analysis. C, cells were treated with 100 Amol/L CDDP for the indicated hours and then analyzed for levels of phosphorylated-ERK, ERK 2, and PARP by Western blot analysis. D, KSR/ and KSR+/+ MEFs were treated with indicated doses of CDDP for 24 hours and analyzed for levels of phosphorylated-ERK and actin.

of PARP precursor following CDDP treatment provides further evidences for induction of apoptosis in MEFs following CDDP treatment. We also examined ERK activation following CDDP treatment in both KSR/ and KSR+/+ MEFs. As shown in Fig. 3D, treatment with 33 and 100 Amol/L CDDP for 24 hours induced


Figure 4. MEK1/2-specific inhibitors PD98059 and U0126 block CDDP-induced ERK activation and apoptosis in both KSR/ and KSR/ MEFs expressing ectopic KSR1. A, top, GFP control vector–transduced KSR/ MEFs (GFP ) were preincubated with the indicated MEK1/2 inhibitor for 3 hours and followed by incubation with 100 Amol/L CDDP for 24 hours. Treated cells were then analyzed for levels of phosphorylated-ERK and ERK-2 by Western blot analysis. Bottom, KSR1/GFP-transduced KSR/ MEFs (KSR1/GFP1) were treated as described above and analyzed for levels of ERK phosphorylation and total ERK-2 protein. B, top, GFP control vector–transduced KSR/ MEFs were treated as described above and apoptotic cells were examined by DAPI staining. Columns, mean of quadruplicate samples; bars, SD. Bottom, KSR1/GFP1-transduced KSR/ MEFs were treated as described above and analyzed for apoptosis by DAPI staining. Columns, mean of quadruplicate samples; bars, SD.

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activation of ERK (top, P-ERK, lanes 2 and 3 versus lane 1). However, treatment of KSR1/GFP-transduced KSR/ MEFs with the same concentration of CDDP resulted in a stronger induction in ERK phosphorylation (bottom, lanes 2 and 3, p-ERK) compared with GFP-transduced KSR/ MEFs (top, lanes 2 and 3, p-ERK). In both cell lines, 3-hour incubation with PD98059 or U0126 before the addition of 100 Amol/L CDDP reduced ERK activity to control levels (Fig. 4A, P-ERK, lanes 5 and 7 versus lane 1). We next examined the effects of the two MEK inhibitors on CDDPinduced apoptosis in GFP- and KSR1/GFP-transduced KSR/ MEFs using DAPI staining analysis. As shown in Fig. 4B, incubation with 100 Amol/L CDDP resulted in greater apoptosis in KSR1/GFPtransduced KSR/ MEFs compared with control GFP-transduced KSR/ MEFs. Furthermore, incubation with PD98059 or U0126 before CDDP exposure resulted in a dose-dependent inhibition of CDDP-induced apoptosis in both GFP- and KSR1/GFP-transduced MEFs (Fig. 4B). Taken together, these results indicate that ERK activation is an important component of CDDP-induced apoptosis in MEFs. Moreover, KSR1 expression in MEFs facilitates CDDPinduced ERK phosphorylation as well as CDDP-induced apoptosis. Ectopic KSR1 expression increases cisplatin sensitivity in cancer cells. To confirm that increased KSR1 expression is associated with changes in sensitivity to CDDP in human cancer cells, we infected MCF-7 breast cancer cells with a recombinant bicistronic retroviral vector expressing both mouse KSR1 and GFP. Following transduction of MCF-7 cells with mouse KSR1/GFP vector, GFP-expressing cells were isolated and the expression of mouse KSR1 measured by Western blot analysis. As shown in Fig. 5A, high levels of mouse KSR1 were expressed in MCF-7 cells transduced with the KSR1/GFP vector, which is not presented in wildtype MCF-7. Reverse transcription-PCR and Western blot analysis using human KSR1 primer and antibody showed that the levels of human KSR1 gene expression remained unchanged following infection with mouse KSR1 expression vector (data not shown). We next studied the effect of mouse KSR1 on the response of MCF-7 human breast cancer cells to CDDP treatment. Both MCF-7 and MCF-7/KSR1 cells incubated with increasing doses of CDDP for 24 hours and then analyzed for ERK phosphorylation. As shown in Fig. 5B, MCF-7 cells–expressed mouse KSR1 (MCF-7/KSR1) displayed an enhanced CDDP-induced ERK phosphorylation (p-ERK) compared with wild-type MCF-7 cells. Furthermore, MCF-7/ KSR1 cells displayed increased CDDP-induced PARP cleavage compared with wild-type MCF-7 cells. Because cleavage of PARP protein by caspases is a hallmark of apoptosis (14), we further examined the influence of KSR1 on CDDP-induced apoptosis in MCF-7 cells using TUNEL assay as well as DAPI staining. Consistent with the finding of enhanced CDDP induced PARP cleavage in MCF-7/KSR1 cells, results from TUNEL assays also showed that ectopic expression of mouse KSR1 in MCF-7 cells enhanced apoptotic response following CDDP treatment relative to wild-type MCF-7 cells (Fig. 5C, MCF-7/KSR1 versus MCF-7). Twenty-four hours following incubation with 30 Amol/L CDDP, there is a marked increase in the mean fluorescence intensity produced by dUTP incorporation in MCF-7/KSR1 cells relative to MCF-7/KSR1 cell incubated with DMSO control (Fig. 5C, right). In contrast, very little increase in fluorescence intensity was observed in wild-type MCF-7 cells incubated with 30 Amol/L CDDP compared with wild-type MCF-7 cells incubated with DMSO control (Fig. 5C, left). We also evaluated CDDP-induced apoptosis by DAPI staining of KSR1-transduced MCF-7 cells and control MCF-7 cells. As shown in

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Fig. 5D, DAPI staining following 24 hours exposure to CDDP revealed a marked increase in CDDP-induced apoptosis in KSR1transduced MCF-7 cells compared with control MCF-7 cells (Fig. 5D). Thus, overexpression of mouse KSR1 in both MEFs and MCF-7 human breast cancer cells is associated with increased CDDP-induced ERK activation and enhanced apoptosis following CDDP exposure.

Discussion MAP kinase (MAPK) signaling pathways are important in regulating cell proliferation and cell survival in response to growth stimulation and stress. MAPKs consist of at least three signal transduction pathways (ERK, JNK and p38). The first class includes ERK1 and ERK2 kinases, whose activation by mitogens leads to the induction of cyclin D1 and the initiation of cell cycle progression. Whereas activation of the Raf/MEK/ERK pathway is involved in cell proliferation (15), JNK/SAPK and p38 kinase pathways are primarily activated by stress signals, including exposure to protein synthesis inhibitors, irradiation, and DNA-damaging agents. In contrast to the Raf/MEK/ERK pathway, activation of the JNK/ SAPK and p38 kinase pathways leads to inhibition of cellular proliferation and/or decreased cell survival (15). CDDP is an effective anticancer agent that acts by forming intrastrand and interstrand DNA cross-links, resulting in cell cycle arrest and/or apoptosis (1). Previous studies have shown that ERK, JNK, and p38 are all activated in response to CDDP treatment. However, inhibitors studies suggest that only the activation of ERK seems to be involved in regulating cell survival following exposure to CDDP (2, 4, 5, 7, 16). ERK inhibition studies indicate that the effect of ERK activation following exposure of cells to CDDP is apparently cell type specific (2, 4, 8, 16). Thus, studies in HeLa and A172 cells indicated that ERK activation following CDDP treatment is correlated with enhanced sensitivity to CDDP (4, 5), whereas studies in A2780 ovarian cancer cells reveal that CDDPinduced ERK activation is associated with increased resistance to CDDP-mediated cytotoxicity (2, 8, 16). Whereas previous reports have suggested ERK activation as a primary response to CDDP exposure (4, 17, 18), the precise regulation of this signaling pathway in response to CDDP treatment remains unclear. Because KSR1 has been previously identified as an important positive regulator of the Raf/MEK/ERK kinase cascade (9), we, therefore, decided to explore the possible influence of KSR1 expression on the response of cells to CDDP treatment and the induction of apoptosis. Using MEFs deficient in KSR1 expression (KSR/ MEFs), we found that loss of KSR1 expression in MEFs was associated with decreased ERK activation following CDDP exposure compared with wild-type MEFs (KSR+/+ MEFs). Concomitant with the decrease in CDDP-induced activation of ERK in KSR/ MEFs, KSR1-deficient MEFs displayed a reduced sensitivity to CDDP compared with KSR+/+ MEFs. Results in this report also indicated that KSR1 plays an important role in the regulation of CDDP-induced ERK activation and CDDP-induced apoptosis. Furthermore, MEK1/2 inhibitor studies showed that ERK inhibition by PD98059 as well as U0126 resulted in decreased apoptosis following CDDP treatment in both KSR+/+ and KSR/ MEFs. Thus, CDDP-activated ERK signaling in MEFs is a key determinant of cellular response to CDDP treatment. To further define the role of KSR1 in CDDP sensitivity, KSR/ MEFs were stably transduced with a KSR1 expression vector, which expresses exogenous KSR1 at a level that restores physiologic ERK

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activation but does not enhance cell proliferation (19). CDDPinduced ERK activation as well as apoptosis were examined in KSR/ cells and in KSR1 reconstituted cells. The studies showed that restoration of KSR1 expression in KSR/ MEFs resulted in an increase in CDDP-induced ERK activation. Furthermore, these studies also indicate that KSR1 expression by itself was sufficient to restore cellular sensitivity to CDDP-induced apoptosis in KSR1deficient MEFs. Thus, KSR1 expression can influence the cellular response to CDDP, at least in part, through the enhancement of ERK activation in response to CDDP exposure. Previous studies have implicated several mammalian proteins including KSR1, MP-1, and RKIP, as scaffolds proteins that facilitate signaling through the Ras/Raf/ERK pathway (20). Although the binding of each of these proteins, including KSR1, to components of Ras/Raf/ERK pathway has been documented, the precise function of these proteins to facilitate signaling through the ERK pathway in vivo remains largely unresolved. For example, Nguyen et al. (10) reported that ERK activation in response to multiple stimuli was attenuated but not abolished in the KSR/ MEFs. Thus, KSR1 expression is a modulator of ERK activation. Another study showed that loss of KSR1 expression through genetic deletion attenuated signaling through EGF/Ras/ERK pathway and abolished the capability of oncogenic Ras (v-Ha-ras) to induce skin cancer in KSR/ mice (21). Recent studies by Yan et al. (22, 23) showed that KSR1 expression can influence the cellular sensitivity to tumor necrosis factor (TNF). In these studies, expression of a dominant-negative KSR1 or antisense KSR1 in mouse intestinal epithelial cells (YAMC; ref. 22), or loss of KSR1 in mouse colonic epithelial cells (23) attenuated TNF-mediated ERK activation and enhanced TNF-induced apoptosis. These workers also found that inhibition of KSR-mediated signaling in those cells also inhibited TNF-induced NF-nB activation and cIAP2 expression. Whereas ERK inhibition by itself had no effect on TNF-mediated apoptosis in YAMC cells, inactivation of ERK in combination with inhibition of NF-nB nuclear translocation enhanced TNF-induced apoptosis in YAMC cells. These studies indicate that alteration in the level or function of KSR can affect multiple downstream regulatory pathways that can influence both proapoptotic and antiapoptotic signaling in cells. Differences in the regulation of downstream pathways following ERK activation could explain the differential on cell sensitivity to various agents including CDDP. Furthermore, the studies by Yan et al. (22) indicated that expression of dominant-negative KSR1, which carries mutations in the kinase domain of KSR1 (D683A/D700A; ref. 24), enhances TNFinduced apoptosis in YAMC cells, suggesting an important role of the kinase domain of KSR1 in the determination of TNF-mediated

cytotoxicity. It is not known, at the present time, whether the kinase domain of KSR1 plays a role in CDDP-induced ERK activation as well as cytotoxicity. Additional studies with the kinase domain mutated KSR1 are required for fully address this issue. Recent studies have also implicated phosphorylation of KSR1 as another determinant of KSR1 function (25). Brennan et al. (25) observed that phosphorylation of KSR1 was associated with translocation of KSR1 into nucleus and resulted in MEK activation.

Figure 5. Introduction of exogenous KSR1 into MCF-7 cells enhanced CDDP-induced ERK phosphorylation and increased CDDP-induced apoptosis. A, levels of mouse KSR1 in MCF-7 cells (MCF-7 ) and MCF-7 cells transduced with mouse KSR1 (MCF-7/KSR1 ) were measured by Western blot analysis. For a comparison, levels of mouse KSR1 in KSR/ MEFs transduced with GFP control vector (MEF/GFP ) or KSR1/GFP vector (MEF/KSR1 ) were also evaluated in the same Western analysis. B, MCF-7 or MCF-7/KSR1 cells were treated with the indicated doses of CDDP for 24 hours and then cells analyzed by Western blot analysis for levels of phosphorylated-ERK, PARP, and actin. C, MCF-7 and MCF-7/KSR1 cells were incubated with either DMSO (gray lines ) or 30 Amol/L CDDP (black lines ) for 24 hours and the cells analyzed for apoptosis using TUNEL assays. D, after 24 hours of exposure to CDDP at the indicated doses, MCF-7 and MCF-7/KSR1 cells were analyzed for apoptosis by DAPI staining. The percentage of apoptosis is shown as the mean (columns ) F SD (bars ) of quadruplicate samples.


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Cancer Research

Furthermore, Ory et al. (26) showed that KSR1 is associated with the serine/threonine protein phosphatase PP2A and that specific inhibition of PP2A prevented the growth factor–induced recruitment of KSR1 to membranes and blocked activation of KSR1-associated MEK and ERK. These studies suggest that KSR1mediated MEK/ERK signaling activation is not only determined by the level of KSR1 protein in cells, but that factors that influence KSR1 phosphorylation may also influence KSR1 function in cells. In the present studies, we show that CDDP-induced ERK activation was significantly enhanced by the presence of KSR1 protein expression. To better understand the role of KSR1 in cellular response to CDDP, additional studies on the regulation of posttranslational modification of KSR1 and its intracellular distribution following CDDP treatment are needed. KSR1 apparently exists in a high-molecular-weight complex in cells in association with both MEK and ERK (27). Other studies have suggested that KSR1 facilitates MAPK activation by preassembling components and/or by assisting the delivery of cytoplasmic MEK and ERK to Ras and Raf at the plasma membrane (28). Furthermore, other proteins, including MP1 and MEKK1, have

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Cancer Res 2005; 65: (10). May 15, 2005

also been shown to interact with components of the ERK signaling cascade and future studies are needed to address the possible role of these other proteins in ERK signaling and cellular sensitivity in response to CDDP. In summary, results in this report have implicated KSR1 expression in MEFs and in MCF-7 human breast cancer cells as a determinant of cellular response to CDDP. In both models, KSR1 expression enhances CDDP-induced activation of ERK and is associated with increased cellular sensitivity to CDDP. These results suggest a potential role of KSR1 modulating the intracellular response to CDDP and raises the possibility that alterations in KSR1 expression or function could be associated with the development of clinical resistance to anticancer drugs, including CDDP.

Acknowledgments Received 7/29/2003; revised 2/13/2005; accepted 3/18/2005. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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