FHL3 promotes pancreatic cancer invasion and metastasis through preventing the ubiquitination degradation of EMT associated transcription factors

Pancreatic ductal adenocarcinoma (PDAC) is intractable due to its strong invasiveness and metastatic ability. Epithelial-mesenchymal transition (EMT) is the pivotal driver of tumor invasion and metastasis. The four-and-a-half LIM domain (FHL) family is involved in regulating transforming growth factor (TGF)-β and Ras signaling, which might control the EMT process. In this study, we found that higher expression of four-and-a-half LIM domains 3 (FHL3) predicted poor prognosis in PDAC. The decreasing of FHL3 changed the EMT phenotype by blocking the TGFβ/Atk/GSK3β/ubiquitin pathways. Interestingly, the GSK3β inhibitor could abrogate the role of FHL3 in the regulation of snail1 and twist1 expression, which implied that GSK3β plays a pivotal role in the FHL3-mediated EMT process. Furthermore, we found that FHL3 can directly bind to GSK3β, which weakened the interaction between GSK3β and snail1/twist1. We also found that the LIM-3 domain of FHL3 was required for the binding of FHL3 to GSK3β. Collectively, our study implied that FHL3, as a binding partner of GSK3β, promoted tumor metastasis in PDAC through inhibiting the ubiquitin-degradation of snail1 and twist1.


INTRODUCTION
Pancreatic ductal adenocarcinoma (PDAC) is an extremely malignant disease that is rarely diagnosed in an early stage, and the 5-year survival rate is lower than approximately 15% [1]. The surrounding tissue invasion characteristic of PDAC is an obstacle for clinical R0 resection, and chemotherapy or radiotherapy regimens cannot efficiently curtail the invasion lesions [2].
In this study, we investigated FHL3 expression in 49 paired PDAC samples. Then, we explored the effects of FHL3 on the EMT process and the underlying mechanisms of FHL3 in pancreatic cancer (PC) cell lines. Our aims were to clarify the role of FHL3 in oncogenesis in PDAC and to clarify the relationship between FHL3 and EMT.
FHL3 regulated EMT process through TGFβ1/ Akt/GSK3β/ubiquitin process but not through TGFβ1/smad2/3/smad4 pathway Next, our study found that the FHL3 knockdown made significant downregulation of TGFβ1 (>60% in PANC1_KD1 and BXPC3_KD1, p<0.001, Figure 4A1-2), smad2/3 (>50% in PANC1_KD1 and BXPC3_KD1, p<0.001, Figure 4A1-2) and smad4 (about 50% in  PANC1_KD1 and BXPC3_KD1, p<0.001, Figure 4A1-2). However, our study found there was almost no influence in the expression of smad2/3 in nucleus/cytoplasm rate in PANC1 and BXPC3 cell lines, and just not more than 20% decreasing of smad4, in nucleus/cytoplasm rate, as FHL3 knockdown ( Figure 4B1-2). As previous studies implied, snail1 was regulated by GSK3β-mediated ubiquitin degradation. Therefore, we explored the following research. In the following study, we found FHL3 knockdown changed the TGFβ1/Akt/GSK3β/ubiquitin pathway. As Figure 4C1-2 showed, FHL3 knockdown not only decreased the absolute level of phosphorylated Akt (ser473-Akt) more than 50%, but also relative level of phosphorylated Akt (ser473-Akt/Akt) more than 50% in PANC1_KD1 and BXPC3_KD1. Meantime, FHL3 knockdown was accompanied with more than 50% downregulation of phosphorylated GSK3β (ser9-GSK3β) and more than 2-fold upregulation of phosphorylated GSK3β (try216-GSK3β), whatever in absolute expression level or relative expression (p-GSK3β/GSK3β) ( Figure  4C1-2). This study implied that FHL3 knockdown decreased the TGFβ1 level, weakened the activity of Akt, result of which further enhanced the activity of GSK3β. In order to verify our study, we use GSK3β inhibitor, 1-Azakenpaullone, for next experiments. On the one hand, as Figure 4D1 showed, the activity of GSK3β was decreased with the increasing dose of inhibitor both in PANC1_KD1 and BXPC3_KD1. And, the level of snail1 and twist1 were also increased after treatment of GSK3β inhibitor ( Figure 4D1). Finally, EMT maker E-cadherin was significantly downregulated ( Figure 4D1). On the other hand, GSK3β inhibitor absolutely reversed the pancreatic cells migration ability which was blocked by FHL3 knockdown both in PANC1_KD1 and BXPC3_KD1 ( Figure 4D2).

FHL3 competitively binded to GSK3β by LIM-3 domain to inhibit ubiquitin process for maintaining the level of EMT associated transcriptional factors
In order to thoroughly determine the roles of FHL3 in the ubiquitin mediated degradation process of EMT associated TFs, we explored the physical interactions between FHL3 and proteins in the ubiquitin process. Coimmunoprecipitation (CO-IP) and mass analysis showed that FHL3 could interact with GSK3β (data not provided) and E3 ligase (RNF146) (data not provided). Based on these results, we performed experiments in HEK293T cells. As our study showed, the expression level of snail1 and twist1 were upregulated about 2-fold with an increased transfection dose of the FHL3-HA plasmid (0μg, 5μg and 15μg, Figure 5A1). Furthermore, as the transfection dose of the FHL3-HA plasmid increased, more FHL3 bound to GSK3β with less snail1/twist1 bound to GSK3β (decreased more than 50%, Figure 5A2); few FHL3 molecules bound to the negative control ( Figure 4A2). These results implied that FHL3 was directly involved in the ubiquitin mediated degradation of snail1 and twist1 through competitively binding to GSK3β to weaken the interaction between GSK3β and snail1/twist1. Furthermore, we explored the GSK3β binding ability of the pivotal domain in FHL3. We designed six truncated forms, as shown in Figure  4B, and transfected plasmids into HEK293T cells. Our study showed that only TF-3, TF-4 and TF-5, all of which contained the LIM-3 domain, could bind to GSK3β. In addition, due to LIM-3 domain deletion, TF-1, TF-2 and TF-6 could not bind to GSK3β ( Figure 5C). These results implied the LIM-3 domain was required for FHL3 binding to GSK3β.

FHL3 knockdown curbed pancreatic cancer cells growth and metastasis in vivo
Then, we validated the role of FHL3 in growth and migration of pancreatic cancer cells in vivo. As Figure  6A showed, 4 weeks after the orthotopic transplantation or tail intravenous injection of pancreatic cancer cells, the liver and lungs were harvested for tumor detection. During our study in vivo, there was a SCID mice died of unknown cause. In our result, we found orthotopic transplantation tumor of PANC1_KD1 cell was smaller more than 50% as compared with PANC1_NC cell (p<0.01, Figure 6B). And HIC staining of Ki67 for those tumor slices showed that PANC1_KD1 cell tumor held stronger staining signal than PANC1_NC cell tumor ( Figure 6C and Supplementary Figure 1). In addition, we also verified the expression level of FHL3 by HIC, which implied PANC1_KD1 cells exactly significantly lose the FHL3 ( Figure 6E and Supplementary Figure 1). Next, in the tumor metastasis model experiments, as Figure 6F1-a, b showed, green area were metastatic tumor from circular pancreatic cancer cells, which were from tail vein injection, and lung metastasis occurred in 1 of the 8 SCID mouse in PANC1_KD1 cells (12.5%), which occurred in 6 of 8 SCID mouse in PANC1_NC cells (75%), that mean FHL3 knockdown decreased the lung metastasis from circular tumor cells more than 50%. And we got the same result in BXPC3_KD1 cells ( Figure 6F1a, b). During the experiments of hepatic metastasis model, our study found occurrent rate of hepatic metastasis was 100% (8/8) in PANC1_NC cells group and 87.5% in BXPC3_NC cells group with which was only 37.5% in PANC1_KD1 cells group and 25% in BXPC3_KD1 cells group ( Figure 6F2-a, b). Those data showed that FHL3 maintained the invasion and metastasis ability in pancreatic cancer cell lines.

DISCUSSION
Pancreatic ductal adenocarcinoma (PDAC) is a malignant tumor with strong invasive ability and few AGING  (B1 and B2) WB assay of nucleuscytoplasm protein showed that FHL3 knockdown hardly changed the expression level of smad2/3 and smad4 in nucleus in PANC1_KD1 and BXPC3_KD1 cells. (C1 and C2) In PANC1_KD1 and BXPC3_KD1 cells, FHL3 knockdown exactly downregulated the absolute and relative expression of phosphorylated AKT (ser473-AKT) more than 50%, p<0.01; and also downregulated the absolute and relative expression of phosphorylated GSK3β (ser9-GSK3β) more than 50%, p<0.01; and upregulated the absolute and relative expression of phosphorylated GSK3β (try216-GSK3β) more than 2-fold, p<0.001. (D1) 0.25μM and 0.50μM GSK3β inhibitor almost eliminated the effect, promoting the TGFβ1/AKT/GSK3β/ubiquitin process, caused by FHL3 knockdown. As treated with GSK3β inhibitor, GSK3β (try216-GSK3β) and E-cadherin were downregulated, snail1 and twist1 were upregulated. (D2) 0.25μM and 0.50μM GSK3β inhibitor reversed the migration ability of PANC1_KD1 and BXPC3_KD1 cells. early diagnosis techniques, which make for tough obstacles to PDAC treatment. FHL proteins, which mediate protein-protein interactions, play paradoxical roles in tumor growth, invasion and chemoradiotherapy resistance. However, in our study, we showed that 1. FHL3 was a biomarker of progression in PDAC; 2. FHL3 plays a role in tumor growth; and 3. FHL3 promotes metastasis by upregulating the expression of EMT associated transcription factors in pancreatic cancer cell lines.
LIM domain-containing proteins, including the FHL family and lim only protein (LMO) family, both of which consist of only evolutionarily conserved LIM domains, play paradoxical roles in tumors. As previous studies have shown, FHL can restrain the expression of cyclinA/B/D/E, upregulate p21 and p27 expression, or inhibit the effect of CDC25 by directly binding to CDC25, and all of these effects initiate G1/2 phase arrest, which endows chemoradiotherapy resistance [12,15,16].
In addition, FHLs can enhance the transcriptional activation of TGF-β and smad2/3/4, and it can also directly enhance the phosphorylation of smad2/3 with the assistance of CK1δ, all of which increases the nuclear translocation of the smad2/3/4 complex [9]. The upregulated TGFβ pathway promotes EMT-TF expression dependent on smad2/3/4 at the transcriptional level [25]. In our study, our data showed the downregulation of TGFβ, smad2/3 and smad4 expression in the total cell lysates ( Figure 4A1-2), but there were few changes in nuclear translocation of smad2/3/4 ( Figure  4B1-2), both effects of which were triggered by FHL3 knockdown. We found weaker metastatic ability in pancreatic cancer cells (PANC1_KD1 and BXPC3_KD1 cells), both in vitro and in vivo experiments, after FHL3 knockdown ( Figure 3C, 3D; Figure 6F1-2). These results suggest that FHL3 regulated EMT process and tumor metastasis were not through TGFβ pathway. (+:5ug, +++:15ug), snail1 and twist1 in HEK293T for 48h followed by CO-IP assay, which showed the higher FHL3 expression was accompanied with the higher expression of snail1 and twist1, p<0.01; meantime, the higher FHL3 expression made less snail1 and twist1 which binding with GSK3β, p<0.001. (B) Truncated forms form FHL3. (C) Transfection with GSK3β and truncated forms for 48h in HEK293T cells, and the CO-IP assay showed only truncated forms which containing LIM-3 domain could bind with GSK3β. AGING  (F1a, F1b) lung metastasis from circular pancreatic tumor cells, and FHL3 knockdown made the lower occurrent rate of lung metastasis in PANC1_KD1 and BXPC3_KD1 groups, p<0.05. (F2a, F2b) hepatic metastasis from orthotopic transplantation tumor, and FHL3 knockdown made the lower occurrent rate of hepatic metastasis in PANC1_KD1 and BXPC3_KD1 groups, p<0.05. AGING However, we found that the mRNA levels of snail1 and twist1 were upregulated (data were not showed here), rather than downregulated, after FHL3 knockdown (data was not in here). Therefore, there was another pathway by which FHL3 regulated the expression of snail1 and twist1. As previous studies showed, FHLs can strengthen Akt expression and activity at the transcriptional level in TGFα-dependent and TGFαindependent ways [28]. Furthermore, FHLs is involved in Ras signaling [12,13], which is linked to complex cell biology processes, including the PI3K/Akt/mTOR, Wnt/β-catenin, and TGFβ/smad pathways [29]. Furthermore, GSK3β, a downstream target of these pathways, has been shown to be a pivotal player in snail1 and twist1 degradation [26,27,30]. Therefore, based on the above findings, our study explored the roles of FHL3 in the ubiquitin-mediated degradation of snail1 and twist1. As our study showed, FHL3 knockdown slightly downregulated Akt expression and significantly weakened Akt activity by strongly abrogating the phosphorylation of ser473 ( Figure 4C1-2). Accordingly, the enhanced activity of GSKβ was shown by the upregulation of the phosphorylation of try216 and the downregulation of ser9, accompanied by the upregulation of snail1 and twist1 expression ( Figure  3E1-2), which was also validated in the context of FHL3 overexpression ( Figure 5A1). Furthermore, we also found that the ability of FHL3 to regulate snail1 and twist1 was almost completely eliminated by a GSK3β inhibitor ( Figure 4D1-2). Furthermore, we found that FHL3 could interact with GSK3β and that FHL3 could compete with snail1 and twist1 to bind to GSK3β in concentration-dependent ways ( Figure 5A2). Then, we found that LIM-3 was the pivotal domain which was required for the combination of FHL3 and GSK3β, and LIM-3 domain might mediate the physical interaction between snail1/twist1, GSK3β and FHL3 ( Figure 5C). Therefore, we believe that FHL3 enhanced the stability of snail1 and twist1 through the TGFβ/Akt /GSK3β/ubiquitin pathway.
The upregulation of TGFβ/smad signaling can inhibit tumor growth in early phase of tumor, but promote tumor progression in middle-later phase of tumor. And, previous studies have shown the tumor-growth-inhibition effects of FHL1 and FHL2 was mediated by TGFβ/smad signaling [9]. In addition, FHL2 can also promote tumor growth by upregulating Ras signaling [12]. In our study, we found that decreased TGFβ/smad signaling failed to promote the tumor growth, instead of restraining tumor growth, induced by FHL3 knockdown.
Generally, our study showed the following highlights: 1) FHL3 lead to PDAC progression; 2) FHL3 maintained tumor growth in pancreatic cancer; 3) FHL3 elevated EMT-TFs to promote EMT process through the TGFβ/Akt/GSK3β/ubiquitin pathways but not TGFβ/smad2/3/4 pathway (Figure 7). In addition, we believe that FHL3 may be a risk factor for PDAC and that the LIM-3 domain may be used to restrain PDAC metastasis, which will supply a new treatment strategy for PDAC.

MATERIALS AND METHODS
Purpose for this study is to explore the roles of FHL3 in proliferation and metastasis about pancreatic cancer. All experiments were repeated, respectively, at least three times for gaining reliable data.

Pancreatic cancer samples preparation
This study was approved by The First Affiliated Hospital of Anhui Medical University Review Board and the ethics committees of Anhui Medical University. 49 matched paraffin-embedded tissue sections, 6 tumor paraffin-embedded tissue sections and 8 paired fresh frozen tissue were collected from tissue bank from January 2011 to January 2018. All patients with pancreatic ductal adenocarcinoma were confirmed by at least two pathologists.

Cell culture
Pancreatic cancer cell lines (PANC1, MIAPACA2, CFPAC1, and BXPC3) and normal pancreas ductal epithelial cell (HPDE) were gained from the cell bank of Chinese academy of science in October 2017 with STR matching analysis. PANC1 and MIAPACA2 were cultured in DMEM (Gibco, USA), CFPAC1 was cultured in IMDM (Gibco, USA), BXPC3 and HPDE was cultured in RPIM-1640 (Gibco, USA). All types of culture media were supplemented with 10% fetal calf serum and 100 units/mL penicillin and streptomycin, but 20% fetal calf serum for CFPAC1.

Cell proliferation and cytotoxicity assays
The cell proliferation was quantified by standard curve (0.1, 0.2, 0.4, 0.8, 1.0, 1.5, 2.0, 3.0×10 4 cells were detected optical density (OD) via cell counting kit-8 (Japan) after 24h transplanted into 96-wells plates, and then fit linear standard curve between log [cell quantity] and OD), cell cytotoxicity assays was performed via MTT assay, and the detail protocol described in our previous study (PMID29331423 and PMID29800682).

Quantitative real-time PCR (qRT-PCR)
Trizol RNA solation system (Invitrogen, USA) was used for total RNA extraction. The cDNA templates were synthesized through PrimeScript RT Reagent Kit (TaKaRa, China), and qRT-PCR was performed with a 7500 Fast™ System (Applied Biosystems, USA) using the Sensi Mix SYBR Kit (Bio-Rad, USA). The mRNA level was calculated via using (=2 -ΔΔCt ), and normalized to GAPDH. All of the sequences of primer were designed by Primer 5 soft, see in Supplementary Table 1.

Western blot analysis
Total protein extraction Cells were harvested by cytology brush, and lysed with RIPA lysis buffer (Sigma, USA) supplemented with phosphorylase and protease inhibitor mixture (Thermo, USA), quantified by the BCA assay. AGING

Cytoplasmic and nucleus protein extraction
Cells were harvested by Tyrisin (Invitrogen), then cytoplasmic and nucleus protein was extracted by Cytoplasmic and Nucleus Protein Extraction Kit (Thermal Scientific, USA) according to its protocol, quantified by the BCA assay.

Small interfering RNA (siRNA) and recombination plasmid (RP)
Small interfering RNA (siRNA) experiments 5 × 10 5 pancreatic cancer cells were transplanted into 6 wells plates for 24h, and then cells were transfected with three different sequences FHL3 siRNA (GenePharma, Shanghai, China) for 48h, 72h and 96h with Lipofectamine 3000 reagent (Invitrogen, USA) and Opti-MEM (Life Technologies, USA), according to the manufacturer's instructions for gaining the best transfection efficiency. Three siRNA sequences for FHL3 were listed in Supplementary Table 2.

Recombination plasmid experiments
Primers of FHL3, GSK3β, Snail1, Twist1 and RNF146, inserted into plasmid pcDNA 3.1(-) (Addgene), were designed with Primer 5 soft, see in Supplementary  Table 3. Briefly, cDNA templates were synthesized through PrimeScript RT Reagent Kit (TaKaRa, China); CDS of genes were amplified with PrimeSTAR® GXL DNA Polymerase (TaKaRa, China); thirdly, products were purified through SanPrep Column DNA Gel Extraction Kit (Sangon Biotech, China); fourthly, the purified products and plasmid were treated with restriction endonuclease (Xho1, EcoR5 and Xba1 were purchased from NEB, USA) respectively; fifthly, recombination of plasmids were performed through homologous recombination with Hieff Clone TM Plus One Step Cloning Kit (Yeasen Biotech, China). All primers were listed in Supplementary Table 3. 5 × 10 5 cells were transplanted into 6 wells plates for 24h, and then cells were transfected with RP for 48h, 72h and 96h with Hieff Trans TM Liposomal Transfection Reagent (Yeasen Biotech, China) for the best transfection efficiency, according to the manufacturer's instructions.

Immunohistochemistry staining and scoring standard
Experiments procedure of paraffin embedding, tissue section, hematoxylineosin (HE) staining and immunohistochemistry for FHL3 expression level were performed as previously described (PMID: 23200678 and 20571492). What more, the work concentration of antibody against FHL3 (Proteintech, China) was 1:150, and 1:200 for Ki67 proliferation index (Abcam). The protein expression level was assessed by Mean of Integrated Option Density (IOD) with Image-Pro R Plus. Briefly, all of the Immunohistochemical sections were photographed for three yields in the same standard, and then select Area of Interesting (AOI) and detect IOD to gain Mean of IOD (IOD/AOI, MI), normalized to positive control (vascular smooth muscle cells). Finally, FHL3 expression level was divided into high and low group according to Mean of MI.

Immunoprecipitation
1×107 Cells were harvested by cytology brush, and lysed with RIPA lysis buffer (Yeasen Biotech, 20118ES60) for protein supernatant, followed by adding immune magnetic beads (Anti-Myc, Anti-HA and Anti-Flag, Bimake) for continuous slight mixing in 4°C for 24h. And then gain immune magnetic beads with Magnetic frame (Bimake), followed by TBS washing. Finally, products were boiled before dissolved in 5x SDS (Yeasen) for 5-10 minutes for western blot assay.

Migration ability assay
Migration ability assays contain transwell and wound healing assay. For transwell, 5 × 10 4 cells, with special treatments or not, were transplanted into transwell plates (24-well, 8.0μm, Corning Incorporated, Corning, NY, USA) with 10% gradient of fetal calf serum for AGING 48h. And the detection procedure was same as our previous study (PMID29331423). Quantification of passed cell area was performed by Image-Pro R Plus. For wound healing assay, cells were seeded at least 90% fusion in 6-well plates, and scratched by 200ul pipette tip, then washed with PBS to remove shed cells for extra 96h culture (PMID29331423). Scratch area was quantified with Image-Pro R Plus.

Tumor growth experiments in vivo
Female athymic nude mice (4 weeks), gained from the SLAC (Shanghai, China), were randomly divided into four groups. 1 × 10 6 cells (PANC1_NC/KD1), in 100ul PBS, were injected into the tail of pancreas. All mouse was sacrificed and the orthotopic pancreatic tumors were harvested for detecting tumor volume (MaA×MiA 2 / 2; MaA=Major axis, MiA=Minor axis), and followed by being processed into frozen sections for HE staining, immunofluorescence staining and Ki67 staining.

Tumor metastasis experiments in vivo
Female SCID mice (4 weeks), gained from the SLAC (Shanghai, China), were randomly divided into eight groups. 1 × 10 6 cells (PANC1_NC/KD1, BXPC3_NC/ KD1), in 100ul PBS, were injected into pancreas tail vein for orthotopic-liver-metastasis tumor model, then all mouse were sacrificed and livers were harvested for HE staining and immunofluorescence staining after 4 weeks. For lung metastasis experiments, 1 × 10 6 cells (PANC1_NC/KD1, BXPC3_NC/KD1), in 100ul PBS, were injected into tail vein, then all mouse was sacrificed and lungs were harvested for HE staining and immunofluorescence staining after 4 weeks.

Statistics
All experimental data were presented as the means ± SD. Statistical Package for the Social Sciences version 21.0 (SPSS Inc., USA) was used for statistical analyses. ANOVA, paired t-test, Chi-square (x 2 ) test and nonparametric test (Mann Whitney U) for statistical analysis of different situations. Statistical significance was considered when p < 0.05 (*p < 0.05; **p < 0.01; ***p < 0.001). All histograms and curves were constructed with GraphPad Prism 6 software (GraphPad Software, La Jolla, CA, USA).