(20S)G-Rh2 Inhibits NF-κB Regulated Epithelial-Mesenchymal Transition by Targeting Annexin A2.

(1) Background: Epithelial-mesenchymal transition (EMT) is an essential step for cancer metastasis; targeting EMT is an important path for cancer treatment and drug development. NF-κB, an important transcription factor, has been shown to be responsible for cancer metastasis by enhancing the EMT process. Our previous studies showed that (20S)Ginsenoside Rh2 (G-Rh2) inhibits NF-κB activity by targeting Anxa2, but it is still not known whether this targeted inhibition of NF-κB can inhibit the EMT process. (2) Methods: In vivo (20S)G-Rh2-Anxa2 interaction was assessed by cellular thermal shift assay. Protein interaction was determined by immuno-precipitation analysis. NF-κB activity was determined by dual luciferase reporter assay. Gene expression was determined by RT-PCR and immuno-blot. EMT was evaluated by wound healing and Transwell assay and EMT regulating gene expression. (3) Results: Anxa2 interacted with the NF-κB p50 subunit, promoted NF-κB activation, then accelerated mesenchymal-like gene expression and enhanced cell motility; all these cellular processes were inhibited by (20S)G-Rh2. In contrast, these (20S)G-Rh2 effect were completely eliminated by overexpression of Anxa2-K301A, an (20S)G-Rh2-binding-deficient mutant of Anxa2. (4) Conclusion: (20S)G-Rh2 inhibited NF-κB activation and related EMT by targeting Anxa2 in MDA-MB-231 cells.


Introduction
Playing key roles in embryonic development, epithelial-mesenchymal transition (EMT) facilitates body formation and tissue differentiation [1][2][3][4]. EMT is triggered by a genetic phenotype shift from epithelial-like to mesenchymal-like in response to pleiotropic signal; cells acquire the migratory and invasive properties by modifying adhesion molecules [3][4][5]. During this process, EMT specific transcription factors (EMT-TFs), in company with other regulatory factors like histone modifier and non-coding RNA, modify the gene expression through different states along EMT [6][7][8][9][10][11]. With the transcriptional activation of EMT-TFs, the expression of adherent junction components and tight junction components are negative-regulated, followed by the alteration in cadherin intermediate filament composition and cellular adhesion status [1, 3,8,9]. EMT is also implicated in other physiological and pathological processes including would healing, tissue repair, fibrosis and cancer [1- 6,11,12]. Tumor metastasis is the leading cause for cancer associated mortality; EMT is the main impetus and the essential access within this duration [11,12]. Despite the enhanced migration capability and invasiveness, EMT protects cancer cells from senescence, apoptosis and immuno-response by enhancing stem-cell properties and triggers the metastasis and dissemination to long distance through

Transfection
Lipo3000 (Invitrogen) was used for lentivirus package, transient transfection in MCF-7 cells and dual luciferase reporter assay according to the reagent protocol.
For lentivirus transfection, pLVX, pMD2.G and psPAX2 were co-transfected into HEK-293-T cells and supernatant for 24 h and 48 h was collected then added into MDA-MB-231 cells.

Immuno-Precipitation
50 µL of Protein A/G Magnetic Beads (MCE, K0202) was washed with 400 µL of IP lysis buffer (Pierce) for 3 times. 5 µg of antibody for immune-precipitation was diluted with 500 µL of IP lysis buffer, then added to the prepared beads, followed by a rotation for 2 h at 4 • C. MDA-MB-231 cells and MCF-7 cells were collected and lysed with IP lysis buffer supplemented with Protease Inhibitor Cocktail (Roche, 04693124001) and 1-mM phenylmethanesulfonyl fluoride (PMSF). Cell lysis was centrifuged with 12,000× g for 20 min at 4 • C and the supernatant was collected. The antibody bonded beads were then collected and combined with cell lysis containing 500 µg of protein with a final volume of 400 µL, followed by another rotation for 2 h at 4 • C. The beads were then washed with IP lysis buffer for 3 times and collected for immuno-blot analysis.

Cellular Thermal Shift Assay
MDA-MB-231 cells and MCF-7 cells were cultured in 100-mm culture plates until the confluence reached 90%. The culture medium was then replaced with new medium supplemented with 15-µM (20S)G-Rh2 (~10 µg/mL) followed by an incubation for 1 h in a humidified 5% CO 2 atmosphere at 37 • C. After digested with trypsin (0.25%, w/v in PBS) and counted, cells were collected with centrifugation at 400× g for 5 min and re-suspended with PBS containing 1-mM PMSF to a final cell density of 2 × 10 7 cells/mL. Each 100 µL of cell suspension was added to a 200-µL tube and heated at indicated temperature for 3 min and incubated at 4 • C for another 2 min. After 2-time rapid freeze-thawing from −80 • C to 25 • C, cell suspension was centrifuged with 20,000× g for 20 min at 4 • C, the supernatant was collected for immune-blot analysis.

Dual Luciferase Reporter Assay
pNF-κB-TA-luc and pRL-CMV (10:1, w:w) were co-transfected into MDA-MB-231 cells and MCF-7 cells with Lipo3000 and cultured for 24 h before chemical treatment. The activity of luciferase was determined with Dual-Luciferase®Reporter Assay System (Promega, E1910) according to the manufacture's protocol. Luminescence generated by luciferase was collected via Infinite F200 Pro (TECAN).

Real-Time Polymerase Chain Reaction
Whole-cell RNA was isolated with TRIzol (Invitrogen). 2 µg of whole-cell RNA was proceeded with High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, 4368814) for cDNA synthesis followed by real-time PCR analysis via PowerUp SYBR Green Master Mix (Applied Biosystems, A25742) and 7500 Real-Time PCR System (Applied Biosystems). The primers used were shown in Table 2. Gene expression was normalized to that of GAPDH and visualized in histogram format.

Cell Migration Analysis
A culture-insert 2 well (Ibidi, 81176) was utilized for would healing assay to determine the migration capability of MDA-MB-231 cells. MDA-MB-231 cells were digested and re-suspended to a final density of 5 × 10 5 cells/mL. Cell suspension was added into chambers as well as outer area and cultured for 24 h for cell adhesion before the inserts were removed. Then the culture medium was replaced with new medium supplemented with indicated chemicals. Then the wound healing status was recorded by microscopy at 12 h, 24 h and 48 h after culture medium alteration.

Cell Invasion Analysis
A Matrigel Transwell invasion assay was performed to determine the invasiveness of MDA-MB-231 cells. A total of 10 4 cells in serum-free DMEM medium were seeded into upper chamber (8-µm pore, Corning, 3464) pre-coated with 1 mg/mL Matrigel Matrix (Corning, 354234) and DMEM medium containing 20% FBS was added into 24-well plate. After incubation for 24 h in a humidified 5% CO 2 atmosphere at 37 • C, cells were fixed with pre-cooled methanol for 30 min and stained with 0.1% crystal violet (w/v) in PBS for 10 min. Images were collected by microscopy to determine the cells passing through Matrigel basement.

Statistical Analysis
All data were obtained from independent triple-replicated experiments and presented as the mean ± standard deviation (SD). Significance was determined by a two-tail Student's t-test via GraphPad Prism v.6 (GraphPad Inc., USA) and data with p-value < 0.05 were considered of statistical significance.

Anxa2 Bound to NF-κB p50 Subunit in MDA-MB-231 Cells and MCF-7 Cells
It has been well-established that Anxa2 binds to p50 to promote NF-κB activation and cell survival in hepatocellular carcinoma and pancreatic cancer cell lines [28,29]. In order to tell the activity of Anxa2 in NF-κB associated EMT in breast cancer, we first assessed the interaction of Anxa2 with NF-κB p50 subunit in breast cancer MDA-MB-231 and MCF-7 cells by coIP analysis ( Figure 1A). As the N-terminus of Anxa2 is responsible for its interaction with p50 [29], we transiently transfected C-terminus myc-tagged full-length Anxa2 and an N-terminus deleted truncated version of Anxa2 (Anxa2-dN). Precipitated by anti-myc antibody, Anxa2-dN failed to interact with p50, demonstrating Anxa2 bound to p50 via its N-terminus in breast cancer cells as does in Hepatoma cells ( Figure 1B).

Anxa2 Promoted NF-κB Activation and Associated EMT in Invasive Breast Cancer Cells
We co-transfected cells with Anxa2 and dual luciferase reporter system for NF-κB activity analysis, the transfection of full-length Anxa2 enhanced NF-κB activity whereas that of Anxa2-dN failed (

Anxa2 Promoted NF-κB Activation and Associated EMT in Invasive Breast Cancer Cells
We co-transfected cells with Anxa2 and dual luciferase reporter system for NF-κB activity analysis, the transfection of full-length Anxa2 enhanced NF-κB activity whereas that of Anxa2-dN failed (Figure 2A Wound healing assay and Transwell invasion assays were then performed with Anxa2-over-expressing MDA-MB-231 cells. Full-length Anxa2 over-expression enhanced the wound healing efficiency and invasiveness through Matrigel basement whereas Anxa2-dN truncation over-expression showed no facilitation ( Figure 2D,E).

Discussion
Cancer metastasis present responsibility for over 90% cancer mortality. EMT-one of the main generators for cancer metastasis-has become an assignable factor in both cancer research and treatment [1,11]. Derived by EMT promoting genes, epithelial-like cancer cells re-program gene expression phenotype, lose epithelial feature, dissociate from primary tumor tissue and spread with circulatory system [2,[4][5][6]. A variety of gene transcriptional regulation was involved in this program, among which EMT-TFs and up-stream transcriptional factors like NF-κB and STAT3, directly alter the expression of key effector molecules [22,23,30,31].
Among them, multi-functional protein Anxa2 behaves as an implicated promoter for EMT and related cellular event. Anxa2 was first associated with cell migration for its heterotetramer with S100A10 [32,33], which promotes extracellular matrix degradation by activating plasmin and following researches indicate Anxa2 is responsible for membrane dynamics in invasive cells by binding to other S100 protein like S100A4 [34], S100A6 [35] and S100A11 [36,37]. Implicated in signaling transduction, phosphorylated Anxa2 at Tyr23 binds to STAT3 and promotes its phosphorylation at Tyr705 as well as following dimerization and nuclear translocation, resulting in up-regulated MMPs and enhanced EMT [38,39]. In addition, couple of researches have demonstrated Anxa2 content is correlated to cell migration capability [40][41][42][43].
It has been well-established that NF-κB bound to the promoter region of ZEB-1/2, SNAIL, SLUG, SIP1 and TWIST1 and the transcription of ZEB-1/2, SLUG, SIP1 and TWIST1 accords with NF-κB activation [22,23]. Anxa2, acting as an NF-κB co-activator in hepatocellular carcinoma and pancreatic cancer [28,29], was first identified bound to p50 subunit of NF-κB in breast cancer cells ( Figure 1A,B). Over-expression of Anxa2 in invasive breast cancer MDA-MB-231 cells accelerated NF-κB activation ( Figure 2A) and the expression EMT-TFs including SLUG, SIP1 and TWIST1 ( Figure 2B,C), followed by the down-regulation of E-cadherin and up-regulation of N-cadherin ( Figure 2B,C). Though containing NF-κB binding site, SNAIL somehow failed to follow NF-κB regulation, presenting differential variation trend as other EMT-TFs ( Figures 2B and 4B) [23]. MMPs responsible for the degradation of extracellular matrix and promote cell motility appeared to be up-regulated in according with EMT phenotype in Anxa2-over-expressing cells ( Figure 2B,C). These gene expression alteration resulted in the enhanced migratory properties and invasiveness ( Figure 2D,E). In contrast, no altered expression of EMT-TFs was observed in company with the activation of NF-κB in less aggressive MCF-7 cells (Figure 2A,B), indicating EMT-TFs expression requires additional regulatory mechanism, which is necessary to perform further investigation in the future study.
(20S)G-Rh2 is one of the most famous ginsenosides from ginseng extract for its remarkable anti-cancer activity.

Conclusions
Our data suggest that Anxa2 is an important co-activator of NF-κB in breast cancer cells. Over-expression of Anxa2 promoted NF-κB activation and pro-EMT gene expression. With the up-regulation of EMT-TFs and MMPs, MDA-MB-231 cells presented a mesenchymal-like phenotype and achieved enhanced migratory properties as well as invasiveness. (20S)G-Rh2 induced NF-κB inhibition by targeting Anxa2 and led to the suppression in pro-EMT gene expression, resulting in the EMT arrest at the epithelial state. Over expression of Anxa2-K301A maintained NF-κB activation and the mesenchymal-like phenotype under (20S)G-Rh2 treatment in MDA-MB-231 cells. Taken together, (20S)G-Rh2 targets Anxa2 in invasive breast cancer cells, inhibits NF-κB activity and EMT and may be a potent candidate for breast cancer treatment and related research.

Conflicts of Interest:
All authors declare no conflict of interest.