FHL3 Contributes to EMT and Chemotherapy Resistance Through Inhibiting Ubiquitination of Slug and Activating TGFβ/Smad-Independent Pathways in Gastric Cancer


 Background: Gastric cancer presents high risk of metastasis and chemotherapy resistance. Hence, the mechanistic understanding of the tumor metastasis and chemotherapy resistance is quietly important.Methods: TCGA database and clinical samples are used for exploring the role of FHL3 in disease progression and prognosis. The roles of FHL3 in metastasis and chemotherapy resistance are explored in vitro and in vivo by siRNA or shRNA treatment. Finally, we explore the FHL3-mediated EMT and chemotherapy resistance.Results: mRNA and protein level of FHL3 is significantly up-regulated in gastric cancer tissues when compares with it in adjacent tissue. Higher expression level of FHL3 companies with worse overall survival in gastric cancer. OPH resistance cells show higher level of FHL3 and mesenchymal phenotype. Knockdown of FHL3 slightly inhibits the cell growth, while it obviously sensitizes the chemotherapy in vivo and in vitro. In addition, down-regulation of FHL3 decreases the mesenchymal markers, such as Slug, Snail, Twist1, and vimentin, while increases the epithelial marker E-cadherin. For mechanism study, FHL3 knockdown down-regulates the expression level or activity of MAPK/ERK pathway and TGFβ/PI3K/Akt/GSK3β-RNF146/ubiquitin pathway in OPH resistance cells. Mesenchymal phenotype cells hold higher level of MDR1, and the FHL3 knockdown reverts the MDR1 in this type cell. Conclusion: FHL3 is a risk of disease progression in gastric cancer. MAPK and PI3K pathways were activated when FHL3 induces EMT and drug resistance process, but the TGFβ/Smad -dependent pathway did not participate in the process. FHL3 competitively bond the ubiquitin complex (slug/GSK3β/RNF146) with slug, inhibit ubiquitination of Slug.

Interestingly, the recent studies point out that EMT is not necessary for tumor invasion and metastasis, but is responsible for chemotherapy resistance [12,13]. And, the mechanism of EMT-associated chemotherapy is complex. Cancer stem cell (CSC) is a feature in mesenchymal phenotype tumor cells, which is closely related to chemotherapy resistance [14][15][16]. In addition, some regulatory factors directly related to EMT can promote chemotherapy resistance. EMT-TFs including Twist, Snail, and FOXC2 regulate chemoresistance by increasing the expression of ATP-binding cassette (ABC) transporters in breast cancer, which is important for chemoresistance [17,18]. And Slug can inhibit the activity of caspase-9 to endow chemoresistance to tumor cells [19]. However, single inhibition of EMT-TFs has few effects in reverting the chemoresistance. And it may be caused by other pathways which participate in the regulation of EMT process. According to previous studies, some EMT-associated pathways are also important in chemoresistance, such as PI3K/AKT pathway, MAPK pathway, and hypoxia pathway, through which chemotherapy resistance is probably induced by up-regulation of ABC transporters and down-regulation of apoptosis [20][21][22][23][24][25]. Thus, the mechanism of EMT-associated chemotherapy resistance is complicated and unknown, and more works are needed to gure out the mechanism of EMT-associated chemotherapy resistance.
LIM-only protein FHLs, including FHL1, FHL2, and FHL3, are characterized by evolutionarily conserved LIM domains and one conserved LIM superfamily domain [26]. FHLs are reported as the transcriptional factors, which participate in lots of signaling pathways. Simply, FHL1-2 are reported in regulation of TGFβ/Smad-independent pathway, such as PI3K/Akt, Wnt/β-catenin, and MAPK/ERK pathways, through which FHLs participate in EMT process and chemo-radio-therapy resistance in pancreatic cancer, breast cancer, and osteosarcoma [27,7,26]. Besides, FHL1-3 are considered as inhibitors of cell cycle checkpoints CDC25, through which they lead to radioresistance in pancreatic cancer and HeLa cell line [28]. In addition, some studies show that FHLs can interact with ER-α to make tumor progression in breast cancer [29,30]. Another study shows FHL2 can directly interact with epithelial phenotype marker ZO-1 to promote tumor invasion in breast cancer [31]. And in our previous study, FHL3 also acts as a regulator in ubiquitin degradation process of EMT-TFs through Akt/GSK3β/ubiquitin pathway in pancreatic cancer [7].
However, some previous studies have suggested that FHL1-3 perform as a tumor repressor in lung cancer, liver cancer, and breast cancer. As far as now, here without enough evidence to reveal the role of FHL3 in chemoresistance and metastasis in gastric cancer.
In fact, our previous study has shown that FHL3 is an important role in the regulation of EMT. In this study, we explore the potential relationship between FHL3 and EMT and chemotherapy resistance in gastric cancer. Simply, we explore the role of FHL3 in disease progression and overall prognosis by investigating TCGA database and clinical samples in GC. Then, we explored the effects of FHL3 on proliferation, metastasis, and chemotherapy resistance in GC cells in vitro and in vivo experiments. In addition, we investigate the underlying mechanisms of FHL3-mediated EMT process and FHL3-mediated chemotherapy resistance in GC cell lines.

Material And Methods
Gastric cancer sample preparation This study was approved by The First A liated Hospital of Anhui Medical University Review Board and the ethics committees of Anhui Medical University. 120 matched para n-embedded tumor tissue sections and 16 paired fresh frozen tissue were collected. All patients who underwent total or partial gastrectomy at the First A liated Hospital of Anhui Medical University from 2013 to 2016. All patients with gastric cancer were con rmed by at least two pathologists. Follow-up time was estimated from the date of surgical treatment to that of an event (i.e., patient death or tumor recurrence) or withdrawal.

Bioinformatic analysis
Gene pro les of GC and non-tumor adjacent tissues based on microarray were downloaded from Gene Expression Omnibus database (GEO; https://www.ncbi.nlm.nih.gov/geo/). TCGA and GTEx RNA sequencing FPKM data of GC, non-tumor adjacent tissues, and normal stomach tissues were downloaded from the UCSC Xena database (https://xenabrowser.net/hub/). The proteomic data of GC and non-tumor adjacent tissues were downloaded from PRIDE Archive under the accession number PXD011821. KEGG pathway analysis was performed using R clusterPro ler package. Gene Set Enrichment Analysis (GSEA) was performed by the JAVA program using gene sets collection (c2.cp.v7.1.symbols.gmt) from the MsigDB.
Cell culture and OPH-resistance cell lines Gastric cancer cell lines (SGC-7901, MGC-823, AGS, and N87) and normal gastric epithelial cells (GES-1) were obtained from the cell bank of the Chinese Academy of Science. All cell lines were cultured in RPMI-1640 medium (Gibco, USA). All culture media were supplemented with 10% fetal calf serum and 100 units/mL penicillin and streptomycin. Gastric cancer cell lines (HGC and SGC) are rstly detected the IC50 of OPH. Secondly, the cells are screened in this dose-treatment of OPH for at least 3 generates. Then, the OPH IC50 of those cells is re-detected, and the cells are screened in the this IC50 OPH again. The screen cycle is performed at least 6 times.

Western blot analysis
Total protein extraction: Cells were harvested with a cytology brush, lysed with RIPA lysis buffer (Sigma-Aldrich, USA) supplemented with a phosphorylase and protease inhibitor mixture (Thermo Fisher Scienti c, USA), and quanti ed by a BCA assay.
The standard detailed experimental process used for Western blotting was the same as that in our previous study. Western blot bands were quanti ed with ImageJ software (NIH, USA).

MTT assay
The cells in the logarithmic phase were plated onto 96-well plates at a density of 5000 cells per well in 200 µl of culture medium and incubated for 24, 48, and 96 h at 37 °C with 5% CO 2 . A volume of 20 µl MTT solution (5 mg/ml; Solarbio Science & Technology, Beijing, China) was added into each well and incubated for another 4 h. The MTT solution was then removed and 100 µl dimethyl sulfoxide (Sigma) was added to each well. The relative optical density (OD) was measured at 570 nm and the experiment was repeated three times.

Immuno uorescence
Brie y, 2.5 × 10 4 cells were seeded in 24-well plates for 24 h, xed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, and blocked with 5% BSA (Sigma-Aldrich) for 1 h at 37 °C. The samples were incubated with a primary antibody (FHL3, 1:200, Proteintech) overnight at 4 °C. Subsequently, the cells were washed with PBS and incubated with secondary antibodies for 1 h at room temperature before being washed again. Finally, the nuclei were stained with 15 µl DAPI (Sigma-Aldrich, USA) before detection with a uorescence microscope (Carl Zeiss, Germany).
Colony forming e ciency assay Firstly, cells were seeded in 6-well plates at a density of 1500 cells per well and incubated in 37 o C for 10 days. Then cells were washed with PBS and xed with 1 mL 4% formaldehyde solution. Then 1 mL crystal violet staining solution was added and washed with PBS for 3 times after 30 minutes.

Live & death staining
Cells were seeded in a 6-well plate and then the cells were transfected with three FHL3 siRNA for 48 h.
After that, AO and PI solutions were added into plates and maintained for 0.5 h before observed by an inverted uorescent microscope.
Wound-healing assay Cells were cultured in a six-well plate and grown to 90% con uence in 2 ml of culture medium. A 10 µl plastic tip was used to create an arti cial wound. Images were taken at 0, 24, and 48 h after scratching. The cell mobility = (0 h width-the indicated time points width) / 0 h width × 100%.
Transwell (Corning Life Sciences, Bedford, MA, USA) and Matrigel invasion (BD Biosciences) were used to assess GC cell migratory and invasive abilities respectively. For migration assays, 1 × 10 5 cells were added to 200 µl serum-free DMEM in the upper chamber and the lower chamber was lled with 600 µl culture medium. After incubation at 37 °C in an atmosphere containing 5% CO 2 for 24 h, the non-migrated cells were carefully removed with a wet cotton swab. Finally, the cells were stained with Giemsa (Sigma, USA) for 10 min followed by imaging and counting under an inverted microscope (100x magni cation).
The cell invasion assay was carried out similarly, while the invasion assay was coated with Matrigel before cells were seeded on the membrane.

Subcutaneous tumor model
Male nude mice (4 to 6 weeks old) obtained from the SLAC (Shanghai, China) were randomly divided into two groups. A total of 1 × 10 6 cells (FHL3-NC and FHL3-SH1 cells) in 100 µl PBS were injected into the subcutaneous. 4 weeks later, the mother tumors are harvested to make the same volume transplanted tumors. Then the FHL3-NC-derived and FHL3-SH1-derived transplanted tumors are transplanted into mice. The tumor volume is investigated every day. All mice were sacri ced, and the tumors are harvested to determine the tumor volume (MaA MiA 2 / 2; MaA=Major axis, MiA=Minor axis), followed by processing into sections for HE staining and Ki67 staining. Step Cloning Kit (Yeasen Biotech, China). Cells were transplanted into 6 wells plates for 24 h followed by transfection of RP for different times with Hieff TransTM Liposomal Transfection Reagent (Yeasen Biotech, China) to nd out the best transfection e ciency, according to the manufacturer's instructions.
A total of 1 × 10 7 cells were harvested with a cytology brush and lysed with RIPA lysis buffer (Yeasen Biotech, 20118ES60) to isolate the protein supernatant, followed by adding magnetic beads (Anti-Myc, × Anti-HA Bimake and Anti-Flag) with continuous slight mixing at 4 °C for 24 h. Then, the magnetic beads were isolated with a magnet (Bimake), followed by washing with TBS. Finally, the products were boiled before being dissolved in 5x SDS (Yeasen) for 5-10 minutes for Western blot assays.

Statistical analysis
All experimental data are presented as the mean ± SD. Statistical Package for the Social Sciences version 21.0 (SPSS Inc., USA) was used for the statistical analyses. ANOVA, paired t-test, Chi-square ( ) test, and a nonparametric test (Mann-Whitney U) were used for statistical analysis in different situations.

FHL3 is a disease progression factor and prognosis prediction biomarker in GC
To investigate the clinical relevance of FHL3 in GC, we systematically analyze multiple publicly available gene expression datasets (The Cancer Genome Atlas/Genotype Tissue Expression (TCGA/GTEx), GSE13861, GSE13911, GSE19826, GSE29998, GSE54129, and GSE63089), which contain > 1,000 gastric cancer patients. We notice that FHL3 mRNA expression is remarkably up-regulated in GC ( Fig. 1a, b, d, e, f and g, P < 0.001). In line with this observation, FHL3 protein expression is also signi cantly elevated in GC by proteogenomic analysis of Beijing dataset which contains 58 paired gastric tumor samples and nontumor adjacent tissues (PXD011821, Fig. 1h, P < 0.001). Furthermore, we explore the role of FHL3 in the progression of GC. We perform Kaplan-Meier analyses in both TCGA and KM-plotter cohorts, and the survival curves show that patients with higher expression level of FHL3 have shorter overall survival time than those with lower expression levels in TCGA (HR = 1.40, 95% CI = 1.15-1.51, P = 0.039, Fig. 1i) and KM-plotter cohorts (HR = 1.55, 95% CI = 1.31-1.84, P < 0.001, Fig. 1j).
To con rm this observation, we examine the FHL3 level in clinical gastric tumor samples. According to the IHC staining result, up-regulated FHL3 expression is positively associated with the tumor TNM stage (Fig. 2a). Consistently, the expression of FHL3 is dramatically up-regulated GC tissues when compares with it in non-tumor adjacent tissues in both 120 matched para n-embedded tumor tissue sections and 16 paired fresh frozen tissue (Fig. 2b, d and e, P < 0.05). In addition, according to the FHL3 expression and the follow-up time of 120 GC patients, Kaplan-Meier analysis indicates that higher expression of FHL3 leads to worse prognosis in GC patients (P < 0.001, Fig. 2c). In 16 paired GC tissue, higher expression level of FHL3 always accompanies with worse TNM stage (Fig. 2f, P = 0.0484).

FHL3 knockdown reduces OPH resistance in vitro
In order to investigate the relationship between the expression of FHL3 and chemotherapy in GC, we rst screen out the FHL3-high-expression GC cell lines. We detect the FHL3 expression level in a normal gastric cell line (GES-1) and several gastric cancer cell lines (SGC, HGC, AGS and N87). As the results show, FHL3 is high expression in all GC cell lines in contrast with GES-1, while SGC and HGC hold the rst and second highest level of FHL3 respectively (SGC-7901 > HGC > AGS > N87 > GES-1, Fig. 3a). Then we make OPH-resistance (OPH-R) cell lines in SGC and HGC cell lines. As the Fig. 2b and c show, OPH-R cell lines hold higher tolerance in OPH (HGC: P < 0.01, SGC: P < 0.05). Whereafter, we silence the FHL3 by lentivirus and verify the knockdown e ciency by western blot (WB) and immuno uorescence (IF) in those OPH-R cell lines. Both WB and IF assays show the same result that SH1 is most e cient in FHL3 knockdown, and the FHL3-SH1 cell lines join our following experiments (Fig. 3d-f). Colony formation assay and live & death staining assay are performed to detect the effect of FHL3-knockdown on OPH chemotherapy. In the colony formation assay, our study nds that OPH-R FHL3-NC cell lines have similar growth ability with the treatment of OPH and NS (Fig. 3g, i and j), while the FHL3 knockdown slightly decreases the colony unit formation (P < 0.05). Encouragingly, the combination treatment (FHL3 knockdown and OPH) restricts the tumor growth of about 80% in OPH-R HGC/SGC cell lines (Fig. 3g, i and j, P < 0.01). Collectively, FHL3 knockdown signi cantly enhances the OPH-induced tumor growth inhibition rate about 1-fold as comparing with the single OPH treatment group (Fig. g, i and j, P < 0.05). In the live & death staining assay, our study shows that obviously less live cells (green) and more death cells (red) in the combination treatment group, when compares with other groups (Fig. 3h, k and l, P < 0.01).
Down-regulation of FHL3 reverts the EMT phenotype and restricts tumor metastasis To investigate the association of FHL3 and EMT, we analyze the data in TCGA. And our study nd that the expression level of FHL3 is negatively correlated to the expression level of E-cadherin (r=-0.403, P < 0.001, Fig. 4a 1 ), and passively correlated to N-cadherin (r = 0.116, P = 0.025, Fig. 4a  and Slug (r = 0.358, P < 0.001, Fig. 4a 6 ). These data imply that FHL3 may contribute to EMT process. In and ZO-1 more than 1-fold, while it down-regulated the Vimentin, Slug, and Snail more than 50% (Fig. 4b 1 − 7 , P < 0.01). However, the Twist1 gets slight changes with the treatment of FHL3 knockdown. In the following experiments, our study nds that FHL3 knockdown restricts the cell migration more than 50% in HGC cell lines, and about 50% in SGC cell lines ( Fig. 4c and e 1 , P < 0.05). In the wound healing assay, we get similar results (Fig. 4d 2 and e 2 ).
FHL3 knockdown reduces OPH resistance and metastasis in subcutaneous/orthotopic stomach tumor bearing-model and lung metastasis model During our study in vivo, we nd subcutaneous tumors growth speed of OPH-R HGC cells is slower in single FHL3-SH1 group and single OPH group as compared with FHL3-NC group ( Fig. 5a and b, P < 0.05). Interestingly, combination treatment (FHL3-SH1 and OPH) signi cantly inhibits the tumor growth when compares with FHL3-SH1 + NS and FHL3-NC + OHP groups (P < 0.01, Fig. 5a-b). At the end of the test, we detect the tumor volume and weight in vitro. As the Fig. 5c- Then, we validate the role of FHL3 in metastasis of gastric cancer cells in vivo. As Fig. 6a shows, 4 weeks after the orthotopic transplantation and tail intravenous injection of gastric cancer cells, the orthotopic stomach tumors and lungs were harvested for tumor detection. In our result, we nd orthotopic transplantation tumor FHL3-SH1 was smaller more than 50% as compared with FHL3-SH1 when treated with OHP (P < 0.001, Fig. 6b, e, f). According to the white nodules on the surface of the lungs (Fig. 6c), we could easily nd the metastasis situ in mice of the FHL3-NC + OHP group. We made slices from these lung samples and performed immuno uorescence (Fig. 6d, g), among which the green area is metastatic tumor cells from the blood cycle which come from tail vein injection or orthotopic stomach situ. According to our observation, lung metastasis occurs in 1/5 SCID mouse in the FHL3-SH1 + OHP group (20%), while it occurs in 4/5 SCID mice in FHL3-NC + OHP (80%). Those results indicate us FHL3 knockdown decreases the lung metastasis from circular tumor cells more than 60%.

RNF146-mediated ubiquitin participates in FHL3-induced EMT process
In our previous study, we have found that FHL3 participates in Akt/GSK3β pathway-mediated ubiquitination degradation of EMT-TFs, and the E3 ligase RNF146 is rstly reported to interact with FHL3 in pancreatic cancer. Thus, we explore the role of RNF146 in FHL3-mediated stabilization of EMT-TFs in GC. As Fig. 7a shows, overexpression of RNF146 restricts the pcDNA-Slug recombination-plasmidmediated up-regulation of Slug about 50% (Fig. 8a and c, P < 0.01). In addition, overexpression of FHL3 signi cantly enhances pcDNA-Slug recombination-plasmid-mediated up-regulation of Slug ( Fig. 8a and c, P < 0.01). Furthermore, FHL3 overexpression eliminates the RNF146 overexpression-mediated downregulation of Slug ( Fig. 8a and c, P < 0.01). Then the CO-IP assay shows that RNF146 can interact both with FHL3 and Slug (Fig. 8a). Those results imply that RNF146 participates in the ubiquitination degradation of Slug, through which it interferes with EMT process.

FHL3 induces chemoresistance via MDR1
According to the previous studies, TGFβ/Smad-independent pathways containing MAPK/ERK and PI3K/Akt both participate in chemotherapy resistance, and those pathways have been pointed out to be probably associated with OPH resistance in GC basing on our above results. However, more details are needed to be nished to further gure out the FHL3-mediated chemotherapy resistance. So, we detect the  (Fig. 8e). The IHC shows that advanced GC with chemotherapy resistance have higher level of FHL3 and MDR when compare with early GC (Fig. 8f).

Discussion
Despite the morbidity and mortality of GC have declined over the past decade, we still face many problems and challenges in the screening and treatment of GC. TNM staging, encompassing the depth of invasion (T), lymph node metastasis (N), and distant metastasis (M) stages, is regarded as the most signi cant prognostic factor of GC. Because of tumor heterogeneity, even patients with the same pathologic stage of GC may have considerable differences in survival after complete surgical resection, indicating that prognosis cannot be accurately determined based on the current staging system alone.
Even after complete resection or targeted therapy, many advanced GC patients still die of local recurrence and/or distant metastasis. Among which, tumor metastasis and chemotherapy failure are the mainly severe problem all clinical doctors have to face. Although various genes and pathways have been researched in GC, the more powerful mechanisms are still needed to resolve the metastasis and chemotherapy resistance.
Recently, LIM domain-only protein family plays pivotal roles in tumor progression, including radiotherapy resistance and metastasis [7,28]. Some studies showed that FHLs play as a tumor repressor in breast cancer, liver cancer, and lung cancer [32][33][34], while other previous studies pointed out that FHLs promotes paclitaxel resistance and radiotherapy resistance in liver cancer cells and HeLa cells respectively [28,35], promote tumor cell growth in liver cancer, glioma and breast cancer [36,37], and promote metastasis in breast cancer cells and pancreatic cancer [7,30]. However, the role of FHLs in gastric cancer is still unclear. In our study, we nd the expression level of FHL3 is obviously up-regulated in GC both in mRNA and protein by analysis in TCGA, GTEx, and Beijing dataset (P < 0.05, Fig. 1a-h). Meantime, the same results are gained from 16 fresh-frozen tumor tissues and 120 para n-embedded sections (P < 0.01). Then, 120 samples show higher level of FHL3 refers to lower differentiation (P = 0.009, Table 1), metastasis trend (P = 0.002, Table 1) and worse stage of TNM (P = 0.039, Table 1) in GC. Besides, FHL3 is negatively associated with the prognosis in GC through Kaplan-Meier analysis in TCGA, KM-plotter cohorts and 120 GC samples (P < 0.05, Fig. 1i, j and 2c). Furthermore, the univariate analysis shows higher level of FHL3 accompanies with higher risk of GC progression ( Table 2, HR = 2.06, p = 0.005). In other words, those results suggest that FHL3 is a potential predictor of disease progression and prognosis in GC.
In the following experiments, the tumor growth is decreased ~ 50% in HGC and ~ 20% in SGC by the FHL3 knockdown (Fig. 3g, i and j). In subcutaneous tumor model, our study nds that FHL3 knockdown reduces the tumor growth ~ 25% in tumor volume and ~ 25% in tumor weight ( Fig. 5c and d). Besides, FHL3 knockdown enhances the cytotoxicity of OPH both in HGC and SGC (Fig. 3g, i and j), which is similar to the results in subcutaneous tumor model (Fig. 5a-d). In addition, our study nds the level of FHL3 is positive with the level of Slug, Snail, Twist1, N-cadherin and vimentin, while it is negative with the level of E-cadherin (Fig. 4a 1 − 6 ). And FHL3 knockdown obviously decreases the expression level of vimentin, Snail and Slug, and increases the level of E-cadherin and ZO-1 both in HGC and SGC (Fig. 4b 1 − and the level of E-cadherin is considered as the feature in EMT process [6,9]. E-box, a pivotal DNA reading frame in CDH1 sequence, through which some transcriptional factors can directly bind to CDH1 to regulate the expression level of E-cadherin, such as snail1/2, twist1/2, Zeb1 and FOXC2 [9]. Besides, other cellular junction protein also paly roles in tumor metastasis, such as N-cadherin and ZO-1. The regulation of EMT hold some recognized pathways, among which the TGFβ-mediated smad-dependent and smadindependent ways are major ones [9,6]. For TGFβ/smad-dependent way, smad complex directly regulates the expression level of EMT-TFs to promote EMT process [9,6]. Previous studies have showed that FHLs promote phosphorylation of smad 2/3 and directly interact with them to lead nuclear accumulation of smad complex in liver cancer, which is important in EMT induction [32]. For TGFβ/smad-independent way, PI3K/Akt, MAPK, NF-κB, Hedgehog and Wnt/β-catenin pathways are upstream regulators of EMT-TFs [38][39][40][41]. Some studies point out that FHLs can increase the activation and transcription of Akt to promote tumor growth and progression in glioma, breast cancer, and ovarian cancer [42,43,30]. Other published papers show that FHLs interfere the MAPK/ERK pathway to result in radiotherapy resistance in pancreatic cancer [44]. It is reported that FHLs knockdown contributes to tumorigenesis prevention in osteosarcoma by down-regulation of Wnt/β-catenin pathways [27]. And FHLs are also associated with hepatocarcinogenesis by activating NF-κB pathway [45]. Therefore, FHL3 is likely to regulate gastric cancer metastasis through the TGFβ/smad-independent pathway. Encouragingly, our study nds MAPK, PI3K/Akt and TGFβ pathways is close to FHL3 by KEEG and GO analysis ( Fig. 5a and b). And in the following experiments, FHL3 knockdown obviously down-regulates the phosphorylation level of MAPK downstream targets ERK1/2, P38 and JNK in HCG cells (Fig. 5c1-4). Besides, FHL3 knockdown obviously down-regulates the phosphorylation level of PI3K, Akt and GSK3β in HCG cells (Fig. 5d 1 − 4 ). However, FHL3 knockdown induced down-regulation of TGFβ has few effects in the phosphorylation level of Smad4. Collectively, FHL3-induced EMT is associated with the activation of MAPK/ERK/JNK/P38 and PI3K/Akt/GSK3β pathways. Furthermore, based on our previous study that FHL3 regulates the Akt/GSK3β/ubiquitin-Snail1/Twist1 pathway to stabilize the EMT-TFs to promote EMT process in pancreatic cancer, we explore the role of FHL3 in the regulation of ubiquitin-mediated EMT. Interestingly, we nd E3 ligase RNF146 can form a complex with FHL3 and Slug, and the up-regulation of FHL3 establishes the up-regulated RNF146-induced ubiquitination degradation of Slug ( Fig. 8a and b).
Well, the mechanism of chemotherapy resistance is complicated. On the one hand, ABC transporters, especially for MDR1, is a central role in chemo-drug e ux to make chemotherapy resistance in gastric cancer [46,47]. MDR1 is directly regulated by NF-κB pathway. And hypoxia also participates in the regulation of MDR1, through which up-regulation of HIF-α can lead chemotherapy in gastric cancer [23].
On the other hand, previous studies show that the inhibition of apoptosis is also important in chemotherapy, and those apoptosis-inhibition-mediated chemotherapy is regulated by MAPK pathway and PI3K/Akt pathway [20][21][22]. Besides, EMT is also considered to be responsible for chemotherapy resistance in pancreatic cancer and breast cancer [12,13]. Nevertheless, the characteristic changes of some signaling pathways in mesenchymal phenotype may be related to the chemotherapy resistance shown by those EMT phenotype cells. Up-regulation of EMT-TFs, such as Snail and Twist1, simultaneously increase the level of ABC transporters to endow chemotherapy resistance during its up-regulation of EMT process [19,17,18]. In fact, our study nds that down-regulation of FHL3 promotes the mesenchymal-epithelial transition (MET), during which it may reduce chemotherapy resistance in HCG cells. Furthermore, our study nds MDR1 is down-regulated in FHL3-knockdown process (Fig. 8b, e). In other words, those TGFβ/smad-independent pathways are the working regulators in FHL3-mediated chemotherapy resistance.

Conclusion
Collectively, as the scheme of our hypothesis shows (Fig. 8g) Figure 1 FHL3 is an independent risk factor of disease progression in gastric cancer. a-h The level of FHL3 in gastric cancer is analyzed in TCGA database, GTEx, and Beijing dataset; i, j The relationship between FHL3 expression level and prognosis is performed by Kaplan-Meier analysis in TCGA, KM-plotter cohorts.  The e ciency of the four FHL3 knockdown sequences in OPH-resistance cell lines by western blot and immuno uorescence, and the FHL3-SH1 is the most e cient sequence for down-regulation of FHL3; g, i and j OPH-resistance FHL3-NC/SH1 cell lines are treated with NS or OPH for 48h, which is followed by colony formation for 14 days; h, k and l OPHresistance FHL3-NC/SH1 cell lines are treated with NS or OPH for 48h, which is followed by live&death staining assay.    The FHL3-mediated OPH resistance in subcutaneous tumor model. a, b Same volume tumors are transplanted into subcutaneous in 4-weeks-age mouse for 2 weeks, and the tumor volume is detected every one day. c, d 2 weeks later, the mouse is harvested to investigate the tumor volume and weight in vitro; e The ki-67 staining assay is performed.