Deletion of Slc9a1 in Cx3cr1+ cells stimulated microglial subcluster CREB1 signaling and microglia-oligodendrocyte crosstalk.

Microglial Na/H exchanger-1 (NHE1) protein, encoded by Slc9a1, plays a role in white matter demyelination of ischemic stroke brains. To explore underlying mechanisms, we conducted single cell RNA-seq transcriptome analysis in conditional Slc9a1 knockout (cKO) and wild-type (WT) mouse white matter tissues at 3 days post-stroke. Compared to WT, Nhe1 cKO brains expanded a microglial subgroup with elevated transcription of white matter myelination genes including Spp1, Lgals3, Gpnmb, and Fabp5. This subgroup also exhibited more acidic pHi and significantly upregulated CREB signaling detected by ingenuity pathway analysis and flow cytometry. Moreover, the Nhe1 cKO white matter tissues showed enrichment of a corresponding oligodendrocyte subgroup, with pro-phagocytosis and lactate shuffling gene expression, where activated CREB signaling is a likely upstream regulator. These findings demonstrate that attenuation of NHE1-mediated H+ extrusion acidifies microglia/macrophage and may underlie the stimulation of CREB1 signaling, giving rise to restorative microglia-oligodendrocyte interactions for remyelination.

Empagliflozin mitigates cardiac hypertrophy through cardiac RSK/NHE-1 inhibition.

BACKGROUND: SGLT2i reduce cardiac hypertrophy, but underlying mechanisms remain unknown. Here we explore a role for serine/threonine kinases (STK) and sodium hydrogen exchanger 1(NHE1) activities in SGLT2i effects on cardiac hypertrophy. METHODS: Isolated hearts from db/db mice were perfused with 1 µM EMPA, and STK phosphorylation sites were examined using unbiased multiplex analysis to detect the most affected STKs by EMPA. Subsequently, hypertrophy was induced in H9c2 cells with 50 µM phenylephrine (PE), and the role of the most affected STK (p90 ribosomal S6 kinase (RSK)) and NHE1 activity in hypertrophy and the protection by EMPA was evaluated. RESULTS: In db/db mice hearts, EMPA most markedly reduced STK phosphorylation sites regulated by RSKL1, a member of the RSK family, and by Aurora A and B kinases. GO and KEGG analysis suggested that EMPA inhibits hypertrophy, cell cycle, cell senescence and FOXO pathways, illustrating inhibition of growth pathways. EMPA prevented PE-induced hypertrophy as evaluated by BNP and cell surface area in H9c2 cells. EMPA blocked PE-induced activation of NHE1. The specific NHE1 inhibitor Cariporide also prevented PE-induced hypertrophy without added effect of EMPA. EMPA blocked PE-induced RSK phosphorylation. The RSK inhibitor BIX02565 also suppressed PE-induced hypertrophy without added effect of EMPA. Cariporide mimicked EMPA's effects on PE-treated RSK phosphorylation. BIX02565 decreased PE-induced NHE1 activity, with no further decrease by EMPA. CONCLUSIONS: RSK inhibition by EMPA appears as a novel direct cardiac target of SGLT2i. Direct cardiac effects of EMPA exert their anti-hypertrophic effect through NHE-inhibition and subsequent RSK pathway inhibition.

Simultaneous disturbance of NHE1 and LOXL2 decreases tumorigenicity of head and neck squamous cell carcinoma.

OBJECTIVE: Although there have been brilliant advancements in the practical application of therapies targeting immune checkpoints, achieving success in targeting the microenvironment remains elusive. In this study, we aimed to address this gap by focusing on Na+ / H+ exchanger 1 (NHE1) and Lysyl Oxidase Like 2 (LOXL2), which are upregulated in head and neck squamous cell carcinoma (HNSCC) cells. METHODS: The malignancy of a metastatic human HNSCC cell line was assessed in a mouse tongue cancer xenograft model by knocking down (KD) NHE1, responsible for regulating intracellular pH, and LOXL2, responsible for extracellular matrix (ECM) reorganization via cross-linking of ECM proteins. In addition to assessing changes in PD-L1 levels and collagen accumulation following knockdown, the functional status of the PD-L1 / PD-1 immune checkpoint was examined through co-culture with NK92MI, a PD-1 positive phagocytic human Natural Killer (NK) cell line. RESULTS: The tumorigenic potential of each single KD cell line was similar to that of the control cells, whereas the potential was attenuated in cells with simultaneous KD of both factors (double knockdown [dKD]). Additionally, we observed decreased PD-L1 levels in NHE1 KD cells and compromised collagen accumulation in LOXL2 KD and dKD cells. NK92MI cells exhibited phagocytic activity toward HNSCC cells in co-culture, and the number of remaining dKD cells after co-culture was the lowest in comparison to the control and single KD cells. CONCLUSION: This study demonstrated the possibility of achieving efficient anti-tumor effects by simultaneously disturbing multiple factors involved in the modification of the tumor microenvironment.

Myometrium infection decreases TREK1 through NHE1 and increases contraction in pregnant mice.

Intrauterine infection during pregnancy can enhance uterine contractions. A two-pore K+ channel TREK1 is crucial for maintaining uterine quiescence and reducing contractility, with its properties regulated by pH changes in cell microenvironment. Meanwhile, the sodium hydrogen exchanger 1 (NHE1) plays a pivotal role in modulating cellular pH homeostasis, and its activation increases smooth muscle tension. By establishing an infected mouse model of Escherichia coli (E. coli) and lipopolysaccharide (LPS), we used Western blotting, real-time quantitative polymerase chain reaction, and immunofluorescence to detect changes of TREK1 and NHE1 expression in the myometrium, and isometric recording measured the uterus contraction. The NHE1 inhibitor cariporide was used to explore the effect of NHE1 on TREK1. Finally, cell contraction assay and siRNA transfection were performed to clarify the relationship between NHE1 and TREK1 in vitro. We found that the uterine contraction was notably enhanced in infected mice with E. coli and LPS administration. Meanwhile, TREK1 expression was reduced, whereas NHE1 expression was upregulated in infected mice. Cariporide alleviated the increased uterine contraction and promoted myometrium TREK1 expression in LPS-injected mice. Furthermore, suppression of NHE1 with siRNA transfection inhibited the contractility of uterine smooth muscle cells and activated the TREK1. Altogether, our findings indicate that infection increases the uterine contraction by downregulating myometrium TREK1 in mice, and the inhibition of TREK1 is attributed to the activation of NHE1.NEW & NOTEWORTHY Present work found that infection during pregnancy will increase myometrium contraction. Infection downregulated NHE1 and followed TREK1 expression and activation decrease in myometrium, resulting in increased myometrium contraction.

Employing the sustained-release properties of poly(lactic-co-glycolic acid) nanoparticles to reveal a novel mechanism of sodium-hydrogen exchanger 1 in neuropathic pain.

Neuropathic pain is caused by injury or disease of the somatosensory system, and its course is usually chronic. Several studies have been dedicated to investigating neuropathic pain-related targets; however, little attention has been paid to the persistent alterations that these targets, some of which may be crucial to the pathophysiology of neuropathic pain. The present study aimed to identify potential targets that may play a crucial role in neuropathic pain and validate their long-term impact. Through bioinformatics analysis of RNA sequencing results, we identified Slc9a1 and validated the reduced expression of sodium-hydrogen exchanger 1 (NHE1), the protein that Slc9a1 encodes, in the spinal nerve ligation (SNL) model. Colocalization analysis revealed that NHE1 is primarily co-localized with vesicular glutamate transporter 2-positive neurons. In vitro experiments confirmed that poly(lactic-co-glycolic acid) nanoparticles loaded with siRNA successfully inhibited NHE1 in SH-SY5Y cells, lowered intracellular pH, and increased intracellular calcium concentrations. In vivo experiments showed that sustained suppression of spinal NHE1 expression by siRNA-loaded nanoparticles resulted in delayed hyperalgesia in naïve and SNL model rats, whereas amiloride-induced transient suppression of NHE1 expression yielded no significant changes in pain sensitivity. We identified Slc9a1, which encodes NHE1, as a key gene in neuropathic pain. Utilizing the sustained release properties of nanoparticles enabled us to elucidate the chronic role of decreased NHE1 expression, establishing its significance in the mechanisms of neuropathic pain.

Na+ /H+ exchanger NHE1 is active at cell-cell contacts and facilitates cell dissemination during collective migration of melanoma cells.

Tumour cell detachment from the primary tumour is an early and crucial step of the metastatic cascade. At the single cell level, it was already shown that migrating melanoma cells establish both intra- and extracellular pH gradients and that the Na+ /H+ exchanger NHE1 accumulates at the leading edges to strengthen cell-matrix interactions. However, less is known about the role of NHE1 in collective cell migration and the specific pH microenvironment at tumour cell-cell contacts. We used MV3 melanoma cells transfected with a NHE1-expressing vector or a control vector. NHE1 localization at cell-cell contacts was assessed via immunofluorescence imaging. Collective migration was analysed by live-cell imaging. The NHE1 activity and the perimembranous pH were measured both intra- and extracellularly by ratiometric fluorescence microscopy. NHE1 clearly localizes at cell-cell contacts. Its overexpression further increases migratory speed and translocation in multidirectional pathway analyses. NHE1 overexpressing MV3 cells also move further away from their neighbouring cells during wound closure assays. pH measurements revealed that the NHE1 is highly active at cell-cell contacts of melanoma cells. NHE1-mediated pH dynamics at such contact sites are more prominent in NHE1-overexpressing melanoma cells. Our findings highlight the contribution of the NHE1 towards modulation and plasticity of melanoma cell-cell contacts. We propose that its localization and functional activity at cell-cell contacts promotes evasion of single melanoma cells from the primary tumour.

Slc9a1 plays a vital role in chitosan oligosaccharide transport across the intestinal mucosa of mice.

The mechanism underlying the intestinal transport of COS is not well understood. Here, transcriptome and proteome analyses were performed to identify potential critical molecules involved in COS transport. Enrichment analyses revealed that the differentially expressed genes in the duodenum of the COS-treated mice were mainly enriched in transmembrane and immune function. In particular, B2 m, Itgb2, and Slc9a1 were upregulated. The Slc9a1 inhibitor decreased the transport efficiency of COS both in MODE-K cells (in vitro) and in mice (in vivo). The transport of FITC-COS in Slc9a1-overexpressing MODE-K cells was significantly higher than that in empty vector-transfected cells (P < 0.01). Molecular docking analysis revealed the possibility of stable binding between COS and Slc9a1 through hydrogen bonding. This finding indicates that Slc9a1 plays a crucial role in COS transport in mice. This provides valuable insights for improving the absorption efficiency of COS as a drug adjuvant.

Targeting Na-H exchanger 1 overcomes nuclear factor kappa B-mediated tumor resistance to radiotherapy.

Intrinsic or acquired radioresistance often limits the efficacy of radiation therapy (RT), thereby leading to local control failure. Cancerous cells have abnormal pH dynamics due to high metabolic demands, but it is unclear how pH dynamics contribute to radioresistance. In this study, we investigated the role of Na-H exchange 1 (NHE1), the major intracellular pH (pHi) regulator, in RT response. We observed that RT increased NHE1 expression and modulated pHi in MDA-MB-231 human breast cancer cells. When combined with RT, pharmacological NHE1 inhibition by 5-(N-Ethyl-N-isopropyl)amiloride (EIPA) reduced pHi and clonogenic survival. EIPA attenuated radiation-damaged DNA repair, increasing G2/M cell cycle arrest. The combination of EIPA and RT increased apoptotic cell death while decreasing phosphorylation of NF-κB p65. Similarly, the knockdown of NHE1 increased radiosensitivity with lower pHi and increased apoptosis. Consistent with in vitro data, the EIPA plus RT inhibited the growth of MDA-MB-231 xenograft tumors in mice to a greater extent than either EIPA or RT alone. EIPA abrogated the RT-induced increase in NHE1 and phospho-NF-κB p65 expression in tumor tissues. Such coincidence of increased NHE1 level, pHi, and NF-κB activation was also found in radioresistant MDA-MB-231 cells, which were reversed by EIPA treatment. Bioinformatics analysis of RNA sequencing data revealed that inhibiting NHE1 reversed three core gene networks that were up-regulated in radioresistant cells and correlated with high NHE1 expression in patient samples: NF-κB, senescence, and extracellular matrix. Taken together, our findings suggest that NHE1 contributes to RT resistance via NF-κB-mediated signaling networks, and NHE1 may be a promising target for improving RT outcomes.

miRNA-23a modulates sodium-hydrogen exchanger 1 expression: studies in medullary thick ascending limb of salt-induced hypertensive rats.

BACKGROUND: The kidney is the main organ in the pathophysiology of essential hypertension. Although most bicarbonate reabsorption occurs in the proximal tubule, the medullary thick ascending limb (mTAL) of the nephron also maintains acid-base balance by contributing to 25% of bicarbonate reabsorption. A crucial element in this regulation is the sodium-hydrogen exchanger 1 (NHE1), a ubiquitous membrane protein controlling intracellular pH, where proton extrusion is driven by the inward sodium flux. MicroRNA (miRNA) expression of hypertensive patients significantly differs from that of normotensive subjects. The aim of this study was to determine the functional role of miRNA alterations at the mTAL level. METHODS: By miRNA microarray analysis, we identified miRNA expression profiles in isolated mTALs from high sodium intake-induced hypertensive rats (HSD) versus their normotensive counterparts (NSD). In vitro validation was carried out in rat mTAL cells. RESULTS: Five miRNAs involved in the onset of salt-sensitive hypertension were identified, including miR-23a, which was bioinformatically predicted to target NHE1 mRNA. Data demonstrated that miRNA-23a is downregulated in the mTAL of HSD rats while NHE1 is upregulated. Consistently, transfection of an miRNA-23a mimic in an mTAL cell line, using a viral vector, resulted in NHE1 downregulation. CONCLUSION: NHE1, a protein involved in sodium reabsorption at the mTAL level and blood pressure regulation, is upregulated in our model. This was due to a downregulation of miRNA-23a. Expression levels of this miRNA are influenced by high sodium intake in the mTALs of rats. The downregulation of miRNA-23a in humans affected by essential hypertension corroborate our data and point to the potential role of miRNA-23a in the regulation of mTAL function following high salt intake.

Inhibition of c-MYC-miRNA 19 Pathway Sensitized CML K562 Cells to Etoposide via NHE1 Upregulation.

As a previously discovered target of DNA damage, Na+/H+ exchanger 1 (NHE1) plays a role in regulation of intracellular pH (pHi) through the extrusion of intracellular proton (H+) in exchange for extracellular sodium (Na+). Its abnormal expression and dysfunction have been reported in solid tumor and hematopoietic malignancies. Here, we reported that suppression of NHE1 in BCR-ABL+ hematopoietic malignancies' K562 cells treated with Etoposide was manipulated by miR-19 and c-MYC. Inhibition of miR-19 or c-MYC enhanced the expression of NHE1 and sensitized K562 cells to Etoposide in vitro. The in vivo nude mouse transplantation model was also performed to confirm the enhanced sensitivity of K562 cells to Etoposide by inhibiting the miR-19 or c-MYC pathway. TCGA analysis conferred a negative correlation between miR-19 level and leukemia patients' survival. Thus, our results provided a potential management by which the c-MYC-miRNA 19 pathway might have a crucial impact on sensitizing K562 cells to Etoposide in the therapeutic approaches.

Luseogliflozin inhibits high glucose-induced TGF-ß2 expression in mouse cardiomyocytes by suppressing NHE-1 activity.

OBJECTIVE: Sodium-glucose cotransporter-2 (SGLT2) inhibitors exhibit cardioprotective properties in patients with diabetes. However, SGLT2 is not expressed in the heart, and the underlying molecular mechanisms are not fully understood. We investigated whether the SGLT2 inhibitor luseogliflozin exerts beneficial effects on high glucose-exposed cardiomyocytes via the suppression of sodium-hydrogen exchanger-1 (NHE-1) activity. METHODS: Mouse cardiomyocytes were incubated under normal or high glucose conditions with vehicle, luseogliflozin, or the NHE-1 inhibitor cariporide. NHE-1 activity and gene expression were evaluated by the SNARF assay and real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis, respectively. Six-week-old male db/db mice were treated with vehicle or luseogliflozin for 6 weeks, and the hearts were collected for histological, RT-PCR, and western blot analyses. RESULTS: High glucose increased NHE-1 activity and transforming growth factor (Tgf)-ß2 mRNA levels in cardiomyocytes, both of which were inhibited by luseogliflozin or cariporide, whereas their combination showed no additive suppression of Tgf-ß2 mRNA levels. Luseogliflozin attenuated cardiac hypertrophy and fibrosis in db/db mice in association with decreased mRNA and protein levels of TGF-ß2. CONCLUSIONS: Luseogliflozin may suppress cardiac hypertrophy in diabetes by reducing Tgf-ß2 expression in cardiomyocytes via the suppression of NHE-1 activity.

Hypoxia enhances motility and EMT through the Na+/H+ exchanger NHE-1 in MDA-MB-231 breast cancer cells.

Breast cancer metastasis is the leading cause of cancer-related deaths. Hypoxia in the tumor mass is believed to trigger cell migration, which is involved in a crucial process of breast cancer metastasis. However, the molecular mechanisms underlying aggressive behavior under hypoxic conditions have not been fully elucidated. Here, we demonstrate the significant motility of MDA-MB-231 cells cultured under hypoxic conditions compared to that of cells cultured under normoxic conditions. MDA-MB-231 cells under hypoxic conditions showed a significant increase in Na+/H+ exchanger isoform 1 (NHE1) expression level, which was observed to co-locate in lamellipodia formation. Inhibition of NHE1 significantly suppressed the intracellular pH and the expression of mesenchymal markers, thereby blocking the high migration activity in hypoxia. Moreover, treatment with ciglitazone, a potent and selective peroxisome proliferator-activated receptor γ (PPARγ) agonist, modulated hypoxia-enhanced motion in cells via the repression of NHE1. These findings highlight that NHE1 is required for migratory activity through the enhancement of epithelial-mesenchymal transition (EMT) in MDA-MB-231 cells under hypoxic conditions, and we propose new drug repurposing strategies targeting hypoxia based on NHE1 suppression by effective usage of PPARγ agonists.

Permissive role of Na+/H+ exchanger isoform 1 in migration and invasion of triple-negative basal-like breast cancer cells.

In breast cancer, it is the resulting metastasis that is the primary cause of fatality. pH regulatory proteins and the tumor microenvironment play an important role in metastasis of cancer cells and acid-extruding proteins are critical in this process. There are several types of breast cancer and triple-negative breast cancer tends to be more metastatic and invasive and is itself is composed of several types. MDA-MB-468 are a triple-negative breast cancer cell line and are classified as basal-like and basal tumors account for up to 15% of breast cancers. Here we examined the effect of removal of the acid-extruding protein, the Na+/H+ exchanger isoform one, from MDA-MB-468 cells. NHE1 was deleted from these cells using the CRISPR/Cas9 system. Western blotting and measurement of activity confirmed the absence of the protein. In wounding/cell migration experiments, deletion of NHE1 reduced the rate of cell migration in the presence of low- or high-serum concentrations. Anchorage-dependent colony formation was also greatly reduced by deletion of the NHE1 protein. Cell proliferation was not affected by knockout of NHE1. The results demonstrate that NHE1 has an important role in migration and invasion of basal-like triple-negative breast cancer cells.

Role of pH Regulatory Proteins and Dysregulation of pH in Prostate Cancer.

Prostate cancer is the fourth most commonly diagnosed cancer, and although it is often a slow-growing malignancy, it is the second leading cause of cancer-associated deaths in men and the first in Europe and North America. In many forms of cancer, when the disease is a solid tumor confined to one organ, it is often readily treated. However, when the cancer becomes an invasive metastatic carcinoma, it is more often fatal. It is therefore of great interest to identify mechanisms that contribute to the invasion of cells to identify possible targets for therapy. During prostate cancer progression, the epithelial cells undergo epithelial-mesenchymal transition that is characterized by morphological changes, a loss of cell-cell adhesion, and invasiveness. Dysregulation of pH has emerged as a hallmark of cancer with a reversed pH gradient and with a constitutively increased intracellular pH that is elevated above the extracellular pH. This phenomenon has been referred to as "a perfect storm" for cancer progression. Acid-extruding ion transporters include the Na+/H+ exchanger NHE1 (SLC9A1), the Na+HCO3- cotransporter NBCn1 (SLC4A7), anion exchangers, vacuolar-type adenosine triphosphatases, and the lactate-H+ cotransporters of the monocarboxylate family (MCT1 and MCT4 (SLC16A1 and 3)). Additionally, carbonic anhydrases contribute to acid transport. Of these, several have been shown to be upregulated in different human cancers including the NBCn1, MCTs, and NHE1. Here the role and contribution of acid-extruding transporters in prostate cancer growth and metastasis were examined. These proteins make significant contributions to prostate cancer progression.

Sodium hydrogen exchanger (NHE1) palmitoylation and potential functional regulation.

AIMS: Determine the effect of palmitoylation on the sodium hydrogen exchanger isoform 1 (NHE1), a member of the SLC9 family. MAIN METHODS: NHE1 expressed in native rat tissues or in heterologous cells was assessed for palmitoylation by acyl-biotinyl exchange (ABE) and metabolic labeling with [3H]palmitate. Cellular palmitoylation was inhibited using 2-bromopalmitate (2BP) followed by determination of NHE1 palmitoylation status, intracellular pH, stress fiber formation, and cell migration. In addition, NHE1 was activated with LPA treatment followed by determination of NHE1 palmitoylation status and LPA-induced change in intracellular pH was determined in the presence and absence of preincubation with 2BP. KEY FINDINGS: In this study we demonstrate for the first time that NHE1 is palmitoylated in both cells and rat tissue, and that processes controlled by NHE1 including intracellular pH (pHi), stress fiber formation, and cell migration, are regulated in concert with NHE1 palmitoylation status. Importantly, LPA stimulates NHE1 palmitoylation, and 2BP pretreatment dampens LPA-induced increased pHi which is dependent on the presence of NHE1. SIGNIFICANCE: Palmitoylation is a reversible lipid modification that regulates an array of critical protein functions including activity, trafficking, membrane microlocalization and protein-protein interactions. Our results suggest that palmitoylation of NHE1 and other control/signaling proteins play a major role in NHE1 regulation that could significantly impact multiple critical cellular functions.

Citronellal alleviates doxorubicin-induced cardiotoxicity by suppressing oxidative stress and apoptosis via Na+ /H+ exchanger-1 inhibition.

The medical usage of Doxorubicin (DOX) as a chemotherapeutic agent is restricted owing to its cardiotoxic properties. This study was designed to explore the effect and underlying mechanisms of Citronellal (CT) on DOX-related cardiotoxicity in rats. Rats were divided into six groups: control, DOX, CT, Lithium chloride (LiCl) (a Na+/H+exchanger-1 [NHE1] activator), DOX + CT, and DOX + CT + LiCl. To induce cardiotoxicity, a cumulative dose of 15 mg/kg DOX was intraperitoneally injected into rats. CT (150 mg/kg) and LiCl (1 mg/kg) were given daily by oral gavage for 6 weeks. CT improved cardiac functional parameters and attenuated the cardiac pathological changes induced by DOX. Further study indicated that CT administration regulated the levels of oxidative stress and apoptosis-related factors and in myocardial tissues, reducing cell per-oxidative damage and apoptosis. Besides this, CT attenuated DOX-induced NHE1 upregulation, and the preventive effects of CT against DOX-induced cardiotoxicity were abrogated by the concurrent administration of LiCl. These results demonstrate that CT could ameliorate DOX-induced cardiotoxicity by inhibiting the NHE1-mediated oxidative stress, apoptosis in rats.

The positive feedback loop of NHE1-ERK phosphorylation mediated by BRAFV600E mutation contributes to tumorigenesis and development of glioblastoma.

The v-raf murine sarcoma viral oncogene homolog B1 (BRAF) activating mutation V600E (BRAFV600E) is involved in glioblastoma multiforme (GBM). Na/H exchanger 1 (NHE1), a main pH regulator affecting cell microenvironment, is hyper-expressed in GBM. However, the relationship between BRAFV600E signal pathway and NHE1 in GMB cells remains unclear. This study found that NHE1 was a downstream target of BRAFV600E 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 BRAFV600E mutation contributing to the proliferation and invasion of GBM cells. Moreover, the proliferation and invasion abilities of BRAFV600E-mutant and BRAF wild type GBM cells were all suppressed by the NHE1 inhibitor, BRAFV600E 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 BRAFV600E and NHE1 inhibitors could be considered as a new therapeutic regimen for GBM, especially for GBM with BRAFV600E.

Therapeutic efficacy of proton transport inhibitors alone or in combination with cisplatin in triple negative and hormone sensitive breast cancer models.

Triple negative breast cancers (TNBCs) are very aggressive and have a poor prognosis due to lack of efficacious therapies. The only effective treatment is chemotherapy that however is frequently hindered by the occurrence of drug resistance. We approached this problem in vitro and in vivo on a triple negative and a hormone sensitive breast cancer cell lines: 4T1 and TS/A. A main defense mechanism of tumors is the extrusion of intracellular protons derived from the metabolic shift to glycolysis, and necessary to maintain an intracellular pH compatible with life. The resulting acidic extracellular milieu bursts the malignant behavior of tumors and impairs chemotherapy. Therefore, we investigated the efficacy of combined therapies that associate cisplatin (Cis) with proton exchanger inhibitors, such as esomeprazole (ESO) and 5-(N-ethyl-N-isopropyl)amiloride (EIPA). Our results demonstrate that in the 4T1 triple negative model the combined therapy Cis plus EIPA is significantly more effective than the other treatments. Instead, in the TS/A tumor the best therapeutic result is obtained with ESO alone. Remarkably, in both 4T1 and TS/A tumors these treatments correlate with increase of CD8+  T lymphocytes and dendritic cells, and a dramatic reduction of M2 macrophages and other suppressor myeloid cells (MDSC) in the tumor infiltrates.

Roles of the Na+/H+ Exchanger Isoform 1 and Urokinase in Prostate Cancer Cell Migration and Invasion.

Prostate cancer is a leading cause of cancer-associated deaths in men over 60 years of age. Most patients are killed by tumor metastasis. Recent evidence has implicated a role of the tumor microenvironment and urokinase plasminogen activator (uPA) in cancer cell migration, invasion, and metastasis. Here, we examine the role of the Na+/H+ exchanger isoform 1 (NHE1) and uPA in DU 145 prostate cancer cell migration and colony formation. Knockout of NHE1 reduced cell migration. The effects of a series of novel NHE1/uPA hexamethylene-amiloride-based inhibitors with varying efficacy towards NHE1 and uPA were examined on prostate cancer cells. Inhibition of NHE1-alone, or with inhibitors combining NHE1 or uPA inhibition-generally did not prevent prostate cancer cell migration. However, uPA inhibition-but not NHE1 inhibition-prevented anchorage-dependent colony formation. Application of inhibitors at concentrations that only saturate uPA inhibition decreased tumor invasion in vivo. The results suggest that while knockout of NHE1 affects cell migration, these effects are not due to NHE1-dependent proton translocation. Additionally, while neither NHE1 nor uPA activity was critical in cell migration, only uPA activity appeared to be critical in anchorage-dependent colony formation of DU 145 prostate cancer cells and invasion in vivo.

Amino Acids 785, 787 of the Na+/H+ Exchanger Cytoplasmic Tail Modulate Protein Activity and Tail Conformation.

The mammalian Na+/H+ exchanger isoform 1 (NHE1) is a plasma membrane protein ubiquitously present in humans. It regulates intracellular pH by removing an intracellular proton in exchange for an extracellular sodium. It consists of a 500 amino acid membrane domain plus a 315 amino acid, regulatory cytosolic tail. Here, we investigated the effect of mutation of two amino acids of the regulatory tail, Ser785 and Ser787, that were similar in location and context to two amino acids of the Arabidopsis Na+/H+ exchanger SOS1. Mutation of these two amino acids to either Ala or phosphomimetic Glu did not affect surface targeting but led to a slight reduction in the level of protein expressed. The activity of the NHE1 protein was reduced in the phosphomimetic mutations and the effect was due to a decrease in Vmax activity. The Ser to Glu mutations also caused a change in the apparent molecular weight of both the full-length protein and of the cytosolic tail of NHE1. A conformational change in this region was indicated by differential trypsin sensitivity. We also found that a peptide containing amino acids 783-790 bound to several more proximal regions of the NHE1 tail in in vitro protein interaction experiments. The results are the first characterization of these two amino acids and show that they have significant effects on enzyme kinetics and the structure of the NHE1 protein.