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Women's Cancers Section, Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland1
Received 26 July 2004/ Returned for modification 20 August 2004/ Accepted 16 November 2004
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ABSTRACT |
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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subunits, and accessory proteins, such as Hsp90, c-Tak1, PP2A, CNK, and 14-3-3 (2, 6, 40, 42, 46, 65), supporting the scaffold hypothesis. Translocation of the protein complex from the cytoplasm to the plasma membrane to assemble a functional Raf/Mek complex was observed upon growth factor stimulation, controlled by KSR1 serine 392 phosphorylation and 14-3-3 binding status (40, 46) (56). KSR1 transfection has either stimulated or inhibited Erk signaling in cell lines, depending on the level of KSR1 protein expressed (reviewed in reference 39). KSR1 null mice exhibited normal development (29, 42). Crosses of KSR1 null mice to MMTV-polyoma middle T mice (wild-type ras) resulted in delayed tumor formation (42), while crosses to Tg.AC mice (v-Ha-ras mutated at codons 12 and 59) inhibited skin papilloma formation (29). Antisense inhibition of KSR1 also inhibited mutant Ras-dependent pancreatic cancer xenograft growth (76).
Other aspects of KSR1 function remain incompletely understood. KSR1 has also been localized to the nucleus, which may be important for the subcellular routing of Mek (9). KSR1–/– mouse embryo fibroblasts exhibited reduced Erk activation in response to 12-O-tetradecanoylphorbol-13-acetate, but Raf activation of Mek remained normal (29). A kinase function for KSR1 has been debated (77, 80). KSR1 phosphorylation has been reported to be influenced by zinc ion concentrations in C. elegans (81).
We recently reported complex interactions between KSR1 and the Nm23-H1 metastasis suppressor (19). Metastatic disease represents one of the most difficult challenges in cancer therapy. Both positive and negative signaling pathways regulate tumor metastasis, including multiple metastasis suppressor genes (64). When reexpressed in a metastatic cell line, these genes suppress in vivo metastasis without significant effects on the size of the primary tumor. The nm23 gene family has been demonstrated to have suppressive activity for the development of lymph node, lung and/or liver metastases in 11 independent studies (3, 7, 17, 25, 26, 36, 38, 52, 59, 68, 69). The biochemical mechanism of metastasis suppression is thought to involve attenuation of signaling for tumor cell motility, invasion, and colonization. At least four classes of Nm23 biochemical activities may contribute to altered signaling, including protein-protein interactions (4, 12, 16, 28, 44, 47, 51, 55, 57, 58, 66), regulation of GTP-binding protein function (15, 48, 50, 72, 79, 82), DNA-associated activities (14, 53, 54, 63), and histidine-dependent protein phosphotransferase activity (11, 73, 74). A role for KSR1 in Nm23-H1 attenuation of Erk activation was investigated. Transfection of wild-type nm23-H1, but not a control vector or a mutant nm23-H1 incapable of inhibiting motility, was associated with low Erk activation (19). Nm23-H1 coimmunoprecipitated KSR1 from 293T cells and human MDA-MB-435 breast carcinoma cells, and Nm23-H1 phosphorylated KSR1 on serines 392 and 434 of KSR1 in vitro (19). The data suggested the hypothesis that Nm23-H1 overexpression and/or kinase activity altered the scaffold properties of KSR1 and attenuated Erk signaling needed for metastatic invasion and colonization.
In this report, the steady-state scaffold function of KSR1 was investigated in control and Nm23-H1-transfected breast carcinoma cells in response to the milieu of factors present in serum, mimicking conditions in which metastases thrive in vivo. We observed increased binding of Hsp90 to KSR1 in Nm23-H1-transfected cells. To date, Hsp90 has been shown to promote either the folding or the proteasome-mediated degradation of multiple client proteins depending on the complex of bound cochaperone proteins (reviewed in references 21 and 33). We report the downstream consequences of an increased Hsp90-KSR1 interaction in Nm23-H1-overexpressing breast carcinoma cells, including decreased KSR1 stability and increased sensitivity to a Hsp90-directed therapeutic agent.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell lines, transfections, and drug treatments. Human MD-MBA-435 breast carcinoma cells or stable transfectants were previously described (26, 31) and were maintained at 37°C in 5% CO2 in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. Cells were plated at a density of 2 x 106 cells/100-mm plate and transfected with HA-KSR1, Nm23-H1-Flag, or control vectors using Effectene transfection reagent (QIAGEN). The cell culture medium was changed 24 h later, and the cells were harvested 48 h posttransfection.
Reagents. 17-Allylamino-17-demethoxygeldanamycin (17-AAG) was kindly provided by the Cancer Therapy Evaluation Program, National Cancer Institute. A 50 mM stock solution in dimethyl sulfoxide (DMSO) was frozen at –80°C in aliquots and protected from light. Cycloheximide (CHX) was obtained from Sigma. MG-132 proteasome inhibitor and the MEK inhibitor PD90859 were purchased from Calbiochem.
Cell fractionation.
Cells were scraped in ice-cold phosphate-buffered saline, collected by centrifugation, and incubated in hypotonic buffer (5 mM sodium phosphate [pH 7.5], 2 mM MgCl2, complete mini EDTA-free protease inhibitor cocktail [Roche Molecular Biochemicals], dual phosphatase inhibitor cocktails 1 and 2 [Sigma], 1 mM phenylmethylsulfonyl fluoride) for 30 min on ice. Cells were sheared by repeated passage through a 25-gauge needle, and the lysate was precleared by low-speed centrifugation (12,000 x g for 5 min). The pellet was washed with hypotonic buffer, centrifuged at low speed, and lysed with NE-PER extraction reagent (Pierce) including protease and phosphatase inhibitors to obtain nuclear proteins. The supernatant was centrifuged at 100,000 x g for 1 h. The resulting supernatant was used as the cytoplasmic extract. The pellet was washed, recentrifuged, and lysed in hypotonic buffer containing 1% (vol/vol) Triton X-100 as the membrane extract. To verify the purity of each fraction, 30 µg of protein were subjected to sodium dodecyl sulfate (SDS)-polyacrylamide electrophoresis, and a Western blot was probed with the following antibodies: anti-Na, K-ATPase (membrane marker; Novus Biological), anti-
-tubulin (cytoplasmic marker; Oncogene), and anti-c-Jun (nuclear marker; Santa Cruz).
Drug treatments. MDA-MB-435 cells were transfected as described above. Twenty-four hours posttransfection, cells were trypsinized, and 3 x 105 cells were plated in six-well plates. Twelve hours later, 100 µM CHX was added. A similar procedure was used for 17-AAG treatments with the exception that cells were plated in 150-mm dishes, trypsinized, and replated after 16 h into four 100-mm dishes. The reversible proteasome inhibitor MG-132 (Calbiochem) was used at a 15 µM concentration and added to the cells 1 h before 17-AAG.
Coimmunoprecipitations and Western blot analysis. For Western blots, cells were lysed in RIPA buffer (20 mM Tris-HCl [pH 8], 137 mM NaCl, 10% glycerol, 1% Nonidet P-40 [NP-40], 2 mM EDTA, 0.5% sodium deoxycholate, 0.1% SDS), containing complete mini EDTA-free protease inhibitor cocktail (Roche Molecular Biochemicals) and phosphatase inhibitor cocktails 1 and 2 (Sigma). Lysates were normalized to equivalent total protein. The phospho-MAP kinase (P-p44/P-p42) and the total MAP kinase (p44/p42) antibodies were obtained from Cell Signaling. After incubation with horseradish peroxidase-conjugated secondary antibody, proteins were visualized by autoradiography with LumiGlo reagents (Cell Signaling).
For coimmunoprecipitations, cells were lysed in NP-40 buffer (20 mM Tris-HCl [pH 8], 137 mM NaCl, 10% glycerol, 1% NP-40, 2 mM EDTA), containing complete mini EDTA-free protease inhibitor cocktail (Roche Molecular Biochemicals), phosphatase inhibitor cocktails 1 and 2 (Sigma), and 1 mM phenylmethylsulfonyl fluoride, and the lysates were precleared with protein G-agarose (Roche Molecular Biochemicals) for 1 h at 4°C. Eight hundred micrograms of lysate was incubated overnight at 4°C with 5 µg of mouse anti-HA antibody (Covance, Princeton, N.J.) or the same amount of control immunoglobulin G antibody (Santa Cruz Biotechnology). The immunocomplexes were precipitated after incubation with 30 µl of protein-G-agarose for 1 h at 4°C, washed three times with NP-40 buffer and twice with 1x phosphate-buffered saline and resuspended with 30 µl of Laemmli buffer. Proteins were separated by SDS-polyacrylamide electrophoresis and transferred to a nitrocellulose membrane. The membrane was then immunoblotted with anti-HA (Covance), anti-14-3-3ß (K-19; Santa Cruz Biotechnology), anti-Mek-1/2 (Upstate), anti-Hsp90 (H-114; Santa Cruz Biotechnology), or anti-Nm23 (clone 56; BD Transduction Laboratories) (Ab 11; Cymbus Biotechnology). Western blots were processed as described above.
Soft agar assay. Soft agar colonization assays were performed as previously described (49). Briefly, a suspension of 3 x 104 cells, containing 0.3% (wt/vol) soft agar, was mixed with various concentrations of 17-AAG or PD90859 prior to setting. The cell layer was overlaid onto a layer of culture medium containing 0.7% (wt/vol) agar in 24-well plates. Colonies were counted after 3 weeks by using an ocular with a grid. Colonies were defined as having a diameter of 0.125 mm as assessed by the ocular grid. Each point is the mean ± standard error of the mean (SEM) of triplicate determinations.
Statistical analyses. Results shown are representative of three experiments conducted, unless otherwise noted. Differences were tested by a two-tailed Student t test using Instat (GraphPad Software, Inc.).
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RESULTS |
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HA-KSR1 was immunoprecipitated from equivalent amounts of total protein in cytoplasmic lysates of the C-100, H1-177, and S-22 cell lines (Fig. 1B), and the relative amount of Hsp90 bound to KSR1 was determined by Western blotting of the immunoprecipitates. KSR1 was expressed at comparable levels in each cell line. Nm23-H1-overexpressing H1-177 cells exhibited greater Hsp90 binding to KSR1 than the control C-100 or S-22 mutant Nm23-H1-expressing cells. A control immunoglobulin G did not immunoprecipitate KSR1 or coimmunoprecipitate Hsp90. In a separate experiment, the Nm23-H1-overexpressing H1-177 cell line also exhibited higher levels of Hsp90 binding to cytoplasmic KSR1 than a stable transfectant expressing a S120G mutant of nm23-H1, the I-205 cell line, which was also not motility inhibited (data not shown). In the experiment shown in Fig. 1C, control C-100 and Nm23-H1-overexpressing H1-177 cells were first immunoprecipitated with anti-Nm23-H1 and then assessed for binding of KSR1 and Hsp90. A trend similar to that seen in Fig. 1B, increased binding of Hsp90 to KSR1, was demonstrated with H1-177 cells with HA-KSR1 transiently transfected. The data also demonstrate three-way complex formation between Nm23-H1, KSR1, and Hsp90 with either anti-HA-KSR1 or anti-Nm23 as immunoprecipitating antibodies (Fig. 1B and C). Thus, a direct correlation of motility-suppressed behavior and quantitatively higher binding of Hsp90 to the cytoplasmic KSR1 scaffold was observed.
Not all of the known KSR1-associated proteins exhibited altered binding levels between the control C-100 and Nm23-H1-overexpressing H1-177 cell lines. In the experiment shown in Fig. 2A, the cells were transiently transfected with HA-KSR1, and KSR1 was immunoprecipitated from cytoplasmic lysates. Western blot analysis of the KSR1 immunoprecipitations was performed to ascertain relative binding levels of other KSR1 scaffold proteins. Increased binding of Hsp90 to KSR1 was again observed with the H1-177 cell line. In addition, a low level of increased Mek1-2 binding but equivalent 14-3-3ß binding were observed with the H1-177 cells. Binding of Erk proteins to KSR1 was at the lower limit of detection but comparable (data not shown). The data demonstrate specificity in altered KSR1 binding protein patterns. To analyze the potential biological implications of the increased association of Mek with KSR1 in the H1-177 cell line, we performed a soft agar colonization assay. Although nm23-H1 transfection had no effect on anchorage-dependent proliferation in vitro or primary tumor size in vivo, nm23-H1 transfectants exhibited decreased colonization in soft agar, thought to represent the ability to grow in a foreign site (26). C-100 and H1-177 cells transiently transfected with HA-KSR1 were exposed to different concentrations of the Mek inhibitor PD90859, and the results from two independent experiments were plotted in Fig. 2B as a percentage of control colonization (vehicle control). Colonization of both cell lines was inhibited by PD90859 with the same sensitivity, indicating that Mek contributions to colonization were equivalent.
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Although membrane fractions were obtained from each cellular lysate, levels of HA-KSR1 protein were at the limit of detection. Transfection of greater HA-KSR1 was avoided, since it has been proposed to alter the stoichiometry of the KSR1 scaffold to its binding proteins (39). Western blots of total cellular lysates from control C-100 and Nm23-H1-overexpressing H1-177 cell lines contained comparable amounts of total Hsp90 (data not shown). Thus, increased binding of Hsp90 to KSR1 in cells expressing high levels of Nm23-H1 did not reflect overall protein trends but an increased affinity.
Nm23-H1 overexpression promotes KSR1 degradation. To date, Hsp90 has been shown to promote either the folding or the proteasome-mediated degradation of client proteins depending on the complex of bound proteins and cochaperones. Geldanamycin and its derivative form, 17-AAG, are benzoquinone ansamycin antibiotics that stabilize Hsp90 in its ADP-bound conformation with a class of cochaperones that promote client protein degradation. Hsp90 binding to KSR1 in fibroblasts has been previously reported; treatment of cells with 17-AAG accelerated KSR1 degradation (65). These data suggested that KSR1 is a client protein of Hsp90. If elevated Nm23-H1 expression resulted in enhanced binding of Hsp90 to KSR1, it was hypothesized that this interaction may exert functional consequences on KSR1 stability. MDA-MB-435 cells were transiently transfected with HA-KSR1, pEGFP as a transfection control, and either a control Flag or Nm23-H1-Flag construct. Twenty-four hours posttransfection, the cells were trypsinized, and equivalent cell numbers were plated in six-well plates. The cells were permitted to attach for 12 h and then were exposed to 100 µM CHX to inhibit protein synthesis. HA-KSR1 protein levels were determined by Western blotting and normalized to GFP levels (Fig. 4). With protein synthesis blocked, only 25 and 2.5% of KSR1 remained after 3 and 5 h of CHX treatment, respectively, in the Nm23-H1-Flag/HA-KSR1-transfected cells. In comparison, KSR1 was more stable in cells expressing low, endogenous levels of Nm23-H1, with 105 and 92.5% of protein remaining at the same time points. Similar trends were observed using stable transfectants (data not shown).
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0.07 µM, compared to 0.25 µM for the C-100 and S-22 cells, an approximately threefold difference. In experiments not shown, H1-177 cells transiently transfected with a control or HA-KSR1 construct exhibited virtually identical sensitivity to 17-AAG in soft agar colonization, indicating that the experimental procedure of transient HA-KSR1 transfection did not alter the biological sensitivity in this assay. The data indicate that Nm23-H1 overexpression conferred increased sensitivity to an Hsp90-specific agent in anchorage-independent growth assays.
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DISCUSSION |
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The metastasis suppressor Nm23-H1 is shown herein to modulate the stability of the KSR1 Erk MAP kinase scaffold protein via its differential binding of Hsp90 complexes. This new pathway may underlie in part the observed inhibition of Erk MAP kinase pathway activity in cells expressing high levels of Nm23-H1 (19), which is hypothesized to contribute to its metastasis-suppressive activity. Erk activity may be required for outgrowth of metastases in a distant site. Transfection of nm23-H1 into MDA-MB-435 breast carcinoma cells in either a transient or stable fashion resulted in two- to threefold greater binding of Hsp90 to KSR1. Other KSR1 scaffold proteins, such as 14-3-3ß, did not exhibit alterations in binding levels, providing evidence of specificity. The mutant Nm23-H1 transfectants S-22 and I-205, which failed to inhibit in vitro motility upon transfection (31), also exhibited lower levels of Hsp90 binding to KSR1. Hsp90 is a chaperone protein, involved in the maturation and/or degradation of numerous client proteins. Limited evidence has been previously reported indicating that KSR1 is an Hsp90 client protein in 293T cells. The geldanamycins (17-AAG), which stabilize Hsp90 in the ADP-bound state favoring protein degradation, accelerated the degradation of KSR1 in fibroblasts (65). Based on these observations, we asked if elevated binding of Hsp90 to the KSR1 scaffold in Nm23-H1-overexpressing cells resulted in its enhanced degradation. In agreement with this hypothesis, enhanced degradation of KSR1 was observed in either cycloheximide- or 17-AAG-treated H1-177 cells. The degradation of KSR1 in high-Nm23-H1-expression breast carcinoma cells correlated with reduced phospho-Erk expression and increased sensitivity to 17-AAG in soft agar colonization assays. These data support the hypothesis that KSR1 scaffold function can be modulated based on Hsp90 binding and, in turn, metastasis suppressor expression. Several caveats are important. (i) These studies are based on two- to eightfold increases in Nm23-H1 expression among the transient and stable transfectants, levels within the range observed in tumor cohorts where quantitated (10, 18, 71). (ii) Complex formation between Nm23-H1, KSR1, and Hsp90 represented a minor proportion of total protein levels. For Nm23-H1, developmental studies have indicated that only 4% of its protein level can exert phenotypic effects (78). Other functions for these abundant proteins exist and may contribute to the observed colonization phenotype. (iii) It is noteworthy that multiple regulatory points may exist for important signaling proteins, such as KSR1. A Ras-responsive E3 ubiquitin ligase protein, Impedes Mitogenic Signal Propagation protein, was recently reported to be degraded in response to Ras activation, permitting Raf-Mek-KSR1 complex formation (35).
Current experiments are investigating the mechanism of increased Hsp90 binding to KSR1 in Nm23-H1-overexpressing breast carcinoma cells. Our data fail to suggest that the Nm23-H1-induced phosphorylation of KSR1 on serines 392 and 434 accounts for the altered Hsp90 binding, based on site-directed mutagenesis experiments (data not shown). Rather, overall Nm23-H1 protein levels appear to be important. The fact that Nm23-H1 and Hsp90 coimmunoprecipitate regardless of the presence of exogenous KSR1 led us to hypothesize that Nm23-H1 could work as a cochaperone itself to confer specificity to the KSR1-Hsp90 complex and promote the degradation of KSR1. This would explain the fact that we did not find any significant differences in the amount of tested cochaperones (Hsp70/Hsc70, Cdc37, Hop, p23, and BAG-1) bound to KSR1 in C-100 and H1-177 cells after treatment with 17-AAG (data not shown).
Our data also suggest that the KSR1 scaffold may have important functions in the nucleus. Little is known of KSR1 nuclear function. Brennan et al. (9) reported that leptomycin B-treated fibroblasts accumulated nuclear KSR1, consistent with a Crm1-dependent nuclear shuttling process. Mutations in KSR1 affecting its interaction with Mek blocked nuclear import of KSR1, and microinjection of Mek or KSR1 promoted cytoplasmic localization of the other protein. These data suggested the hypothesis that KSR1 serves to regulate the subcellular distribution of Mek. Data from Saccharomyces cerevisiae are also consistent with an important role for scaffolds in the nucleus. Ste5 is the MAP kinase scaffold protein required for mating (13) and continuously shuttles from the cytoplasm to the nucleus but translocates to the plasma membrane upon pheromone stimulation. Nuclear shuttling of Ste5 controls the amount of Ste5 available to bind membrane receptors and enhances pathway activation (32). In our experiments, KSR1 immunoprecipitated from nuclear extracts of MDA-MB-435 breast carcinoma cells bound Erk pathway members and was subject to the same changes in Hsp90 binding between low- and high-Nm23-H1-expression cell lines that were observed in the cytoplasm. Thus, nuclear KSR1 is not merely binding Mek but exists as a more complete scaffold with similar regulatory influences, where a function to directly bind membranous Raf-1 appears unlikely. KSR1 complexes were chromatographed in the 250- to 500-kDa range (42), and it is unknown whether this complex can move in toto from the cytoplasm to the nucleus or has to reform in each compartment. We hypothesize that membrane-associated KSR1 may have a distinct protein binding pattern, since the KSR1 coimmunoprecipitation pattern from total versus cytoplasmic or nuclear lysates of breast carcinoma cells differs (data not shown). This hypothesis could not be tested, however, since the transfection of greater KSR1 to enable detection is thought to upset the stoichiometry of binding proteins, thereby altering the function of the Erk pathway.
Finally, the data speak to the potential importance of metastatic competence as a contributor to therapeutic efficacy. The geldanamycins affect degradation of numerous client proteins; the 17-AAG form is in phase I-II clinical trials and the water-soluble 17-DMAG form has entered phase I trial. Several Hsp90 clients have been identified in breast cancer, including Her-2, Raf-1, mutant p53, Akt, and hormone receptors (1, 5). Our data showing that increased binding of Hsp90 to KSR1 leads to its more-rapid degradation identifies KSR1 as another client in breast cancer. The concentrations of 17-AAG used herein are consistent with KSR1 being a clinically tractable target. Using 17-AAG, we demonstrated that the trend of increased Nm23-H1 expression, increased Hsp90 binding to KSR1, and more-rapid KSR1 degradation has functional consequences; high-Nm23-H1-expression breast cells were approximately threefold more sensitive to 17-AAG inhibition of soft agar colonization. These data suggest that 17-AAG may not be optimally active against low-Nm23-H1-expression, highly metastatic tumor cells and that drug combinations may need to be investigated in the metastatic setting. The 17-AAG sensitivity of tumor cells dichotomized by the expression of other metastasis-associated genes will be of interest to further develop this hypothesis. It should be noted that most drugs have never been tested on low- versus high-metastatic-potential tumor cells before clinical trial, and many others may unfortunately exhibit similar trends. 17-AAG has been previously reported to modulate several metastasis-related proteins and to inhibit the motility of tumor cells (43, 75); to our knowledge the present data are the first comparison of function in cells with various degrees of metastatic competency. Current experiments are addressing 17-AAG combinations with standard and new therapeutic agents for in vitro and in vivo efficacy against highly metastatic breast carcinoma lines. Such an approach may ultimately improve our clinical trial efficacy, in contrast to current situations where drugs are developed on primary xenograft data but then asked to work in a metastatic clinical setting (22).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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