Correlation between PDZK1, Cdc37, Akt and Breast Cancer Malignancy: The Role of PDZK1 in Cell Growth through Akt Stabilization by Increasing and Interacting with Cdc37
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PDZ domain containing 1 (PDZK1) is a scaffold protein that plays a role in the fate of several proteins. Estrogen can induce PDZK1 gene expression; however, our recent report showed that PDZK1 expression in the breast cancer cell line MCF-7 is indirect and involves insulin-like growth factor (IGF)-1 receptor function. Such a relationship was established in cell culture systems and human breast cancer tissues. Here we show that overexpression of PDZK1 promoted an increase in cyclin D1 and enhanced anchorage-independent growth of MCF-7 cells in the absence of 17β-estradiol, suggesting that PDZK1 harbors oncogenic activity. Indeed, PDKZ1 overexpression enhanced epidermal growth factor receptor (EGFR)-stimulated MEK/ERK1/2 signaling and IGF-induced Akt phosphorylation. PDZK1 appeared to play this role, in part, by stabilizing the integrity of the growth promoting factors Akt, human epidermal growth factor receptor 2 (Her2/Neu) and EGFR. Increased Akt levels occurred via a decrease in the ubiquitination of the kinase. PDZK1 overexpression was associated with resistance to paclitaxel/5-fluorouracil/etoposide only at low concentrations. Although the increased stability of Akt was sensitive to heat shock protein 90 (HSP90) inhibition, increased levels of the cochaperone cell division cycle 37 (Cdc37), as well as its ability to bind PDZK1, appear to play a larger role in kinase stability. Using human tissue microarrays, we show strong positive correlation between PDZK1, Akt and Cdc37 protein levels, and all correlated with human breast malignancy. There were no positive correlations between PDZK1 and Cdc37 at the mRNA levels, confirming our in vitro studies. These results demonstrate a relationship between PDZK1, Akt and Cdc37, and potentially Her2/Neu and EGFR, in breast cancer, representing a new axis that can be targeted therapeutically to reduce the burden of human breast cancer.
Breast cancers that develop genetic and/or epigenetic changes increase the incidence of disease recurrence and distant metastases, as well as the development of resistance to standard therapies that ultimately decrease the overall survival of affected patients (1, 2, 3). Changes leading to increased expression of Akt, human epidermal growth factor receptor 2 (HER2/Neu) or epidermal growth factor receptor (EGFR), for example, are associated with highly metastatic and therapy-resistant breast cancers (4,5). The posttranslational fate of these factors is often governed by their association with chaperones such as heat shock protein 90 (Hsp90) (6,7). Inhibition of Hsp90, for instance, was shown to compromise the integrity of several proon-congenic factors, thus making it a rather appealing therapeutic stagey against cancer (6,8). The efficiency of Hsp90 in promoting the stability of client proteins is mediated by cochaperones such as cell division cycle 37 (Cdc37) (9). Cdc37 is primarily responsible for securing the integrity of protein kinases such as Akt and EGFR (7,10). Akt, a serine/threonine kinase, is critical for the mediation of cell signaling initiated by growth factors, cytokines and other cellular stimuli (11). Perturbations in Akt activity are associated with numerous pathologies including breast cancer (11). Akt promotes a reduction of cell cycle inhibitors such as the cyclin-dependent kinase inhibitor 1 (p21/WAF1) and p27Kip1 and an enhancement of cell cycle proteins such as c-Myc and cyclin D1 (12).
PDZ domain containing 1 (PDZK1) is a 70-kDa adapter protein expressed primarily in the apical brush-border membrane of the proximal tubules of the kidney (13) and has an important role in lipid metabolism. PDZK1 helps maintain the integrity of SR-B1 in a posttranscriptional manner (13,14). It is noteworthy that in normal conditions, PDZK1 is marginally expressed in other organs, including the liver and intestine, but is not detected in the breast (13). The PDZK1 gene was initially reported to be estrogen-responsive in the breast cancer cell line MCF-7 (15). Additionally, a significant association between plasma 17β-estradiol (E2) levels and PDZK1 mRNA expression levels in estrogen receptor (ER)-a(+) human breast cancer was established (16), strengthening the relationship between ER-α and PDZK1. In a recent investigation (17), we conducted a series of studies in which we found that E2-induced PDZK1 mRNA expression was slow and was blocked upon inhibition of protein synthesis. These data suggest indirect involvement of ER-α and a requirement for an intermediate ER-regulated gene product. Gene expression array analysis identified insulin-like growth factor 1 receptor (IGF-1R) as a potential candidate. Interestingly, PDZK1 knockdown completely blocked E2-dependent growth of MCF-7 cells and reduced c-Myc expression, suggesting its role during cell proliferation. PDZK1 overexpression stimulated MCF-7 cell proliferation and enhanced E2 growth promotion, potentially through an increase in c-Myc expression. This function may be related to the physical interaction between the PDZK1 and the Src/ER/EGFR complex and enhanced EGFR-mediated signal transduction. This enhancement of the extracellular-signal-regulated kinase 1 and 2 (ERK1/2) pathway may, in part, explain the involvement of PDZK1 in promoting E2-mediated growth of MCF-7 cells. However, these results do not explain the role of PDZK1 in enhancing cell growth in the absence of E2.
The present study was designed to specifically address the mechanism(s) by which PDZK1 enhances growth of breast cancer cells in the absence of E2 by focusing on key factors involved in cell growth, particularly Akt.
Materials and Methods
Dulbecco’s modified Eagle medium (DMEM), penicillin, streptomycin and fetal bovine serum (FBS) were from Invitrogen/Life Technolgies (now Thermo Fisher Scientific, Minneapolis, MN, USA). Charcoal/dextran-treated FBS was purchased from Hyclone (Logan, UT, USA); E2, 5-fluorouracil, paclitaxel and etoposide were from Sigma-Aldrich (St. Louis, MO, USA); recombinant human epidermal growth factor (EGF) was from R&D Systems (Minneapolis, MN, USA), MG132 was from Calbiochem (San Diego, CA, USA); AG1478 was from Cell Signaling Technology (Danvers, MA, USA); and geldanamycin-derivative 17-AAG was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Unless otherwise indicated, all other drugs were purchased from Sigma-Aldrich.
Cell Culture, Cell Proliferation, Cell Survival, Transfection, Immunoblot Analysis and Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)
MCF-7 cells (ATCC, Manassas, VA, USA) were cultured according to ATCC specifications. Before treatment with E2 or EGF, medium was changed to DMEM supplemented with 5% charcoal/dextran-treated FBS. Cell proliferation was measured by MTT assay kit (Roche, Indianapolis, IN, USA). MCF-7 cells were transiently transfected with specific siRNAs targeting Cdc37 (sc-29255 from Santa Cruz Biotechnology or O3 from Thermo Scientific, Pittsburgh, PA, USA) or the negative control (scrambled) siRNA NEG3 (SA Bioscience, Frederick, MD, USA) by using Lipofectamine 2000 (Invitrogen/Life Technologies) according to the manufacturer’s instructions. Cells were also stably transfected with an expression vector encoding human PDZK1 or empty vector (pcDNA3.1), and positive clones were isolated after selection. Clones were combined to avoid clonal differences except where indicated. Cells were then treated as described in the figure legends before being subjected to total RNA or protein preparation. Isolated RNA was reverse-transcribed and the resulting cDNA was subjected to conventional PCR with primer sets (IDT, San Jose, CA, USA) specific to human PDZK1, Cdc37, Akt1 or β-actin. Protein extracts were subjected to immunoblot analysis with antibodies to PDZK1(EPR3751) (Novus Biological), phospho-ERK1/2, ERK1/2 (total), HSP90(C45G5), EGFR, Akt (pan)(C67E7), phospho(Ser473)-Akt (all purchased from Cell Signaling Technology), mono- and polyubiquitinylated conjugates (FK2) (Enzo Life Sciences, Farmingdale, NY, USA), poly (ADP-ribose) polymerase 1 (PARP-1; BD Biosciences, San Jose, CA, USA), the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) (Abcam, Cambridge, MA, USA), Cdc37, Her2/Neu, cyclin D1 or GADPH (all from Santa Cruz Biotechnology). Immune complexes were detected with appropriate secondary antibodies and chemiluminescence reagents (Pierce, Rockford, IL, USA). For cell viability/death, cells were seeded in 96-well plates at a density of 5,000 cells/well and then treated with different concentrations of 5-fluorouracil, paclitaxel or etoposide for 48 h. Cell death was determined by using the MTT assay.
Clonogenic Soft Agar Assay
Anchorage-independent cell growth was measured in six-well plates. A 1-mL layer of 0.5% agar (Gibco/Life Technologies [now Thermo Fisher Scientific]) in tissue culture medium was solidified in the bottom of each well. Cells to be assayed were suspended at 37°C in 1 mL of 0.35% agar in tissue culture medium, and then 2.5 × 103 pcDNA3.1 MCF-7 or PDZK1 over-expressing MCF-7 cells were added per well. Both cells were then added in 2 mL medium to the top of the agar, and the medium was changed every 3 d. After 10 d, all of the colonies were counted under a dissection microscope.
The lysates were initially pre-cleared by incubation with protein A/G plusagarose beads (Santa Cruz Biotechnology) before an overnight incubation at 4°C with the indicated specific antibodies or normal IgG. Protein A/G plus-agarose beads were then added to the lysates, and the mixtures were incubated on a rotating wheel at 4°C for an additional 4 h. The beads were pelleted and washed several times in wash buffer (0.1% NP-40, 50 mmol/L Tris, pH 8.0, 150 mmol/L NaCl, 2 µg/mL leupeptin, 2 µg/mL aprotinin, 1 mmol/L phenylmethylsul-fonyl fluoride, 5 mmol/L NaF and 100 µmol/L Na3VO4). Antibody-antigen complexes were eluted in the sample buffer by boiling and resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (18).
Breast cancer and normal tissue microarray (TMA) sections (US Biomax, Rockville, MD, USA), which included a total of 262 neoplastic tissue samples and 87 normal breast adjacent tissue cores (17), were subjected to immunohisto-chemistry with antibodies to PDZK1 (ProteinTech Group, Chicago, IL, USA), Akt (Cell Signaling Technology) or Cdc37 (Santa Cruz Biotechnology) essentially as described previously (8). TMA slides were processed essentially as recently described (17). Stained slides were scanned using the ×40 objective, and immunoreactivity was analyzed by using Image-Pro Plus software (version 6.0) (Media Cybernetics Inc., Rockville, MD, USA) as described previously (9,10). The measurement parameters included density mean, area sum and integrated optical density. The software system allows a computerized assessment of the density of the staining as a sum of the values for intensity of all the pixels of a counted region in an analyzed area as well as the total area in an unbiased manner. Threshold range of the colors of positive staining was selected in a way that both faint and strong signals were detected without a high background. Density of immunoreactivity was next determined for all areas with a positive signal according to the weighted histoscore method (19): Histoscore = ∑ (% negative staining × 0) + (% weak staining × 1) + (% moderate staining × 2) + (% strong staining × 3).
Data are presented as means ± standard error of the mean from at least three separate experiments. Comparisons between multiple groups were performed with either a Student t test or one-way analysis of variance (ANOVA) with a Bonferroni test. Differences were considered statistically significant at p < 0.05. All statistical summaries and analyses were performed by using GraphPad software, version 5 (GraphPad, La Jolla, CA, USA).
All supplementary materials are available online at https://doi.org/www.molmed.org .
PDZK1 Increases Akt Levels Posttranslationally by Decreasing Its Targeting to the Ubiquitin System
PDZK1 Increases Akt Levels by Increasing and Interacting with the HSP90 Cochaperone Cdc37
It is noteworthy that HSP90 functions as part of a multimeric chaperone complex that is aided by several cofactors (26). A cofactor that prominently secures the interaction of the HSP90 complex with kinases is the cell division cycle 37 homolog (Cdc37) (9). Akt is known to interact with Cdc37 (24), and Cdc37 knock-down reduces Akt levels (10). Consistent with these reports and as shown in Figure 4B, partial knockdown of Cdc37 using two different siRNAs in PDZK1-overexpressing MCF-7 cells reduced the levels of Akt, suggesting a potential connection between PDZK1 and Cdc37. Thus, we speculated that the PDZK1-associated increase in Akt levels might be associated with alterations in Cdc37 levels. We found PDZK1 overexpression was associated with a marked increase in Cdc37 protein levels (Figure 4C) without a concomitant increase in mRNA levels (Figure 4D). To determine whether PDZK1 interacts with Cdc37, we performed pull-down assays using antibodies to PDZK1. Figure 4E shows that PDZK1 interacted with Cdc37 in extracts from PDZK1-overexpressing MCF-7 cells. The interaction was confirmed by using pull-down assays with Cdc37 antibodies, followed by immunoblot analyses with antibodies to PDZK1 (Figure 4F). Interestingly, much greater levels of Akt were coimmunoprecipitated with PDZK1 and Cdc37 in extracts from PDZK1-overexpressing MCF-7 cells compared with extracts from MCF-7 cells expressing control vector. The interaction between the three proteins was also confirmed by coimmunoprecipitation by using antibodies to PDZK1 followed by immunoblot analyses with antibodies to the respective proteins. Together, these findings suggest an important interaction between PDZK1, Cdc37 and Akt that results in increased Akt stability and enhancement of associated signal transduction pathways.
Given the relationship between PDZK1 and Cdc37, it was conceivable to predict a correlation between these two factors and Akt in breast cancer tissues. In serial sections of the tissue microarray described above, Akt protein levels were found to be significantly higher in breast carcinomas compared with that in normal tissues, as assessed by using immunohistochemistry (Figure 5G). Analyses of PDZK1, Akt and Cdc37 respective immunoreactivity revealed a significant positive correlation between PZDK1 and Akt (r = 0.540, p < 0.001) (Figure 5H) and an even stronger correlation between Akt and Cdc37 (r = 0.744, p < 0.001) (Figure 5I). These results clearly support the relationship between the three different proteins suggested by our in vitro studies.
Recently, we found that although PDZK1 expression is not a direct product of ER stimulation, it may influence ER-α function through an interaction with the ER-α/EGFR/Src complex, thereby resulting in elevated levels of c-Myc (17). This observation is supported by the finding that PDZK1 knockdown reduces E2-induced c-Myc expression and EGFR-mediated ERK1/2 activation. The results of the current study show that PDZK1 expression was sufficient to enhance EGF-stimulated ERK1/2 phosphorylation, suggesting that this enhancement was not associated with any of the multiple factors that may be induced by E2 treatment in MCF-7 cells. This enhanced ERK1/2 activation may explain the association between PDZK1 expression and increased c-Myc and cyclin D1 in MCF-7 cells. An important feature of PDZK1 that may contribute to its role in breast cancer cell growth and resistance to chemotherapeutic drugs is its ability to stabilize Akt. Our results suggest that Akt stability may be related to the PDZK1-associated increase in the cochaperone Cdc37. This relationship is clearly displayed by the strong positive correlation between the three different factors. It appears that PDZK1 reduces Akt ubiquitination and thus its degradation by the proteasome system (Figure 6). Cdc37 is critical for the stability of numerous growth-promoting kinases, including Akt, and a reduction in the cochaperone drastically reduces the levels of client proteins (10).
The mechanism by which PDZK1 overexpression increases Cdc37 levels is not clear, but it appears that it occurs by a posttranslational mechanism because Cdc37 mRNA levels were not influenced by PDZK1. Given the ability of PDZK1 to maintain the integrity of the HDL receptor SR-B1 (30) in addition to its ability to interact with the cochaperone Cdc37, it is plausible that PDZK1 increases Cdc37 levels through persistent interaction. Our results do not unequivocally demonstrate a direct interaction between PDZK1 and Cdc37, and it is possible that PDZK1 interacts with any member of the HSP90 complex, including HSP90 itself, Akt or any other protein that might interact with the complex. This result is likely considering the report by Hu et al. (31), where a yeast two-hybrid screen of a random peptide library identified a number of putative proteins that may interact with PDZK1; however, Akt, Her2/Neu and EGFR were not among the candidates. Interestingly, other factors known to be involved in cell proliferation and signaling in breast cancer were identified in their screen, such as cyclin-dependent kinase 4 (CDK4) and insulin-like growth factor-binding protein 5 (IGFBP5) (32,33). PDZK1 function appears to be tissue-specific because gene deletion has been shown to completely inhibit SR-B1 expression exclusively in the liver, which explains the proatherogenic phenotype observed in PDZK1 knockout mice (14,34). It is, however, unclear whether the expression and function of PDZK1 observed in breast cancer could be applicable to other cancers. Therefore, more tissue-specific experimentation is required to clarify the exact function and specificity of PDZK1 in the context of cancer.
It is rather interesting that PDZK1 overexpression and the associated increase in Akt, Her2/Neu and EGFR conferred resistance of MCF-7 cells to only low concentrations of 5-fluorouracil, paclitaxel or etoposide. It is well established that overexpression of the latter factors provide substantial resistance to chemotherapeutic drugs in vitro, in animal models and in human disease. For instance, is the interaction of PDZK1 with Akt a determinant for this phenotype? This is a likely scenario. However, a study by Inoue et al. (35) examined a number of cell lines derived from multiple myelomas with high-level gene amplification at 1q12-q22 corresponding, in part, to PDZK1 with a subsequent overexpression at the protein level. The PDZK1-overexpressing cell lines exhibited resistance to melphalan-, cisplatin- and vincristin-induced death compared with cell lines with no PDZK1. Although the conclusions of the latter study were based on a correlation rather than a direct examination of PDZK1 function, detailed examinations of PDZK1 function in drug resistance are warranted.
Together, our findings provide evidence for a role of PDZK1 in driving growth of breast cancer cells by influencing the fate of critical factors in cell proliferation (Figure 6). This function may be mediated via increased Cdc37. More importantly, we report for the first time that Cdc37 protein is elevated in human breast cancer tissues and such elevation may be associated with increased PDZK1. Much work remains to decipher the exact mechanism by which PDZK1 influences Cdc37 expression in breast cancer cells. Overall, the results of the current study, along with data from our previous recent report (17), support the notion that PDZK1 and Cdc37 may constitute a new axis that can be targeted therapeutically to reduce or eliminate the burden of human breast cancer.
The authors declare that they have no competing interests as defined by Molecular Medicine, or other interests that might be perceived to influence the results and discussion reported in this paper.
This work was supported in part by grant RSG-116608 from the American Cancer Society and grant HL072889 from the National Institutes of Health, as well as funds from the Louisiana Cancer Research Consortium (New Orleans, LA, USA) to AH Boulares.
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