Background
Lung cancer is the leading cause of cancer death in males, and is the second cause of cancer death in females worldwide [
1]. The incidence and mortality of lung cancer are increasing rapidly in recent years, which are mainly caused by environmental pollution and smoking. Non-small cell lung cancer (NSCLC) accounts for about 85% of all lung cancers, and can be any type of epithelial lung cancer other than small cell lung cancer. The most common types of NSCLC include squamous cell carcinoma, large cell carcinoma, and adenocarcinoma. Although there are great developments in NSCLC treatment, patients with advanced NSCLC still have a very low five-year survival rates [
2‐
5]. Therefore, it is of great importance to identify the potential molecular targets for NSCLC therapy.
TIPE3 is the newest member of TIPE (tumor necrosis factor-α-induced protein 8, TNFAIP8) family, which consists of a group of proteins including TIPE, TIPE1 and TIPE2 that regulate tumorigenesis and immunity [
6‐
11]. TIPE3 gene locates human chromosome 15 or murine chromosome 9 [
12‐
14]. TIPE3 protein is extensively expressed in murine organs including uterus, lung, brain, bladder, intestine and colon with various expression levels. Notably, marked increase in TIPE3 expression was detected in human cancer tissues including cervical, colon, lung and esophageal. Murine TIPE3 has been demonstrated to serve as a phosphoinositide carrier to activate PI3K-AKT and MEK-ERK signaling pathways, thus promoting the growth and tumorigenesis of NIH3T3-HRasV12 cells [
6,
8]. Similar to other family members, TIPE3 has a highly conserved TIPE2 homology (TH) domain. The crystal structure showed that the α1-α6 helixes of human TIPE3 TH-domain formed a large hydrophobic cavity, which likely accommodates lipid secondary messengers PIP2 and PIP3 for phosphoinositide signaling [
8]. TIPE3 expression was found in normal human lung tissues, and markedly increased in human lung cancer tissues [
8,
15]. It remains unclear whether and how TIPE3 is involved in human NSCLC. In the present study, we revealed that plasma membrane-localizing TIPE3 was positively correlated with the malignance of NSCLC. Using two different NSCLC cell lines and xenograft tumor models, we demonstrated that endogenous human TIPE3 promoted cell proliferation and migration in NSCLC; while exogenous human TIPE3 produced differential roles in the growth and migration of NSCLC cells based on its different subcellular location.
Methods
Patients
Lung tissue arrays from 48 cases of human primary NSCLC (OUTDO BIOTECH, Shanghai, China) were used to detect the expression of TIPE3. The correlation between TIPE3 expression and clinical features, as well as the correlation of plasma membrane expression of TIPE3 with T stage of NSCLC were analyzed.
Cell culture
The H1975 cell line (human lung adenocarcinoma) (TCHu193) was purchased from Shanghai Cell Bank of Chinese Academy of Sciences (Shanghai, China), and cultured in RPMI 1640 medium (Gibco, CA, USA) containing 10% fetal bovine serum (FBS) (Gibco). The A549 cell line (human lung adenocarcinoma) (GDC063) was purchased from China Center for Type Culture Collection (Wuhan, China), and cultured in F12 K medium (Macgene, Beijing, China) supplemented with 10% FBS.
Plasmids, siRNA and transfection
Plasmid carrying N-terminal flag-tagged human TIPE3 genes was constructed by Sangon Biotech (Shanghai, China). Specific siRNA for human TIPE3 (5’CGCAGCAUGGAUUCGGAUUdTdT3’, 3’dTdTGCGUCGUACCUAAGCCUAA5’; 5’GGAACGUGCUCUCCAAUCUdTdT3’, 3’dTdTCCUUGCACGAGAGGUUAGA5’) was designed and synthesized by RIBOBIO (Guangzhou, China). Tumor cells were transfected with plasmid or siRNA using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocols. Recombinant lentiviral vector carrying C-terminal flag-tagged human TIPE3 gene was constructed by Genechem (Shanghai, China). Lentiviral transfection was performed in tumor cells as per the instruction.
Reverse transcriptional PCR
Total RNA was extracted from cells using Trizol Reagent (Invitrogen). PCR was performed using 2 × Taq PCR MasterMix (TIANGEN, Beijing, China). The primers were as follows: human TIPE3, 5′-GAGGAGCTGGTTATTGTGGAGAA-3′ and 5′-ATCGGCAAAGTGGTTAAAGACG-3′; human GAPDH, 5′-AACGGATTTGGTCGTATTGGG-3′ and 5′-CCTGGAAGATGGTGATGGGAT-3′.
Western-blot
Equal amount of protein was separated by SDS-PAGE and transferred onto PVDF membranes (Millipore, Billerica, MA). Membranes were probed overnight at 4 °C with primary antibodies against human TIPE3 (1:300; BOSTER, Wuhan, China), p-AKT, p-ERK, AKT, ERK (1:1000, Cell Signaling Technology, Beverly, MA), and flag (1:10000, MBL, Nagano, Japan), GAPDH or β-actin (1:1000; ZSGB-Bio, Beijing, China), followed by secondary antibodies (1:2000; ZSGB-Bio) conjugated with peroxidase for 1 h at room temperature. Signals were detected by SuperSignal West Pico Chemiluminescent Substrate (Pierce Biotechnology, Rockford, IL).
Immunohistochemistry
The paraffin slides were stained with rabbit antibody against human TIPE3 (1:200) at 4 °C overnight, followed by HRP-conjugated anti-rabbit IgG using MaxVsion Kit. The 3, 5-diaminobenzidine peroxidase Substrate Kit (Maixin, Fuzhou, China) was used for color detection. The sections were counterstained with hematoxylin. The results were independently assessed by two experienced pathologists. The staining intensity was scored from 0 to 3 (0, no staining; 1, weak; 2, moderate; 3, strong). The staining extent was scored from 0 to 3 based on the percentage of positive cells (0, < 1%; 1, 1%–33%; 2, 34%–66%; 3, 67%–100%). The two scores for each slide were then combined to produce a final grade of TIPE3 expression: 0–2, low; 3–8, high. The average score was used if there were discrepancies in the assessment.
Immunofluorescence
The cells on cover slips were fixed, permeabilized and then were probed with rabbit antibody against human TIPE3 (1:100) and mouse antibody against α1 Na
+
/K
+
-ATPase (1:200; abcam, Cambridge, UK) overnight at 4 °C, followed by Alexa Fluor 594-conjugated goat anti-rabbit IgG (1:300, Proteintech Group, Rocky Hill, NJ) and FITC-conjugated goat anti-mouse IgG (1:300, CWBIO, Beijing, China). Nuclei were stained by 4′, 6-diamidino-2-phenylin-dole (DAPI) (Beyotime, Shanghai, China) for 5 min. Results were analyzed on confocal laser microscopy (Carl Zeiss, LSM780, Oberkochen, Germany).
Cell viability assay
Cells were seeded in 96-well plates at 3000 cells per well and cultured for indicated time periods. Cell viability was evaluated using CCK8 (DOJINDO LABORATORISE, Japan) according to the manufacturer’s instructions. The absorbance was determined at 450 nm.
Cell migration assay
Tumor cell migration was analyzed in 24 well Boyden chambers with 8-μm pore size polycarbonate membranes (Costar, Acton). Cells (4 × 104) were suspended in 200 μl serum-free medium and placed in the upper chamber. The lower compartments were filled with 600 μl medium with 10% FBS. After 10 h of incubation, the cells remaining on the upper surface of the membrane were removed. The cells on the lower surface of the membrane were fixed and stained with crystal violet, and then were counted under light microscope at × 200 magnification.
Establishment of xenograft tumors in nude mice
Male BALB/c nu/nu mice (4–6 week old) were purchased from Chinese Academy of Sciences (Shanghai, China) and maintained in laminar-flow cabinets under specific pathogen-free conditions. A549 cells transfected with mock or recombinant lentivirus were subcutaneously injected into flanks of nude mice. The tumor growth was monitored by calculating the tumor volume (length×width×width). The mice were sacrificed under anesthesia 30 days after inoculation. The tumors were resected and processed for standard histopathological study. All procedures were approved by Ethical Review Board of Shandong University, and carried out in accordance with the Animal [Scientific Procedures] Act 1986 and the institutional guidelines for animal care and utilization.
Statistical analysis
Statistical analysis was performed with GraphPad Prism 5. Chi-square or Fisher’s exact test was used to evaluate correlation; Student’s t test, One-way or Two-way ANOVA were used to evaluate differences. P value < 0.05 was considered statistically significant.
Discussion
Human TIPE3 is highly expressed in lung cancer tissues compared with adjacent non-cancer tissues, indicating the possible involvement of TIPE3 in lung tumorigenesis [
8,
15]. Considering high incidences and low survival rates of NSCLC, we tried to reveal the connection between TIPE3 and NSCLC. Through correlation analysis, we found no correlation of TIPE3 expression with clinical features in patients with NSCLC, but positive correlation of plasma membrane-localizing TIPE3 with T stage of NSCLC. These findings raise the possibility that human TIPE3 affects the tumorigenesis of NSCLC depending on its subcellular location.
Using two NSCLC cell lines A549 and H1975, we showed that TIPE3 was highly expressed in plasma membrane of the lung cancer cells with long pseudopodia, particularly in the position of protrusion. It has been recognized that protrusion formation is essential for cell migration to favor caner dissemination [
16‐
18]. So, in the present study, TIPE3 on plasma membrane may be involved in cell motility and contribute to the growth and migration of lung cancer cells. Next, we showed that silence of endogenous TIPE3 significantly inhibited the growth and migration of NSCLC cells. Consistently, stably overexpression of exogenous TIPE3 via recombinant lentivirus transfection significantly promoted the growth and migration of NSCLC cells. In this stable overexpression system, we found that much TIPE3 scattered in the plasma membrane, although some TIPE3 still existed in the cytoplasm; simultaneously, the expression levels of p-AKT and p-ERK that is critical for cell survival, growth and migration [
19‐
23], were obviously upregulated. These observations support previous finding in NIH3T3 cells that TIPE3 serves as a lipid transfer to activate PI3K-AKT and MEK-ERK pathways [
8], and further suggest that TIPE3 may promote the growth and migration of lung cancer cells through AKT and ERK activation. To confirm the role and subcellular location of TIPE3 in the tumorigenesis of NSCLC, we established xenograft tumor models using nu/nu mice. To minimize the number and suffering of the mice, five mice per group were used in this experiment, and the tumor volume was strictly monitored. Xenograft tumors established with NSCLC cells overexpressing TIPE3 grew faster than those established with control NSCLC cells, leading to increases in tumor size and weight. Immunohistochemistry showed that TIPE3, especially plasma membrane-localizing TIPE3, was obviously increased in the tumor tissue sections from mice received NSCLC cells overexpressing TIPE3 compared with those from mice received control NSCLC cells. These data provide direct evidences for the pro-tumorigenesis of human TIPE3 in NSCLC, and also emphasize the importance of subcellular location for TIPE3 function. More detailed mechanisms remain to be further investigated.
Notably, the essential role of plasma membrane-localizing TIPE3 in promoting cell growth and migration was further clarified in a transient overexpression system using plasmid transfection. In this system, either long or short TIPE3 transfected by plasmids inhibited the growth and migration of NSCLC cells, producing opposite effects to lentivirus transfection system; interestingly, after transient expression, TIPE3 was mainly localized in cytoplasm. These observations confirmed that different subcellular locations caused differential effects of human TIPE3 on the tumorigenesis of NSCLC. Plasma membrane-localizing TIPE3 drove a promotive effect on cell proliferation and migration of NSCLC cells, while cytoplasmic TIPE3 led to an inhibitory effect. Fayngerts et al. reported that N-terminal region was essential for the effects of TIPE3 on cell growth and survival, depletion of N-terminal region elicited a negative effect [
8]. In the present study, TIPE3 with N-terminal flag in transient plasmid transfection system was detained in cytoplasm, while TIPE3 with C-terminal flag in stable lentivirus transfection system was orientated to plasma membrane. Since N-terminal domain, which is involved in signal cleavage, protein folding and modifications like glycosylation, is critical during protein targeting, translocation and insertion into membrane [
24,
25], a very likely explanation for the discrepant effects of exogenous human TIPE3 is that flag at N-terminal domain influences the membrane location and then the function of TIPE3. Murine TIPE3 has been demonstrated to shuttle two lipid second messengers phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] and increase their levels in plasma membrane [
8]. Membrane PtdIns(3,4,5)P3 can recruit its effector protein molecules like AKT from cytoplasm to membrane surface to activate, stabilize, and propagate downstream signaling cascades, thus mediating a variety of physiological processes including cell survival and growth [
26‐
29]. So, it would be necessary for human TIPE3 to orientate in plasma membrane to exert its promotive effect on NSCLC tumorigenesis. As for cytoplasmic TIPE3 binding with lipids, failure to target membrane may lead to decreases in membrane PtdIns(3,4,5)P3 and then the activation of downstream signals, thus mediating the inhibitory effect on NSCLC tumorigenesis. Therefore, appropriate strategies and detailed mechanisms for the application of TIPE3 in NSCLC therapy remain to be further investigated.
Acknowledgments
We thank Microscopy Characterization Facility, Shandong University for providing assistance on the fluorescence imaging work.