Background
Lung carcinoma is the leading cause of cancer-related deaths worldwide [
1]. Although great progress since the introduction of third-generation anti-neoplastic agents and individualized targeted therapy, such as epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors, the overall 5-year survival rate is still modest [
2]. Therefore, an in-depth understanding of potential biomarkers and the therapeutic targets of lung carcinoma will facilitate to the development of novel therapeutic strategy.
Lipid phosphate phosphatases (LPPs), also known as phospholipid phosphatase (PLPP), belong to a superfamily of integral membrane glycoproteins and have six putative transmembrane domains and three highly conserved domains. The conserved domains are paralleled with the transmembrane domains where they form the active sites of the phosphatases [
3,
4]. LPPs exert multifarious functions via catalyzing the dephosphorylation of a number of bioactive lipid mediators including phosphatidic acid (PA), lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P), to attenuate cell activation. Moreover, LPPs can also generate alternative signals with additional biological activities from phosphatidate, sphingosine and ceramide [
3]. Among their versatile biological roles, the fact that LPPs can convert the phosphatidate generated from phosphatidylcholine within the cell by phospholipase D to diacylglycerol is worth noting. This increase in diacylglycerol concentration has been referred to as the second phase of diacylglycerol signaling in which LPPs are believed to play an important role, which further results in the release and influx of intracellular Ca
2+ [
5].Furthermore, accumulating evidence has shown that aberrant expression of LPPs has been implicated in the development and progression of cancer. For example, through analyzing a comparative genomic hybridization array and expression profiling data for a series of 152 ductal breast cancer tissues and 21 breast cancer cell lines, Bernard-Pierrot and colleagues reported that PLPP5 was overexpressed, which contributed to cell survival and cell transformation. Importantly, the oncogenic properties of PLPP5 were further demonstrated by its ability to transform NIH-3 T3 fibroblasts, further inducing their anchorage-independent growth [
6]; in oral squamous cell carcinoma (OSCC) patients, PLPP3 was downregulated in approximately 70% OSCC patients and PLPP3 expression negatively correlated with TNM stage and tumor volume [
7]. Moreover, Mahmood et al. reported that overexpression of PLPP5 caused by amplification of the 8p11-12 chromosomal region, a common genetic event in many epithelial cancers, was found in several cancers, including breast cancer, pancreatic adenocarcinomas and lung carcinoma [
8]. Thus, these studies indicated that different LPPs exert oncogenic or tumor-suppressive functions depending on the tumor types.
In this study, by analyzing the expression levels of LPP family proteins in lung carcinoma RNA expression profile datasets from The Cancer Genome Atlas (TCGA), we found that PLPP4 is dramatically elevated compared with other LPPs in lung carcinoma tissues. Our results further verify that overexpression of PLPP4 is observed in lung carcinoma tissues and cells and positively correlates with advanced clinicopathological features and poor prognosis in lung carcinoma patients. Silencing PLPP4 inhibits the proliferation and tumorigenicity of lung carcinoma cells both in vitro and in vivo. In addition, our findings reveal that downregulating PLPP4 inhibits Ca2+-permeable cationic channel in lung carcinoma cells. Taken together, our findings indicate that PLPP4 plays an important role in the progression of lung carcinoma and suggest that PLPP4 may serve as a potential target for human lung carcinoma treatment.
Methods
Cell culture
The human lung carcinoma cell lines A549, Calu-3, NCI-H226, NCI-H292, NCI-H358, NCI-H1650 and NCI-H1975, and a normal lung epithelial cell line WI-38 were obtained from the Shanghai Chinese Academy of Sciences Cell Bank (China). All cells were cultured in RPMI-1640 medium (Life Technologies, Carlsbad, CA, US) supplemented with penicillin G (100 U/ml), streptomycin (100 mg/ml) and 10% fetal bovine serum (FBS, Life Technologies) and were grown under a humidified atmosphere of 5% CO2 at 37 °C7.
Patients and tumor tissues
Five paired lung adenocarcinoma tissues, 2 paired lung squamous cell carcinoma tissues and 1 lung adenosquamous carcinoma tissue and the 8 corresponding matched adjacent tumor normal tissues, and 43 tissues from benign pulmonary lesions were obtained during surgery, and the clinicopathological features of the patients are summarized in Additional file
1: Table S1 and Additional file
2: Table S2. The total 265 paraffin-embedded, archived non-small cell lung cancer samples were obtained from surgery or needle biopsy, and the clinicopathological features of the patients are summarized in Additional file
3: Table S3. All tissues were collected from the Clinical Biobank of Collaborative Innovation Center for Medical Molecular Diagnostics of Guangdong Province, The Affiliated Jiangmen Hospital of Sun Yat-sen University (Guangdong, China) between January 2016 and December 2016.Patients were diagnosed based on clinical and pathological evidence, and the specimens were immediately snap-frozen and stored in liquid nitrogen tanks. For the use of these clinical materials for research purposes, prior patients’ consents and approval from the Institutional Research Ethics Committee were obtained. The proportions of tumor vs. non-tumor in H&E-stained tissue samples were evaluated by two independent professional pathologists. The tumor proportions in all clinical lung cancer tissue samples analyzed in this study exceeded 75%.
Total RNA from tissues or cells was extracted using the RNA Isolation Kit-miRNeasy Mini Kit (Qiagen, USA) according to the manufacturer’s instructions. Messenger RNA (mRNA) was reverse transcribed from the total mRNA using the Revert Aid First Strand cDNA Synthesis Kit (Thermo, USA) according to the manufacturer’s protocol. Complementary DNA (cDNA) was amplified and quantified on a CFX96 system (BIORAD, USA) using iQ SYBR Green (BIO-RAD, USA). The primers are provided in Additional file
4: Table S4. Real-time PCR was performed according to a standard method, as previously described [
9]. Primers for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were synthesized and purified by RiboBio (Guangzhou, China). GAPDH was used as an endogenous control for mRNA. Relative fold expressions was calculated using the comparative threshold cycle (2
-ΔΔCt) method.
Vectors and retroviral infection
Short hairpin (shRNA) RNA for human PLPP4 was cloned into a phU6-MCS-Ubiquitin -EGFP-IRES-puromycin plasmid (GV280, Genechem, China), and the list of primers for the clone reactions is presented in Additional file
5: Table S5. Transfection of the plasmids was performed using Lipofectamine 3000 reagent (Invitrogen, USA) according to the manufacturer’s instructions. Stable cell lines expressing shPLPP4#1or shPLPP4#2 were generated by lentiviral infection using HEK293T cells, and selected with 0.5 mg/L puromycin for 10 days. The luciferase reporter system of pE2F-luc, pRb-luc and pGL3-luc (Clontech) was used to examine the transcriptional activity of E2F and Rb.
Cell counting kit-8 analysis and colony formation assay
For cell counting kit-8 analysis, cells (2 × 10
3) were seeded into 96 well plates and the specific staining process and methods were performed according to the previous study [
10]. For colony formation assay, cells (0.2 × 10
3) were plated into six well plates and cultured for 10 days. Colonies were then fixed for 15 min with 10% formaldehyde and stained for 30s with 1.0% crystal violet. Plating efficiency was calculated as previously described [
11]. Different colony morphologies were captured under a light microscope (Olympus).
Anchorage-independent growth ability assay
Cells (3 × 10
3) were suspended in 2 ml of complete medium plus 0.3% agar (Sigma-Aldrich, St Louis, MO, US). The agar-cell mixture was plated as a top layer onto a bottom layer of 0.6% complete medium agar mixture as previously described [
12]. After 14 days of culture, the colony size was measured using an ocular micrometer and colonies >0.1 mm in diameter were counted.
Cell cycle analysis
Pretreatment and staining were performed using the Cell Cycle Detection Kit (KeyGEN, China) according to the manufacturer’s instructions. Cells (5 × 105) were harvested by trypsinization, washed with ice-cold phosphate-buffered saline (PBS) and fixed in 75% ice-cold ethanol in PBS. Before staining, cells were gently resuspended in cold PBS, and ribonuclease was added to the cells’ suspension tube, and incubated at 37 °C for 30 min, followed by incubation with propidium iodide(PI) for 20 min at room temperature. Cell samples (2 × 104) were then analyzed by FACSCanto II flow cytometer (Becton, Dickinson and Company, Franklin Lakes, NJ, US) and the data were analyzed using FlowJo 7.6 software (TreeStar Inc., Ashland, OR, US).
High throughput data processing and visualization
The RNA sequencing profiles of lung adenocarcinoma and lung squamous cell carcinoma were downloaded from The Cancer Genome Atlas (TCGA;
https://cancergenome.nih.gov/) and analyzed using Excel 2010 and GraphPad 5 software. The 17 RNA expression profiles of non-small cell lung cancer based on Affymetrix U133 Plus2.0 microarray were downloaded from ArrayExpress (
http://www.ebi.ac.uk/arrayexpress/). Integrated analysis of all data collected from TCGA and ArrayExpress using the YuGene program is summarized in Additional file
6: Table S6 [
13], including 340 normal lung tissues and 2032 lung cancer tissues. The integrative expression profile of lung cancer was named for the AE-meta dataset.
Immunohistochemistry
The immunohistochemistry procedure and scoring of PLPP4 expression were performed as previously described [
14]. The slides were incubated overnight at 4 °C in a humidified chamber with anti-PLPP4 antibodies (Novus Biologicals: NBP2-14545) diluted 1:100 in PBS. Scores given by the two independent investigators were averaged for further comparative evaluation of PLPP4 expression. Tumor cell proportion were scored as follows: 0 (no positive tumor cells); 1 (<10% positive tumor cells); 2 (10–35% positive tumor cells); 3 (35–70% positive tumor cells) and 4 (>70% positive tumor cells). Staining intensity was graded according to the following criteria: 0 (no staining); 1 (weak staining, light yellow); 2 (moderate staining, yellow brown) and 3 (strong staining, brown). The staining index (SI) was calculated as the product of the staining intensity score and the proportion of positive tumor cells. Based on this method of assessment, PLPP4 expression in lung carcinoma samples was evaluated by the SI, with scores of 0, 1, 2, 3, 4, 6, 8, 9 or 12. A SI score of 4 was the median for all sample tissues SI socres. High and low expression of PLPP4 were stratified according to the following criteria: SI ≤ 4 was used to define tumors with low expression of PLPP4, and SI score 6 was used to define tumors with high expression of PLPP4.
Dual luciferase report experiments
Cells (5 × 105) were plated in 60-mm cell culture dishes, and allowed to proliferate to 60–80% confluence after 24 h of culture, and the reporter constructs were transfected into cells using Lipofectamine 3000. After a 12-h incubation, the transfection medium was replaced; cells were harvested and washed with PBS, and lysed with passive lysis buffer (Promega). The cell lysates were analyzed immediately using the Synergy™ 2 microplate system (BioTek, Winooski, VT, US). Luciferase and Renilla luciferase were measured using a Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer’s instructions. The luciferase activity of each lysate was normalized to the Renilla luciferase activity. The relative transcriptional activity was converted to the fold induction above the vehicle control value.
Western blotting
Nuclear/cytoplasmic fractions were separated by using the Cell Fractionation Kit (Cell Signaling Technology, USA) according to the manufacturer’s instructions, and whole cell lysates were extracted using RIPA Buffer (Cell Signaling Technology). Western blots were performed according to a standard method, as previously described [
15]. Antibodies against cyclin D1, cyclin A2 and cyclin B1 were purchased from Cell Signaling Technology (Cyclin Antibody Sampler Kit: Cat#9869) (Danvers, MA, USA), and PLPP4 (Cat#: ab150925), NFAT1 (Cat#: ab49161), p-NFAT1 (Cat#: ab200819) and p84 (Cat#: ab102684) from Abcam. The membranes were stripped and reprobed with an anti–α-tubulin antibody (Cell Signaling Technology. Cat#: 2125) as the loading control.
Statistical analysis
All values are presented as the mean ± standard deviation (SD). Significant differences were determined using GraphPad 5.0 software (USA). Student’s t-test was used to determine significant differences between two groups. One-way ANOVA was used to determine statistical differences between multiple groups. The chi-square test was used to analyze the relationship between PLPP4 expression and clinicopathological characteristics. Survival curves were plotted using the Kaplan-Meier method and compared by log-rank test. P < 0.05 was considered significant. All the experiments were repeated three times.
Discussion
The key findings of the current study show that PLPP4 is dramatically upregulated in lung carcinoma tissues and cells and positively correlates with advanced clinicopathological features, including pathological grade, T category and lung carcinoma stage, and poor prognosis in lung carcinoma patients. Moreover, silencing PLPP4 inhibits the proliferation, cell cycle progression, tumorigenicity and lung metastasis abilities of lung carcinoma cells both in vitro and in vivo. Our findings further reveal that downregulating PLPP4 inhibits Ca2 + −permeable cationic channel in lung carcinoma cells. Therefore, our results elucidate the oncogenic role of PLPP4 in lung carcinoma via elevating intracellular Ca2+.
Numerous studies have reported that altered expression of LPPs has been involved in the development and progression of several human cancers. Flanagan et al. reported that downregulating PLPP2 transcriptionally repressed by p53 impaired anchorage-dependent growth of cancer cell lines MCF7, SK-LMS1, MG63, and U2OS, as well as decreased cell proliferation by delaying entry into the S phase of the cell cycle [
18]. In addition, it has been demonstrated that PLPP3 was downregulated in approximately 70% of oral squamous cell carcinoma (OSCC) patients, and low expression of PLPP3 positively correlated with TNM stage in OSCC patients [
7]. These studies indicated that although belonging to a phosphatidate phosphatase family, different members of the PLPP family function as an oncogene or tumor suppressor in different tumor types. In the current study, we first revealed that PLPP4 was elevated in lung carcinoma tissues. High expression of PLPP4 significantly correlated with advanced clinicopathological features, and poor overall and progression-free survival in lung carcinoma patients. In vitro and in vivo assays demonstrated that silencing PLPP4 inhibited the proliferation and cell cycle progression, and the tumorigenesis of lung carcinoma cells in subcutaneous, as well as in the lung. Thus, our results indicate that high expression of PLPP4 is implicated in the progression of lung carcinoma via promoting the proliferation and cell cycle in lung carcinoma cells.
PLPP4 was first identified by DNA microarray, cDNA dot blot analysis, and reverse transcription-PCR techniques in paired breast cancer tissues [
19]. One year later, Takeuchi et al. found several unknown ESTs via oligonucleotide microarray analysis and cloned two novel cDNAs encoding the putative 264-residue protein PLPP5 and the 271-residue protein PLPP4 [
20]. Furthermore, Manzano and colleagues reported that PLPP4 was upregulated in only the estrogen receptor (ER)-ERBB2+ subgroup, but not in other subtypes of breast cancer, suggesting a specific role of PLPP4 in ER-ERBB2+ subtypes [
21]. However, the clinical significance and biological role of PLPP4 in cancers remain blank. In this study, through analyzing the expression levels of LPPs proteins in the lung carcinoma RNA expression profile datasets from TCGA, we first found that PLPP4 was considerably elevated compared with other LPPs in lung carcinoma tissues. Overexpression of PLPP4 was further observed in our lung carcinoma tissues and cells and positively correlated with advanced clinicopathological features and poor prognosis. Moreover, silencing PLPP4 repressed the proliferation, tumorigenicity and lung metastasis abilities of lung carcinoma cells both in in vitro and in vivo, as well as inhibited Ca
2+-permeable cationic channel in lung carcinoma cells. Therefore, our findings indicate that PLPP4 plays an important role in lung carcinoma.
Numerous studies have demonstrated that LPPs catalyze the conversion of phosphatidic acid generated from phosphatidylcholine to diacylglycerol [
3,
4]. The increment of diacylglycerol concentration could directly activate some transient receptor potential channel (TRPC) family members without depleting intracellular Ca
2+ stores, leading to the release and influx of intracellular Ca
2+ [
22,
23]. As a universal second messenger, calcium is involved in several important cellular processes, including growth, differentiation, and programmed cell death [
24,
25], as well as in pathological diseases, such as cancer development [
5,
26]. Therefore, these studies suggested that LPPs may contribute to the development of cancer via diacylglycerol-gated Ca
2+-permeable cationic channel [
4,
5]. Furthermore, therapies aiming at inhibiting intracellular Ca
2+ may be favorable for the treatment of cancer [
27,
28]. In the current study, our results found that silencing PLPP4 effectively reduced intracellular Ca
2+ and S54 phosphorylated levels of NFAT1, which further inhibited the proliferation and tumorigenesis in lung carcinoma cells. Our findings indicate that inhibition of PLPP4 may be used as a novel therapeutic strategy in the treatment of lung carcinoma.
It has been widely documented that LPPs may serve as a potential marker for the diagnosis of cancer or as a putative therapeutic target in the treatment of cancers. Amin and colleagues have shown that PLPP1, a downstream ErbB2 signaling target, may be used as a diagnostic marker in breast cancer [
29]. Moreover, it was reported that a series of in vitro data, including anchorage-dependent growth and proliferation assays, validated PLPP2 as a putative therapeutic target for treatment of cancer [
18]. In this study, our results implied that high expression of PLPP4 was observed in lung adenocarcinoma and lung squamous cell carcinoma tissues and was positively associated with pathological grade, T category and lung carcinoma stage in patients, suggesting that PLPP4 holds promise as a novel marker for the diagnosis of lung carcinoma. Furthermore, subcutaneous and lung colonization models showed that downregulating PLPP4 dramatically inhibited the tumorigenesis of lung carcinoma cells in a mouse model, indicating that PLPP4 may hold potential applicable value as a therapeutic target against lung carcinoma. However, further validation needs to be warrant in a large series of studies.