Tissue microarray containing a total of 132 NSCLC cases was obtained from Outdo Biotech Co. (Shanghai, China). The age of these patients ranged from 20 to 84 (mean 60.5) and a 5-year follow-up was completed. The study was approved by the Ethics Committee of Heilongjiang Province Hospital.

NSCLC cell lines, including A549, H1299 and NCI-H460 were obtained form Cell Bank (SIBS, China). All cells were maintained in Dulbecco's modified Eagle's medium (DEME), supplemented with 10% fetal bovine serum and antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin). PDK1 overexpression plasmid pcDNA3.1-PDK1 and empty plasmid were transiently transfected into cells using Lipofectamine 2000 (Invitrogen, USA). Lentivirus containing PDK1 shRNA (RiboBio, China) were used to infect cells to establish the stable knock-down cell lines.

The tissue microarray slides were deparaffinized, rehydrated in xylene and graded ethanol. Then antigens were retrieved at 95°C for 30 min. After cooling to room temperature, the slides were incubated with 3% hydrogen peroxide to block the endogenous peroxidase activity. Anti-PDK1 antibody was added to the slides overnight. After washing, the slides were incubated with HRP-labeled secondary antibody and stained using diaminobenzidine solution and counterstained with hematoxylin.

The IHC results were measured using a semi-quantitative score for both intensity and percentage of positive staining. Briefly, the intensity of staining was scored as: 1 (no stain), 2 (weak stain), 3 (medium stain), 4 (strong stain) and the percentage was scored as: 1 (<25%), 2 (25–50%), 3 (50–75%), 4 (>75%). Then, the final score was calculated by multiplying the two scores and the overall score of 8 was the cut-off score which classified them into low expression and high expression groups.

Cell lysates were prepared using RIPA buffer with protease inhibitor and phosphatase inhibitor cocktail. Total protein (70 µg) was loaded to 10% SDS-PAGE and transferred onto PVDF membranes. After blocking with 5% BSA, the membranes were blotted with primary antibody at 4°C overnight. Membranes were then incubated with peroxidase-conjugated secondary antibody at 37°C. The blotting was visualized by enhanced chemiluminescence detection system. The antibodies used in this study were as followed: anti-PDK1 (10026-1-AP) from ProteinTech Group, Inc. (USA), anti-p-PDH (ab177461), anti-β-actin (ab8226) from Abcam (Cambridge, MA, USA) and anti-PDH (#3205) from Cell Signaling Technology (MA, USA).

Total RNA was extracted by TRIzol reagent (Invitrogen) according to the manufacturer's insturctions. The first-strand cDNA was synthesized by PrimeScript RT Reagent kit (Takara, Japan). Quantitative real-time PCR was performed by SYBR Green method in ABI7500 system. The primers used in the study were as follows: PDK1, forward, 5′-CTCAGGACACCATCCGTTCA-3′; reverse, 5′-ACCATGTTCTTCTAGGCCTTTCAT-3′. β-actin, forward, 5′-TGATGATATCGCCGCGCTC-3′; reverse, 5′-CCATCACGCCCTGGTGC-3′. β-actin was used as internal control and the level of PDK1 was normalized using 2^−∆∆ct method.

For analyzing the viability of PDK1 overexpressing or knockdown cells, 2×10³ cells/well were seeded in 96-well plates. Cell Counting Kit-8 (CCK-8, Dojindo, Japan) was used to measure the cell viability at 24, 48, 72 and 96 h according to the manufacturer's insturctions. The absorbance was tested at 450 nm. The experiments were repeated at least three times.

Indicated cells were seeded into the 6-well plate at 500 cells per well after 48 hours of transfected with vector, PDK1 plasmid or infected with PDK1 shRNA. The medium was changed every four days. After 2 weeks of incubation, colonies were fixed with 4% paraformaldehyde and stained with crystal violet. The colony number was measured under a microscope. All of the experiments were performed at least three times.

The apoptosis of cells with PDK1 ectopic expression or knockdown was analyzed using Annexin V/PI apoptosis detection kit (BD Bioscience, San Diego, CA, USA). Cells were trypsinized, collected in PBS, and resuspended in binding buffer. After incubation with Annexin V/PI for 30 min the proportion of Annexin V-positive cells were analyzed by FACSCalibur flow cytometer. All of the experiments were performed at least three times.

To detect cell migration ability, indicated cells (10⁴ in serum-free medium) were seeded into the top chamber of Transwell. The bottom chamber contained complete medium with 20% serum. After 48 h of incubation at 37°C, cells on the lower membrane were fixed and stained with 0.1% crystal violet and imaged.

For lactate production measurement, cells were seeded into 96-well plates with phenol red-free medium and determined using Lactate assay kit (BioVision, CA, USA). For glucose uptake assay, the ³H-2-deoxyglucose was detected. Cells were cultured in 6-well plates and ³H-2-deoxyglucose was added to the cells and then washed with PBS and lysed in SDS. The radioactivity of cell lysates were detected. For oxygen consumption assay, the consumption rate was measured with MitoCell system (Strathkelvin Instruments, UK). ATP levels were measured using CellTiter-Glo Luminescent Viability Assay kit (Promega).

Statistical analysis was performed with SPSS 21.0 software (IBM, Chicago, IL, USA). The correlation between PDK1 expression and clinicopathological parameters was measured by Chi-square test. Survival rate was measured by Kaplan-Meier method and the significance was calculated with log-rank test. Univariate and multivariate analysis were performed by Cox regression model. Student's t-test was used to compare the groups in the cell experiments. Data are presented as the mean ± SEM of three independent experiments. A P-value of <0.05 was considered statistically significant.

To explore the relationship between the important enzymes involved in glycolysis and NSCLC progression, we analyzed the previous studies that identified numerous deregulated genes in NSCLC and focused on the glycolytic enzymes. We noted that PDK1 was upregulated in several studies which presented expression profiling by array. According to the GSE19188, containing a cohort of 91 tumor- and 65 adjacent normal lung tissue samples, PDK1 was significantly higher in tumor tissues compared to normal tissues (Fig. 1A). More importantly, the PDK1 expression in individual subtype of lung cancer, including adenocarcinoma (ADC), squamous cell carcinoma (SCC) and large cell carcinoma, was also enhanced (Fig. 1B). Furthermore, we employed another dataset to validate the hypothesis. As shown in Fig. 1C, the PDK1 expression in GSE10072 containing 180 cases of paired tumor samples was in conformity with our observation.

Then we examined the protein level of PDK1 in 132 pairs of NSCLC samples by immunohistochemical staining. The results revealed that PDK1 levels were significantly higher in tumor tissues than that in non-tumor tissues (Fig. 1D). We also analyzed the relationship between PDK1 and clinicopathological features. The results indicated that PDK1 expression was correlated with T stage (P=0.019) (Table I). These data suggested that PDK1 expression increased with the NSCLC development.

We explored the prognostic significance of PDK1 expression in NSCLC patients. All 132 NSCLC patients were divided into two groups: high expression (high PDK1, n=93) and low expression (low PDK1, n=38). Using Kaplan-Meier methods, we determined that patients with PDK1 high staining revealed significantly shorter survival rate compared with those with PDK1 low staining (Fig. 2A, P<0.0001, log-rank test). We also performed univariate analysis to evaluate the associations between prognosis and clinicopathological features including age, gender, T stages, N stages, histological differentiation, tumor size and PDK1 expression. Among these features, advanced T stages (P=0.003), advanced N stages (P<0.0001) and PDK1-positivity (P=0.001) were significantly correlated with poor prognosis. Further multivariate regression analysis revealed that advanced N stages (P=0.001) and PDK1-positivity (P=0.001) were independent prognostic factors (Table II).

We then explored the correlations between PDK1 expression and prognosis using various subset analyzes. The results revealed that PDK1 was associated with shorter survival regardless of T stages or N stages (Fig. 2B-E). Moreover, we analyzed the PDK1 expression in GSE30219 and demonstrated that PDK1 could be a prognostic factor of NSCLC.

For subsequent experiments, we performed ectopic expression of PDK1 in A549 and H1299 cell lines and knockdown of PDK1 in H460 cells. The colony formation assay was carried out to evaluate the functions of PDK1 on proliferation of NSCLC cell lines. As shown in Fig. 3A and B, the statistical results indicated that overexpression of PDK1 could significantly promote cell growth while knockdown inhibited it. Moreover, the CCK-8 assay was used to further determine the growth effect of PDK1. The results of CCK-8 assay is shown in Fig. 3C and D, and were similar with colony formation assays. Also we performed FACS to explore whether the growth suppression was caused by apoptosis. Cell apoptosis was decreased after PDK1 overexpression (Fig. 3E). Conversely, knocking down of PDK1 markedly increased the percentage of Annexin V-positive cells (Fig. 3F). In addition, we employed Transwell assay to examine the effect of PDK1 on cell mobility (Fig. 3G and H). The number of migratory cells with overexpression of PDK1 was increased significantly compared to control cells. Collectively, we suggested that PDK1 played oncogenic roles in NSCLC cells.

As PDK1 is a key enzyme in modulating tumor cell glycolysis, we explored the function of PDK1 in regulating the Warburg effect. Western blot analysis was performed to measure the PDK1 overexpression or knockdown effects (Fig. 4A and B). Besides, p-PDH expression was increased by PDK1 phosphorylation. Measurement of metabolic parameters revealed that overexpression of PDK1 significantly increased cellular glucose uptake and lactate production (Fig. 4C and E), while knockdown of PDK1 decreased both glucose uptake and lactate production (Fig. 4D and F). Moreover, forced expression of PDK1 led to increased ATP levels and reduced O₂ consumption (Fig. 4G and I). Contrary results were observed when we knocked down PDK1 (Fig. 4H and J). Collectively, we suggested that PDK1 could promote Warburg effect to facilitate cancer cell proliferation.