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

doi:10.21203/rs.3.rs-523299/v1

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The Positive Feedback Loop of NHE1-ERK Phosphorylation Mediated by BRAF^V600E Mutation Contributes to Tumorigenesis and Development of Glioblastoma

https://doi.org/10.21203/rs.3.rs-523299/v1

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published 01 Dec, 2021

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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 BRAFwild 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

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 pathway³. In gliomas, the expression level and activity of BRAF protein are positively correlated with the malignant degree of gliomas⁴. 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 metabolism³. BRAF^V600E inhibitors Vemurafenib (PLX4032) and dabrafenib (GSK21118436) have been approved for the treatment of BRAF^V600E mutant melanoma⁵. 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 pH⁸. 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 ischemia⁹. 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 metastasis¹⁰ and induce apoptosis of leukemia cells¹¹. 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 concerned¹⁸: 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 cells²⁰. 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 cells²¹. 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.

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% CO₂.

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 1.0x10⁴ cells/well and cultured for 24 h at 37℃ in an incubator containing 5% CO₂. 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 10⁷ 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 3x10⁶ 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 1x10⁵ 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 previously³⁵. 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, 1×10⁷/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.

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. 1A). 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. 1B, 1C). The FIR value of AM38 cells, which could indirectly reflect NHE1 activity, was markedly higher than that of U251 cells (P = 0.005) (Fig. 1D). 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. 2A) 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. 2B). 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. 2B). 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. 2C), 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. 2D). 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. 2D), but did not affected the NHE1 expression and phosphorylation (P = 0.961, P = 0.198, respectively) (Fig. 2C). 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. 2E). 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. 3A, 3B). 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. 3C, 3D). 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. 3C, 3D). 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. 4A 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. 4A 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. 4A 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. 4B, 4C). 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. 4B, 4C). 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. 4D).

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. 5A 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. 5A 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. 5A 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. 5B left; invasion: F = 30.379, P < 0.001; P = 0.003, P = 0.699, Fig. 5C 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. 5B right; invasion: F = 89.933, P < 0.001; P < 0.001, P < 0.001, P < 0.001, Fig. 5C 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. 5A 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. 5D 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. 5D 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. 5E 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. 5E 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. 5F 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. 5F 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. 5E right; Vimentin: P = 0.013, P = 0.030, Fig. 5F 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 6A 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. 6B). 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. 6C). 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. 6C). 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. 6C). 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. 6D 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. 6D, 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. 6D, 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. 6D 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.

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 pH²². 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/ERK²³ 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 activity³². 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.

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.

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).

No competing interests reported.

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The Positive Feedback Loop of NHE1-ERK Phosphorylation Mediated by BRAF^V600E Mutation Contributes to Tumorigenesis and Development of Glioblastoma

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