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Metastatic cancer cells are highly plastic for the expression of different tumor phenotype hallmarks and organotropism. This plasticity is highly regulated but the dynamics of the signaling processes orchestrating the shift from one cell phenotype and metastatic organ pattern to another are still largely unknown. The scaffolding protein NHERF1 has been shown to regulate the expression of different neoplastic phenotypes through its PDZ domains, which forms the mechanistic basis for metastatic organotropism. This reprogramming activity was postulated to be dependent on its differential phosphorylation patterns. Here, we show that NHERF1 phosphorylation on S279/S301 dictates several tumor phenotypes such as in vivo invasion, NHE1-mediated matrix digestion, growth and vasculogenic mimicry. Remarkably, injecting mice with cells having differential NHERF1 expression and phosphorylation drove a shift from the predominantly lung colonization (WT NHERF1) to predominately bone colonization (double S279A/S301A mutant), indicating that NHERF1 phosphorylation also acts as a signaling switch in metastatic organotropism.

Tumor cell invasion depends largely on degradation of the extracellular matrix (ECM) by protease-rich structures called invadopodia, whose formation and activity requires the convergence of signaling pathways engaged in cell adhesion, actin assembly, membrane regulation and ECM proteolysis. It is known that β1-integrin stimulates invadopodia function through an invadopodial p(T567)-ezrin/NHERF1/NHE1 signal complex that regulates NHE1-driven invadopodia proteolytic activity and invasion. However, the link between β1-integrin and this signaling complex is unknown. In this study, in metastatic breast (MDA-MB-231) and prostate (PC-3) cancer cells, we report that integrin-linked kinase (ILK) integrates β1-integrin with this signaling complex to regulate invadopodia activity and invasion. Proximity ligation assay experiments demonstrate that, in invadopodia, ILK associates with β1-integrin, NHE1 and the scaffold proteins p(T567)-ezrin and NHERF1. Activation of β1-integrin increased both invasion and invadopodia activity, which were specifically blocked by inhibition of either NHE1 or ILK. We conclude that ILK integrates β1-integrin with the ECM proteolytic/invasion signal module to induce NHE1-driven invadopodial ECM proteolysis and cell invasion.

Triple negative breast cancer (TNBC) patients cannot be treated with endocrine therapy or targeted therapies due to lack of related receptors. These patients overexpress the epidermal growth factor receptor (EGFR), but are resistant to tyrosine kinase inhibitors (TKIs) and anti-EGFR therapies. Mechanisms suggested for resistance to TKIs include EGFR independence, mutations and alterations in EGFR and in its downstream signalling pathways. Ligand-induced endocytosis and degradation of EGFR play important roles in the downregulation of the EGFR signal suggesting that its activity could be regulated by targeting its trafficking. Evidence in normal cells showing that the scaffolding protein Na+/H+ exchanger regulatory factor 1 (NHERF1) can associate with EGFR to regulate its trafficking, led us to hypothesize that NHERF1 expression levels could regulate EGFR trafficking and functional expression in TNBC cells and, in this way, modulate its role in progression and response to treatment. We investigated the subcellular localization of NHERF1 and its interaction with EGFR in a metastatic basal like TNBC cell model, MDA-MB‑231, and the role of forced NHERF1 overexpression and/or stimulation with EGF on the sensitivity to EGFR specific TKI treatment with gefitinib. Stimulation with EGF induces an interaction of NHERF1 with EGFR to regulate its localization, degradation and function. NHERF1 overexpression is sufficient to drive its interaction with EGFR in non-stimulated conditions, inhibits EGFR degradation and increases its retention time in the plasma membrane. Importantly, NHERF1 overexpression strongly sensitized the cell to the pharmacological inhibition by gefitinib of EGFR-driven growth, motility and invadopodia-dependent ECM proteolysis. The further determination of how the NHERF1‑EGFR interaction is regulated may improve our understanding of TNBC resistance to the action of existing anticancer drugs.

Degradation of the extracellular matrix (ECM) is a critical step of tumor cell invasion and requires protease- dependent proteolysis focalized at the invadopodia where the proteolysis of the ECM occurs. Most of the extracellular proteases belong to serine- or metallo-proteases and the inva- dopodia is where protease activity is regulated. While recent data looking at global protease activity in the growth medium reported that their activity and role in invasion is dependent on Na + /H + exchanger 1 (NHE1)-driven extracellular acidifica - tion, there is no data on this aspect at the invadopodia, and an open question remains whether this acid extracellular pH (pHe) activation of proteases in tumor cells occurs pref- erentially at invadopodia. We previously reported that the NHE1 is expressed in breast cancer invadopodia and that the NHE1-dependent acidification of the peri-invadopodial space is critical for ECM proteolysis. In the present study, using, for the first time, in situ zymography analysis, we demonstrated a concordance between NHE1 activity, extracellular acidifi - cation and protease activity at invadopodia to finely regulate ECM digestion. We demonstrated that: (i) ECM proteolysis taking place at invadopodia is driven by acidification of the peri-invadopodia microenvironment; (ii) that the proteases have a functional pHe optimum that is acidic; (iii) more than one protease is functioning to digest the ECM at these invadopodial sites of ECM proteolysis; and (iv) lowering pHe or inhibiting the NHE1 increases protease secretion while blocking protease activity changes NHE1 expression at the invadopodia.

BACKGROUND INFORMATION: CF (cystic fibrosis) is a disease caused by mutations within the CFTR (CF transmembrane conductance regulator) gene. The most common mutation, DeltaF508 (deletion of Phe-508), results in a protein that is defective in folding and trafficking to the cell surface but is functional if properly localized in the plasma membrane. We have recently demonstrated that overexpression of the PDZ protein NHERF1 (Na(+)/H(+)-exchanger regulatory factor 1) in CF airway cells induced both a redistribution of DeltaF508CFTR from the cytoplasm to the apical membrane and the PKA (protein kinase A)-dependent activation of DeltaF508CFTR-dependent chloride secretion. In view of the potential importance of the targeted up-regulation of NHERF1 in a therapeutic context, and since it has been demonstrated that oestrogen treatment increases endogenous NHERF1 expression, we tested the hypothesis that oestrogen treatment can increase NHERF1 expression in a human bronchiolar epithelial CF cell line, CFBE41o(-), with subsequent rescue of apical DeltaF508CFTR chloride transport activity. RESULTS: We found that CFBE41o(-) cells do express ERs (oestrogen receptors) in the nuclear fraction and that beta-oestradiol treatment was able to significantly rescue DeltaF508CFTR-dependent chloride secretion in CFBE41o(-) cell monolayers with a peak between 6 and 12 h of treatment, demonstrating that the DeltaF508CFTR translocated to the apical membrane can function as a cAMP-responsive channel, with a significant increase in chloride secretion noted at 1 nM beta-oestradiol and a maximal effect observed at 10 nM. Importantly, knock-down of NHERF1 expression by transfection with siRNA (small interfering RNA) for NHERF1 inhibited the beta-oestradiol-dependent increase in DeltaF508CFTR protein expression levels and completely prevented the beta-oestradiol-dependent rescue of DeltaF508CFTR transport activity. CONCLUSIONS: These results demonstrate that beta-oestradiol-dependent up-regulation of NHERF1 significantly increases DeltaF508CFTR functional expression in CFBE41o(-) cells.

There is evidence that cystic fibrosis transmembrane conductance regulator (CFTR) interacting proteins play critical roles in the proper expression and function of CFTR. The Na(+)/H(+) exchanger regulatory factor isoform 1 (NHERF1) was the first identified CFTR-binding protein. Here we further clarify the role of NHERF1 in the regulation of CFTR activity in two human bronchial epithelial cell lines: the normal, 16HBE14o-, and the homozygous DeltaF508 CFTR, CFBE41o-. Confocal analysis in polarized cell monolayers demonstrated that NHERF1 distribution was associated with the apical membrane in 16HBE14o- cells while being primarily cytoplasmic in CFBE41o- cells. Transfection of 16HBE14o- monolayers with vectors encoding for wild-type (wt) NHERF1 increased both apical CFTR expression and apical protein kinase A (PKA)-dependent CFTR-mediated chloride efflux, whereas transfection with NHERF1 mutated in the binding groove of the PDZ domains or truncated for the ERM domain inhibited both the apical CFTR expression and the CFTR-dependent chloride efflux. These data led us to hypothesize an important role for NHERF1 in regulating CFTR localization and stability on the apical membrane of 16HBE14o- cell monolayers. Importantly, wt NHERF1 overexpression in confluent DeltaF508 CFBE41o- and DeltaF508 CFT1-C2 cell monolayers induced both a significant redistribution of CFTR from the cytoplasm to the apical membrane and a PKA-dependent activation of CFTR-dependent chloride secretion.

Recent research has highlighted the fundamental role of the tumour's extracellular metabolic microenvironment in malignant invasion. This microenvironment is acidified primarily by the tumour-cell Na(+)/H(+) exchanger NHE1 and the H(+)/lactate cotransporter, which are activated in cancer cells. NHE1 also regulates formation of invadopodia - cell structures that mediate tumour cell migration and invasion. How do these alterations of the metabolic microenvironment and cell invasiveness contribute to tumour formation and progression?

Although Cystic fibrosis transmembrane conductance regulator (CFTR) has been shown to regulate the activity of NHE3, the potential reciprocal interaction of NHE3 to modulate the protein kinase A (PKA)-dependent regulation of CFTR in epithelial cells is still unknown. In the present work, we describe experiments to define the interactions between CFTR and NHE3 with the regulatory, scaffolding protein, NHERF that organize their PKA-dependent regulation in a renal epithelial cell line that expresses endogenous CFTR. The expression of rat NHE3 significantly decreased PKA-dependent activation of CFTR without altering CFTR expression, and this decrease was prevented by mutation of either of the two rat NHE3 PKA target serines to alanine (S552A or S605A). Inhibition of CFTR expression by antisense treatment resulted in an acute decrease in PKA-dependent regulation of NHE3 activity. CFTR, NHE3, and ezrin were recognized by NHERF-2 but not NHERF-1 in glutathione S-transferase pull-down experiments. Ezrin may function as a protein kinase A anchoring protein (AKAP) in this signaling complex, because blocking the binding of PKA to an AKAP by incubation with the S-Ht31 peptide inhibited the PKA-dependent regulation of CFTR in the absence of NHE3. In the A6-NHE3 cells S-Ht31 blocked the PKA regulation of NHE3 whereas it now failed to affect the regulation of CFTR. We conclude that CFTR and NHE3 reciprocally interact via a shared regulatory complex comprised of NHERF-2, ezrin, and PKA.

In recent years an increasing number of publications have emphasized the growing importance of hydrogen ion dynamics in modern cancer research, from etiopathogenesis and treatment. A proton [H+]-related mechanism underlying the initiation and progression of the neoplastic process has been recently described by different research groups as a new paradigm in which all cancer cells and tissues, regardless of their origin and genetic background, have a pivotal energetic and homeostatic disturbance of their metabolism that is completely different from all normal tissues: an aberrant regulation of hydrogen ion dynamics leading to a reversal of the pH gradient in cancer cells and tissues (↑pHi/↓pHe, or “proton reversal”). Tumor cells survive their hostile microenvironment due to membrane-bound proton pumps and transporters, and their main defensive strategy is to never allow internal acidification because that could lead to their death through apoptosis. In this context, one of the primary and best studied regulators of both pHi and pHe in tumors is the Na+/H+ exchanger isoform 1 (NHE1). An elevated NHE1 activity can be correlated with both an increase in cell pH and a decrease in the extracellular pH of tumors, and such proton reversal is associated with the origin, local growth, activation and further progression of the metastatic process. Consequently, NHE1 pharmaceutical inhibition by new and potent NHE1 inhibitors represents a potential and highly selective target in anticancer therapy. Cariporide, being one of the better studied specific and powerful NHE1 inhibitors, has proven to be well tolerated by humans in the cardiological context, however some side-effects, mainly related to drug accumulation and cerebrovascular complications were reported. Thus, cariporide could become a new, slightly toxic and effective anticancer agent in different human malignancies.

The cystic fibrosis transmembrane conductance regulator (CFTR) mutation ΔF508CFTR still causes regulatory defects when rescued to the apical membrane, suggesting that the intracellular milieu might affect its ability to respond to cAMP regulation. We recently reported that overexpression of the Na+/H+ exchanger regulatory factor NHERF1 in the cystic fibrosis (CF) airway cell line CFBE41o-rescues the functional expression of ΔF508CFTR by promoting F-actin organization and formation of the NHERF1–ezrin–actin complex. Here, using real-time FRET reporters of both PKA activity and cAMP levels, we find that lack of an organized subcortical cytoskeleton in CFBE41o-cells causes both defective accumulation of cAMP in the subcortical compartment and excessive cytosolic accumulation of cAMP. This results in reduced subcortical levels and increased cytosolic levels of PKA activity. NHERF1 overexpression in CFBE41o-cells restores chloride secretion, subcortical cAMP compartmentalization and local PKA activity, indicating that regulation of ΔF508CFTR function requires not only stable expression of the mutant CFTR at the cell surface but also depends on both generation of local cAMP signals of adequate amplitude and activation of PKA in proximity of its target. Moreover, we found that the knockdown of wild-type CFTR in the non-CF 16HBE14o-cells results in both altered cytoskeletal organization and loss of cAMP compartmentalization, whereas stable overexpression of wt CFTR in CF cells restores cytoskeleton organization and re-establishes the compartmentalization of cAMP at the plasma membrane. This suggests that the presence of CFTR on the plasma membrane influences the cytoskeletal organizational state and, consequently, cAMP distribution. Our data show that a sufficiently high concentration of cAMP in the subcortical compartment is required to achieve PKA-mediated regulation of CFTR activity.

Several studies have demonstrated that Na(+) /H(+) exchanger regulatory factor 1 (NHERF1) protein, which is overexpressed and heterogeneously distributed in different stages of breast cancer, could be used as a tumour marker for prognosis in molecular detection strategies. We observed that tumour-infiltrated lymphocytes in the tumour tissue display a high level of NHERF1 staining, in contrast to those present in the contiguous non-involved tissue. Hypothesizing that cancer cells elicit a specific T-cell response associated with the characteristics of the solid tumour, our aim was to evaluate NHERF1 in peripheral lymphocytes from healthy donors and breast cancer patients.NHERF1 levels were analysed in 55 breast cancer patients and 40 healthy donors, and these levels were compared with clinical pathological features. NHERF1 was overexpressed in circulatory peripheral lymphocytes from patients as compared with those from healthy subjects. Furthermore, in both circulatory lymphocytes and tissues, NHERF1 was positively associated with tumour grade, Nottingham Prognostic Index and oestrogen receptor, whereas there was no association with other clinical parameters in either tissue.We propose that NHERF1 measurements in circulatory lymphocytes of breast cancer patients may be a valid method for the prediction of breast cancer occurrence and prognosis, and may have value in the management of cancer patients.

Previous studies have shown that the PDZ-binding motif of the E6 oncoprotein from the mucosal high-risk (HR) human papillomavirus (HPV) types plays a key role in HPV-mediated cellular transformation in in vitro and in vivo experimental models. HR HPV E6 oncoproteins have the ability to efficiently degrade members of the PDZ motif-containing membrane-associated guanylate kinase (MAGUK) family; however, it is possible that other PDZ proteins are also targeted by E6. Here, we describe a novel interaction of HPV type 16 (HPV16) E6 with a PDZ protein, Na + /H + exchange regulatory factor 1 (NHERF-1), which is involved in a number of cellular processes, including signaling and transformation. HPV16 E6 associates with and promotes the degradation of NHERF-1, and this property is dependent on the C-terminal PDZ-binding motif of E6. Interestingly, HPV16 E7, via the activation of the cyclin-dependent kinase complexes, promoted the accumulation of a phosphorylated form of NHERF-1, which is preferentially targeted by E6. Thus, both oncoproteins appear to cooperate in targeting NHERF-1. Notably, HPV18 E6 is not able to induce NHERF-1 degradation, indicating that this property is not shared with E6 from all HR HPV types. Downregulation of NHERF-1 protein levels was also observed in HPV16-positive cervical cancer-derived cell lines, such as SiHa and CaSki, as well as HPV16-positive cervical intraepithelial neoplasia (CIN). Finally, our data show that HPV16-mediated NHERF-1 degradation correlates with the activation of the phosphatidylinositol-3′-OH kinase (PI3K)/AKT signaling pathway, which is known to play a key role in carcinogenesis.

Extracellular matrix (ECM) degradation is a critical process in tumor cell invasion and requires membrane and released proteases focalized at membrane structures called invadopodia. While extracellular acidification is important in driving tumor invasion, the structure/function mechanisms underlying this regulation are still unknown. Invadopodia are similar in structure and function to osteoclast podosomes responsible for bone degradation, and extracellular acidification is central to podosome action, suggesting that it could also be for invadopodial function. Here, utilizing a novel system for in situ zymography in native matrices, we show that the Na(+)/H(+) exchanger (NHE1) and NHE1-generated extracellular acidification are localized at and necessary for invadopodial-dependent ECM degradation, thereby promoting tumor invasion. Stimulation with EGF increased both NHE1-dependent proton secretion and ECM degradation. Manipulation of the NHE1 expression by RNA interference or activity via either transport-deficient mutation or the specific inhibitor cariporide confirmed that NHE1 expression and activity are required for invadopodia-mediated ECM degradation. Taken together, our data show a concordance among NHE1 localization, the generation of a well-defined acidic extracellular pH in the nanospace surrounding invadopodia, and matrix-degrading activity at invadopodia of human malignant breast carcinoma cells, providing a structural basis for the role of NHE1 in invasion and identifying NHE1 as a strategic target for therapeutic intervention.

NHERF1 overexpression increases functional apical expression of F508del CFTR in CFBE41o- cells. Here, we show that this occurs via the formation of the multiprotein complex NHERF1-phosphoezrin-actin, which provides a regulated linkage between F508del CFTR and the actin cytoskeleton resulting in an increased F508del CFTR stability in the membrane., We have demonstrated that Na+/H+ exchanger regulatory factor 1 (NHERF1) overexpression in CFBE41o- cells induces a significant redistribution of F508del cystic fibrosis transmembrane conductance regulator (CFTR) from the cytoplasm to the apical membrane and rescues CFTR-dependent chloride secretion. Here, we observe that CFBE41o- monolayers displayed substantial disassembly of actin filaments and that overexpression of wild-type (wt) NHERF1 but not NHERF1-Δ Ezrin-Radixin-Moesin (ERM) increased F-actin assembly and organization. Furthermore, the dominant-negative band Four-point one, Ezrin, Radixin, Moesin homology (FERM) domain of ezrin reversed the wt NHERF1 overexpression-induced increase in both F-actin and CFTR-dependent chloride secretion. wt NHERF1 overexpression enhanced the interaction between NHERF1 and both CFTR and ezrin and between ezrin and actin and the overexpression of wt NHERF1, but not NHERF1-ΔERM, also increased the phosphorylation of ezrin in the apical region of the cell monolayers. Furthermore, wt NHERF1 increased RhoA activity and transfection of constitutively active RhoA in CFBE41o- cells was sufficient to redistribute phospho-ezrin to the membrane fraction and rescue both the F-actin content and the CFTR-dependent chloride efflux. Rho kinase (ROCK) inhibition, in contrast, reversed the wt NHERF1 overexpression-induced increase of membrane phospho-ezrin, F-actin content, and CFTR-dependent secretion. We conclude that NHERF1 overexpression in CFBE41o- rescues CFTR-dependent chloride secretion by forming the multiprotein complex RhoA-ROCK-ezrin-actin that, via actin cytoskeleton reorganization, tethers F508del CFTR to the cytoskeleton stabilizing it on the apical membrane.

BACKGROUND:Neoplastic transformation originates from a large number of different genetic alterations. Despite this genetic variability, a common phenotype to transformed cells is cellular alkalinization. We have previously shown in human keratinocytes and a cell line in which transformation can be turned on and followed by the inducible expression of the E7 oncogene of human papillomavirus type 16 (HPV16), that intracellular alkalinization is an early and essential physiological event driven by the up-regulation of the Na/(+)H(+) exchanger isoform 1 (NHE1) and is necessary for the development of other transformed phenotypes and the in vivo tumor formation in nude mice. METHODOLOGY:Here, we utilize these model systems to elucidate the dynamic sequence of alterations of the upstream signal transduction systems leading to the transformation-dependent activation of NHE1. PRINCIPAL FINDINGS:We observe that a down-regulation of p38 MAPK activity is a fundamental step in the ability of the oncogene to transform the cell. Further, using pharmacological agents and transient transfections with dominant interfering, constitutively active, phosphorylation negative mutants and siRNA strategy to modify specific upstream signal transduction components that link HPV16 E7 oncogenic signals to up-regulation of the NHE1, we demonstrate that the stimulation of NHE1 activity is driven by an early rise in cellular cAMP resulting in the down-stream inhibition of p38 MAPK via the PKA-dependent phosphorylation of the small G-protein, RhoA, and its subsequent inhibition. CONCLUSIONS:All together these data significantly improve our knowledge concerning the basic cellular alterations involved in oncogene-driven neoplastic transformation.

Many physiological and pathophysiological processes, such as embryogenesis, immune defense, wound healing, or metastasis, are based on cell migration and invasion. The activity of the ubiquitously expressed NHE1 isoform of the plasma membrane Na + /H + exchanger is one of the requirements for directed locomotion of migrating cells. The mechanisms by which NHE1 is involved in cell migration are multiple. NHE1 contributes to cell migration by affecting the cell volume, by regulating the intracellular pH and thereby the assembly and activity of cytoskeletal elements, by anchoring the cytoskeleton to the plasma membrane, by the organization of signal transduction and by regulating gene expression. The present review focuses on two additional, extracellular mechanisms by which NHE1 activity contributes to cell migration and invasion. Protons extruded by the NHE1 lead to local, extracellular acidification which, on the one hand, can create pH optima needed for the activity of proteinases at invadopodia/podosomes necessary for extracellular matrix digestion and, on the other hand, facilitates cell/matrix interaction and adhesion at the cell front.

Understanding the signal transduction systems governing invasion is fundamental for the design of therapeutic strategies against metastasis. Na+/H+exchanger regulatory factor (NHERF1) is a postsynaptic density 95/disc-large/zona occludens (PDZ) domain-containing protein that recruits membrane receptors/transporters and cytoplasmic signaling proteins into functional complexes. NHERF1 expression is altered in breast cancer, but its effective role in mammary carcinogenesis remains undefined. We report here that NHERF1 overexpression in human breast tumor biopsies is associated with metastatic progression, poor prognosis, and hypoxia-inducible factor-1α expression. In cultured tumor cells, hypoxia and serum deprivation increase NHERF1 expression, promote the formation of leading-edge pseudopodia, and redistribute NHERF1 to these pseudopodia. This pseudopodial localization of NHERF1 was verified in breast biopsies and in three-dimensional Matrigel culture. Furthermore, serum deprivation and hypoxia stimulate the Na+/H+exchanger, invasion, and activate a protein kinase A (PKA)-gated RhoA/p38 invasion signal module. Significantly, NHERF1 overexpression was sufficient to induce these morphological and functional changes, and it potentiated their induction by serum deprivation. Functional experiments with truncated and binding groove-mutated PDZ domain constructs demonstrated that NHERF1 regulates these processes through its PDZ2 domain. We conclude that NHERF1 overexpression enhances the invasive phenotype in breast cancer cells, both alone and in synergy with exposure to the tumor microenvironment, via the coordination of PKA-gated RhoA/p38 signaling.

Metastasis results from a sequence of selective events often involving interactions with elements of the tumor-specific physiological microenvironment. The low-serum component of this microenvironment confers increased motility and invasion in breast cancer cells by activating the Na+/H+exchanger isoform 1 (NHE1). The present study was undertaken to characterize the signal transduction mechanisms underlying this serum deprivation-dependent activation of both the NHE1 and the concomitant invasive characteristics such as leading edge pseudopodia development and penetration of matrigel in breast cancer cell lines representing different stages of metastatic progression. Using pharmacological and genetic manipulation together with transport and kinase activity assays, we observe that the activation of the NHE1 and subsequent invasion by serum deprivation in metastatic human breast cells is coordinated by a sequential RhoA/p160ROCK/p38MAPK signaling pathway gated by direct protein kinase A phosphorylation and inhibition of RhoA. Fluorescence resonance energy transfer imaging of RhoA activity and immunofluorescence analysis of phospho-RhoA and NHE1 show that serum deprivation dynamically remodels the cell, forming long, leading edge pseudopodia and that this signal module is preferentially compartmentalized in these leading edge pseudopodia, suggesting a tight topographic relation of the signaling module to an invasion-specific cell structure.

The molecular signal components essential to paclitaxel-dependent apoptosis in breast cancers are potential targets for combined therapy. However, the signal mechanisms underlying paclitaxel action still need to be better defined.In a breast cancer cell line, pharmacological agents and transient transfection with dominant interfering and constitutive active mutants were used to identify the signal transduction module involved in the regulation of paclitaxel-induced apoptosis and to evaluate its potential as a therapeutic target.In MDA-MB-435 cells, paclitaxel treatment stimulated the activity of both protein kinase A and p38, and inhibited the activity of the Na(+)/H(+) exchanger isoform 1 (NHE1) with similar IC(50) concentrations as for its activation of apoptosis. Activation and inhibition experiments demonstrated that protein kinase A and p38 participate sequentially upstream of the NHE1 in regulating the paclitaxel-induced apoptotic pathway. Importantly, concurrent specific inhibition of the NHE1 with paclitaxel treatment resulted in a synergistic induction of apoptosis and a reduction in the paclitaxel IC(50) for apoptosis. This sensitization of paclitaxel apoptotic action by specific inhibition of NHE1 was verified in breast cancer cell lines with different paclitaxel sensitivity.We have, for the first time, identified NHE1 as an essential component of paclitaxel-induced apoptosis in breast cancer cells and, importantly, identified that simultaneous inhibition of the NHE1 results in a synergistic potentiation of low-dose paclitaxel apoptotic action. As specific NHE1 inhibitors have finished Phase II/Phase III clinical trials for myocardial protection, there is the possibility for a rapid biological translation of this novel therapeutic strategy to a clinical setting.

The change of a normal, healthy cell to a transformed cell is the first step in the evolutionary arc of a cancer. While the role of oncogenes in this ‘passage’ is well known, the role of ion transporters in this critical step is less known and is fundamental to our understanding the early physiological processes of carcinogenesis. Cancer cells and tissues have an aberrant regulation of hydrogen ion dynamics leading to a reversal of the normal tissue intracellular to extracellular pH gradient (ΔpHito ΔpHe). When this perturbation in pH dynamics occurs during carcinogenesis is less clear. Very early studies using the introduction of different oncogene proteins into cells observed a concordance between neoplastic transformation and a cytoplasmic alkalinization occurring concomitantly with a shift towards glycolysis in the presence of oxygen, i.e. ‘Warburg metabolism’. These processes may instigate a vicious cycle that drives later progression towards fully developed cancer where the reversed pH gradient becomes ever more pronounced. This review presents our understanding of the role of pH and the NHE1 in driving transformation, in determining the first appearance of the cancer ‘hallmark’ characteristics and how the use of pharmacological approaches targeting pH/NHE1 may open up new avenues for efficient treatments even during the first steps of cancer development.