Background
Research interest in the cell biology of lactate has been re-vitalized by a recent boom of investigation on the role of lactate-enriched microenvironment in tumorigenesis and tumor progression [
1‐
4]. Unlike their normal counterparts, cancer cells reprogram their metabolism to rely on mainly on glycolysis regardless of oxygen availability [
5,
6]. This phenomenon, known as Warburg effect, is characterized by increased glucose uptake and lactate production, which leads to acidification of tumor environment. Previous in vitro studies have shown that acidic environment is associated with certain key features of tumor progression including invasion, immune evasion, angiogenesis and resistance to therapy [
7‐
10]. Furthermore, pretreatment of tumor cells with acid before tail vein injection increased experimental metastases [
11], and inhibition of this acidity by oral NaHCO
3 reduced the incidence of in vivo metastases [
12]. In addition, the acidification of tumor extracellular space promotes secretion of proteolytic enzymes that are involved in tissue remodeling and degradation of basement membrane (11, 12), thus facilitating tumor cells invasion and metastasis.
The increased invasiveness and motility of tumor cells induced by lactate are reminiscent of the EMT processes, during which epithelial cells lose polarity and intercellular adhesion, and acquire a highly mesenchymal phenotype. EMT is a strictly controlled process mediate by multiple signal pathways including TGF-β. TGF-β is secreted as a latent complex tightly bound to extracellular matrix [
13]. Activation of TGF-β signaling pathway is primarily regulated by conversion of latent TGF-β to active TGF-β. Many factors can influence the liberation of TGF-β from the latent complex such as proteases, integrins, reactive oxygen species (ROS) and low pH [
14]. Once latent TGF-β activated, TGF-β exerts its effect by binding to type I and type II (TGF-βRI/II) serine/threonine receptor. The activated TGF-βRI/II then phosphorylated receptor-regulated R-Smads (Smad2 and Smad3), R-Smad complex with common-mediator Smad4 translocated into nucleus, where they, in junction with other transcription factors, regulate genes involved in induction of EMT [
15,
16]. Most prominent of target genes by TGF-β is a zinc-containing transcription factor Snail [
17,
18]. Expression of Snail suppresses E-Cadherin expression and induces EMT in a variety of cancer cells including lung cancer cells. In addition to regulating EMT, overexpression of Snail induces resistance to apoptosis and tumor recurrence [
19]. Importantly, activation of EMT by Snail has also been associated with acquisition of stem cell traits in normal and neoplastic cells [
20], suggesting that EMT program plays a critical role in many stages of tumor progression. The study by Ansieau group showed that EMT-inducer Twist collaborating with Ras oncogene bypass senescence [
21], a state of irreversible proliferative arrest as a consequence of genomic damage [
22]. Senescence can also be induced prematurely by oncogenes and has been observed in various human premalignant lesions [
23]. However, the mechanisms by which how senescence is subverted during development of malignancy remain poorly understood. An intriguing relationship between glycolytic pathway and cellular senescence has recently been reported [
24‐
26]. Here, we present evidence of TGF-β-initiated EMT program induced by lactate-enriched microenvironment not only relevant in advanced tumor cells for metastasis also in early stage of tumor transformation. We demonstrated that TGF-β-induced Snail protein is required for escaping senescence induced by oncogene. Our findings might provide valuable clues to the suspected connections of early metabolic reprograming in premalignant lesions to tumor initiation and progression.
Methods
Cell culture, antibodies, reagents and plasmids
A549 (human lung adenocarcinoma), H1299 (human lung adenocarcinoma) and BEAS-2B (B2B) (normal lung epithelial) cells were cultured with DMEM (Hyclone, Logan, UT, USA) containing 10% fetal bovine serum (FBS, Gibco BRL, Grand Island, NY, USA) at 37 °C in a humidified atmosphere of 5% CO2. Anti-Ras (no. 8955), anti-p21 (no. 2947), anti-PTEN (no. 9188), anti-phospho-Rb (no. 85165), anti-N-cadherin (no. 4061), anti-Snail (no. 3895), anti-LDHA (no. 3582), anti-B-Raf (no. 9433), anti-Slug (no. 9585), anti-Caveolin-1 (no. 3267), anti-p27 (no. 2552), anti-Phospho-Smad3 (no. 9520), anti-Phospho-Smad2 (no. 3101) were obtained from Cell Signaling Technology (Danvers, MA, USA). Anti-gamma H2AX (phospho S139) (ab11174), anti-CDKN2A/ p16INK4a (ab108349), anti-Fibronectin (ab299), anti-GPCR GPR81 (ab124010) were purchased from Abcam (Cambridge, UK). Anti-β-Actin (A1978) and anti-Vimentin (V6630) were purchased from Sigma (Sigma, Victoria, BC, Canada). Twist (sc-81,417) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). E-cadherin (13–1700) was obtained from Thermo Fisher Scientific (Waltham, MA, USA). Lactate, a-cyano-4-hydroxycinnamate (CHC), or LY2159299 was purchased from Roche (San Francisco, CA, USA), Sigma-Aldrich (Sigma, Victoria, BC, Canada) or SB505124(Selleck Chemicals, Houston, Texas, United States), respectively. The pCMV-PTEN-C124S, pBabe B-Raf (V600E), pCMV p16INK4a were purchased from Addgene (Cambridge, MA, USA). The pRL-CMV vector was purchased from Promega (Madison, WI, USA).
Cloning and DNA construction
To construct different length of p16
INK4a promoters or Snail promoter, fragments were amplified from B2B genome DNA by PCR (the primers are listed in Table
1) and were then cloned into pGL3-Basic Vector (Promega, Madison, WI, USA) at the Kpn I and Hind III sites. Point mutations in the p16
INK4a promoter was generated by site-specific mutagenesis using the overlap PCR extension method and the longest p16
INK4a promoter was used as the template and the primers are listed in Table
1. GPR81 cDNA was amplified using total reverse-transcribed cDNA as the template. The amplified PCR fragments were digested with KpnI/EcoRI restriction enzymes and inserted into the pcDNA3. 1 (+) vector.
Table 1
Primers used for PCR amplifications
p16 | NM_001195132.1 | Forward: GGGGTACC AGGGAGTAAGTTCTTCTTGGTCTTTC |
| | Reverse: CCCAAGCTTCTATTAACTCCGAGCACTTAGCGAAT |
p16 | NM_001195132.1 | Forward: GGGGTACCGCGGATAATTCAAGAGCTAACAGGTA |
| | Reverse: CCCAAGCTTCTATTAACTCCGAGCACTTAGCGAAT |
p16 | NM_001195132.1 | Forward: GGGGTACCATACTTTCCCTATGACACCAAACAC |
| | Reverse: CCCAAGCTTCTATTAACTCCGAGCACTTAGCGAAT |
p16 | NM_001195132.1 | Forward: CACTTTCTAGTCGTATACGGGATTTCGATTCTCGGT |
| | Reverse: ACCGAGAATCGAAATCCCGTATACGACTAGAAAGTG |
GPR81 | NM_032554.3 | Forward: GGGGTACCATGTACAACGGGTCGTGCTG |
| | Reverse: GGAATTCTCAGTGCCACTCAACAATGT |
Ras activity assay
Ras activation status of the cells was determined using the Ras Assay Kit (Abcam, ab128504) according to the manufacturer’s protocol. In brief, 106 B2B cells transfected with K-Ras (G12S)-expressing plasmid were lysed in 1 ml of ice-cold kit-provided lysis buffer containing protease inhibitors. Fifty microliters of lysate were added to 10 μl of 6× protein loading buffer (Beyotime Institute of Biotechnology, Shanghai, China); This represents the total Ras load. The remaining lysates were incubated with 40 μl of GST–RBD fusion protein-linked glutathione sepharose beads, which had been pre-equilibrated with lysis buffer, under constant mixing for 30 min at 4 °C. After centrifugation, the beads were washed 2 times with 1 ml of ice-cold lysis buffer. Beads were drained well and fifty microliters of 1× Laemmli sample buffer were added to the samples, this represents the Ras-GTP pull-down. The total Ras load and Ras-GTP pull-down were resolved on a 10% SDS PAGE gel. Primary antibody detecting pan-Ras was provided in the kit and secondary antibody goat-anti-mouse IgG conjugated to HRP and Commassie Blue Fast Staining Solution (Beyotime Institute of Biotechnology, Shanghai, China) were used.
Senescence-associated β-galactosidase staining
β-Galactosidase activity of the cells was determined using a Senescence β-Galactosidase Staining Kit (Beyotime Institute of Biotechnology, Shanghai, China) according to the manufacturer’s protocol with minor modifications. In brief, Cells were washed three times with PBS and fixed with stationary liquid provided in the kit for 45 min at room temperature. Next, the cells were incubated overnight at 37 °C in darkness with the working solution containing 0.05 mg/ml 5-bromo-4-chloro-3-indolyl-b-d-galactopyranoside (X-gal)). The population of SA-β-gal-positive cells was determined by counting 100 cells per field and photographs were taken using Cytation™ 5 Cell Imaging Multi-Mode Reader (BioTek, Winooski, VT, United States). The proportions of cells positive for the SA-β-gal activity are given as percentage of the total number of cells counted in each well. The results are expressed as mean of triplicates±SD.
Immunofluorescence assay
BEAS-2B cells were grown on coverslips and transfected with K-Ras(G12S). Different concentrations of lactate were added for 3 h after 48 h transfection. Cells were fixed with 4% paraformaldehyde and permeabilized with 0. 25% Triton X-100 for 5 min at room temperature. After subsequent blocking with 2% bovine serum albumin for 15 min, cells were incubated with primary antibodies against anti-γH2AX (phospho-S139) (ab11174; Abcam) at 4 °C with gently shaking overnight, and then incubated with fluorescein isothiocyanate-conjugated anti-rabbit antibody (no. 8889 s; Cell Signaling Technology) for 1 h at room temperature. 4′, 6-Diamidino-2-phenylindole (Sigma, Victoria, BC, Canada) was used to visualize the nuclei. Immunofluorescence was detected by fluorescence microscopy (Leica TCS SP8; Leica Microsystems, Mannheim, Germany).
Quantitative real-time RT–PCR analysis
Cells were treated as indicated and total mRNA was isolated using TRIzol according to the manufacturer’s protocols. The obtained RNA was re-transcribed using PrimeScript First Strand cDNA Synthesis Kit (TaKaRa Bio, DaLian, China). The cDNA was mixed with ABI SYBR Green Master Mix (Applied Biosystems, Carlsbad, CA, USA), and the mixture was subjected to amplification using an ABI 7500 Real-time PCR System (Applied Biosystems). The primers used were: SNAIL forward: 5′- AGGCAGCTATTTCAGCCTCC-3′; SNAIL reverse:5’-CACATCGGTCAGACCAG AGC-3′; GAPDH forward: 5′- GACCCCTTCATTGACCTCAAC-3′; GAPDH reverse:5’-CTTCTCCATGGTGGTGAAGA-3′; p16INK4a forward:5’-GTCCCCTTGCCTGGAAAGAT-3′; p16INK4a reverse:5′- CACCTCCTCTACCCGACCC-3′; β-actin forward: 5′- CCTTCCTGGGCATGGAGTCCT-3′; β-actin reverse: 5’-GGAGCAATGATCTTG ATCTTC-3′. Each sample was repeated in triplicate and analyzed using the Relative Quantification Software (Applied Biosystems).
Dual luciferase reporter assays
Cells were co-transfected with experimental reporter. After 48 h, cells were lysed and activities of firefly luciferase and Renilla luciferase were analyzed following the manufacturer’s instruction. Each experiment was repeated in triplicate using a multimode microplate reader (TriStar LB941; Berthold Technologies, Bad Wildbad, Germany). The results are expressed as mean of triplicates ± SD.
Chromatin immunoprecipitation (ChIP) assay
B2B Cells were transfected with Snail cDNA. ChIP on B2B cells (1 × 10 8) were performed using the SimpleChIP Enzymatic Chromatin IP kit (Cell signaling technology, No. 9002) according to the manufacturer’s instructions with minor modifications. The cells were fixed in DMEM medium containing 1% formaldehyde for 15 min at room temperature, and the reaction was stopped by glycine quenching (125 mM final concentration). Nuclei were collected and digested with micrococcal nuclease (0.5 μl, provided by the SimpleChIP kit) followed by 2 min of sonication (3 cycles of 10 s of sonication and 30 s without sonication) using a vibra cell VCX 130 (Sonics & Materials, Inc., NEWTOWN, CT, USA). Pull downs were performed on DNA fragments (ranging from 150 to 900 bp) using anti-FLAG (M2) antibody SIGMA, No. F1804). The immunoprecipitated DNA and input DNA were extracted by reversing the crosslinks. Standard PCR and qRT-PCR were performed with purified DNA as templates. The primers used were: p16INK4a forward: 5′- AGGGTTTCTGACTTAGTGAA -3′; p16INK4a reverse:5′- TTCCTAGTTGTGAGAGCC -3′. The standard PCR products were run on a 1% agarose gel and scanned under UV using FluorChem FC3 (ProteinSimple, San Jose, CA, USA), and qRT-PCR results were analyzed according to the protocols.
Wound healing assay
Cells were seeded in a 6-well plate at a concentration of 1 × 106 cells/well and allowed to form a confluent monolayer for 24 h. Cells were then treated for 24 h with fresh medium added 0, 10, 20 mM lactate. Then the monolayer cells were scratched with 1 mL pipette tips, washed with PBS to remove floating cells and photographed by a phase-contrast microscope at 100 magnification (Olympus, Shinjuku-ku, Tokyo, Japan) (time 0). Cells were further incubated with DMEM for 48 h or 72 h and photographed again (time 48 h, 72 h). The numbers of cells migrated to time 0 wound area were counted.
Cell invasion assays
For assessment of cell motility, the CHEMICON Cell invasion assay was performed in an Invasion Chamber (Millipore, Billerica, MA, USA). Cells were seeded in triplicate at a density of 3. 0 × 105 cells/ chamber. After 48 h, cells which had not moved to the lower wells were removed from the upper face of the filters using cotton swabs, and cells that had moved to the lower surface of the filter were stained by using a Cell Invasion Assay Kit. (CHEMICON, No. ECM550). Cell migration was quantified by visual counting after being photographed by a phase-contrast microscope at 100 magnification (Olympus, Shinjuku-ku, Tokyo, Japan). Experiments were performed in triplicate. Mean values for three random fields were obtained for each well.
Plasmid and short interfering RNA (siRNA) transfection
Cells seeded in plates were grown to 70%–90%confluence before plasmids transfection and transfection of plasmids was done with PolyJet DNA Transfection Reagent (SignaGen Laboratories, Gaithersburg, MD, USA) according to the manufacturer’s instructions. The transfection with siRNA using GenMute siRNA Transfection Reagent (SignaGen Laboratories) when cells seeded in plates were grown to 30%–50% confluence. All the siRNAs were purchased from RiboBio Company (Guangzhou, China). After transfection for 48 h, cells were deprived of serum and growth factors for 12 h and then treated with lactate (Roche, San Francisco, CA, USA) for 3 h and harvested. The sequences of the siRNAs are listed in Table
2.
Table 2
Sequences of siRNA
GPR81 | NM_032554.3 | CTGCTAGACTCTATTTCCT |
LDHA | NM_001165414.1 | GCCAUCAGUAUCUUAAUGATT |
SNAIL | NM _005985.3 | CAAATACTGCAACAAGGAA |
Western blot
Cells were scraped and homogenized with Sample Buffer, Laemmli 2 × Concentrate(S3401; SIGMA). The total or membrane protein concentration was isolated by Membrane and Cytosol Protein Extraction Kit (Beyotime Institute of Biotechnology, Shanghai, China). Protein per sample was separated by polyacrylamide gel electrophoresis and then transferred to nitrocellulose (NC) membrane (GE Healthcare, Piscataway, NJ, USA) and detected with the antibodies. The signals were scanned by FluorChem FC3 (ProteinSimple, San Jose, CA, USA).
Enzyme linked immunosorbent assay (ELISA)
ELISA was used to detect TGF-β1 in culture supernatant of A549 and H1299 cells that were treated with lactate (20 mM) or medium titrated with HCI for 3 h to lower PH, according to the manufacturer’s instructions (NeoBioscience Technology, Shenzhen, China). The culture supernatant of A549 and H1299 cells that were treated with PBS alone served as the control groups. The absorbance at 450 nm was measured using Cytation™ 5 Cell Imaging Multi-Mode Reader (BioTek, Winooski, VT, United States). According to the standard curve, the sample concentration was calculated.
Lactate determination
Cells (2 × 105) were treated with glucose (0, 2. 7 and 4. 5 μg/ μl) for 3 h. Lactate in the Culture medium was measured using the Lactate Assay Kit (BioVision, Milpitas, CA, USA) according to the manufacturer’s instructions. The concentration of lactate was determined using Lactate Standard Curve.
RNA-seq number analyses in human NSCLC tissues
The gene correlations were analyzed using the Cancer Genome Atlas (TCGA) data (RNA-Seq-HTSeq-FPKM-UQ)in Lung adenocarcinoma(
n = 181) and Lung squamous(
n = 155) (
http://tcga-data.nci.nih.gov). RNA-seq number values were matched with the gene expression. Subsequently, the Spearman’s rank correlation coefficient (rho) between SNAI1 gene expression and CDKN2A was calculated. All statistical analyses and data generation were carried out using R version 3.4.3 (
http://www.r-project.org) (Table
3).
Table 3
List of assayable 2 genes and correlation
Gene title | Gene symbol |
P value
| Spearman’s rank correlation coefficient |
P value
| Spearman’s rank correlation coefficient |
Snail family transcriptional repressor 1 | SNAI1 | < 2.2e-16 | - 0.729 | < 2.2e-16 | - 0.906 |
Cyclin dependent kinase inhibitor 2A | CDKN2A | | | | |
Statistical analysis
Statistical analyses were performed with analysis of variance (ANOVA) using SPSS 13.0 Statistical Software (SPSS Inc., Chicago, IL, USA) and are presented as mean ± s.d. from triplicated independent experiments. A significant difference was considered when the P-value from a two-tailed test was < 0.05.
Discussion
The preferential use of aerobic glycolysis by tumor cells resulted in high amounts of lactate in tumor microenvironment. Elevated lactate concentration is correlated with increased metastasis and poor prognosis for overall survival. Here, we showed that lactate is a crucial regulator of EMT. Mechanistically, lactate mediates extracellular matrix remodeling by releasing TGF-β1, a major inducer of EMT. The present study clear shows that lactate dose-dependently increased snail expression and defines snail as a key contributor to lactate-induced EMT in lung cancer cells. Furthermore, our data argues that a key function of snail induced by lactate is also required for suppression of oncogene-induced senescence in premalignant cells.
Despite initial lack of appreciation, lactate is now considered to play a significant role in cancer progression. Previous work has strongly implicated that lactate promotes tumor cells migratory and invasive activity and is associated with higher incidence of metastases in cancer patients. Our study clearly showed that the invasive and migratory potential was significantly enhanced by lactate in lung cancer cell lines in a dose-dependent manner. The increased invasiveness and motility of tumor cells is directly linked to snail activity induced by lactate. Our finding is also consistent with previous works showing that acidic extracellular pH stimulates tumor cells migration and invasion in vitro and promotes experimental metastasis in nude mice. Several approaches employed by present study, including inhibition of MCT1 by CHC or knockdown of GPR81 using siRNA or direct modulation of extracellular pH, demonstrated that extracellular acidification itself is a direct cause of the increased snail expression and physiologically coupled to LDHA-dependent conversion of pyruvate to lactate. Taken together, these results provided significant evidence that acidity-induced upregulation of proteins known to promote invasive growth and metastasis is a possible mechanism for lactate-induced metastasis.
Tumor invasiveness is also dependent on extracellular matrix remodeling, which facilitates proteases cleavage of ECM barriers and promotes angiogenesis. The ECM remodeling process can be induced by low pH. Low pH has been shown to stimulate the release of Cathepsin B and MMP9, both of which accelerate tumor cell invasion. TGF-β is secreted as a latent complex that is tightly bound to extracellular matrix. Liberation and activation of TGF-β from the latent complex is stimulated by a variety of activators, including proteases, TSP-1 and low pH. Acidification in tumor environment probably through denaturing LAP disrupts the interaction of LAP and TGF-β and releases and activated TGF-β. Here we showed that lactate exerts its central function in induction of EMT by directly remodeling ECM and releasing activated TGF-β.