The Positive Feedback Loop of NHE1-ERK Phosphorylation Mediated by BRAFV600E Mutation Contributes to Tumorigenesis and Development of Glioblastoma

The Positive Feedback Loop of NHE1-ERK Phosphorylation Mediated by BRAFV600E Mutation Contributes to Tumorigenesis and Development of Glioblastoma | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The Positive Feedback Loop of NHE1-ERK Phosphorylation Mediated by BRAF V600E Mutation Contributes to Tumorigenesis and Development of Glioblastoma Yuhui Li, Dan Li, Yankun Liu, Shuqing Wang, Mingyang Sun, Zhongyuan Zhang, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-523299/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 01 Dec, 2021 Read the published version in Biochemical and Biophysical Research Communications → Version 1 posted You are reading this latest preprint version Abstract The occurrence rate of v-raf murine sarcoma viral oncogene homolog B1 (BRAF) activating mutation V600E (BRAF V600E ) in glioblastoma multiforme (GBM) is more than 50%. Na/H exchanger 1 (NHE1), a main pH regulator affecting cell microenvironment, is hyper-expressed in GBM. However, the relationship between BRAF V600E signal pathway and NHE1 in GMB cells remains unclear. This study found that NHE1 was a downstream target of BRAF V600E and an upstream factor of extracellular signal-regulated kinase (ERK). In addition, there was a positive feedback loop between NHE1-ERK phosphorylation under regulation of BRAF V600E mutation contributing to the proliferation and invasion of GBM cells. Moreover, the proliferation and invasion abilities of BRAF V600E -mutant and BRAF wild type GBM cells were all suppressed by the NHE1 inhibitor, BRAF V600E inhibitor and combination of them. The inhibitory effect of combination of the two inhibitors was better than each single drug both in vitro and in vivo . Combination of BRAF V600E and NHE1 inhibitors could be considered as a new therapeutic regimen for GBM, especially for GBM with BRAF V600E . Neurosurgery Nuclear Medicine & Medical Imaging glioblastoma BRAFV600E mutation NHE1 ERK inhibitor Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Glioma accounts for about 45% of all intracranial tumors, of which more than half are glioblastoma multiforme (GBM). GBM patients often have a poor prognosis due to the invasive growth and prone to relapse after surgery of GBM 1 – 2 . The invasion and occurrence of GBM are complex processes with multiple factors involved. The v-raf murine sarcoma viral oncogene homolog B1 (BRAF) is an important transduction factor of downstream mitogen activated protein kinase (MAPK) signaling pathway 3 . In gliomas, the expression level and activity of BRAF protein are positively correlated with the malignant degree of gliomas 4 . The 600th amino acid residue gene of BRAF changes from valine to glutamic acid to form continuous activated BRAF V600E , which leads to the continuous activation of MAPK signaling pathway and changes of tumor cell proliferation and metabolism 3 . BRAF V600E inhibitors Vemurafenib (PLX4032) and dabrafenib (GSK21118436) have been approved for the treatment of BRAF V600E mutant melanoma 5 . However, studies in gliomas have shown that the inhibition of MAPK signaling pathway by BRAF V600E inhibitor alone is not persistent, which may be due to drug resistance or activation of compensatory mechanism 6 – 7 . Further search for new therapeutic targets could provide new ideas for the treatment of BRAF V600E mutant GBM. Na/H exchanger 1 (NHE1) is an important transmembrane protein that regulates the intracellular pH 8 . Our research has shown that NHE1 is the main cause contributing to contraction of rat brain penetrating arteries under the condition of intracellular alkalosis caused by transient cerebral ischemia 9 . NHE1 gene is highly expressed in malignant tumors such as breast cancer, leukemia and GBM 10 – 12 . Down-regulating the expression of NHE1 can inhibit the intracellular pH adjustment to inhibit breast cancer cell metastasis 10 and induce apoptosis of leukemia cells 11 . In addition, studies have found that NHE1 protein is activated in various malignant tumor cells such as breast cancer cells, renal cancer cells and melanoma cells 13 – 15 . Cancer cells produce a large amount of lactic acid due to the enhancement of anaerobic glycolysis. Overexpressed and activated NHE1 can excrete H + and result in extracellular acidic microenvironment, which is conducive to the proliferation and invasion of cancer cells 16 – 17 . The anti-tumor effects of NHE1 inhibitors are increasingly concerned 18 : Cariporide can inhibit the proliferation and invasion of breast cancer cells 15 , 19 , also inhibit the proliferation of cholangiocarcinoma cells and induce apoptosis of cholangiocarcinoma cells 20 . These show that inhibition the expression and activity of NHE1 will provide a new method of tumor treatment. In addition, KarkiP's study has shown that BRAF protein directly binds to NHE1 and enhances its activity in cervical cancer and renal cancer cells 21 . However, the regulation mechanism of NHE1 activation in GMB cells is still unknown. This study combined microenvironment regulator NHE1 and MAPK tumorigenic signaling pathway and aimed to investigate whether BRAF activates NHE1 in GBM cells and whether NHE1 mediates MAPK signaling pathway. In addition, the inhibition effects of NHE1 inhibitor alone or in combination with BRAF V600E inhibitor on GBM were analyzed. Materials And Methods Cell culture Human glioblastoma cell lines U251 and AM38 were purchased from Saier Biotechnology Co., Ltd. (Tianjin, China). The U251 cells were cultured in DMEM/F12 medium (GIBCO BRL., Ltd., USA), the AM38 cells were cultured in RPMI 1640 medium (GIBCO BRL., Ltd., USA), supplemented with 10% fetal bovine serum (GIBCO BRL., Ltd., USA), 100 IU/ml of penicillin and 100 µg/ml of streptomycin (Beijing Dingguochangsheng Biotechnology Co., Ltd., Beijing, China). All cells were cultured at 37℃ in a humidified incubator containing 5% CO 2 . Cell proliferation assays Cell proliferation assays were performed using MTT (Tianjin Saier Biotechnology Co., Ltd., Tianjin, China). The cells were seeded into 96-well microtiter plates (Orange Scientific, Ltd., Belgium) at a density of 1.0x10 4 cells/well and cultured for 24 h at 37℃ in an incubator containing 5% CO 2 . The cells were then respectively treated with HOE-642 (0.16 nM, CAS: 159138-80-4) or SB590885 (0.16 nM, CAS: 405554-55-4) for 24 h. Subsequently, 10 µL MTT (5 mg/mL) was added to each well. After termination of culture, the absorbance was measured at 570 nm using a microplate reader (Bio-tek, Ltd., USA). Each experiment was carried out in three replicate wells and was repeated three times. Intracellular pH measurements For digital imaging of cytosolic pH, cell suspensions were prepared with HEPES at the cell concentration of 4 x 10 7 cells/ml. Adding BCECF-AM/DMSO solution to the cell suspension to a final concentration of 3 µM. After incubating at 37℃ for 30 min, the cells were washed 3 times with HEPES buffer to prepare a cell suspension of 3x10 6 cells/ml. The fluorescence values of OD1 (440 nm) and OD2 (490 nm) were detected by a multifunctional microplate reader. The fluorescence ratio (FIR = OD2/OD1) reflects the pH and thus the activity of NHE1 in the cell. Plasmids and transfection The whole gene synthesis film BRAF V600E was purchased from Saier Biotechnology Co., Ltd. (Tianjin, China). The outer film and the expression vector pcDNA3.1(+) were double-digested and ligated, and positive clones were selected after transformation with XL1-blue competent E. coli (Stratagene, Co., Ltd., USA). Then, the plasmid was extracted in small amounts by alkaline lysis. After digestion and identification of the recombinant plasmid, the digested product was subjected to 0.8% agarose gel electrophoresis to identify and record the correct recombinant plasmid (5.4 kb and 2.3 kb fragment bands). All plasmids were verified by sequencing. Then, the plasmid was extracted and purified using the B-type plasmid small volume rapid extraction kit (Bodatec, Co., Ltd., Beijing, China). The U251 cells were seeded in 6-well plates and cultured for 24 h. Subsequently, the cells were respectively transfected with the pcDNA3.1(+), pcDNA3.1(+)/BRAF V600E using Lipofectamine 2000 Reagent (Invitrogen, Co., Ltd., USA), according to the manufacturer's protocol. Western blot Total protein was collected using protein lysis buffer containing 1 µl protease inhibitor (Beijing Suo Laibao Technology Co., Ltd., Beijing, China) and 1 ml radioimmunoprecipitation assay (RIPA) lysis buffer at 14,000xg for 30 min at 4℃. The total protein concentration was calculated using the BCA assay according to the kit instructions (Merck, Co., Ltd., USA). The proteins were boiled at 99℃ for 5 min. The samples were transferred to PVDF membranes (Millipore, Co., Ltd., Shanghai, China) following SDS-PAGE. Subsequently, the membranes were incubated for 1 h in blocking buffer (containing 5% skim milk), then incubated with the following primary antibodies overnight at 4℃: anti-BRAF antibody (1:1000; Rabbit. no. ab33899; Abcam, Co., Ltd., USA), anti-BRAF V600E antibody (1:500; Rabbit. no. ab200535; Abcam, Co., Ltd., USA), anti-NHE1 antibody (1:1000; Rabbit. no. 67363-1-Ig; abbexa, Ltd., USA), anti-Phospho-(Ser) 14-3-3 binding motif antibody (1:500; Rabbit. no. 9601S; CST, Co., Ltd., USA), anti-extracellular signal-regulated kinase (ERK) antibody (1:2000; Rabbit. no. 51068-1-AP; proteintech, Co., Ltd., USA), anti-p-ERK antibody (1:500; Rabbit. no. 9101S; CST, Co., Ltd., USA), anti-E-cadherin antibody (1:500; Rabbit. no. SRP05266; Saier Biotechnology Co., Ltd., Tianjin, China), anti-Vimentin antibody (1:500; Rabbit. no. SRP01327; Saier Biotechnology Co., Ltd., Tianjin, China), anti-RSK antibody (1:500; Rabbit. no. SRP08000; Saier Biotechnology Co., Ltd., Tianjin, China), p-RSK antibody (1:500; Rabbit. no. 9341; CST, Co., Ltd., USA), anti-GAPDH antibody (1:1000; Rabbit. no. SRP13406; Saier Biotechnology Co., Ltd., Tianjin, China). The membranes were washed 4 times in Tris-buffered saline solution with 1X TBST and incubated with horseradish peroxidase-conjugated goat-anti-rabbit antibody (Amresco Co., Ltd., USA) for 1.5 h at RT. Following a final wash with 1X TBST, immunoreactive bands were detected using the ChampGel automatic gel imaging analyzer (Beijing SageCreation Science Co., Ltd., China). Optical band density was quantified using Image J (National Institutes of Health, Bethesda, MD, USA). Transwell invasion assay The cell invasion capability was detected using transwell chamber culture systems. A total of 1x10 5 cells were placed onto a Matrigel-coated transwell chamber with serum-free opti-MEM medium (Thermo Fisher Scientific, Inc., USA). The DMEM medium containing 20% FBS was added to the lower chamber as a chemoattractant. After 24 h, the cells attached to the lower surface of the insert filter were fixed with 33% (v/v) acetic acid (glacial acetic acid: methyl alcohol = 1: 3) and stained with crystal violet and counted. Immunoprecipitation (IP) and Western blot IP assays and western blot were performed as described previously 35 . Briefly, the cell extracts were incubated with magnetic beads antibody complex for 5 h at 4°C. Then the complex was washed and the immunoprecipitated proteins were analyzed by SDS/PAGE, transferred onto PVDF membrane and detected using each antibody. Animal experiment Nude mice were obtained from Tianjin Saier Biotechnology Co., Ltd. For the subcutaneous and metastatic models, 1×10 7 /ml U251 and AM-38 cells suspended in 100 µl medium were injected subcutaneously on the left back of nude mice. SB590885 (50mg/kg) or HOE-642 (15mg/kg) were injected into the abdominal cavity per mouse every 2 days. After 3 weeks of observation, the two groups of nude mice with different tumor formation were anesthetized and photographed. All studies performed with mice were approved by the Animal Care Committee of North China University of Science and Technology. All experiments involving mice complied with local and international regulations, ethical guidelines and the ARRIVE guidelines. Statistical analysis All statistical analysis was performed using SPSS software version 17.0 (SPSS, Inc., Chicago, IL, USA). The data are presented as the mean ± standard deviation (SD). One-way analysis of variance was used to analyze differences between groups. Scheffe post hoc testing was used to determine pairwise differences between means. P < 0.05 was considered to indicate a statistically significant difference. Results NHE1 was activated in BRAF V600E -mutant AM38 cells The protein level of BRAF and BRAF V600E in AM38 cells were significantly higher than these in U251 cells ( P < 0.001, P < 0.001) (Fig. 1 A). It confirmed that AM38 cell contains BRAF V600E -mutant. Both the expression and phosphorylation levels of NHE1 in AM38 cells were obviously higher than those in U251 cells ( P = 0.022, P = 0.001, respectively) (Fig. 1 B, 1 C). The FIR value of AM38 cells, which could indirectly reflect NHE1 activity, was markedly higher than that of U251 cells ( P = 0.005) (Fig. 1 D). These data suggested that the expression, phosphorylation level and activity of NHE1 might be related to BRAF V600E mutation. NHE1 was a downstream factor of BRAF V600E and an upstream regulator of ERK To explore the relationship between BRAF V600E and NHE1, the BRAF V600E -overexpressed pcDNA3.1(+) plasmids were constructed (Fig. 2 A) and the overexpression of BRAF V600E in U251 cells at 48 h post-transfection were confirmed ( F = 1971.738, P < 0.001; pcDNA3.1(+)/BRAF V600E group vs pcDNA3.1(+) group P < 0.001) (Fig. 2 B). The protein levels of BRAF V600E were not affected by NHE1 inhibitor HOE-642 (pcDNA3.1(+)/BRAF V600E group vs pcDNA3.1(+)/BRAF V600E + HOE-642 group P = 0.083) (Fig. 2 B). This showed that BRAF V600E was not affected by NHE1. However, both NHE1 expression ( F = 29.765, P < 0.001) and phosphorylated NHE1 (p-NHE1) levels ( F = 45.887, P < 0.001) (Fig. 2 C), as well as the FIR value (NHE1 activity) ( F = 214.093, P < 0.001), in BRAF V600E -overexpressed U251 cells were significantly upregulated compared with those of U251 cells (pcDNA3.1(+)/BRAF V600E group vs pcDNA3.1(+) group: P = 0.001, P = 0.001, P = 0.001, respectively) (Fig. 2 D). HOE-642 dramatically reversed the effect of BRAF V600E overexpression on NHE1 activity (pcDNA3.1(+)/BRAF V600E group vs pcDNA3.1(+)/BRAF V600E + HOE-642 group P = 0.002) (Fig. 2 D), but did not affected the NHE1 expression and phosphorylation ( P = 0.961, P = 0.198, respectively) (Fig. 2 C). These suggested that NHE1 was a downstream factor of BRAF V600E . Moreover, the phosphorylated ERK (p-ERK) level was markedly increased by BRAF V600E -overexpression, while was partially decreased by HOE-642 treatment ( F = 160.760, P = 0.001; P < 0.001, P < 0.001, respectively), while total ERK levels were not affected ( F = 2.396, P = 0.172) (Fig. 2 E). This suggested that NHE1 inhibitor could inhibit ERK phosphorylation. These data indicated that NHE1 was involved in BRAF/ERK signal pathway as an upstream regulator of ERK. The proliferation and invasion abilities of U251 cells enhanced by BRAF V600E overexpression were inversed by NHE1 inhibitor Both the proliferation and invasion abilities of BRAF V600E -overexpressed U251 cells were significantly enhanced compared with those of U251 at 48 h post-transfection, while the effects were significantly reversed by HOE-642 treatment (proliferation: F = 62.197, P < 0.001; P < 0.001, P = 0.002, respectively) (invasion: F = 125.601, P < 0.001; P < 0.001, P < 0.001, respectively) (Fig. 3 A, 3 B). In addition, the proliferation marker Ki67 and the mesenchymal cell marker Vimentin were all significantly higher in BRAF V600E -overexpressed U251 cells, whereas epithelial cell marker E-cadherin was markedly lower, than those in U251 cells (for Ki67: F = 277.911, P < 0.001; P < 0.001; for Vimentin: F = 76.854, P < 0.001; P < 0.001; for E-cadherin: F = 7208.162, P < 0.001; P < 0.001) (Fig. 3 C, 3 D). NHE1 inhibitor HOE-642 dramatically inversed the effects of BRAF V600E overexpression on protein levels of Ki67 ( P = 0.001) and Epithelial-Mesenchymal Transition (EMT) markers ( P < 0.001, P < 0.001, respectively) (Fig. 3 C, 3 D). These data indicated that NHE1 inhibitor could repress proliferation and invasion of BRAF V600E GBM cells by affecting Ki67 and EMT. The phosphorylation and activity of NHE1 were positively regulated by p-ERK There was no significantly difference of total ribosomal S6 kinase (RSK) between U251 cells and AM38 cell ( F = 3.750, P = 0.060, P = 0.845) (Fig. 4 A left). However, the level of phosphorylated RSK (p-RSK), an active marker of ERK signal pathway, was significantly higher in AM cells than that in U251 cells ( F = 53.847, P = 0.060, P < 0.001) (Fig. 4 A right). Total ESK levels in U152 and AM38 cells were not affected by ERK agonist Honokiol and ERK inhibitor SCH772984, respectively ( P = 0.986, P = 0.275) (Fig. 4 A left). Whereas p-RSK was markedly increased in Honokiol-treated U251 cells compared with that in untreated U251 cells ( P = 0.001), and was significantly decreased in SCH772984-treated AM38 cells compared with that in untreated AM38 cells ( P = 0.001). These confirmed the effectiveness of Honokiol and SCH772984 on ERK activity. In addition, it was found that FIR value and p-NHE1 level in Honokiol-treated U251 cells were significantly upregulated compared those in untreated U251 cells (for FIR: F = 47.399, P < 0.001; P < 0.001; for p-NHE1: F = 133.299, P < 0.001; P < 0.001), while there was no significant difference of total NH1 among indicated groups ( F = 1.384, P = 0.316) (Fig. 4 B, 4 C). Whereas FIR value and p-NHE1 level in AM38 cells treated with SCH772984 were dramatically decreased than those in untreated AM38 cells ( P = 0.005, P < 0.001, respectively) (Fig. 4 B, 4 C). These data suggested that NEH1 could be phosphorylated and activated by p-ERK. Co-IP analysis confirmed that NHE1 directly interacted with BRAF V600E in AM38 cells, but not with wild type BRAF (BRAF WT ) and ERK (Fig. 4 D). The combination of NHE1 inhibitor and BRAF V600E inhibitor had better inhibitory effects on proliferation and invasion abilities of GBM cells with BRAF V600E The NHE1 activities (FIR) in U251 were suppressed by NHE1 inhibitor HOE-642, but not by the BRAF V600E inhibitor SB590885 ( F = 14.950, P = 0.001, HOE-642 group vs. DMSO group P = 0.011, SB590885 group vs. DMSO group P = 0.892) (Fig. 5 A left). The NHE1 activities in AM38 cells were suppressed by HOE-642, SB590885, as well as combination of them, respectively ( F = 101.013, P < 0.001, HOE-642 group vs. DMSO group P < 0.001, SB590885 group vs. DMSO group P < 0.001, HOE-642 + SB590885 group vs. DMSO group P < 0.001) (Fig. 5 A right). The combination of HOE-642 and SB590885 had better inhibitory effects on NHE1 activities than HOE-642 or SB590885 alone in AM38 cells (HOE-642 + SB590885 group vs. HOE-642 group P = 0.011; vs SB590885 group P = 0.030) (Fig. 5 A right). Both the proliferation and invasion abilities of U251 cells were inhibited by HOE-642, but not by SB590885 (proliferation: F = 12.109, P = 0.002; P = 0.037, P = 0.979,Fig. 5 B left; invasion: F = 30.379, P < 0.001; P = 0.003, P = 0.699, Fig. 5 C left). Both the proliferation and invasion abilities of AM38 cells were suppressed by HOE-642, SB590885, as well as combination of them, respectively (proliferation: F = 33.877, P < 0.001; P = 0.007, P = 0.002, P < 0.001, Fig. 5 B right; invasion: F = 89.933, P < 0.001; P < 0.001, P < 0.001, P < 0.001, Fig. 5 C right). The combination of HOE-642 and SB590885 had better inhibitory effects both on proliferation and invasion of AM38 cells than HOE-642 or SB590885 alone (proliferation: HOE-642 + SB590885 group vs. HOE-642 group P = 0.009; vs. SB590885 group P = 0.043; invasion: HOE-642 + SB590885 group vs. HOE-642 group P < 0.001; vs. SB590885 group P = 0.044) (Fig. 5 A right). The Ki67 levels in U251 cells treated with HOE-642, SB590885 and combination of them were respectively suppressed by 41.5%, 47.5% and 63.0% compared with DMSO group ( F = 22.672, P = 0.006; P = 0.030, P = 0.019, P = 0.007), while combination of HOE-642 and SB590885 has no advantage than each alone ( P = 0.208, P = 0.402) (Fig. 5 D left). The Ki67 levels in AM38 cells of the three groups were respectively suppressed by 28.5%, 33.1% and 55.0% compared with DMSO group ( F = 51.534, P = 0.001; P = 0.014, P = 0.008, P = 0.001), while the inhibition effect of the combination group was better than each drug alone ( P = 0.036) (Fig. 5 D right). For the EMT marker, the E-cadherin in U251 cells were markedly upregulated by HOE-642, but not by SB590885 ( F = 45.423, P = 0.002; P = 0.006, P = 0.764, Fig. 5 E left), while E-cadherin in AM38 cells were significantly increased by HOE-642, SB590885, as well as combination of them, respectively ( F = 191.572, P < 0.001; P = 0.024, P = 0.003, P < 0.001, Fig. 5 E right). On the other hand, the Vimentin in U251 cells were markedly downregulated by HOE-642, but not by SB590885 ( F = 38.388, P = 0.002; P = 0.031, P = 0.895, Fig. 5 F left), while those in AM38 cells were not changed by HOE-642 or SB590885 alone ( F = 28.796, P = 0.004; P = 0.266, P = 0.083, Fig. 5 F right). The effects of combination of HOE-642 and SB590885 on E-cadherin and Vimentin in AM38 cells were better than each single drug (E-cadherin: P < 0.001, P = 0.001, Fig. 5 E right; Vimentin: P = 0.013, P = 0.030, Fig. 5 F right). These data indicated that the combination of BRAF V600E inhibitor and NHE1 inhibitor has better inhibitory effects on proliferation and invasion abilities of GBM cells with BRAF V600E . The combination of BRAF V600E inhibitor and NHE1 inhibitor had better inhibitory effects on GBM cells with BRAF V600E in vivo To further explore the inhibitory effects of combined BRAF V600E inhibitor and NHE1 inhibitor on GBM cells, nude mice tumorigenesis experiments were conducted. All nude mice were in good health and activity before treatment and did not die until the end of the experiment. Figure 6 A shows tumor formation in nude mice in each group. There were no significantly difference of body weight among all the indicated groups (U251: F = 0.452, P = 0.718; AM38: F = 0.293, P = 0.830) (Fig. 6 B). The U251 tumor volumes were markedly downregulated by HOE-642, but not by SB590885 ( F = 22.387, P < 0.001; P = 0.007, P = 0.954) (Fig. 6 C). The AM38 tumor volumes were significantly decreased by HOE-642, SB590885, as well as combination of them, respectively ( F = 22.823, P < 0.001; P = 0.001, P = 0.001, P < 0.001) (Fig. 6 C). The inhibitory effects of combination of HOE-642 and SB590885 on tumor volumes of U251 and AM38 cells were better than each single drug (U251: P = 0.040, P < 0.001, AM38: P = 0.049, P = 0.026, Fig. 6 C). These indicated that the inhibitory effects of combination of HOE-642 and SB590885 on tumor volumes of U251cells and AM38 cells were better than each single drug. In addition, the AM38 tumor weight was markedly higher than U251 tumor weight (Fig. 6 D left, middle). The U251 tumor wrights treated with HOE-642, SB590885 and combination of them were respectively suppressed by 41.9%, 30.0% and 64.2% compared with DMSO group, respectively ( F = 30.620, P < 0.001; P < 0.001, P = 0.003, P < 0.001) (Fig. 6 D, right). The AM38 tumor weights of the three groups were respectively suppressed by 63.1%, 62.9% and 78.8% compared with DMSO group, respectively ( F = 107.482, P < 0.001; P < 0.001, P < 0.001, P < 0.001) (Fig. 6 D, right). Moreover, the inhibitory effects of combination of HOE-642 and SB590885 on tumor weights of U251 cells and AM38 cells were all better than each single drug (U251: P = 0.033, P = 0.001; AM38: P = 0.030, P = 0.029) (Fig. 6 D right). These data confirmed that the combination of BRAF V600E inhibitor and NHE1 inhibitor has better inhibitory effects on proliferation of U251 cells than each single drug in vivo . Discussion The present study found that the expression, phosphorylation and activity of NHE1 in BRAF V600E -mutant AM38 cells were all higher than those in BRAF WT U251 cells. Overexpression of BRAF V600E activated the ERK pathway and upregulated the expression, phosphorylation and activity of NHE1, as well as the cell viabilities, invasion abilities and corresponding markers. Those effects of BRAF V600E overexpression were reversed by NHE1 inhibitor HOE-642. These data indicated that NHE1 is a downstream target of BRAF V600E and an upstream factor of ERK. The microenvironment factor NHE1 interacts with BRAF V600E /ERK oncogenic signaling pathway in GBM cells. This work also found that NHE1 was directly interacted with BRAF V600E in BRAF V600E GBM cells. This is consistent with the report in malignant melanoma cells, in which stimulated NHE1 by BRAF V600E induced to aberrant pH 22 . There was speculated that a similar mechanism may exist in cancer cells with BRAF V600E mutation. Moreover, the transport activity of NHE1 regulated by multiple intracellular signaling molecules, including MAPK/ERK 23 and PI3K/AKT kinases 24 – 25 , those interact with different site of serine residues of the cytosolic C-terminus and phosphorylate NHE1. Additionally, we found that both the phosphorylation and activity of NHE1 were positively regulated by ERK activator and inhibitor in GBM cells, but not NHE1 expression. These suggested that there is a positive feedback loop between NHE1-ERK phosphorylation under regulation of BRAF V600E mutation contributing to the proliferation and invasion of GBM cells (Fig. 7 ). However, we did not detected interaction between NHE1 and ERK in BRAF V600E GBM cells. We speculated that BRAF V600E may have the advantage structure to combine with NHE1 in compared with BRAF and ERK in GBM cells. Evidences have suggested the ERK, MEK, RSK, RAF-1, PAK5, and 14-3-3 exist as complexes in some cell types 26 – 31 . The interaction between NHE1 and PI3K/AKT signaling pathways in GBM cells should be considered in the further study. In addition, the proliferation and invasion abilities of BRAF V600E -mutant and BRAF WT GBM cells, as well as the corresponding markers, were all suppressed by the NHE1 inhibitor, BRAF V600E inhibitor and combination of them. The inhibitory effect of combination of the two inhibitors in BRAF V600E -mutant cells was better than each single drug both in vitro and in vivo . This probably partly due to the inhibition of some compensation mechanism by the two inhibitors in BRAF V600E -mutant GBM cells. For the persistence of suppressive effect of combination treatment, this work only observed data by day 21. Further study should be performed. There have several selective inhibitors to be invested and used for clinical treatment of tumors with BRAF V600E mutation: (1) Vemurafenib (PLX4032) could selectively bind to the ATP-binding site of BRAF V600E and inhibits its activity 32 . A clinical case report showed that children GBM with BRAF V600E mutation received Vemurafenib treatment and achieved clinical complete remission (Robinson et al., 2014). (2) UAI-201 promotes GBM cell cycle inhibition and autophagy by blocking BRAF V600E /MEK/ERK pathway in glioma cells with BRAF V600E 4 . (3) Dabrafenib (GSK2118436) was approved for unresectable or metastatic melanoma and anaplastic thyroid cancer harbouring the BRAF V600E mutation as monotherapy or in combination with trametinib (a MAPK kinase inhibitor) 5 , 34 . Our work showed that SB590885 is also an effective inhibitor for GBM with BRAF V600E mutation in vivo and in vitro, which provides a basis for clinical application. Moreover, HOE-642 is another candidate drug for GBM cells BRAF V600E mutation and has good synergistic effect with BRAF V600E inhibitor. These data could enlighten treatment for breast cancer and cholangiocarcinoma 19 , 20 , 35 . To conclude, the present study clarified that microenvironment factor NHE1 involves in BRAF V600E /ERK oncogenic signaling pathway and contributes to the proliferation and invasion of GBM cells. Combination of BRAF V600E and NHE1 inhibitors probably considered as a new therapeutic regimen for future research and clinical application of GBM with BRAF V600E . Declarations Author Contribution YHL and DL performed most of the experiments and wrote the manuscript. YKL and SQW guided experimental technology. MYS performed some of the experiments. ZYZ and XZ guided and performed statistical analysis. YFL and JWL conceived the project and supervised the experiments. All authors read and approved the final manuscript. Acknowledgements This work was supported by Science and Technology Innovation Team Training Plan Project Fund of Tangshan (18130203B), 2019 City Talent Project Fund of Tangshan (A201905007) and Fund of Key Laboratory of Hebei Province (SZX2020043). Conflicts interest The authors declare no competing interests. References Liu, R. Z. et al. Association between cytoplasmic CRABP2, altered retinoic acid signaling, and poor prognosis in glioblastoma. Glia. 64 , 963–976 (2016). Jiang, J. et al. Regorafenib induces lethal autophagy arrest by stabilizing PSAT1 in glioblastoma. Autophagy. 16 , 106–122 (2020). Davies, H. et al. Mutations of the BRAF gene in human cancer. Nature. 417 , 949–954 (2002). Ahn, J. H., Lee, Y. W., Ahn, S. K. & Lee, M. Oncogenic BRAF inhibitor UAI-201 induces cell cycle arrest and autophagy in BRAF mutant glioma cells. Life Sci. 104 , 38–46 (2014). Jang, S. & Atkins, M. B. Treatment of BRAF-mutant melanoma: The role of vemurafenib and other therapies. Clin Pharmacol Ther. 95 , 24–31 (2014). Yao, T. W. et al. EGFR blockade prevents glioma escape from BRAFV600E targeted therapy. Oncotarget. 6 , 21993–22005 (2015). Zhang, J. et al. Combined BRAF V600E and MEK blockade for BRAF V600E -mutant gliomas. Neurooncol . 131 ,495–505. (2017). Stock, C. & Pedersen, S. F. Roles of pH and the Na+/H + Exchanger NHE1 in Cancer: From Cell Biology and Animal Models to an Emerging Translational Perspective. Semin Cancer Biol. 43 , 5–16 (2017). Li, Y. H., Horiuchi, T., Murata, T. & Hongo, K. Mechanism of alkalosis-induced constriction of rat cerebral penetrating arterioles. Neuroscience Research. 70 , 98–103 (2011). Chen, Q. et al. Increased NHE1 expression is targeted by specific inhibitor cariporide to sensitize resistant breast cancer cells to doxorubicin in vitro and in vivo. BMC Cancer. 19 , 211–223 (2019). Rich, I. N., Worthington-White, D., Garden, O. A. & Musk, P. Apoptosis of leukemic cells accompanies reduction in intracellular pH after targeted inhibition of the Na+/H + exchanger. Blood. 95 , 1427–1434 (2000). Guan, X. D. et al. Elevated Na/H Exchanger 1 (SLC9A1) Emerges as a Marker for Tumorigenesis and Prognosis in Gliomas. J Exp Clin Cancer Res. 37 , 255–270 (2018). Ludwig, F. T., Schwab, A. & Stock, C. The Na+ /H+ -Exchanger (NHE1) Generates pH Nanodomains at Focal Adhesions. J Cell Physiol. 228 , 1351–1358 (2013). Karumanchi, S. A. et al. VHL tumor suppressor regulates Cl–/HCO3– exchange and Na+/H + exchange activities in renal carcinoma cells. Physiol Genomics. 5 , 119–128 (2001). Wang, J. et al. CIAPIN1 targets Na(+)/H(+) exchanger 1 to mediate MDA-MB-231 cells' metastasis through regulation of MMPs via ERK1/2 signaling pathway. Exp Cell Res. 333 , 60–72 (2001). Neri, D. & Supuran, C. T. Interfering with pH regulation in tumors as a therapeutic strategy. Nat Rev Drug Discov. 10 , 767–777 (2001). Brisson, L., Reshkin, S. J., Goré, J. & Roger, S. pH regulators in invadosomal functioning: proton delivery for matrix tasting. Eur J Cell Biol. 91 , 847–860 (2012). Harguindey, S. et al. Cariporide and other new and powerful NHE1 inhibitors as potentially selective anticancer drugs an integral molecular/biochemical/metabolic/clinical approach after one hundred years of cancer research. J Transl Med. 11 , 282–298 (2013). Lin, Y. N. et al. NHE1 mediates MDA-MB-231 cells invasion through the regulation of MT1-MMP. Exp Cell Res. 317 , 2031–2040 (2011). Sario, A. D. et al. Selective inhibition of ion transport mechanisms regulating intracellular pH reduces proliferation and induces apoptosis in cholangiocarcinoma cells. Dig Liver Dis. 39 , 60–69 (2007). Karki, P., Li, X. J., Schrama, D. & Fliegel, L. B-Raf associates with and activates the NHE1 isoform of the Na+/H + exchanger. J Biol Chem. 286 ,13096–13105. Cong, D. et al. 2014. Upregulation of NHE1 protein expression enables glioblastoma cells to escape TMZ-mediated toxicity via increased H(+) extrusion, cell migration and survival. Carcinogenesis . 35 , 2014–2024. (2011). Bandyopadhyay, S. et al. A human MAP kinase interactome. Nat Methods. 7 , 801–805 (2010). Meima, M. E., Webb, B. A., Witkowska, H. E. & Barber, D. L. The sodium-hydrogen exchanger NHE1 is an Akt substrate necessary for actin filament reorganization by growth factors. J Biol Chem. 284 , 26666–26675 (2009). Wang, H., Cai, J., Du, S. X., Wei, W. & Shen, X. H. LAMC2 modulates the acidity of microenvironments to promote invasion and migration of pancreatic cancer cells via regulating AKT-dependent NHE1 activity. Exp Cell Res. 391 , 111984 (2020). Lundquist, J. J. & Dudek, S. M. Differential activation of extracellular signal-regulated kinase 1 and a related complex in neuronal nuclei. Brain Cell Biol. 35 , 267–281 (2006). Kolch, W. Coordinating ERK/MAPK signaling through scaffolds and inhibitors. Nat Rev Mol Cell Biol. 6 , 827–837 (2005). Wang, X. & Studzinski, G. P. Raf-1 signaling is required for the later stages of 1,25-dihydroxyvitamin D3-induced differentiation of HL60 cells but is not mediated by the MEK/ERK module. J Cell Physiol. 209 , 253–260 (2006). Wu, X., Carr, H. S., Dan, I. & Ruvolo, P. P. & Frost J. A. p21 activated kinase 5 activates Raf-1 and targets it to mitochondria. J Cell Biochem. 105 , 167–175 (2008). Edmunds, J. W. & Mahadevan, L. C. MAP kinases as structural adaptors and enzymatic activators in transcription complexes. J Cell Sci. 117 , 3715–3723 (2004). Ren, J. G., Li, Z. & Sacks, D. B. IQGAP1 modulates activation of B-Raf. Proc Natl Acad Sci U S A. 104 , 10465–10469 (2007). Garbe, C., Eigentler, T. K. & Vemurafenib Recent Results Cancer Res. 211 , 77–89 (2018). Robinson, G. W., Orr, B. A. & Gajjar, A. Complete clinical regression of a BRAF V600E-mutant pediatric glioblastoma multiforme after BRAF inhibitor therapy. BMC Cancer. 14 , 258–262 (2014). Puszkiel, A. et al. Clinical Pharmacokinetics and Pharmacodynamics of Dabrafenib. Clin Pharmacokinet. 58 , 451–467 (2019). Wang, Y. et al. Williams syndrome transcription factor is a target of pro-oncogenic Ser158 phosphorylation mediated by Ras-MAPK pathway in human breast cancer. Int J Clin Exp Pathol. 2 , 1668–1675 (2016). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 01 Dec, 2021 Read the published version in Biochemical and Biophysical Research Communications → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-523299","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":28062380,"identity":"07f9a27c-6c67-45fe-99e2-11386d48adcf","order_by":0,"name":"Yuhui Li","email":"","orcid":"","institution":"Tangshan People’s Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yuhui","middleName":"","lastName":"Li","suffix":""},{"id":28062381,"identity":"03a9403a-0830-4aa0-a90c-a0b9ec75a4ea","order_by":1,"name":"Dan Li","email":"","orcid":"","institution":"The Cancer Institute, Tangshan People’s Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Dan","middleName":"","lastName":"Li","suffix":""},{"id":28062384,"identity":"5bafeb7b-dcb4-4996-a931-eb8c4f8fd17a","order_by":2,"name":"Yankun Liu","email":"","orcid":"","institution":"The Cancer Institute, Tangshan People’s Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yankun","middleName":"","lastName":"Liu","suffix":""},{"id":28062385,"identity":"938eede5-dbf1-4de7-b4a8-066383d2303c","order_by":3,"name":"Shuqing Wang","email":"","orcid":"","institution":"Hospital of North China University of Science and Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Shuqing","middleName":"","lastName":"Wang","suffix":""},{"id":28062387,"identity":"640a63d0-db1b-42be-ab95-e6423f8f5b1e","order_by":4,"name":"Mingyang Sun","email":"","orcid":"","institution":"Tangshan People’s Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Mingyang","middleName":"","lastName":"Sun","suffix":""},{"id":28062389,"identity":"10dbc012-3fb7-4a48-9ffe-b7931e5cfa27","order_by":5,"name":"Zhongyuan Zhang","email":"","orcid":"","institution":"Zunhua People’s Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zhongyuan","middleName":"","lastName":"Zhang","suffix":""},{"id":28062391,"identity":"478d8288-babf-4ae2-a7c9-b05d30d6555c","order_by":6,"name":"Xuan Zheng","email":"","orcid":"","institution":"Nuclear Medicine Clinical Laboratory, Tangshan People’s Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xuan","middleName":"","lastName":"Zheng","suffix":""},{"id":28062394,"identity":"b92e0572-02cf-450c-9f3a-148d9412286f","order_by":7,"name":"Jingwu Li","email":"","orcid":"","institution":"The Cancer Institute, Tangshan People’s Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jingwu","middleName":"","lastName":"Li","suffix":""},{"id":28062396,"identity":"d67ebded-831d-4449-bf80-99c03d518354","order_by":8,"name":"Yufeng Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzUlEQVRIiWNgGAWjYFACxgcgUoaNvYFoLcwGIJKHjecwqVoYJJKJ1GBwI5nxccEvBh4+yfeHP1fmHE5sYD97gJAWZuOZfUCHSSezSZ7dBtTCk5dAQEv+MWneHogWxkaQFgkeA0K2sP8Ga5E8zPyRWC1szDw/gFokmBkkidIieeYxszRvAyiQk82AWtKN23hy8GvhO57M+JnnD4OcfPvBx0CHWcv2s5/Br0XhAJBgbPsP4zc7tuFVDwTyDSDyD5xfZ09IxygYBaNgFIw8AADuo0AyKdq5PQAAAABJRU5ErkJggg==","orcid":"","institution":"The Cancer Institute, Tangshan People’s Hospital","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Yufeng","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2021-05-13 13:14:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-523299/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-523299/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1016/j.bbrc.2021.11.104","type":"published","date":"2021-12-01T07:54:44+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":9685648,"identity":"f3db8242-ca3b-44da-9604-0aa4c0283fab","added_by":"auto","created_at":"2021-05-27 22:05:33","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":495628,"visible":true,"origin":"","legend":"NHE1 was activated in BRAFV600E-mutant AM38 cells\n(A) The relative proteins levels of BRAF and BRAFV600E in U251 and AM38 cells were analyzed by Western blot with GAPDH as internal control. The relative proteins levels of (B) NHE1 and (C) p-NHE1 in U251 and AM38 cells were analyzed by Western blot with GAPDH as internal control. (D) The NHE1 activity of U251 and AM38 cells were detected by BCECF-AM fluorescent probe. The Fluorescence Intensity Ratio (FIR, FIR=OD2/OD1) at 440 nm and 490 nm was measured by UV-Visible. FIR reflects the pH value and the activity of NHE1 in the cell. The experiments were repeated three times independently and the results were expressed as mean±standard deviation (χ̅ ±SD).","description":"","filename":"fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-523299/v1/3873323f53395210ebebeec7.png"},{"id":9685299,"identity":"0a933c0f-8804-45df-b3e5-28f359ac1740","added_by":"auto","created_at":"2021-05-27 21:59:32","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1444130,"visible":true,"origin":"","legend":"NHE1 was at downstream of BRAFV600E and at upstream of ERK \n(A) Schematic diagram of the recombinant pcDNA3.1(+)/BRAFV600E plasmids construction. (B) The relative protein levels of BRAFV600E in U251 cells of each group at 48 h post-transfection were verified by Western blot with GAPDH as internal control. (C) The relative protein levels of NHE1 and phosphorylation NHE1 (p-NHE1) in U251 cells of each group were analyzed by Western blot with GAPDH as internal control. (D) The NHE1 activity of U251 cells in each group detected by BCECF-AM fluorescent probe. (E) The relative protein levels of ERK and p-ERK in U251 cells of each group were analyzed by Western blot with GAPDH as internal control. HOE-642 is a NHE1 inhibitor. The experiments were repeated three times independently and the results were expressed as χ̅ ±SD.","description":"","filename":"fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-523299/v1/1689c256e108087cfe764ba2.png"},{"id":9685151,"identity":"fcaf13f7-e000-436a-948b-c2cb51111939","added_by":"auto","created_at":"2021-05-27 21:56:32","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1014793,"visible":true,"origin":"","legend":"The enhanced expression and invasion abilities of U251 cells induced by BRAFV600E overexpression were inversed by HOE-642 \nThe proliferation (A) and invasion abilities (B) of each group cells at 48 h post-transfection were analyzed by MTT (optical density, OD) and matrigel-transwell assay, respectively. The relative protein levels of Ki67 (C) and Vimentin and E-cadherin (D) in U251 cells of each group were analyzed by Western blot with GAPDH as internal control. HOE-642 is a NHE1 inhibitor. The experiments were repeated three times independently and the results were expressed as χ̅ ±SD.","description":"","filename":"fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-523299/v1/2d514e6223f3059889a404ba.png"},{"id":9685550,"identity":"708fae5f-ff92-4acc-8212-8706e6116d29","added_by":"auto","created_at":"2021-05-27 22:02:33","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1004309,"visible":true,"origin":"","legend":"The phosphorylation and activity of NHE1 were positively regulated by ERK activity \n(A) The relative protein levels of RSK and p-RSK in U251 cells of each group were analyzed by Western blot with GAPDH as internal control. Honokiol is an ERK agonist with anticancer activity, and SCH772984 is a novel specificity ERK inhibitor. (B) The NHE1 activities in U251 and AM38 cells were detected by BCECF-AM fluorescent probe. (C) The NHE1 and p-NHE1 levels in U251 and AM38 cells were analyzed by Western blot with GAPDH as internal control. The experiments were repeated three times independently and the results were expressed as χ̅ ±SD. (D) The interactions of NHE1 between BRAF, BRAFV600E and ERK in AM38 cells were analyzed by immunoprecipitation and Western blot.","description":"","filename":"fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-523299/v1/a88de3e40f6271eeb63cdb34.png"},{"id":9685549,"identity":"084bf3b1-365e-4028-ab4f-17721b01303c","added_by":"auto","created_at":"2021-05-27 22:02:33","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1684837,"visible":true,"origin":"","legend":"The proliferation and invasion abilities of GBM cells were inhibited by BRAFV600E inhibitor and NHE1 inhibitor alone and combined \n(A) The NHE1 activity of U251 and AM38 cells were detected by BCECF-AM fluorescent probe. The Fluorescence Intensity Ratio (FIR, FIR=OD2/OD1) at 440 nm and 490 nm was measured by UV-Visible. FIR reflects the pH value and the activity of NHE1 in the cell. HOE-642 is a NHE1 inhibitor, SB590885 is a BRAF inhibitor. (B) The invasion abilities of each group cells at 48 h post-transfection were analyzed by matrigel-transwell assay. The relative protein levels of Ki67 (C) and E-cadherin (D) and Vimentin (E) in U251 cells of each group were analyzed by Western blot with GAPDH as internal control. The experiments were repeated three times independently and the results were expressed as χ̅ ±SD.","description":"","filename":"fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-523299/v1/53119b8f7f3108573b3f6d46.png"},{"id":9685303,"identity":"8334d565-b7b3-4e89-89e0-bbf69fd03afc","added_by":"auto","created_at":"2021-05-27 21:59:33","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":6605965,"visible":true,"origin":"","legend":"(A) Tumor photos and body photos of nude mice in each group. (B) The nude mouse body weight growth curve and (C) tumor growth curve. The long diameter and short diameter of the tumor were measured and weighed every 3 days, and the growth curve of the transplanted tumor was drawn based on the average tumor volume of each group of animals. (D) Statistics of tumor weight in each group of nude mice. The experiments were repeated three times independently and the results were expressed as χ̅ ±SD.","description":"","filename":"fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-523299/v1/c875f092c96a7b8cfa9b5b1b.png"},{"id":9685300,"identity":"0357c654-01f2-49c5-a9f4-69433d515b70","added_by":"auto","created_at":"2021-05-27 21:59:33","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":701900,"visible":true,"origin":"","legend":"Schematic diagram of the positive feedback between NHE1 and ERK phosphorylation in GBM Cells with BRAFV600E mutation.","description":"","filename":"fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-523299/v1/600164613a0d9b82f2e3d820.png"},{"id":16055889,"identity":"f480daa6-cd5e-4b5c-a009-e98c1d556982","added_by":"auto","created_at":"2021-12-01 07:54:48","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2461941,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-523299/v1/51fd4a8f-275d-4087-ac88-fcd7d04371ff.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eThe Positive Feedback Loop of NHE1-ERK Phosphorylation Mediated by BRAF\u003csup\u003eV600E\u003c/sup\u003e Mutation Contributes to Tumorigenesis and Development of Glioblastoma\u003c/p\u003e","fulltext":[{"header":"Introduction","content":" \u003cp\u003eGlioma accounts for about 45% of all intracranial tumors, of which more than half are glioblastoma multiforme (GBM). GBM patients often have a poor prognosis due to the invasive growth and prone to relapse after surgery of GBM\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. The invasion and occurrence of GBM are complex processes with multiple factors involved.\u003c/p\u003e \u003cp\u003eThe v-raf murine sarcoma viral oncogene homolog B1 (BRAF) is an important transduction factor of downstream mitogen activated protein kinase (MAPK) signaling pathway\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. In gliomas, the expression level and activity of BRAF protein are positively correlated with the malignant degree of gliomas\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. The 600th amino acid residue gene of BRAF changes from valine to glutamic acid to form continuous activated BRAF\u003csup\u003eV600E\u003c/sup\u003e, which leads to the continuous activation of MAPK signaling pathway and changes of tumor cell proliferation and metabolism\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. BRAF\u003csup\u003eV600E\u003c/sup\u003e inhibitors Vemurafenib (PLX4032) and dabrafenib (GSK21118436) have been approved for the treatment of BRAF\u003csup\u003eV600E\u003c/sup\u003e mutant melanoma\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. However, studies in gliomas have shown that the inhibition of MAPK signaling pathway by BRAF\u003csup\u003eV600E\u003c/sup\u003e inhibitor alone is not persistent, which may be due to drug resistance or activation of compensatory mechanism\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Further search for new therapeutic targets could provide new ideas for the treatment of BRAF\u003csup\u003eV600E\u003c/sup\u003e mutant GBM.\u003c/p\u003e \u003cp\u003eNa/H exchanger 1 (NHE1) is an important transmembrane protein that regulates the intracellular pH\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Our research has shown that NHE1 is the main cause contributing to contraction of rat brain penetrating arteries under the condition of intracellular alkalosis caused by transient cerebral ischemia\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eNHE1\u003c/em\u003e gene is highly expressed in malignant tumors such as breast cancer, leukemia and GBM\u003csup\u003e\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Down-regulating the expression of NHE1 can inhibit the intracellular pH adjustment to inhibit breast cancer cell metastasis\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e and induce apoptosis of leukemia cells\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. In addition, studies have found that NHE1 protein is activated in various malignant tumor cells such as breast cancer cells, renal cancer cells and melanoma cells\u003csup\u003e\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Cancer cells produce a large amount of lactic acid due to the enhancement of anaerobic glycolysis. Overexpressed and activated NHE1 can excrete H\u0026thinsp;+\u0026thinsp;and result in extracellular acidic microenvironment, which is conducive to the proliferation and invasion of cancer cells\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. The anti-tumor effects of NHE1 inhibitors are increasingly concerned\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e: Cariporide can inhibit the proliferation and invasion of breast cancer cells\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e, also inhibit the proliferation of cholangiocarcinoma cells and induce apoptosis of cholangiocarcinoma cells\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. These show that inhibition the expression and activity of NHE1 will provide a new method of tumor treatment.\u003c/p\u003e \u003cp\u003eIn addition, KarkiP's study has shown that BRAF protein directly binds to NHE1 and enhances its activity in cervical cancer and renal cancer cells\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. However, the regulation mechanism of NHE1 activation in GMB cells is still unknown. This study combined microenvironment regulator NHE1 and MAPK tumorigenic signaling pathway and aimed to investigate whether BRAF activates NHE1 in GBM cells and whether NHE1 mediates MAPK signaling pathway. In addition, the inhibition effects of NHE1 inhibitor alone or in combination with BRAF\u003csup\u003eV600E\u003c/sup\u003e inhibitor on GBM were analyzed.\u003c/p\u003e "},{"header":"Materials And Methods","content":"\u003cp\u003eCell culture\u003c/p\u003e\n\u003cp\u003eHuman glioblastoma cell lines U251 and AM38 were purchased from Saier Biotechnology Co., Ltd. (Tianjin, China). The U251 cells were cultured in DMEM/F12 medium (GIBCO BRL., Ltd., USA), the AM38 cells were cultured in RPMI 1640 medium (GIBCO BRL., Ltd., USA), supplemented with 10% fetal bovine serum (GIBCO BRL., Ltd., USA), 100 IU/ml of penicillin and 100 \u0026micro;g/ml of streptomycin (Beijing Dingguochangsheng Biotechnology Co., Ltd., Beijing, China). All cells were cultured at 37℃ in a humidified incubator containing 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\n\u003cp\u003eCell proliferation assays\u003c/p\u003e\n\u003cp\u003eCell proliferation assays were performed using MTT (Tianjin Saier Biotechnology Co., Ltd., Tianjin, China). The cells were seeded into 96-well microtiter plates (Orange Scientific, Ltd., Belgium) at a density of 1.0x10\u003csup\u003e4\u003c/sup\u003e cells/well and cultured for 24 h at 37℃ in an incubator containing 5% CO\u003csub\u003e2\u003c/sub\u003e. The cells were then respectively treated with HOE-642 (0.16 nM, CAS: 159138-80-4) or SB590885 (0.16 nM, CAS: 405554-55-4) for 24 h. Subsequently, 10 \u0026micro;L MTT (5 mg/mL) was added to each well. After termination of culture, the absorbance was measured at 570 nm using a microplate reader (Bio-tek, Ltd., USA). Each experiment was carried out in three replicate wells and was repeated three times.\u003c/p\u003e\n\u003cp\u003eIntracellular pH measurements\u003c/p\u003e\n\u003cp\u003eFor digital imaging of cytosolic pH, cell suspensions were prepared with HEPES at the cell concentration of 4 x 10\u003csup\u003e7\u003c/sup\u003e cells/ml. Adding BCECF-AM/DMSO solution to the cell suspension to a final concentration of 3 \u0026micro;M. After incubating at 37℃ for 30 min, the cells were washed 3 times with HEPES buffer to prepare a cell suspension of 3x10\u003csup\u003e6\u003c/sup\u003e cells/ml. The fluorescence values of OD1 (440 nm) and OD2 (490 nm) were detected by a multifunctional microplate reader. The fluorescence ratio (FIR\u0026thinsp;=\u0026thinsp;OD2/OD1) reflects the pH and thus the activity of NHE1 in the cell.\u003c/p\u003e\n\u003cp\u003ePlasmids and transfection\u003c/p\u003e\n\u003cp\u003eThe whole gene synthesis film BRAF\u003csup\u003eV600E\u003c/sup\u003e was purchased from Saier Biotechnology Co., Ltd. (Tianjin, China). The outer film and the expression vector pcDNA3.1(+) were double-digested and ligated, and positive clones were selected after transformation with XL1-blue competent E. coli (Stratagene, Co., Ltd., USA). Then, the plasmid was extracted in small amounts by alkaline lysis. After digestion and identification of the recombinant plasmid, the digested product was subjected to 0.8% agarose gel electrophoresis to identify and record the correct recombinant plasmid (5.4 kb and 2.3 kb fragment bands). All plasmids were verified by sequencing. Then, the plasmid was extracted and purified using the B-type plasmid small volume rapid extraction kit (Bodatec, Co., Ltd., Beijing, China). The U251 cells were seeded in 6-well plates and cultured for 24 h. Subsequently, the cells were respectively transfected with the pcDNA3.1(+), pcDNA3.1(+)/BRAF\u003csup\u003eV600E\u003c/sup\u003e using Lipofectamine 2000 Reagent (Invitrogen, Co., Ltd., USA), according to the manufacturer's protocol.\u003c/p\u003e\n\u003cp\u003eWestern blot\u003c/p\u003e\n\u003cp\u003eTotal protein was collected using protein lysis buffer containing 1 \u0026micro;l protease inhibitor (Beijing Suo Laibao Technology Co., Ltd., Beijing, China) and 1 ml radioimmunoprecipitation assay (RIPA) lysis buffer at 14,000xg for 30 min at 4℃. The total protein concentration was calculated using the BCA assay according to the kit instructions (Merck, Co., Ltd., USA). The proteins were boiled at 99℃ for 5 min. The samples were transferred to PVDF membranes (Millipore, Co., Ltd., Shanghai, China) following SDS-PAGE. Subsequently, the membranes were incubated for 1 h in blocking buffer (containing 5% skim milk), then incubated with the following primary antibodies overnight at 4℃: anti-BRAF antibody (1:1000; Rabbit. no. ab33899; Abcam, Co., Ltd., USA), anti-BRAF\u003csup\u003eV600E\u003c/sup\u003e antibody (1:500; Rabbit. no. ab200535; Abcam, Co., Ltd., USA), anti-NHE1 antibody (1:1000; Rabbit. no. 67363-1-Ig; abbexa, Ltd., USA), anti-Phospho-(Ser) 14-3-3 binding motif antibody (1:500; Rabbit. no. 9601S; CST, Co., Ltd., USA), anti-extracellular signal-regulated kinase (ERK) antibody (1:2000; Rabbit. no. 51068-1-AP; proteintech, Co., Ltd., USA), anti-p-ERK antibody (1:500; Rabbit. no. 9101S; CST, Co., Ltd., USA), anti-E-cadherin antibody (1:500; Rabbit. no. SRP05266; Saier Biotechnology Co., Ltd., Tianjin, China), anti-Vimentin antibody (1:500; Rabbit. no. SRP01327; Saier Biotechnology Co., Ltd., Tianjin, China), anti-RSK antibody (1:500; Rabbit. no. SRP08000; Saier Biotechnology Co., Ltd., Tianjin, China), p-RSK antibody (1:500; Rabbit. no. 9341; CST, Co., Ltd., USA), anti-GAPDH antibody (1:1000; Rabbit. no. SRP13406; Saier Biotechnology Co., Ltd., Tianjin, China). The membranes were washed 4 times in Tris-buffered saline solution with 1X TBST and incubated with horseradish peroxidase-conjugated goat-anti-rabbit antibody (Amresco Co., Ltd., USA) for 1.5 h at RT. Following a final wash with 1X TBST, immunoreactive bands were detected using the ChampGel automatic gel imaging analyzer (Beijing SageCreation Science Co., Ltd., China). Optical band density was quantified using Image J (National Institutes of Health, Bethesda, MD, USA).\u003c/p\u003e\n\u003cp\u003eTranswell invasion assay\u003c/p\u003e\n\u003cp\u003eThe cell invasion capability was detected using transwell chamber culture systems. A total of 1x10\u003csup\u003e5\u003c/sup\u003e cells were placed onto a Matrigel-coated transwell chamber with serum-free opti-MEM medium (Thermo Fisher Scientific, Inc., USA). The DMEM medium containing 20% FBS was added to the lower chamber as a chemoattractant. After 24 h, the cells attached to the lower surface of the insert filter were fixed with 33% (v/v) acetic acid (glacial acetic acid: methyl alcohol\u0026thinsp;=\u0026thinsp;1: 3) and stained with crystal violet and counted.\u003c/p\u003e\n\u003cp\u003eImmunoprecipitation (IP) and Western blot\u003c/p\u003e\n\u003cp\u003eIP assays and western blot were performed as described previously\u003csup\u003e35\u003c/sup\u003e. Briefly, the cell extracts were incubated with magnetic beads antibody complex for 5 h at 4\u0026deg;C. Then the complex was washed and the immunoprecipitated proteins were analyzed by SDS/PAGE, transferred onto PVDF membrane and detected using each antibody.\u003c/p\u003e\n\u003cp\u003eAnimal experiment\u003c/p\u003e\n\u003cp\u003eNude mice were obtained from Tianjin Saier Biotechnology Co., Ltd. For the subcutaneous and metastatic models, 1\u0026times;10\u003csup\u003e7\u003c/sup\u003e/ml U251 and AM-38 cells suspended in 100 \u0026micro;l medium were injected subcutaneously on the left back of nude mice. SB590885 (50mg/kg) or HOE-642 (15mg/kg) were injected into the abdominal cavity per mouse every 2 days. After 3 weeks of observation, the two groups of nude mice with different tumor formation were anesthetized and photographed. All studies performed with mice were approved by the Animal Care Committee of North China University of Science and Technology. All experiments involving mice complied with local and international regulations, ethical guidelines and the ARRIVE guidelines.\u003c/p\u003e\n\u003cdiv\u003e\n\u003cp\u003eStatistical analysis\u003c/p\u003e\n\u003cp\u003eAll statistical analysis was performed using SPSS software version 17.0 (SPSS, Inc., Chicago, IL, USA). The data are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). One-way analysis of variance was used to analyze differences between groups. Scheffe post hoc testing was used to determine pairwise differences between means. \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered to indicate a statistically significant difference.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results","content":" \u003cp\u003eNHE1 was activated in BRAF\u003csup\u003eV600E\u003c/sup\u003e-mutant AM38 cells\u003c/p\u003e \u003cp\u003eThe protein level of BRAF and BRAF\u003csup\u003eV600E\u003c/sup\u003e in AM38 cells were significantly higher than these in U251 cells (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). It confirmed that AM38 cell contains BRAF\u003csup\u003eV600E\u003c/sup\u003e-mutant. Both the expression and phosphorylation levels of NHE1 in AM38 cells were obviously higher than those in U251 cells (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.022, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001, respectively) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). The FIR value of AM38 cells, which could indirectly reflect NHE1 activity, was markedly higher than that of U251 cells (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.005) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). These data suggested that the expression, phosphorylation level and activity of NHE1 might be related to BRAF\u003csup\u003eV600E\u003c/sup\u003e mutation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNHE1 was a downstream factor of BRAF\u003csup\u003eV600E\u003c/sup\u003e and an upstream regulator of ERK\u003c/p\u003e \u003cp\u003eTo explore the relationship between BRAF\u003csup\u003eV600E\u003c/sup\u003e and NHE1, the BRAF\u003csup\u003eV600E\u003c/sup\u003e-overexpressed pcDNA3.1(+) plasmids were constructed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) and the overexpression of BRAF\u003csup\u003eV600E\u003c/sup\u003e in U251 cells at 48 h post-transfection were confirmed (\u003cem\u003eF\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1971.738, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; pcDNA3.1(+)/BRAF\u003csup\u003eV600E\u003c/sup\u003e group vs pcDNA3.1(+) group \u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). The protein levels of BRAF\u003csup\u003eV600E\u003c/sup\u003e were not affected by NHE1 inhibitor HOE-642 (pcDNA3.1(+)/BRAF\u003csup\u003eV600E\u003c/sup\u003e group vs pcDNA3.1(+)/BRAF\u003csup\u003eV600E\u003c/sup\u003e\u003cem\u003e+\u003c/em\u003eHOE-642 group \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.083) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). This showed that BRAF\u003csup\u003eV600E\u003c/sup\u003e was not affected by NHE1.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eHowever, both NHE1 expression (\u003cem\u003eF\u003c/em\u003e\u0026thinsp;=\u0026thinsp;29.765, \u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001) and phosphorylated NHE1 (p-NHE1) levels (\u003cem\u003eF\u003c/em\u003e\u0026thinsp;=\u0026thinsp;45.887, \u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), as well as the FIR value (NHE1 activity) (\u003cem\u003eF\u003c/em\u003e\u0026thinsp;=\u0026thinsp;214.093, \u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001), in BRAF\u003csup\u003eV600E\u003c/sup\u003e-overexpressed U251 cells were significantly upregulated compared with those of U251 cells (pcDNA3.1(+)/BRAF\u003csup\u003eV600E\u003c/sup\u003e group vs pcDNA3.1(+) group: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001, respectively) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). HOE-642 dramatically reversed the effect of BRAF\u003csup\u003eV600E\u003c/sup\u003e overexpression on NHE1 activity (pcDNA3.1(+)/BRAF\u003csup\u003eV600E\u003c/sup\u003e group vs pcDNA3.1(+)/BRAF\u003csup\u003eV600E\u003c/sup\u003e\u003cem\u003e+\u003c/em\u003eHOE-642 group \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.002) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD), but did not affected the NHE1 expression and phosphorylation (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.961, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.198, respectively) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). These suggested that NHE1 was a downstream factor of BRAF\u003csup\u003eV600E\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eMoreover, the phosphorylated ERK (p-ERK) level was markedly increased by BRAF\u003csup\u003eV600E\u003c/sup\u003e-overexpression, while was partially decreased by HOE-642 treatment (\u003cem\u003eF\u003c/em\u003e\u0026thinsp;=\u0026thinsp;160.760, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001; \u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001, \u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001, respectively), while total ERK levels were not affected (\u003cem\u003eF\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.396, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.172) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). This suggested that NHE1 inhibitor could inhibit ERK phosphorylation. These data indicated that NHE1 was involved in BRAF/ERK signal pathway as an upstream regulator of ERK.\u003c/p\u003e \u003cp\u003eThe proliferation and invasion abilities of U251 cells enhanced by BRAF\u003csup\u003eV600E\u003c/sup\u003e overexpression were inversed by NHE1 inhibitor\u003c/p\u003e \u003cp\u003eBoth the proliferation and invasion abilities of BRAF\u003csup\u003eV600E\u003c/sup\u003e-overexpressed U251 cells were significantly enhanced compared with those of U251 at 48 h post-transfection, while the effects were significantly reversed by HOE-642 treatment (proliferation: \u003cem\u003eF\u003c/em\u003e\u0026thinsp;=\u0026thinsp;62.197, \u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001; \u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.002, respectively) (invasion: \u003cem\u003eF\u003c/em\u003e\u0026thinsp;=\u0026thinsp;125.601, \u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001; \u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001, \u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001, respectively) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). In addition, the proliferation marker Ki67 and the mesenchymal cell marker Vimentin were all significantly higher in BRAF\u003csup\u003eV600E\u003c/sup\u003e-overexpressed U251 cells, whereas epithelial cell marker E-cadherin was markedly lower, than those in U251 cells (for Ki67: \u003cem\u003eF\u003c/em\u003e\u0026thinsp;=\u0026thinsp;277.911, \u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001; \u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001; for Vimentin: \u003cem\u003eF\u003c/em\u003e\u0026thinsp;=\u0026thinsp;76.854, \u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001; \u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001; for E-cadherin: \u003cem\u003eF\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7208.162, \u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001; \u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). NHE1 inhibitor HOE-642 dramatically inversed the effects of BRAF\u003csup\u003eV600E\u003c/sup\u003e overexpression on protein levels of Ki67 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001) and Epithelial-Mesenchymal Transition (EMT) markers (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, \u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001, respectively) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). These data indicated that NHE1 inhibitor could repress proliferation and invasion of BRAF\u003csup\u003eV600E\u003c/sup\u003e GBM cells by affecting Ki67 and EMT.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe phosphorylation and activity of NHE1 were positively regulated by p-ERK\u003c/p\u003e \u003cp\u003eThere was no significantly difference of total ribosomal S6 kinase (RSK) between U251 cells and AM38 cell (\u003cem\u003eF\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3.750, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.060, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.845) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA left). However, the level of phosphorylated RSK (p-RSK), an active marker of ERK signal pathway, was significantly higher in AM cells than that in U251 cells (\u003cem\u003eF\u003c/em\u003e\u0026thinsp;=\u0026thinsp;53.847, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.060, \u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA right). Total ESK levels in U152 and AM38 cells were not affected by ERK agonist Honokiol and ERK inhibitor SCH772984, respectively (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.986, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.275) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA left). Whereas p-RSK was markedly increased in Honokiol-treated U251 cells compared with that in untreated U251 cells (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001), and was significantly decreased in SCH772984-treated AM38 cells compared with that in untreated AM38 cells (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001). These confirmed the effectiveness of Honokiol and SCH772984 on ERK activity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn addition, it was found that FIR value and p-NHE1 level in Honokiol-treated U251 cells were significantly upregulated compared those in untreated U251 cells (for FIR: \u003cem\u003eF\u003c/em\u003e\u0026thinsp;=\u0026thinsp;47.399, \u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001; \u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001; for p-NHE1: \u003cem\u003eF\u003c/em\u003e\u0026thinsp;=\u0026thinsp;133.299, \u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001; \u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001), while there was no significant difference of total NH1 among indicated groups (\u003cem\u003eF\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1.384, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.316) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Whereas FIR value and p-NHE1 level in AM38 cells treated with SCH772984 were dramatically decreased than those in untreated AM38 cells (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.005, \u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001, respectively) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). These data suggested that NEH1 could be phosphorylated and activated by p-ERK. Co-IP analysis confirmed that NHE1 directly interacted with BRAF\u003csup\u003eV600E\u003c/sup\u003e in AM38 cells, but not with wild type BRAF (BRAF\u003csup\u003eWT\u003c/sup\u003e) and ERK (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eThe combination of NHE1 inhibitor and BRAF\u003csup\u003eV600E\u003c/sup\u003e inhibitor had better inhibitory effects on proliferation and invasion abilities of GBM cells with BRAF\u003csup\u003eV600E\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eThe NHE1 activities (FIR) in U251 were suppressed by NHE1 inhibitor HOE-642, but not by the BRAF\u003csup\u003eV600E\u003c/sup\u003e inhibitor SB590885 (\u003cem\u003eF\u003c/em\u003e\u0026thinsp;=\u0026thinsp;14.950, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001, HOE-642 group vs. DMSO group \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.011, SB590885 group vs. DMSO group \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.892) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA left). The NHE1 activities in AM38 cells were suppressed by HOE-642, SB590885, as well as combination of them, respectively (\u003cem\u003eF\u003c/em\u003e\u0026thinsp;=\u0026thinsp;101.013, \u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001, HOE-642 group vs. DMSO group \u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001, SB590885 group vs. DMSO group \u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001, HOE-642\u0026thinsp;+\u0026thinsp;SB590885 group vs. DMSO group \u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA right). The combination of HOE-642 and SB590885 had better inhibitory effects on NHE1 activities than HOE-642 or SB590885 alone in AM38 cells (HOE-642\u0026thinsp;+\u0026thinsp;SB590885 group vs. HOE-642 group \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.011; vs SB590885 group \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.030) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA right).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBoth the proliferation and invasion abilities of U251 cells were inhibited by HOE-642, but not by SB590885 (proliferation: \u003cem\u003eF\u003c/em\u003e\u0026thinsp;=\u0026thinsp;12.109, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.002; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.037, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.979,Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB left; invasion: \u003cem\u003eF\u003c/em\u003e\u0026thinsp;=\u0026thinsp;30.379, \u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.003, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.699, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC left). Both the proliferation and invasion abilities of AM38 cells were suppressed by HOE-642, SB590885, as well as combination of them, respectively (proliferation: \u003cem\u003eF\u003c/em\u003e\u0026thinsp;=\u0026thinsp;33.877, \u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.007, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.002, \u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB right; invasion: \u003cem\u003eF\u003c/em\u003e\u0026thinsp;=\u0026thinsp;89.933, \u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001; \u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001, \u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001, \u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC right). The combination of HOE-642 and SB590885 had better inhibitory effects both on proliferation and invasion of AM38 cells than HOE-642 or SB590885 alone (proliferation: HOE-642\u0026thinsp;+\u0026thinsp;SB590885 group vs. HOE-642 group \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.009; vs. SB590885 group \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.043; invasion: HOE-642\u0026thinsp;+\u0026thinsp;SB590885 group vs. HOE-642 group \u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001; vs. SB590885 group \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.044) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA right).\u003c/p\u003e \u003cp\u003eThe Ki67 levels in U251 cells treated with HOE-642, SB590885 and combination of them were respectively suppressed by 41.5%, 47.5% and 63.0% compared with DMSO group (\u003cem\u003eF\u003c/em\u003e\u0026thinsp;=\u0026thinsp;22.672, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.006; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.030, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.019, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.007), while combination of HOE-642 and SB590885 has no advantage than each alone (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.208, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.402) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD left). The Ki67 levels in AM38 cells of the three groups were respectively suppressed by 28.5%, 33.1% and 55.0% compared with DMSO group (\u003cem\u003eF\u003c/em\u003e\u0026thinsp;=\u0026thinsp;51.534, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.014, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.008, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001), while the inhibition effect of the combination group was better than each drug alone (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.036) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD right).\u003c/p\u003e \u003cp\u003eFor the EMT marker, the E-cadherin in U251 cells were markedly upregulated by HOE-642, but not by SB590885 (\u003cem\u003eF\u003c/em\u003e\u0026thinsp;=\u0026thinsp;45.423, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.002; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.006, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.764, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE left), while E-cadherin in AM38 cells were significantly increased by HOE-642, SB590885, as well as combination of them, respectively (\u003cem\u003eF\u003c/em\u003e\u0026thinsp;=\u0026thinsp;191.572, \u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.024, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.003, \u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE right). On the other hand, the Vimentin in U251 cells were markedly downregulated by HOE-642, but not by SB590885 (\u003cem\u003eF\u003c/em\u003e\u0026thinsp;=\u0026thinsp;38.388, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.002; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.031, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.895, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF left), while those in AM38 cells were not changed by HOE-642 or SB590885 alone (\u003cem\u003eF\u003c/em\u003e\u0026thinsp;=\u0026thinsp;28.796, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.004; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.266, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.083, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF right). The effects of combination of HOE-642 and SB590885 on E-cadherin and Vimentin in AM38 cells were better than each single drug (E-cadherin: \u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE right; Vimentin: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.013, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.030, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF right). These data indicated that the combination of BRAF\u003csup\u003eV600E\u003c/sup\u003e inhibitor and NHE1 inhibitor has better inhibitory effects on proliferation and invasion abilities of GBM cells with BRAF\u003csup\u003eV600E\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe combination of BRAF\u003csup\u003eV600E\u003c/sup\u003e inhibitor and NHE1 inhibitor had better inhibitory effects on GBM cells with BRAF\u003csup\u003eV600E\u003c/sup\u003e in vivo\u003c/p\u003e \u003cp\u003eTo further explore the inhibitory effects of combined BRAF\u003csup\u003eV600E\u003c/sup\u003e inhibitor and NHE1 inhibitor on GBM cells, nude mice tumorigenesis experiments were conducted. All nude mice were in good health and activity before treatment and did not die until the end of the experiment. Figure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA shows tumor formation in nude mice in each group. There were no significantly difference of body weight among all the indicated groups (U251: \u003cem\u003eF\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.452, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.718; AM38: \u003cem\u003eF\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.293, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.830) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). The U251 tumor volumes were markedly downregulated by HOE-642, but not by SB590885 (\u003cem\u003eF\u003c/em\u003e\u0026thinsp;=\u0026thinsp;22.387, \u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.007, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.954) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). The AM38 tumor volumes were significantly decreased by HOE-642, SB590885, as well as combination of them, respectively (\u003cem\u003eF\u003c/em\u003e\u0026thinsp;=\u0026thinsp;22.823, \u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001, \u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). The inhibitory effects of combination of HOE-642 and SB590885 on tumor volumes of U251 and AM38 cells were better than each single drug (U251: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.040, \u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001, AM38: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.049, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.026, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). These indicated that the inhibitory effects of combination of HOE-642 and SB590885 on tumor volumes of U251cells and AM38 cells were better than each single drug.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn addition, the AM38 tumor weight was markedly higher than U251 tumor weight (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD left, middle). The U251 tumor wrights treated with HOE-642, SB590885 and combination of them were respectively suppressed by 41.9%, 30.0% and 64.2% compared with DMSO group, respectively (\u003cem\u003eF\u003c/em\u003e\u0026thinsp;=\u0026thinsp;30.620, \u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001; \u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.003, \u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD, right). The AM38 tumor weights of the three groups were respectively suppressed by 63.1%, 62.9% and 78.8% compared with DMSO group, respectively (\u003cem\u003eF\u003c/em\u003e\u0026thinsp;=\u0026thinsp;107.482, \u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001; \u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001, \u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001, \u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD, right). Moreover, the inhibitory effects of combination of HOE-642 and SB590885 on tumor weights of U251 cells and AM38 cells were all better than each single drug (U251: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.033, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001; AM38: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.030, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.029) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD right). These data confirmed that the combination of BRAF\u003csup\u003eV600E\u003c/sup\u003e inhibitor and NHE1 inhibitor has better inhibitory effects on proliferation of U251 cells than each single drug \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e "},{"header":"Discussion","content":" \u003cp\u003eThe present study found that the expression, phosphorylation and activity of NHE1 in BRAF\u003csup\u003eV600E\u003c/sup\u003e-mutant AM38 cells were all higher than those in BRAF\u003csup\u003eWT\u003c/sup\u003e U251 cells. Overexpression of BRAF\u003csup\u003eV600E\u003c/sup\u003e activated the ERK pathway and upregulated the expression, phosphorylation and activity of NHE1, as well as the cell viabilities, invasion abilities and corresponding markers. Those effects of BRAF\u003csup\u003eV600E\u003c/sup\u003e overexpression were reversed by NHE1 inhibitor HOE-642. These data indicated that NHE1 is a downstream target of BRAF\u003csup\u003eV600E\u003c/sup\u003e and an upstream factor of ERK. The microenvironment factor NHE1 interacts with BRAF\u003csup\u003eV600E\u003c/sup\u003e/ERK oncogenic signaling pathway in GBM cells.\u003c/p\u003e \u003cp\u003eThis work also found that NHE1 was directly interacted with BRAF\u003csup\u003eV600E\u003c/sup\u003e in BRAF\u003csup\u003eV600E\u003c/sup\u003e GBM cells. This is consistent with the report in malignant melanoma cells, in which stimulated NHE1 by BRAF\u003csup\u003eV600E\u003c/sup\u003e induced to aberrant pH\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. There was speculated that a similar mechanism may exist in cancer cells with BRAF\u003csup\u003eV600E\u003c/sup\u003e mutation. Moreover, the transport activity of NHE1 regulated by multiple intracellular signaling molecules, including MAPK/ERK\u003csup\u003e23\u003c/sup\u003e and PI3K/AKT kinases\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e, those interact with different site of serine residues of the cytosolic C-terminus and phosphorylate NHE1. Additionally, we found that both the phosphorylation and activity of NHE1 were positively regulated by ERK activator and inhibitor in GBM cells, but not NHE1 expression. These suggested that there is a positive feedback loop between NHE1-ERK phosphorylation under regulation of BRAF\u003csup\u003eV600E\u003c/sup\u003e mutation contributing to the proliferation and invasion of GBM cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). However, we did not detected interaction between NHE1 and ERK in BRAF\u003csup\u003eV600E\u003c/sup\u003e GBM cells. We speculated that BRAF\u003csup\u003eV600E\u003c/sup\u003e may have the advantage structure to combine with NHE1 in compared with BRAF and ERK in GBM cells. Evidences have suggested the ERK, MEK, RSK, RAF-1, PAK5, and 14-3-3 exist as complexes in some cell types\u003csup\u003e\u003cspan additionalcitationids=\"CR27 CR28 CR29 CR30\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. The interaction between NHE1 and PI3K/AKT signaling pathways in GBM cells should be considered in the further study.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn addition, the proliferation and invasion abilities of BRAF\u003csup\u003eV600E\u003c/sup\u003e-mutant and BRAF\u003csup\u003eWT\u003c/sup\u003e GBM cells, as well as the corresponding markers, were all suppressed by the NHE1 inhibitor, BRAF\u003csup\u003eV600E\u003c/sup\u003e inhibitor and combination of them. The inhibitory effect of combination of the two inhibitors in BRAF\u003csup\u003eV600E\u003c/sup\u003e-mutant cells was better than each single drug both in \u003cem\u003evitro\u003c/em\u003e and in \u003cem\u003evivo\u003c/em\u003e. This probably partly due to the inhibition of some compensation mechanism by the two inhibitors in BRAF\u003csup\u003eV600E\u003c/sup\u003e-mutant GBM cells. For the persistence of suppressive effect of combination treatment, this work only observed data by day 21. Further study should be performed.\u003c/p\u003e \u003cp\u003eThere have several selective inhibitors to be invested and used for clinical treatment of tumors with BRAF\u003csup\u003eV600E\u003c/sup\u003e mutation: (1) Vemurafenib (PLX4032) could selectively bind to the ATP-binding site of BRAF\u003csup\u003eV600E\u003c/sup\u003e and inhibits its activity\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. A clinical case report showed that children GBM with BRAF\u003csup\u003eV600E\u003c/sup\u003e mutation received Vemurafenib treatment and achieved clinical complete remission (Robinson et al., 2014). (2) UAI-201 promotes GBM cell cycle inhibition and autophagy by blocking BRAF\u003csup\u003eV600E\u003c/sup\u003e/MEK/ERK pathway in glioma cells with BRAF\u003csup\u003eV600E \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. (3) Dabrafenib (GSK2118436) was approved for unresectable or metastatic melanoma and anaplastic thyroid cancer harbouring the BRAF\u003csup\u003eV600E\u003c/sup\u003e mutation as monotherapy or in combination with trametinib (a MAPK kinase inhibitor)\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Our work showed that SB590885 is also an effective inhibitor for GBM with BRAF\u003csup\u003eV600E\u003c/sup\u003e mutation in vivo and in vitro, which provides a basis for clinical application. Moreover, HOE-642 is another candidate drug for GBM cells BRAF\u003csup\u003eV600E\u003c/sup\u003e mutation and has good synergistic effect with BRAF\u003csup\u003eV600E\u003c/sup\u003e inhibitor. These data could enlighten treatment for breast cancer and cholangiocarcinoma\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTo conclude, the present study clarified that microenvironment factor NHE1 involves in BRAF\u003csup\u003eV600E\u003c/sup\u003e/ERK oncogenic signaling pathway and contributes to the proliferation and invasion of GBM cells. Combination of BRAF\u003csup\u003eV600E\u003c/sup\u003e and NHE1 inhibitors probably considered as a new therapeutic regimen for future research and clinical application of GBM with BRAF\u003csup\u003eV600E\u003c/sup\u003e.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYHL and DL performed most of the experiments and wrote the manuscript. YKL and SQW guided experimental technology. MYS performed some of the experiments. ZYZ and XZ guided and performed statistical analysis. YFL and JWL conceived the project and supervised the experiments. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Science and Technology Innovation Team Training Plan Project Fund of Tangshan (18130203B), 2019 City Talent Project Fund of Tangshan (A201905007) and Fund of Key Laboratory of Hebei Province (SZX2020043).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts interest \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLiu, R. 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Na/H exchanger 1 (NHE1), a main pH regulator affecting cell microenvironment, is hyper-expressed in GBM. However, the relationship between BRAF\u003csup\u003eV600E\u003c/sup\u003e signal pathway and NHE1 in GMB cells remains unclear. This study found that NHE1 was a downstream target of BRAF\u003csup\u003eV600E\u003c/sup\u003e and an upstream factor of extracellular signal-regulated kinase (ERK). In addition, there was a positive feedback loop between NHE1-ERK phosphorylation under regulation of BRAF\u003csup\u003eV600E\u003c/sup\u003e mutation contributing to the proliferation and invasion of GBM cells. Moreover, the proliferation and invasion abilities of BRAF\u003csup\u003eV600E\u003c/sup\u003e-mutant and BRAF\u003csup\u003e \u003c/sup\u003ewild type GBM cells were all suppressed by the NHE1 inhibitor, BRAF\u003csup\u003eV600E\u003c/sup\u003e inhibitor and combination of them. The inhibitory effect of combination of the two inhibitors was better than each single drug both in \u003cem\u003evitro \u003c/em\u003eand in \u003cem\u003evivo\u003c/em\u003e. Combination of BRAF\u003csup\u003eV600E\u003c/sup\u003e and NHE1 inhibitors could be considered as a new therapeutic regimen for GBM, especially for GBM with BRAF\u003csup\u003eV600E\u003c/sup\u003e.\u003c/p\u003e","manuscriptTitle":"The Positive Feedback Loop of NHE1-ERK Phosphorylation Mediated by BRAFV600E Mutation Contributes to Tumorigenesis and Development of Glioblastoma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-05-27 21:56:30","doi":"10.21203/rs.3.rs-523299/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"200bc745-3c11-4d70-9108-049dc3545376","owner":[],"postedDate":"May 27th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":4555399,"name":"Neurosurgery"},{"id":4555400,"name":"Nuclear Medicine \u0026 Medical Imaging"}],"tags":[],"updatedAt":"2021-12-01T07:54:44+00:00","versionOfRecord":{"articleIdentity":"rs-523299","link":"https://doi.org/10.1016/j.bbrc.2021.11.104","journal":{"identity":"biochemical-and-biophysical-research-communications","isVorOnly":true,"title":"Biochemical and Biophysical Research Communications"},"publishedOn":"2021-12-01 07:54:44","publishedOnDateReadable":"December 1st, 2021"},"versionCreatedAt":"2021-05-27 21:56:30","video":"","vorDoi":"10.1016/j.bbrc.2021.11.104","vorDoiUrl":"https://doi.org/10.1016/j.bbrc.2021.11.104","workflowStages":[]},"version":"v1","identity":"rs-523299","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-523299","identity":"rs-523299","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}