Representative images of DUSP5P1 expression detected by RNA FISH assay were shown in Fig. 1A. DUSP5P1 expression was significantly increased in primary gastric tumors as compared with adjacent non-tumor tissues (P < 0.01, Fig. 1B). DUSP5P1 expression was also significantly higher in metastatic lesions as compared to primary gastric tumors (P < 0.05, Fig. 1C). To evaluate the prognostic value of DUSP5P1, its expression was further examined in two cohorts of primary gastric tumor tissues by qRT-PCR (Cohort I: n = 112, Cohort II: n = 106). The optimal cutoff value of DUSP5P1 expression was determined by ROC curve analysis (P = 0.007, Fig. S1). High DUSP5P1 expression was found in 33.04% (37/112) of primary gastric tumors in cohort I, and 36.79% (39/106) in cohort II, respectively.
Fig. 1: DUSP5P1 upregulation in primary gastric cancers are associated with poor prognosis of gastric cancer patients.A Representative images of DUSP5P1 positive staining by RNA FISH in GC tissue. B DUSP5P1 expression was significantly upregulated in gastric tumors as compared to adjacent non-tumor tissues by RNA FISH. C DUSP5P1 expression was upregulated in metastasis lesions as compared to primary tumor tissues. D Kaplan–Meier survival analysis in GC patients with different DUSP5P1 expression status in cohort I. E Kaplan–Meier survival analysis in GC patients with different DUSP5P1 expression in cohort II. F Kaplan–Meier survival analysis in GC patients with different DUSP5P1 expression in TCGA dataset. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.001.
Kaplan-Meier survival curve indicated that patients with high DUSP5P1 expression had significantly shorter overall survival (OS) than those with low or silenced DUSP5P1 expression in Cohort I (P = 0.0118) (Fig. 1D) and Cohort II (P = 0.005) (Fig. 1E, left panel). High DUSP5P1 expression predicted a higher risk of cancer-related death by univariate Cox regression analysis (Cohort I: P = 0.014; Cohort II: P = 0.006) (Table S4). Multivariate Cox regression analysis showed that DUSP5P1 high expression was an independent poor prognostic factor for GC patients (Cohort I: P = 0.021; Cohort II: P = 0.002) (Table S5). Moreover, DUSP5P1 high expression was also associated with shortened progression-free survival (PFS) (P = 0.0154) (Fig. 1E, right panel). After stratification by tumor staging, high DUSP5P1 predicted poor prognosis in stage I-II GC patients both in Cohort I (P = 0.0201) (Fig. 1D) and in Cohort II (OS, P = 0.0203; PFS, P = 0.0482) (Fig. 1E).
Our findings were then validated in TCGA cohort (N = 406). Consistently, Kaplan-Meier survival analysis demonstrated that high DUSP5P1 expression predicted shortened OS and PFS for GC patients (OS: P = 0.0005; PFS: P = 0.0017) (Fig. 1F), especially for stage I-II GC patients (OS: P = 0.0103; PFS: P = 0.0391) (Fig. 1F). High DUSP5P1 expression was associated with poor prognosis of GC patients by univariate Cox regression analysis (P = 0.001, Table S6) and multivariate Cox regression analysis (P = 0.002, Table S7). These results indicated that high DUSP5P1 expression predicted poor prognosis in GC patients at an early stage.
DUSP5P1 promotes migration and invasion abilities of GC cellsDUSP5P1 was highly expressed in 5 out of 8 GC cell lines (AGS, HGC27, MKN74, NCI-N87, and MKN45), but was silenced in the normal gastric epithelial cell line GES1 (Fig. 2A). Ectopic expression of DUSP5P1 in GES1, BGC823, and MGC803 cells promoted cell migration (Fig. 2B1) and invasion (Fig. 2B2). Moreover, we further co-cultured GES1, BGC823, and MGC803 cells expressing control vector or DUSP5P1 together with red fluorescence protein (RFP) with normal liver cells LO2, respectively. Ectopic expression of DUSP5P1 in GES1, BGC823, and MGC803 cells led to RFP + colonies significantly larger in diameter as compared with control cells (Fig. 2B3). Conversely, DUSP5P1 knockdown in MKN74, MGC803, and AGS cells suppressed cell migration and invasion (Fig. 2C1 and C2). These phenotypic effects were confirmed by Western blot showing that DUSP5P1 enhanced the protein expression of epithelial-mesenchymal transition (EMT) markers β-catenin, Snail, Slug, and Claudin-1, whilst expression of E-cadherin was reduced (Fig. 2B4). In contrast, silencing of DUSP5P1 mediated opposite effects on these EMT markers (Fig. 2C3). These results suggested that DUSP5P1 played an important role in promoting pro-metastatic properties of GC cells.
Fig. 2: DUSP5P1 promotes GC cell migration, invasion in vitro and metastasis in vivo.A DUSP5P1 was expressed in GC cell lines but not in the normal gastric cell line GES-1 by RT-PCR. B Representative images of migration and Matrigel invasion transwell assay revealed that ectopic expression of DUSP5P1 promoted cell migration (B1), cell invasion (B2) and RFP-tagged GC cell colonies in co-cultures with LO2(B3) in GES1, BGC823, and MGC803 cells, accompanied by enhanced protein levels of β-catenin, Snail, Slug and Claudin-1, and reduced level of E-cadherin (B4). C Representative images of migration and Matrigel invasion transwell assays revealed that knockdown of DUSP5P1 inhibited cell migration (C1) and invasion in MKN74, MGC803 and AGS cells (C2), accompanied by reduced protein levels of β-catenin, Snail, Slug and Claudin-1, and enhanced level of E-cadherin (C3). D Representative macroscopic appearance and histological confirmation by HE staining of lung metastasis of GC cells. DUSP5P1 expression in BGC823 cells significantly increased the number of metastatic lesions in the lungs. E Representative macroscopic appearances of peritoneal surfaces implanting and HE stained images injected with BGC823 cells transfected DUSP5P1 and control vector were shown. F Representative macroscopic appearances of lung metastasis and HE stained images of the lungs injected with MKN45 cells transfected shDUSP5P1 and shNC were shown. G Representative macroscopic appearances of peritoneal surfaces implanting and HE stained images injected with MKN45 cells transfected shDUSP5P1 and shNC were shown. H Representative HE stained images of the liver were shown. Silencing of DUSP5P1 in MKN45 cells significantly reduced the number of metastatic lesions in the liver. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.001.
DUSP5P1 promotes GC cells metastasis to the peritoneum, lung, and liver in vivoTo investigate the tumorigenic ability of DUSP5P1 in vivo, we injected BGC823 cells with stably expressing DUSP5P1 or control vector into the tail vein of nude mice. After four weeks, histological examinations demonstrated that confirmed that 90% (9/10) of the mice bearing BGC823-DUSP5P1 produced lung metastases, while only 40% (4/10) of the mice in BGC823-control vector cells exhibited lung metastases (P < 0.05) (Figs. 2D, S2). Moreover, the number of metastatic lesions was significantly higher in DUSP5P1-overexpressing group than in control vector group (P < 0.05) (Fig. 2D). We further assessed the effect of DUSP5P1 on peritoneum metastasis in vivo through abdominal implantation. Consistently, more tumor nodules were observed in the peritoneum of the DUSP5P1 group as compared to control group (Figs. 2E, S3).
To confirm the effect of DUSP5P1 on metastasis, we evaluated the metastatic capacity of MKN45 cells with or without DUSP5P1 knockdown in three experimental metastasis models using tail vein (lung), abdominal (peritoneum) or intrasplenic (liver) injection, respectively. At the end of experiment, fewer tumor nodules were observed on the lung (Figs. 2F, S4) and peritoneal surfaces (Figs. 2G, S5) in shDUSP5P1 group as compared to shNC group. Histological examination showed that the average number of metastatic lesions in the lung, liver and peritoneum from shDUSP5P1 group was significantly decreased as compared with shNC group (Fig. 2F–H). Collectively, these results indicated the important role of DUSP5P1 in promoting GC metastasis.
DUSP5P1 induces focal adhesion and MAPK signaling cascadesTo understand the molecular mechanisms of DUSP5P1 in promotion of GC metastasis, global gene transcriptional profiling was analyzed by RNA-sequencing in DUSP5P1-ectopic expressing MGC803 cells and their control counterparts. Our result showed that ectopic expression of DUSP5P1 significantly dysregulated twelve signaling pathways including focal adhesion, MAPK signaling, pathway in cancer, FOXO signaling, Cell adhesion molecules, tight junction, p53 signaling, platinum drug resistance, Ras signaling, AMPK signaling, mTOR signaling and Hippo signaling pathways. Among them, focal adhesion and MAPK signaling were the most enriched pathways (Fig. 3A). The major differentially expressed genes were MAPK8, ARHGAP5, PDPK1, MAPK14, PDGFRB, LAMA1, CAV1, THBS1, MYLK3, COL1A1, COL4A4, PAP1B (focal adhesion) and MAP3K13, MAPK8, PDGFRB, HSPA1A, DUSP3, RAP1B (MAPK signaling) (Fig. 3B). Consistently, DUSP5P1 increased protein expression of key regulators of focal adhesion and MAPK signaling pathways, including Paxillin, FAK, p-ERK1/2, p-p38, and MYC in MGC803, BGC823 and GES1 cells (Fig. 3C). On the contrary, DUSP5P1 knockdown significantly inhibited protein expression of these factors in MGC803, MKN74, and AGS cells (Fig. 3D). We then tested whether the inhibition of focal adhesion or MAPK pathways could blunt the tumor-promoting effect of DUSP5P1. As shown in Fig. 3E, treatment with focal adhesion inhibitor (FAK inhibitor) abolished the promoting effect of DUSP5P1 on cell migration in BGC823, MGC803, and GES1 cells. Consistent results were obtained using MAPK pathway inhibitors targeting p38 and ERK (Figs. 3F1 and F2). Hence, DUSP5P1 exerts the tumor-promoting effect by activating focal adhesion and MAPK signaling.
Fig. 3: DUSP5P1 activates focal adhesion and MAPK signaling pathway.A KEGG pathways enriched by differentially expressed genes affected by DUSP5P1. B Enriched differentially expressed genes of focal adhesion and MAPK signaling pathway. C DUSP5P1 re-overexpression promoted focal adhesion and MAPK pathway as evidenced by protein expression of factors in MAPK signaling pathway in AGS and GES1 cells by Western blot. D DUSP5P1 knockdown inhibited the MAPK pathway as evidenced by decreased protein expression of focal and MAPK signaling pathway. E Effect of DUSP5P1 on gastric cell migration in the presence or absence of FAK inhibitor (VS6063) in GES1, MGC803, and BGC823 cells (20μmol/L). F Effect of DUSP5P1 on gastric cell migration in the presence or absence of MAPK inhibitor. Effect of DUSP5P1 on gastric cell migration in the presence or absence of ERK inhibitor (PD98059) in GES1 (100 μmol/L), MGC803 (150 μmol/L) and BGC823 (150 μmol/L); Effect of DUSP5P1 on gastric cell migration in the presence or absence of p38 inhibitor (BIRB796) in GES1, MGC803, and BGC823 (100 μmol/L). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.001.
ARHGAP5 is a direct downstream target of DUSP5P1We next analyzed the cellular localization of DUSP5P1 by RNA FISH after nuclear and cytoplasmic fractionation. Twenty-six probes of DUSP5P1 were designed for RNA FISH (Table S2). RT-PCR after RNA nuclear and cytoplasmic fractionation and RNA FISH results showed that DUSP5P1 is mainly located in the nucleus of GC cells (Fig. 4A1 and A2). To identify the genomic interactions of DUSP5P1 with its downstream effectors, ten probes complementary to the lncRNA transcript used to purify chromatin bound DUSP5P1 were designed and divided into odd and even groups. ChIRP-seq was then performed in MGC803 cells transfected with DUSP5P1 vector. Eighty-nine DUSP5P1-binding DNA candidates were identified in both odd and even probes group (Fig. 4B). The dysregulated genes were mainly enriched in cell proliferation, cell cycle, cell apoptosis, cell invasion, which involved in MAPK, WNT and focal adhesion pathways (Fig. 4C, D). Among the candidate genes, eighty-nine genes were found to be regulated by DUSP5P1 in RNA-seq analysis (Fig. 4D). Representative visual images of the enrichment feature of the target regions are shown in Fig. 4E1. Conventional ChIRP-PCR assay confirmed that DUSP5P1 bound to DNA motifs within ARHGAP5, COL4A4, NRTN, and PIP5K1B genes (randomly selected region of GAPDH as control) (Fig. 4E2), indicating that DUSP5P1 may regulate transcription of these candidate genes. Based on the results of both RNA-seq and KEGG pathway analysis, ARHGAP5 were chosen for further investigation. Motif analysis was conducted using the MEME online motif comment tool to discover binding motifs. Typical NFAT ‘core’ binding motifs (and their reversed strands) were identified in odd or even probes group, respectively. Among them, four motifs were identified, including TCAAGT/CGA, TATGTG, ATGATG, and CT/CGT/CCTC (Fig. 4F). DUSP5P1-binding sites in ARHGAP5 DNA sequence were mapped to the promoter region with two binding motifs (TATGTG, CT/CGT/CCTC) (Fig. 4G1). To further validate the regulatory effect of DUSP5P1, we cloned these binding motifs into the promoter region of the pGL3 reporter for luciferase assay. As shown in Fig. 4G2, DUSP5P1 significantly altered the activity of TATGTG motif in MKN45 and BGC823 cells, while motif deletion significantly diminished DUSP5P1-mediated the activity of TATGTG as evidenced by luciferase reporter assay. These results demonstrated that DUSP5P1 directly binds to the promoter of ARHGAP5 and upregulates its transcription.
Fig. 4: ARHGAP5 is the direct downstream target of DUSP5P1.A Analysis of DUSP5P1 location. A1: DUSP5P1 was mainly located in the nucleus of GC cells by RT-PCR after nuclear and cytoplasmic fractionation. A2: Representative images of nuclear localization of DUSP5P1 in GC tissues by RNA FISH. A Nuclear localization of DUSP5P1 in DUSP5P1 overexpressed BGC823, GES1 and MGC803 cells following DUSP5P1 transfection by RNA FISH. B Flow chart of ChIRP-sequencing assay and RNA-sequencing data analysis of binding site for DUSP5P1 to select the direct downstream target genes of DUSP5P1. C The function and pathway enrichment of DUSP5P1 regulated target genes. D Downstream targets of DUSP5P1 identified by ChIRP-sequencing and RNA-sequencing. D1: The enrichment fold change of even and odd probes by ChIRP-sequencing and D2: the corresponding change of mRNA expression, respectively. E Validation of DUSP5P1 and binding target region of the downstream genes. E1: Representative visual images of enrichment feature of the target regions; E2: ChIRP-PCR was performed to determine the interaction between DUSP5P1 and target region of the downstream genes. F Motif analysis of target sequence by ChIRP-sequencing. G Analysis of motif ARHGAP5 binded. G1: The binding region and the motif analysis of the ARHGAP5 gene. G2: Luciferase reporter assay showed that DUSP5P1 binds to the “TATGTG” motif. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.001.
Pro-metastatic function of DUSP5P1 is dependent on upregulation of ARHGAP5Ectopic expression of DUSP5P1 significantly upregulated transcription and increased the expression of ARHGAP5 in BGC823, MGC803, and GES1 cells (Fig. 5A, B). Moreover, DUSP5P1 expression was positively correlated with the mRNA expression of ARHGAP5 (P < 0.001, Fig. 5C). ARHGAP5 protein expression was significantly higher in primary gastric tumors as compared with adjacent non-tumor tissues by immunohistochemical (IHC) staining (P < 0.05, Fig. 5D). Depletion of ARHGAP5 in MKN74 and AGS cells with normal endogenous DUSP5P1 expression (Fig. 5E) significantly decreased cell migration and invasion (Fig. 5F). We then assessed whether the pro-metastatic function of DUSP5P1 in GC was dependent on ARHGAP5. ARHGAP5 knockdown in DUSP5P1-overexpressing BGC823 and MGC803 cells significantly blunted the promoting effects of DUSP5P1 on cell migration and invasion (Fig. 5G1), while ARHGAP5 overexpression significantly diminished DUSP5P1 KO-mediated inhibition of migration in MGC803 and BGC823 cells (Fig. 5G2), indicating that the tumor-promoting effect of DUSP5P1 is at least in part dependent on ARHGAP5 in GC.
Fig. 5: DUSP5P1 exerts tumor-promoting function partially depending on the ARHGAP5.A, B ARHGAP5 mRNA A and protein expression B were correlated with the DUSP5P1 expression. Ectopic expression of DUSP5P1 increased the mRNA and protein expression of ARHGAP5 in BGC823, MGC803 and GES1 by qRT-PCR and Western blot. C DUSP5P1 expression positively correlated with ARHGAP5. D Representative images of ARHGAP5 protein expression in primary GC tissues by IHC (left panel). ARHGAP5 expression significantly higher in the GC tissue as compared with adjacent non-cancer tissues (right panel). E Successful knockdown of ARHGAP5 in AGS and MKN74 cells was confirmed by RT-PCR and Western blot. F ARHGAP5 knockdown inhibited cell migration and invasion. G Pro-metastatic function of DUSP5P1 is dependent on ARHGAP5. (G1) Knockdown of ARHGAP5 significantly blunted the promoting effects of DUSP5P1 on cell migration and invasion. (G2) Effect of DUSP5P1 knockdown on migration ability with or without ARHGAP5 overexpression was investigated. ARHGAP5 overexpression significantly blunted the inhibiting effects of DUSP5P1 knockdown on cell migration. H ARHGAP5 activated MAPK signaling. H1: ARHGAP5 activated MAPK signaling as evidenced by SRE luciferase reporter assay (left panel); H2: The effectors of MAPK signaling pathway regulated by ARHGAP5 identified by MAPK signaling pathway PCR array; H3: Correlation analysis of ARHGAP5 and effector genes form TCGA database. I Effect of DUSP5P1 on MAPK pathway with or without ARHGAP5 knockdown was detected by SRE luciferase reporter assay. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.001.
Given that focal adhesion promotes MAPK pathway, the effect of ARHGAP5 on MAPK pathway was investigated by SRE luciferase reporter assay. ARHGAP5 depletion reduced MAPK activities in both AGS and MKN74 cells (Fig. 5H1). The effects of ARHGAP5 on MAPK signaling components are investigated using MAPK pathway PCR array by qRT-PCR (Fig. 5H2). The major differentially expressed genes such as NLK, NRAS, MAPK9, MAPK14, RPS6KB1, CREB1, ATF2, and MAPK1 were further confirmed by TCGA dataset (Fig. 5H3). Moreover, knockdown of ARHGAP5 significantly diminished DUSP5P1-mediated MAPK signaling activation as evidenced by luciferase reporter assay (Fig. 5I).
DUSP5P1 is a potential therapeutic targetPrevious reports indicated that focal adhesion molecules, MAPK signaling pathway and specific lncRNA in tumor cells induced resistance to chemotherapy [13, 14]. Our results also showed that DUSP5P1 significantly dysregulated platinum drug resistance pathway (Fig. 3A). We thus asked whether DUSP5P1 affects chemotherapeutic efficacy in GC cells. GES1 and BGC823 cells stably transfected with DUSP5P1 were treated with Oxaliplatin (5μmol for 6 days). We found that DUSP5P1 overexpression partially counteracted the cytotoxic effect of Oxaliplatin in both GES1 and AGS cells (Fig. 6A1). Moreover, DUSP5P1 ectopic expressing GC cells also demonstrated Oxaliplatin resistance over a range of drug dosages (Fig. 6A2). To test if DUSP5P1 is a potential therapeutic target for platinum drug-resistant GC, we established five personalized platinum drugs resistant PDO models generated from advanced GC cases with different clinicopathological features (Table S8). As shown in Fig. 6B, knockdown of DUSP5P1 significantly suppressed the GC cell growth in the three of five platinum drugs resistant PDO models (Fig. S6). Moreover, ARHGAP5 overexpression significantly diminished DUSP5P1 KO-mediated inhibition of cell growth after Oxaliplatin treated (Fig. S7). Our findings suggested that DUSP5P1 may serve as a potential target in platinum drugs resistant GC patients.
Fig. 6: DUSP5P1 is a potential therapy target in platinum drugs resistant GC cells.A DUSP5P1 transfected GC cells was partially resistant to the chemotherapy effect of Oxaliplatin by MTT assay. B DUSP5P1 Knockdown suppressed the GC PDO cell growth in the three of five platinum drugs resistant models. C Combination therapy of Oxaliplatin and DUSP5P1 depletion significantly inhibited the migration of the GC cells. D Representative HE staining image of the lung metastasis revealed metastasis synergistic inhibiting effect of Oxaliplatin and DUSP5P1 depletion in vivo. E Proposed mechanistic scheme of DUSP5P1 promoting the GC progression. DUSP5P1 promoted the expression of ARHGAP5, by directly binding to promoter region, which further promotes the activity of focal adhesion and MAPK signaling pathway. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.001.
The synergistic effect of the combination of DUSP5P1 depletion and Oxaliplatin on cell migration was then investigated. MKN74, MGC803 and AGS cells with stable knockdown of DUSP5P1 were treated with Oxaliplatin at 25 μmol for 36 h. Combined Oxaliplatin treatment and shDUSP5P1 showed a synergistic effect on suppressing the cell migration as compared with DUSP5P1 depletion alone (Fig. 6C). To confirm the synergistic effect of DUSP5P1 on metastasis in vivo, we inoculated nude mice with MKN45 cells administration stably transfected with shDUSP5P1 or shNC via tail vein, and portal vein, and treated with 5 mg/kg Oxaliplatin (i.p.) or 5% glucose (i.p., vehicle), respectively. Combination of Oxaliplatin and shDUSP5P1 showed a synergistic effect on suppressing lung metastasis as compared with DUSP5P1 depletion or Oxaliplatin treatment alone (Figs. 6D, S8). Collectively, these results demonstrated the synergistic effect of DUSP5P1 depletion and Oxaliplatin on inhibiting GC metastasis.
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