Background
Pancreatic cancer is the third leading cause of cancer-related death in the United States [
1]. Pancreatic cancer is a rapidly fatal disease with a 5-year survival rate of less than 5%. Pancreatic ductal adenocarcinoma (PDAC) accounts for 95% of pancreatic cancer [
2]. There is still no effective treatment for advanced PDAC [
3]. A better understanding of the role of driver mutations in the development of PDAC is likely to find new therapeutic targets.
More than 90% of PDACs carry mutated
KRAS alleles [
4]. KRAS mutations have been shown to play a key role in the development of PDAC [
5]. The most common mutation is the constitutively active KRAS
G12D allele. KRAS
G12D mutation is essential for the initiation and maintenance of pancreatic cancer [
6]. Although KRAS mutations have been identified as a driver of PDAC, KRAS targeted therapy has not been successfully developed. Direct inhibition of KRAS has proven clinically challenging. Inhibition of KRAS downstream targets is an effective strategy for targeting KRAS mutations. KRAS activates different downstream effectors in a context specific manner. The KRAS-driven signal network is different between PDAC, non-small cell lung cancer (NSCLC) and colon cancer [
7]. Therefore, it is necessary to clarify the precise molecular mechanism of KRAS in the development of pancreatic cancer.
Transgelin-2 belongs to the family of actin binding proteins (ABPs) and has been characterized as a smooth muscle cytoskeletal protein. In recent years, dysregulated expression of transgelin-2 has been reported in different types of cancers. Up-regulation of transgelin-2 was observed in pancreatic cancer [
8], colorectal cancer [
9], lung adenocarcinoma [
10,
11] and cervical squamous cell carcinoma [
12]. Previously, we found that transgelin-2 is highly expressed in PDAC tissues compared with adjacent normal tissues. High level of transgelin-2 is associated with poor prognosis in patients with PDAC [
8]. In contrast, down-regulation of transgelin-2 was observed in the tissues of Barrett’s adenocarcinoma patients [
13]. Therefore, specific upstream factors are involved in regulating the context-dependent expression of transgelin-2. Driver gene mutations play a key role in tumorigenesis. In general, cancer contain 2–8 of these key mutations [
14]. Although transgelin-2 is known to be involved in the development of cancer [
15], the relationship between transgelin-2 and driver gene mutation is not fully understood.
In the present study, we analyzed the relationship between KRAS and transgelin-2 in PDAC. We found that the protein stability of transgelin-2 was regulated by KRAS. ERK-mediated phosphorylation resulted in accumulation of transgelin-2 protein. These findings indicate transgelin-2 is a downstream target of KRAS signaling. KRAS-ERK-transgelin-2 axis may be explored for targeted therapy of PDAC.
Methods
Patients
This work was done with the approval of the Ethics Committee of Zhongshan Hospital. A total of 114 patients diagnoses with pancreatic cancer between 2003 and 2009 were enrolled in the study. Clinical characteristics including age, gender, anatomical location of tumor, histology of the tumor, lymph node involvement and metastasis status, were obtained from patient records. Patients who did not reach the outcome under study were censored at the date of their last visit. For the analyses of overall survival, each patient’s time began on the date of diagnosis and ended on the date of death or on the date last seen alive.
Immunohistochemical staining
Immunohistochemical staining of paraffin sections for transgelin-2 or SREBP-1 protein was performed with an LSAB kit (DAKO, Marseilles, France), using p-145-transgelin-2 antibody (dilution, 1:500) The sections were incubated in 3,3′ diaminobenzide tetrahydrochloride with 0.05% H2O2 for 3 min. Immunostaining scores were independently evaluated by three pathologists. Semi-quantitative scores were used to analyze antibody immunostaining. Intensity of staining was categorized into −, +, ++ or +++, denoting negative (0), weak (1), moderate (2) or strong staining (3). Extent of immunostaining was categorized into 0 (< 10%), 1 (10–25%), 2 (26–50%) or 3 (> 50%) on the basis of the percentage of positive cells. Three random microscopic fields per tissue were calculated. The final score of expression level was determined by the formula: final score = intensity score × percentage score. The final score was ranged from 0 to 9. The final score of ≤3 was defined as low expression, and > 3 as high expression.
KRAS mutation analysis
KRAS mutation was assessed using the Sanger sequencing. Formalin-fixed, paraffin-embedded tissue were taken, and 2 to 3 unstained 10-μm sections were used for DNA extraction. Genomic DNA was extracted using QIAamp DNA FFPE Tissue kit (Qiagen, Hilden, Germany). DNA were amplified in a HotStarTaq Master Mix (Qiagen) using the primers 5’-AAAAGGTACTGGTGGAGTATTTGA-3′ and 5’-CATGAAAATGGTCAGAGAAACC-3′. Cycling conditions of the PCR were as follows: initial denaturation at 95° for 5 min, followed by 35 cycles of 95 °C for 45 s, 58 °C for 45 s, 72 °C for 1 min and a final extension at 72 °C for 5 min. The purified PCR product was used for sequencing.
Cell lines and transfection
The cell lines BxPC-3 and HPDE6-C7 were purchased from the cell bank of the Chinese Academy of Sciences (Shanghai, China), and the other cells were obtained from the ATCC (Manassas, VA, USA).. The PDAC cell lines were maintained in DMEM or RPMI 1640 medium (Invitrogen) supplemented with 10% FBS (Invitrogen), 100 U/ml penicillin (Invitrogen), and 100 mg/ml streptomycin (Invitrogen). All transfections were performed with Lipofectamine 2000 (Thermo Scientific, Waltham, MA, USA) or Lipofectamine RNAiMAX (Thermo Scientific) transfection reagents. Cells were cultured until 40–50% confluence at the time of transfection. At 24–48 h after transfection, cells were harvested for quantitative PCR or western blot analysis. For gene knockdown experiments, control cells were incubated with OPTI-MEM and transfection reagent (vehicle group), or with OPTI-MEM and transfection reagent plus no-silencing siRNA (siRNA-NC group). The KRAS siRNA sequences are as follows: sense CAGCUAAUUCAGAAUCAUU, antisense AAUGAUUCUGAAUUAGCUG.
Real-time PCR
Total RNA was isolated and purified using an TRIZOL Reagent (Invitrogen). RNA quality was assessed using NanoDrop 2000 (Thermo Fisher Scientific, USA) and RNA integrity was assessed using electrophoresis through an agarose gel. The first strand cDNA was synthesized using 1 μg of RNA and SuperScript® III Reverse Transcriptase (Invitrogen). qRT-PCR was performed with SYBR Green PCR reagents on an ABI Prism 7900 detection system (Applied Biosystems, CA, USA). RNA levels were normalized to the level of β-actin or GAPDH and calculated as delta-delta threshold cycle (ΔΔCT). The primer sequences were as follows: transgelin-2 forward: GGAGATCTCTCCCCGCA, reverse TCCACTGGATCAGGATCTGC; KRAS forward: -TGACCTGCTGTGTCGAGAAT, reverse TTGTGGACGAATATGATCCAA.
Cell proliferation
Cell proliferation assay was performed as described previously [
16]. Briefly, 10
4 cells/well were seeded into six-well plates after 24 h transfection. Cell numbers were counted every 24 h. At least three independent experiments were performed. Growth curve assays were performed by counting live cells using trypan blue exclusion.
Western blotting
Cells were lysed in ice-cold lysis buffer (50 mM Tris-HCl, pH 7.4, containing 150 mM NaCl, 1% Triton X-100, and protease inhibitor cocktail) for 30 min on ice. Lysates were centrifuged at 2,0000 g for 30 min at 4 °C. The supernatant was mixed with SDS loading buffer (100 mM Tris-HCl, pH 6.8, 4% SDS, and 20% glycerol with bromophenol blue) and heated for 5 min. Proteins were separated by 12% SDS-PAGE gel and transferred to PVDF membranes. The membrane was blocked in 5% non-fat milk, and incubated with the intended primary antibody in TBS containing 0.1% Tween 20 (TBS-T) for 3 h. After washing with TBST-T, the membrane was incubated with HRP-conjugated secondary antibody for 1 h. After three washes with TBST-T, bands were visualized by chemiluminescence. The primary antibodies used in this study were: as follow anti-transgelin-2 (Novus Biologicals, CO, USA), anti-β-actin (Cell Signaling Technology, MA, USA), anti-Flag (Sigma-Aldrich, MO, USA), anti-GFP (Abcam, MA, USA), anti-ERK1/2 (Cell Signaling Technology, MA, USA), anti-phospho-ERK1/2-T202/y204 antibody(Cell Signaling Technology, MA, USA), anti-β-tubulin antibody (Abcam, MA, USA), anti-GST antibody (Abcam, MA, USA).
Co-immunoprecipitation
Collected cells were extracted for 30 min in ice-cold lysis buffer (50 mM Tris-HCl, pH 7.4, containing 150 mM NaCl, 1% Triton X-100) containing protease inhibitor cocktail (Sigma-Aldrich, MO, USA). After centrifuge at 2,0000 g for 30 min at 4 °C, supernantant was pre-cleared by incubation with Protein A/G-Sepharose (Sigma-Aldrich, MO, USA). Pre-cleared supernatants were incubated with transgelin-2 antibody or control IgG for 3 h at 4 °C. Then Protein A/G Sepharose was added to pull down the immune-complex. After washing, the immunoprecipiated proteins were analyzed by SDS-PAGE.
Fusion protein purification
E. coli strain BL21(DE3) was transformed with plasmid. While OD600 = 1.6, protein expression was induced by addition of IPTG (1 mM) for 4 h at 30 °C. Bacteria were harvested by centrifugation at 4, 000 g for 10 min at 4 °C. The cell pellets were suspended in GST extraction buffer (20 mM HEPES,pH 7.6,0.5 M NaC,0.5uM EDTA,10% Glycerol,0.5% NP-40) containing protease inhibitors. The supernatant was added with glutathione–conjugated bead slurry to incubate 3 h at 4 °C. After extensive washing with GST wash buffer. The fusion proteins were eluted by glutathione. The purified proteins were analyzed SDS-PAGE with coomassie staining.
In vitro kinase assay
Hek293T cells were transfected with Flag-ERK2 for 48 h. Then cells were lysed in buffer (50 mM Tris-HCl, pH 7.4, containing 150 mM NaCl, 1% Triton X-100). The cleared supernatant was incubated with ANTI-FLAG M2 Affinity Agarose Gel (Sigma-Aldrich, MO, USA) for 3 h at 4 °C. Then Immune complexes were washed 3 times with lysis buffer and assay buffer (50 mM Tris-HCl pH 7.4, 50 mM NaCl, 10 mM MgCl2, 1 mM MnCl2 and 10% glycerol). The GST-transgelin-2 fusion protein was purified as described previously [
17] Kinase reactions were carried out in 50 μl of assay buffer with 50 mM ATP at 30 °C for 1 h. The reactions were stopped by addition of 5X sample buffer, followed by boiling for 2 min The supernatants were analyzed by SDS-PAGE.
Antibody preparation
The p-S145-transgelin-2 antibody was raised against the synthetic phosphopeptide antigen encompassing ARDDGLFS*GDPNWFP, where S* represent phosphoserine. The peptide was conjugated to keyhole limpet hemocyanin and used to immunize rabbits. Phosphopeptide-reactive rabbit antiserum was first purified by protein A chromatography. The purified antibodies then were passed through a column coupled with the unphosphorylated peptide to deplete antibodies that react with unphosphorylated transgelin-2. The specificity of each antibody was confirmed by ELISA assay.
Xenograft tumor studies
BALB/c severe combined immunodeficiency mice were purchased from Shanghai Laboratory Animal Center. All experimental procedures using animals were in accordance with the guidelines provided by the Animal Ethics Committee of Zhongshan Hospital. Nude mice were subcutaneously injected with 5 × 106 cells expressing transgelin-2 wild-type and S145A mutant in conjunction with stable knockdown of endogenous transgelin-2 by shRNA on the dorsal flanks, respectively. Tumor growth was assessed with caliper measurement. Tumor volume was calculated according to the following formula: V = (Length x Width2)/2.
Statistical analysis
Normality of variables was tested with Shapiro–Wilk test. Normally distribute continuous variables were expressed as mean ± SD, and categorical variables were summarized as median with interquartile range. Quantitative variables with normal distribution were analyzed with one-way ANOVA with Tukey HSD as a post-hoc test. Comparisons between groups with categorical variables were evaluated by Kruskal-Wallis followed by Dunn test. Correlation analyses between continuous or categorical variables were performed by Pearson’s or Spearman’s, respectively. The association between non-parametric variables was assessed with Chi-square test. Parametric variables were compared with the independent samples t-test. P < 0.05 was considered to indicate a statistically significant difference.
Discussion
Despite intensive research efforts to target KRAS, the impact of redundancy and compensation pathways limits the clinical use of these drugs in PDAC. KRAS mutations drive the oncogenesis of PDAC through a constitutively activated MAPK pathway. During PDAC development, different downstream effectors are activated or repressed by the MAPK pathway. Our results provide a novel mechanism of KRAS addiction in PDAC and link the ability of mutant KRAS to promote pancreatic cancer cell proliferation with transgelin-2.
Oncogenic KRAS signaling is the main driving force of PDAC development. KRAS signaling is highly complex and dynamic, involving a variety of downstream effectors. The oncogenic KRAS signaling in PDAC is believed to through the three major pathways: Raf/Mek/Erk, PI3K/Pdk1/AKT and Ral guanine nucleotide exchange factor (RalGEF) pathways [
7]. Genetic inactivation of PDK1 is able to block KRAS-driven PDAC formation [
32]. Activation of Raf/Mek/Erk by Braf
V600E with KRAS
G12D mutation results in a more aggressive phenotype with more PanINs compared with KRAS
G12D alone [
33]. RalGEFs, which load GTP to small GTPases of the RAS superfamily, are necessary for KRAS-induced transformation of PDAC [
34]. Here, our data indicate that KRAS downstream effector, ERK2, directly binds and phosphorylates transgelin-2. The protein stability of transgelin-2 is regulated by KRAS through ERK. It should mentioned that other KRAS downstream effectors may be also involved in regulation of transgelin-2.
Transgelin-2 is one of the homologues of transgelin, an early marker of smooth muscle cell (SMC) differentiation. Transgelin-2 in humans has 64% amino acid sequence homology to transgelin [
35]. They have different cell-specific expression specificity. Transgelin is abundant in SMCs and fibroblasts, whereas transgelin-2 is predominantly expressed in epithelial cells. In PDAC tissues, both transgelin and transgelin-2 showed higher expression levels compared with adjacent normal tissues [
8,
36,
37]. These studies establish a close relationship between the transgelin family and the development of PDAC. Due to the high similarity of protein sequences, it is worth further studying whether transgelin play similar biological roles as transgelin-2 in PDAC. Here, we found that the turnover of transgelin-2 protein is regulated through ERK-mediated phosphorylation upon KRAS mutation. The S145 residue in transgelin-2 is unique compared with its homolog, and so it is unlikely that transgelin is regulated by KRAS signaling in this way. Upregulation of transgelin in PDAC may be regulated by other upstream factors.
Several studies have shown that high levels of transgelin-2 in cancer tissues are due to down-regulation of specific microRNAs. The transgelin-2 gene is a target gene for miRNA-1 and miRNA-133a [
38‐
41]. Most of these miRNAs are described as tumor suppressors and have the ability to inhibit cell proliferation by inhibiting transgelin-2. We have previously found that the transgelin-2 gene is a downstream gene for the SREBP-1 transcription factor. We observed that the inhibition of transgelin-2 gene transcription only partially inhibited the increase of transgelin-2 protein after KRAS activation. Therefore, the regulation of mRNA level does not completely explain the increase of transgelin-2 protein in PDAC cells. In fact, the transcription level in many cases is not sufficient to predict the protein level [
42]. Here, we found that KRAS stabilized transgelin-2 protein by inhibiting proteasome-mediated degradation. Further evidence is needed to reinforce the existence of this posttranslational machinery in other types of cancer. Gene transcription, miRNA regulation, or protein stability regulation may have different effects on transgelin-2 in different types of cancers.
The function of transgelin-2 in cancer cells is largely unknown. However, in other types of cells, transgelin-2 can exert its function of stabilizing actin. Transgelin-2 is highly expressed in both T-cells and B-cells. In addition, transgelin-2 levels can be used to differentiate B cell subsets [
43]. In T-cells, transgelin-2 stabilizes F-actin at the immunological synapse, thereby enhancing T cell activation and effector functions [
44]. In B cells, transgelin-2 also participates in T cell activation by stabilizing T cell-B cell conjugation [
45]. Transgelin-2 is also associated with nonalcoholic fatty liver disease (NAFLD), type 2 diabetes and hyperlipidemia. The levels of transgelin-2 are correlates with the severity of NAFLD [
46,
47]. We have previously identified that transgelin-2 is highly expressed in PDAC patients with type 2 diabetes. Transgelin-2 is also a target of lipid master regulator SREBP-1 [
8]. It appears that transgelin-2 is involved in the lipid metabolism of normal and malignant cells. Therefore, more insight should be provided on the molecular mechanism by which transgelin-2 regulates lipid metabolism. It is well-known that the homologue of transgelin-2, transgelin (SM22), plays an important role in migration and differentiation [
48]. Several drugs including statins, Salvianolic acid A, Paeonol and SB-T-121205 regulate cancer cell metastasis and are associated with transgelin-2 expression [
49‐
52]. Transgelin-2 is also involved in endothelial cell motility and tube formation, which involves the phosphorylation of myosin light chain followed by actin-myosin interaction [
49]. In addition, transgelin-2 is up-regulated in stromal cells of lymph node-positive breast cancer [
53]. Knockdown of transgelin-2 significantly inhibits invasion and metastasis of GBM cells. Mesenchymal related gene signatures are highly enriched in high transgelin-2 expression GBM tissues. And the mesenchymal phenotype of GBM cells can be reversed by transgelin-2 silencing [
54]. This is consistent with our observation that lymph node involvement correlates with high levels of S145 phosphorylation of transgelin-2 in PDAC tissues. However, in multivariate analysis, there was no significant correlation between S145 phosphorylation of transgelin-2 levels and lymph node involvement. Tumor size remained significantly correlated with S145 phosphorylation of transgelin-2. Thus, transgelin-2 plays a dominant role in the regulation of cell proliferation in PDAC, which may be different in other types of cancer.
In summary, we found that transgelin-2 expression was regulated by KRAS in PDAC. KRAS mutation led to accumulation of transgelin-2 protein through phosphorylation of S145 residue by ERK. The S145 phosphorylation of transgelin-2 was a prognostic marker of PDAC. High level of S145 phosphorylation predicts poor prognosis in patients with PDAC. In addition, targeting KRAS-ERK-trasngelin2 can be utilized for PDAC treatment in the future.