A functional genomic approach integrating microarray and proteomic analyses done in our laboratory has identified 14-3-3ζ as a putative oncogene whose activation was common and driven by its genomic amplification in lung adenocarcinomas. 14-3-3ζ is believed to function in cell signaling, cycle control, and apoptotic death. Following our initial finding, here, we analyzed its expression in lung tumor tissues obtained from 205 patients with various histologic and stage non–small cell lung cancers (NSCLC) using immunohistochemistry and then explored the effects of specific suppression of the gene in vitro and in a xenograft model using small interfering RNA. The increased 14-3-3ζ expression was positively correlated with a more advanced pathologic stage and grade of NSCLCs (P = 0.001 and P = 0.006, respectively) and was associated with overall and cancer-specific survival rates of the patients (P = 0.022 and P = 0.018, respectively). Down-regulation of 14-3-3ζ in lung cancer cells led to a dose-dependent increased sensitivity to cisplatin-induced cell death, which was associated with the inhibition of cell proliferation and increased G2-M arrest and apoptosis. The result was further confirmed in the animal model, which showed that the A549 lung cancer cells with reduced 14-3-3ζ grew significantly slower than the wild-type A549 cells after cisplatin treatment (P = 0.008). Our results suggest that 14-3-3ζ is a potential target for developing a prognostic biomarker and therapeutics that can enhance the antitumor activity of cisplatin for NSCLC. [Cancer Res 2007;67(16):7901–6]

Non–small cell lung cancer (NSCLC) is the leading cause of cancer death in the United States. NSCLC consists of adenocarcinoma (ACC), squamous cell carcinoma (SCC), and large cell carcinoma (LC; refs. 1, 2). Identification of precise prognostic marker and effective therapeutic target for NSCLC is pivotal in the management of the deadliest form of cancer. Using a functional genomic approach that simultaneously integrated genomic and transcript microarrays, proteomics, and tissue microarray analyses (TMA), we have found that the genomic amplification and increased transcript level of 14-3-3ζ were associated with its high protein expression in stage I lung adenocarcinomas, suggesting that 14-3-3ζ activation might be involved in the development of lung cancer (3).

14-3-3 proteins are a family of highly conserved cellular proteins and play an important role in a wide variety of cellular processes (4). These include signal transduction, cell cycle regulation, apoptosis, genotoxic stress response, cellular metabolism, cytoskeleton organization and malignant transformation. Recent studies have shown that 14-3-3ζ interacted with other key cellular proteins involved in the tumor development and progression (4). For example, 14-3-3ζ is one of the major transforming growth factor-β–induced proteins, which can promote epithelial-mesenchymal transition of epithelial cells in cancer cell transformation (5). Furthermore, 14-3-3ζ can regulate raf-1 activity by interacting with the Raf-1 cysteine-rich domain to cause subsequent events necessary for the full activation of Raf-1 in tumorigenesis (6). In addition, 14-3-3ζ can interact with the tumor suppressor tuberin to negatively regulate phosphoinositide-3′-kinase signaling downstream of Akt (7). Moreover, 14-3-3ζ could bind to Cdc25C in irradiated A549 cancer cells, and suppression of 14-3-3ζ resulted in a decrease in Cdc25C localization in cytoplasm and Cdc2 phosphorylation, thus sensitizing the cancer cells to ionizing radiation (8). Most recently, up-regulation of 14-3-3ζ was found to associate with features of biologically aggressive oral carcinoma (9). These lines of evidence strongly suggest that dysregulation of 14-3-3ζ may be specifically activated in tumor and contributes to cancer cell growth. Therefore, the prognostic value of altered 14-3-3ζ expression in NSCLCs and its use as a potential target for cancer therapy warrants further research.

Here, we found that increased 14-3-3ζ protein levels were positively associated with stage and grading of NSCLC and inversely related to poor outcomes of the patients. We also found that down-regulation of 14-3-3ζ in NSCLC cells made them more sensitive to cisplatin in vitro that was associated with the inhibition of cell proliferation, additive G2-M cell cycle arrest, and increased apoptosis. The result was confirmed in the animal model, which showed that the A549 lung cancer cells with reduced 14-3-3ζ grew significantly slower than the wild-type A549 cells after cisplatin treatment. Our data suggested that 14-3-3ζ might be a significant prognostic factor and an attractive target in sensitizing cancer cells to cisplatin in treating NSCLC.

Tissue specimens and immunohistochemical analysis. First, to determine correlation between 14-3-3ζ expression and clinicopathologic features in NSCLCs, we used lung TMA (Express Biotech) constructed from lung cancer tissues of 57 NSCLC patients with different stages and histologies (Table 1). Second, to evaluate the associations between 14-3-3ζ expression and the outcomes of NSCLC patients, we used lung TMAs consisting of tumor specimens obtained from 148 stage I NSCLC patients, from whom we had complete medical records and follow-up data (Table 2; ref. 10). Immunohistochemistry (IHC) staining was done on the TMA sections using rabbit polyclonal antibody against 14-3-3ζ (Santa Cruz Biotechnology) and DAKO LSAB+ and 3, 3′-diaminobenzidine as the chromogen as described in our previous reports (3, 10). Each batch of slides contained a positive control and a negative control. The intensity of reactivity was scored using a four-tier system: 0, indicated no staining; 1, weak staining; 2, moderate staining; and 3, strong staining. The cells with grades 1, 2, and 3 staining intensity were considered as positive cells for 14-3-3ζ. The immunoreactive score of the sample was determined by the percentage of positive cells. Measurements were normalized to these controls (negative control = 0, positive control = 100%). The percentage of cell expressing 14-3-3ζ of all the normal control tissues was ≤10%. Therefore, the score of 10% as a cutoff value was chosen for continuing following analyses and to define higher or lower expression of 14-3-3ζ in tumors.

Table 1.

Clinicopathologic characteristics of the NSCLC patients with different stages and histologies by 14-3-3ζ expression

CharacteristicsTotal patientsPatients with positive 14-3-3ζ expressionP
All cases 57 45  
Age (y)   0.964 
    ≤60 21 17  
    >60 36 28  
Gender   0.542 
    Female 13 10  
    Male 44 35  
Histology   0.089 
    ACC 30 23  
    SCC 25 20  
    LC  
Stage (tumor-node-metastasis)   0.001 
    I 17 10  
    II 16 13  
    III 15 13  
    IV  
Grade (differentiation)   0.006 
    Well 15 11  
    Moderate 22 15  
    Poor 20 19  
CharacteristicsTotal patientsPatients with positive 14-3-3ζ expressionP
All cases 57 45  
Age (y)   0.964 
    ≤60 21 17  
    >60 36 28  
Gender   0.542 
    Female 13 10  
    Male 44 35  
Histology   0.089 
    ACC 30 23  
    SCC 25 20  
    LC  
Stage (tumor-node-metastasis)   0.001 
    I 17 10  
    II 16 13  
    III 15 13  
    IV  
Grade (differentiation)   0.006 
    Well 15 11  
    Moderate 22 15  
    Poor 20 19  

NOTE: P values ≤0.05 were considered statistically significant.

Table 2.

Clinicopathologic characteristics of stage I NSCLC patients by 14-3-3ζ expression

CharacteristicsNumber of patients14-3-3ζ expression
P
PositiveNegative
All cases 148 94 54  
Age (y)    0.672 
    ≤60 46 29 17  
    >60 102 65 37  
Gender    0.863 
    Female 59 38 21  
    Male 89 56 33  
Histology    0.077 
    ACC 60 38 22  
    SCC 84 54 30  
    Other  
Grade (differentiation)    0.004 
    Well 12  
    Moderate 45 21 24  
    Poor 91 69 22  
CharacteristicsNumber of patients14-3-3ζ expression
P
PositiveNegative
All cases 148 94 54  
Age (y)    0.672 
    ≤60 46 29 17  
    >60 102 65 37  
Gender    0.863 
    Female 59 38 21  
    Male 89 56 33  
Histology    0.077 
    ACC 60 38 22  
    SCC 84 54 30  
    Other  
Grade (differentiation)    0.004 
    Well 12  
    Moderate 45 21 24  
    Poor 91 69 22  

NOTE: P values ≤0.05 were considered statistically significant. Other histologic types: large cell (three patients) and bronchioalveoli (one patient).

Cell lines and culture conditions. A549, H358, and H460 lung cancer cell lines and RetroPack PT67 cells were purchased from the American Type Culture Collection. The cells were maintained in RPMI 1640 or DMEM (Sigma) supplemented with 5% to 10% FCS and penicillin/streptomycin (Sigma).

Generation of stable short hairpin small-interfering RNA-14-3-3ζ transfectants. To obtain stable expression of the short hairpin small-interfering RNA (shRNA) sequences to specifically reduce the expression of 14-3-3ζ, three oligonucleotide pairs directed against three different regions (nucleotides 728–746, 502–520, and 177–195) of the human 14-3-3ζ mRNA (NM_003406) were selected for use in the pSilencer 5.1-U6 Retro vector system (Ambion). They are referred to as shRNA-14-3-3ζ 1, 2, and 3, respectively. Two types of scrambled shRNAs were designed and used to control for nonspecific silencing events: one is general scrambled shRNA that had no known homology with any mammalian gene, and the other is a mutated shRNA that contains the same nucleotides as the shRNA-14-3-3ζ2, but in an irregular sequence. Plasmids were transfected into the PT67 cells by using LipofectAMINE 2000 (Invitrogen) according to the manufacturer's instructions. Cells were then grown in selective media for 2 weeks. Infectious retroviruses were harvested and filtered through 0.45-μm cellulose acetate filters.

Transfection of NSCLC cells. NSCLC cell lines, A549, H358, and H460 were transfected with 2 μg retro-shRNA-14-3-3ζs using LipofectAMINE (Invitrogen) per instructions of the manufacturer. Meanwhile, the cells were also transfected in parallel with retro-scrambled shRNAs or PBS (mock) to serve as transfection controls. At 48 h following transfection, cells were passaged into the medium containing 2 μg/mL puromycin and allowed to grow until distinct colonies could be distinguished. Ten single colonies of 14-3-3ζ, or scrambled-vector cell transfectants, were isolated and expanded.

Assessment of cell viability by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Exponentially growing cells were plated in microtiter plates and incubated at 37°C in 5% CO2. On the following day, cells were pretreated with different concentrations of cisplatin (0, 2, 4, 6, 8, and 10 μm; Sigma). Doses of cisplatin were chosen based on previous experimental report (11). After 48 h, the number of viable cells was determined using the dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) as described in our previous report (12). Each condition was done in triplicate wells, and each experiment was repeated at least thrice.

Cell cycle analysis and apoptosis assays. Cell cycle progression was investigated using the flow cytometry method, as described in our previous study (12). Cell apoptosis was determined by flow cytometry using an Annexin V apoptosis kit (Roche) according to instructions outlined by the manufacturer. The fluorescence emitted by cells was analyzed using a flow cytometer (Becton Dickinson).

Western blot analysis. Western blotting was done as described previously (3, 12). Antibodies used were obtained as follows: 14-3-3ζ (C-16), 14-3-3 β (C-20), 14-3-3γ (A-12), and 14-3-3ε (T-16), Cdc2, and β-actin from Santa Cruz Biotechnology; and caspase-3, Cdc2(06-923), phospho-cdc2 (Thr161) (9114) from Upstate Biotechnology.

In vivo tumor model. Tumors were induced in athymic Swiss nu/nu/Ncr nude (nu/nu) mice by inoculating 1 million of A549 lung cancer cells, A549 cells transfected with scrambled shRNA and A549 cells transfected with 14-3-3 shRNAs per mouse, respectively. There were 10 mice for each group. After 17 days, five mice of each group were randomly selected and received i.p. injection of cisplatin at a dose of 3 mg/kg for 5 consecutive days. After a 9-day cessation of treatment, a second round of 5-day treatment was administered followed by another 9-day cessation. Tumor volumes were determined by direct measurement with calipers and calculated using the formula: (larger diameter) × (small diameter)2/2.

Statistical analysis. Overall survival time was calculated from the date of first surgery to the date of death from any cause. Cancer-specific survival time was calculated from the date of surgery to death from cancer-related causes. Survival curves were calculated by using the product-limit estimate (Kaplan-Meier method), and curves were examined by using the log-rank test. Univariate and multivariate analyses were done using the Cox's proportional hazards model to determine which independent factors might have a joint significant influence on survival.

14-3-3ζ overexpression is associated with aggressive biological behavior of NSCLC. We first did immunohistochemistry staining on cancer tissues obtained from a cohort of 57 NSCLC patients with different clinical stages and histologic types and grades (Table 1). Positive immunohistochemistry staining for 14-3-3ζ was found in the cytoplasm and confined almost exclusively to the tumor cells (Fig. 1). Overexpression of 14-3-3ζ was found in 45 of 57 NSCLCs (79%), occurred 23 of 30 adenocarcinoma (77%) and 20 of 25 SSC (70%), 2 of 2 LC (100%), suggesting that there was no significant difference of 14-3-3ζ overexpression between different histologic types (P = 0.089). High 14-3-3ζ expression was significantly correlated with histologic grade and clinical stage of those with NSCLC (all P < 0.05). We subsequently analyzed the relationship between 14-3-3ζ expression and outcomes of NSCLC patients in the lung TMAs consisting of stage I NSCLC tissues (Table 2). The probability of overall survival at 5 years after surgery were 0.36 [95% confidence interval (95% CI), 0.54–0.67] for patients whose tumors showed positive 14-3-3ζ expression compared with 0.68 (95% CI, 0.63–0.79) for patients whose tumors showed negative 14-3-3ζ expression. The probability of cancer-specific survival were 0.60 (95% CI, 0.49–0.62) for patients whose tumors showed positive 14-3-3ζ expression compared with 0.95 (95% CI, 0.65–0.84) for patients whose tumors showed negative 14-3-3ζ expression. The data suggest that the overall and cancer-specific survival probability were significantly different between the two groups (P = 0.0007 and P = 0.0009, respectively, log-rank test; Fig. 2). By performing multivariate analysis (age, gender, histologic type, and smoking status) using the Cox model, we found that the overexpression of 14-3-3ζ was the only independent predictor for disease-free and cancer-specific survival among the clinical and histologic parameters tested (P = 0.022 and P = 0.018, respectively, log-rank test). Collectively, the observation that increased 14-3-3ζ expression was significantly correlated with a more advanced pathologic grade and stage and poor clinical outcome of NSCLCs implied that 14-3-3ζ could play an important role in the progression of NSCLCs and detection of the protein expression might be used as prognostic biomarkers for the disease.

Figure 1.

14-3-3ζ expression in NSCLC as assessed by immunohistochemistry. A, immunohistochemical staining of an adenocarcinoma using 14-3-3ζ antibody showed that most cancer cells expressed 14-3-3ζ in the cytoplasm. B, immunohistochemistry staining of an adenocarcinoma without incubation with the primary antibody was used as negative control. Original magnification, ×200.

Figure 1.

14-3-3ζ expression in NSCLC as assessed by immunohistochemistry. A, immunohistochemical staining of an adenocarcinoma using 14-3-3ζ antibody showed that most cancer cells expressed 14-3-3ζ in the cytoplasm. B, immunohistochemistry staining of an adenocarcinoma without incubation with the primary antibody was used as negative control. Original magnification, ×200.

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Figure 2.

Probability of (A) overall survival and (B) cancer-specific survival by levels of 14-3-3ζ expression in stage I NSCLC. The Kaplan-Meier method was used to determine the survival probability, and the log-rank test was used to compare the survival curves between groups.

Figure 2.

Probability of (A) overall survival and (B) cancer-specific survival by levels of 14-3-3ζ expression in stage I NSCLC. The Kaplan-Meier method was used to determine the survival probability, and the log-rank test was used to compare the survival curves between groups.

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Down-regulation of 14-3-3ζ sensitizes lung cancer cells to cisplatin in vitro. NSCLC cells, A549, H460, and H358 cells were infected with high titers of retroviruses encoding the shRNAs against 14-3-3ζ, which were referred to as sh14-3-3ζ1, sh14-3-3ζ2, and sh14-3-3ζ3. sh14-3-3ζ2 was the most effective among the three shRNA constructs because the levels of 14-3-3ζ in the cells transfected with it always dropped by at least 75% (Fig. 3A). Therefore, the stable cell lines transfected with sh14-3-3ζ2 were used in the following experiments. Furthermore, this down-regulation was specific because there is no significant change of 14-3-3ζ protein in the mock cells and the cells infected with the two types of scrambled shRNAs (control cells) and no changes in the expression of the other members of 14-3-3 family in the cells transfected with shRNA–14-3-3ζ2. We then assessed the effects of reduced expression of 14-3-3ζ on cell growth by comparing the 14-3-3ζ knockdown cells with the control cells for the proliferation and apoptosis. We did not observe a significant difference in cell proliferation and apoptosis between the cells transfected with sh14-3-3ζ2 and the controls.

Figure 3.

Down-regulation of 14-3-3ζ in lung cancer cells in vitro. A, Western blot showed expression of 14-3-3ζ in mock, scrambled shRNAs, and three shRNAs for 14-3-3ζ in A549 cells. Actin expression was used as a control. B, effects of 14-3-3ζ suppression on the growth of lung cancer cells treated with various doses of cisplatin for 48 h. C, effects of 14-3-3ζ suppression on the G2-M arrest induced by cisplatin. All three cancer cell lines (A549, H358, and H460) were used in the in vitro experiments, and only A549 is shown in the figure. Each data point represented the mean value from three independent experiments.

Figure 3.

Down-regulation of 14-3-3ζ in lung cancer cells in vitro. A, Western blot showed expression of 14-3-3ζ in mock, scrambled shRNAs, and three shRNAs for 14-3-3ζ in A549 cells. Actin expression was used as a control. B, effects of 14-3-3ζ suppression on the growth of lung cancer cells treated with various doses of cisplatin for 48 h. C, effects of 14-3-3ζ suppression on the G2-M arrest induced by cisplatin. All three cancer cell lines (A549, H358, and H460) were used in the in vitro experiments, and only A549 is shown in the figure. Each data point represented the mean value from three independent experiments.

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Cisplatin has been the standard of care for the treatment of NSCLCs (13). We therefore evaluated whether the specific 14-3-3ζ knockdown could alter the sensitivity of NSCLC cells to cisplatin. The stable cell lines with suppression of 14-3-3ζ (A549/sh-14-3-3ζ2, H358/sh14-3-3ζ2, and H460/sh14-3-3ζ2) and their corresponding control cells were exposed to increasing concentrations of cisplatin for 48 h, and cell survival was determined by MTT assay. As shown in Fig. 3B, cisplatin treatment resulted in a dose-dependent decrease in proliferative capacity in all cancer cells. However, inhibition of tumor cell growth by cisplatin was more significantly enhanced in the cells with specific suppression of 14-3-3ζ than the control cells (all P < 0.05). The data therefore implied that 14-3-3ζ knockdown could enhance tumor cell sensitivity to cisplatin in a dose-dependent manner.

One of the key pathways in cisplatin-mediated tumor killing is by inducing arrest of cell cycle checkpoints, whereas 14-3-3ζ has been suggested to play an important role in the regulation of cell division (13, 14). To determine effect of 14-3-3ζ suppression on cisplatin-induced cell cycle arrest, we evaluated the cell cycle status of the 14-3-3ζ knockdown cancer cells when treated with cisplatin. Cisplatin treatment of all cancer cells led to a dose-dependent increase in the percentage of cells in G2-M phase. However, as shown in Fig. 3C, the cancer cells lacking 14-3-3ζ expression were more sensitive to the G2-M arrest induced by cisplatin, with a dramatic increase in the population of the cells in G2-M, as compared with the cells without 14-3-3ζ knockdown. This observation suggested that the suppression of 14-3-3ζ could enhance G2-M arrest in the cancer cells induced by cisplatin. To better understand the mechanism behind the observation, the status of Cdc2 phosphorylation, a critical biochemical process in the DNA damage G2-M checkpoint, was examined by Western blotting using anti-Cdc2 antibody and anti–phospho-Cdc2 antibody (Thr161). Western blotting showed a statistical increase of the phosphorylated form of Cdc2 rather than Cdc2 in the cells with reduced 14-3-3ζ expression (Fig. 4A), which was concomitant with G2-M arrest determined by flow cytometry, suggesting that the reduction of 14-3-3ζ sensitized the cisplatin-induced G2-M arrest through a mechanism of activation Cdc2 (phosphorylation of Cdc2).

Figure 4.

Down-regulation of 14-3-3ζ in lung cancer cells in vitro and in vivo. A, Western blot showed increased Cdc2 (Thr161) phosphorylation (P-Cdc2) in 14-3-3ζ down-regulation cells following cisplatin treatments. The cells were treated with various doses of cisplatin for 48 h. B, Western blot showed the activation of caspase-3 in the 14-3-3 suppression cells compared with the controls when treated with 6 μmol/L or higher cisplatin. All three cancer cell lines (A549, H358, and H460) were used in the in vitro experiments, and only A549 is shown in (A) and (B). Each data point represents the mean value from three independent experiments. C, effects of 14-3-3ζ knockdown on lung tumor growth in nude mice in combination with cisplatin treatment. Columns, mean of tumor mass from 10 mice; bars, SE.

Figure 4.

Down-regulation of 14-3-3ζ in lung cancer cells in vitro and in vivo. A, Western blot showed increased Cdc2 (Thr161) phosphorylation (P-Cdc2) in 14-3-3ζ down-regulation cells following cisplatin treatments. The cells were treated with various doses of cisplatin for 48 h. B, Western blot showed the activation of caspase-3 in the 14-3-3 suppression cells compared with the controls when treated with 6 μmol/L or higher cisplatin. All three cancer cell lines (A549, H358, and H460) were used in the in vitro experiments, and only A549 is shown in (A) and (B). Each data point represents the mean value from three independent experiments. C, effects of 14-3-3ζ knockdown on lung tumor growth in nude mice in combination with cisplatin treatment. Columns, mean of tumor mass from 10 mice; bars, SE.

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Another major mechanism of cisplatin to kill tumor cells is by inducing apoptotic cell death. 14-3-3ζ has been suggested to be implicated in the activation of the apoptosis pathway (15). To determine whether 14-3-3ζ knockdown can affect the cisplatin-induced apoptosis of cancer cells, we first evaluated the apoptosis of the cells with specific suppression of 14-3-3ζ when treated with cisplatin using the annexin V staining assay. Cisplatin treatment resulted in a dose-dependent increase in apoptosis in both the cells with reduced level of 14-3-3ζ2 and control cells. Interestingly, 14-3-3ζ depletion cells were noted in 40% or more increase of cells that entered apoptosis than that of mock and scrambled shRNA cells at 6 μmol/L or higher cisplatin. Furthermore, activation of caspase-3 was clearly increased in the 14-3-3 suppression cells compared with controls when treated with 6 μmol/L or higher cisplatin (Fig. 4B). Therefore, the finding implies that the inhibition of 14-3-3ζ can sensitize lung cancer cells to cisplatin-induced apoptosis, and the effect might be through a caspase-dependent mechanism.

Knockdown of 14-3-3ζ increases sensitivity of cancer cells to cisplatin in vivo. A549 cancer cells, scrambled shRNA A549 cells, and A549/sh14-3-3ζ2 cells were inoculated in nude mice, respectively. After 17 days, the tumor volume induced by A549 cells, scrambled shRNA A549 cells, and A549/sh14-3-3ζ2 cells were 169.49 ± 20.61, 154.54 ± 20.06, and 151.49 ± 34.78 mm3, respectively (P = 0.091). By 28 days after the initiation of cisplatin treatment, the ratio of tumor growth among A549 and scrambled shRNA A549 tumors was 54% and 50%, respectively, whereas it was 22% for A549/sh14-3-3ζ2 (P = 0.008; Fig. 4C). The observation suggests that despite showing a similar growth pattern as did the tumors induced by controls, the tumors induced by the specific suppression of 14-3-3ζ A549 cells were more sensitive to cisplatin treatment than the tumors induced by the control cells without 14-3-3ζ depletion. These in vivo data are consistent with the results obtained in vitro and support the suggestion that the inhibition of expression of 14-3-3ζ can sensitize the tumor to the action of the cisplatin.

The ability to identify NSCLC patients at high risk of recurrence following surgery is important because such a test would identify patients who might benefit from adjuvant therapies, whereas sparing patients who are at low risk of recurrence from toxic treatment. However, the current prognostic assessments of NSCLC do not allow a precise distinction between the patients who will benefit from therapy and those who will not (16). In the present study, the up-regulation of 14-3-3ζ was only observed in lung cancer tissues but absent in the nonmalignant tissues and is positively correlated with stage and grading of NSCLCs. Furthermore, we found that the 14-3-3ζ expression inversely correlated with survival. More importantly, 14-3-3ζ functions as an independent prognostic factor for predicting outcome in early-stage patients with lung cancer in a multivariate analysis. Our clinical evidence clearly supports the notion that the increased expression of 14-3-3ζ contributes to lung cancer development and progression, and the detection of the 14-3-3ζ aberrations might be a useful biomarker to identify poor prognoses in patients with NSCLC.

Cisplatin is included in most protocols employed to date for the treatment of NSCLC (13). However, the patient's response to this drug is often far from optimum because of the dose-dependent side effects and the development of resistance to the chemotherapeutic agent (17). We considered that sensitizing the cancer cells to cisplatin would be a way of obtaining a better response from the tumor to the drug and also of combating any resistance by the lung cancer cells by reducing the dose. In the present study, although all the NSCLC cell lines tested are sensitive to cisplatin, the sensitivity of the 14-3-3ζ depletion cells to cisplatin significantly increases, as represented by increased cancer cell growth inhibition in vitro. Consistently, tumors induced by the cancer cells lacking 14-3-3ζ expression in xenograft models showed a significantly slower kinetics of growth than the ones induced by the cancer cells without reduced 14-3-3ζ when treated by cisplatin. Interestingly, the enhanced sensitivity of cancer cells to cisplatin is in a dose-dependent manner. Therefore, our study suggests that using RNA interference to inhibit 14-3-3ζ may be beneficial in reducing the required dose of cisplatin for the same effect, thus reducing the extensive problems associated with toxicity. This strategy may also be able to reduce the occurrence of drug resistance. The investigation of whether 14-3-3ζ suppression can also enhance the anticancer effect of cisplatin analogues is ongoing in the laboratory.

It is accepted that the cytotoxicity of cisplatin is due to DNA damage through mediating the induction of cell cycle arrest (13). After DNA damage, the G2-M transition is regulated by maturation-promoting factors, such as Cdc2, which can determine entry into mitosis. Especially, elevated Cdc2 phosphorylation can arrest cells in the G2 phase (18). In the present study, the 14-3-3ζ knockdown enhanced cell accumulation at the G2-M phase after cisplatin treatment; concurrently, Cdc2 (Thr161) phosphorylation was significantly induced in 14-3-3ζ down-regulation cells following cisplatin treatment. In contrast, the phosphorylated form of Cdc2 in control cells was not induced. The observation is consistent with the report of others that 14-3-3ζ plays an important role in G2-M phase checkpoints by interacting with cell cycle regulatory proteins, including Cdc2, especially the phosphorylation of Cdc2 (8). Therefore, the results of our studies indicated that the sensitization of cancer cells by specific suppression of 14-3-3ζ to cisplatin might be through an increased cell cycle arrest at G2-M phase mediated by elevated Cdc2 phosphorylation.

It is also believed that cisplatin kills cancer cells through apoptotic mechanism (15). 14-3-3ζ was proposed to be involved in apoptosis through multiple interactions with proteins of the core mitochondrial machinery, proapoptotic transcription factors, and their upstream signaling pathways (8, 1922). In the current study, the cancer cells, with the inactivation of 14-3-3ζ, had increased apoptosis accompanied with higher amount of the cleaved caspase-3 than did control cells after exposure to cisplatin. Therefore, the results suggest that the chemosensitization effect of the down-regulation of 14-3-3ζ involved the activation of the apoptosis pathway alterations of cell proliferation in a highly caspase-3–dependent manner. Taken together, our result might reveal that the suppression of 14-3-3ζ mediates sensitivity to chemotherapy by a mechanism of introducing the arrest of G2-M phase checkpoint and apoptosis. Further investigations of the relationship between 14-3-3ζ and other factors (e.g., p53) and the biological significance in the DNA damage cell cycle response and apoptosis are ongoing in the laboratory.

In summary, 14-3-3ζ may not only be a useful molecular marker for selecting patients with poor prognosis to receive more aggressive preoperative or adjuvant therapy in the setting of a clinical trial, but may also be an effective therapeutic target for NSCLC. Nevertheless, a longitudinal study in a large population to validate its prognostic value and further molecular biological analysis of the gene to develop a novel strategy for improving treatment efficiencies of certain anticancer drugs will be needed.

Grant support: National Cancer Institute grant CA-113707 (F. Jiang).

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.

We thank Mildred Michalisko of the Department of Pathology for the editorial review of this manuscript.

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