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 ([upwards arrow]pHi/[downwards arrow]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.

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.

Cancer cells acquire an unusual glycolytic behavior to a large extent relative to an intracellular alkaline (pHi). This effect is part of the metabolic alterations found in most, if not all, cancer cells to deal with unfavorable conditions, mainly hypoxia and low nutrient supply, in order to preserve its evolutionary trajectory with the production of lactate after ten steps of glycolysis. Thus, cancer cells reprogram their cellular metabolism in a way that gives them their evolutionary and thermodynamic advantage. Tumors exist within a highly heterogeneous microenvironment, and cancer cells survive within any of the different habitats that lie within tumors thanks to the overexpression of different membrane-bound proton transporters. This creates a highly abnormal and selective proton reversal in cancer cells and tissues that is involved in local cancer growth and in the metastatic process. Because of this environmental heterogeneity, cancer cells within one part of the tumor may have a different genotype and phenotype than within another part. This phenomenon has frustrated the potential of single-target therapy of this type of reductionist therapeutic approach over the last decades. Here, we present a detailed biochemical framework on every step of tumor glycolysis and then propose a new paradigm and therapeutic strategy based upon the dynamics of the hydrogen ion in cancer cells and tissues in order to overcome the old paradigm of one enzyme-one target approach to cancer treatment. Finally, a new and integral explanation of the Warburg effect is advanced.

BACKGROUND INFORMATION: P2×7R is a member of the ionotropic family of purinergic receptors activated by millimolar concentrations of extracellular ATP such as induced by inflammatory stimuli. The receptor is widely expressed in cells of haematopoietic origin such as monocytes, macrophages and microglia. There is growing interest in anta-gonist compounds of the P2×7R since it has been demonstrated to be a viable therapeutic target for inflammatory diseases. Here, we tested the possible P2×7 antagonist effect of MED1101, a newly synthesised dialdehydic compound on U937 monocyte cells. RESULTS: Human U937 cells express the full-length P2×7A receptor isoform. Treatment with lipopolysaccharide (LPS), a potent inducer of inflammation, significantly increased the expression of the receptor in the plasma membrane. Importantly, MED1101 induced internalisation of the P2×7R already after 30 min incubation in both physiological conditions and in presence of the inflammatory stimulus (LPS) and this effect was observable for up to 12 h after its removal. Moreover, MED1101 induced an impairment of monocyte migration/transmigration through direct P2×7R antagonism and subsequent inhibition of the intracellular signal transduction processes of Ca(2+) influx and MAPK phosphorylation. CONCLUSIONS: Our results clearly demonstrate that in U937 monocyte cells MED1101 acts as a P2×7R antagonist through the induction of receptor internalisation and subsequent inhibition of down-stream signal transduction pathways that regulate monocyte migration/transmigration, thus playing a potential therapeutic role in inflammatory diseases.

Aims: 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. Methods and results: 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. Conclusions: 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.

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- Ezrin-Radixin-Moesin (ERM) increased F-actin assembly and organization. Furthermore, the dominantnegative 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- ERM, 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 CFBE41ocells 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 RhoAROCK-ezrin-actin that, via actin cytoskeleton reorganization, tethers F508del CFTR to the cytoskeleton stabilizing it on the apical membrane.

Cancer cells and tissues, regardless of their origin and genetic background, have an aberrant regulation of hydrogen ion dynamics leading to a reversal of the intracellular to extracellular pH gradient (∆pHi to ∆pHe) in cancer cells and tissue as compared to normal tissue. This perturbation in pH dynamics rises very early in carcinogenesis and is one of the most common patho-physiological hallmarks of tumors. Recently, there has been a very large increase in our knowledge of the importance and roles of pHi and pHe in developing and driving a series of tumor hallmarks. This reversed proton gradient is driven by a series of proton export mechanisms that underlie the initiation and progression of the neoplastic process. 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). The NHE1 is an integral membrane transport protein involved in regulating pH and in tumor cells is a major contributor to the production and maintainance of their reversed proton gradient. It is activated during oncogene-dependent transformation resulting in cytosolic alkalinization which the drives subsequent hallmark behaviors including growth factor- and substrate-independent growth, and glycolytic metabolism. It is further activated by various growth factors, hormone, the metabolic microenvironment (low serum, acidic pHe and hypoxia) or by ECM receptor activation. This review will present the recent progress in understanding the role the NHE1 in determining tumor progression and invadopodia-guided invasion/metastasis and recent patents for NHE1 inhibitors and novel therapeutic protocols for anti-NHE1 pharmacological approaches. These may represent a real possibility to open up new avenues for wide-spread and efficient treatments against cancer.

Metastatic cells are highly plastic for differential expression of tumor phenotype hallmarks and metastatic organotropism. The signaling proteins orchestrating the shift of one cell phenotype and organ pattern to another are little known. Na(+)/H(+) exchanger regulatory factor (NHERF1) is a molecular pathway organizer, PDZ-domain protein that recruits membrane, cytoplasmic, and cytoskeletal signaling proteins into functional complexes. To gain insight into the role of NHERF1 in metastatic progression, we stably transfected a metastatic breast cell line, MDA-MB-231, with an empty vector, with wild-type NHERF1, or with NHERF1 mutated in either the PDZ1- or PDZ2-binding domains to block their binding activities. We observed that NHERF1 differentially regulates the expression of two phenotypic programs through its PDZ domains, and these programs form the mechanistic basis for metastatic organotropism. The PDZ2 domain promotes visceral metastases via increased invadopodia-dependent invasion and anchorage-independent growth, as well as by inhibition of apoptosis, whereas the PDZ1 domain promotes bone metastases by stimulating podosome nucleation, motility, neoangiogenesis, vasculogenic mimicry, and osteoclastogenesis in the absence of increased growth or invasion. Collectively, these findings identify NHERF1 as an important signaling nexus for coordinating cell structure with metastatic behavior and identifies the "mesenchymal-to-vasculogenic" phenotypic transition as an essential step in metastatic progression.

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.-Busco, G., Cardone, R. A., Greco, M. R., Bellizzi, A., Colella, M., Antelmi, E., Mancini, M. T., Dell'Aquila, M. E., Casavola, V., Paradiso, A., Reshkin, S. J. NHE1 promotes invadopodial ECM proteolysis through acidification of the peri-invadopodial space

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.

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 takes place. Most of the extracellular proteases belong to serine- or metallo-proteases and the invadopodia is where protease activity is regulated. While recent data looking at global protease activity in the growth medium has reported that their activity and role in invasion is dependent on Na+/H+ exchanger (NHE1)-driven extracellular acidification, there is no data on this aspect at the invadopodia, an open question remains whether this acid pHe activation of proteases in tumor cells occurs preferentially at invadopodia. We have 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. Here, using, for the first time an in situ zymography analysis, we demonstrate that a concordance between NHE1 activity, extracellular acidification and protease activity at invadopodia to finely regulate ECM digestion. We demonstrate 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.

Extracellular matrix (ECM) degradation is a critical process in tumor cell invasion and requires matrix degrading protrusions called invadopodia. The Na(+)/H(+) exchanger (NHE1) has recently been shown to be fundamental in the regulation of invadopodia actin cytoskeleton dynamics and activity. However, the structural link between the invadopodia cytoskeleton and NHE1 is still unknown. A candidate could be ezrin, a linker between the NHE1 and the actin cytoskeleton known to play a pivotal role in invasion and metastasis. However, the mechanistic basis for its role remains unknown. Here, we demonstrate that ezrin phosphorylated at T567 is highly overexpressed in the membrane of human breast tumors and positively associated with invasive growth and HER2 overexpression. Further, in the metastatic cell line, MDA-MB-231, p-ezrin was almost exclusively expressed in invadopodia lipid rafts where it co-localized in a functional complex with NHE1, EGFR, ß1-integrin and phosphorylated-NHERF1. Manipulation by mutation of ezrins T567 phosphorylation state and/or PIP2 binding capacity or of NHE1s binding to ezrin or PIP2 demonstrated that p-ezrin expression and binding to PIP2 are required for invadopodia-mediated ECM degradation and invasion and identified NHE1 as the membrane protein that p-ezrin regulates to induce invadopodia formation and activity.

Cancer cells and tissues, regardless of their origin and genetic background, have an aberrant regulation of hydrogen ion dynamics leading to a reversal of the intracellular to extracellular pH gradient (ΔpHi to ΔpHe) as compared to normal tissues. This perturbation in pH dynamics occurs very early in carcinogenesis and is one of the most common pathophysiological hallmarks of tumors. Recently, there has been quite a significant increase in our knowledge of the importance and roles of pHi and pHe in developing and driving a series of tumor hallmarks. This reversed proton gradient is driven by a series of proton export mechanisms that underlie the initiation and progression of the neoplastic process. 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). The NHE1 is an integral membrane transport protein involved in regulating pH and in tumor cells is a major contributor to the production and maintenance of their reversed proton gradient. It is activated during oncogene-dependent transformation resulting in cytosolic alkalinization which drives subsequent hallmark behaviors including growth factor- and substrate-independent growth, and glycolytic metabolism. It is further activated by various growth factors, hormones, the metabolic microenvironment (low serum, acidic pHe and hypoxia) or by ECM receptor activation. This chapter will present the recent progress in understanding the role of the NHE1 in determining tumor progression and invadopodia-guided invasion/metastasis and recent patents for NHE1 inhibitors and novel therapeutic protocols for anti-NHE1 pharmacological approaches. These may represent a real possibility to open up new avenues for wide-spread and efficient treatments against cancer.

Triple negative breast cancer (TNBC) patients cannot be treated with endocrine therapy or targeted therapies due to lack of related receptors. These patients overexpress 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 down-regulation 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.

BACKGROUND: The aim of this study was to evaluate the effects of vitrification on morpho-functional parameters (blastomere/chromatin integrity and bioenergy/oxidative potential) of mouse preimplantation embryos. METHODS: In vivo produced mouse (4/16-cell, morulae and blastocyst-stage) embryos were randomly divided into vitrification and control groups. For vitrification, embryos were exposed to a 2-step loading of ethylene glycol and propylene glycol, before being placed in a small nylon loop and submerged into liquid nitrogen. After warming, the cryoprotectants were diluted by a 3-step procedure. Embryo morphology, chromatin integrity and energy/oxidative status were compared between groups. RESULTS: Vitrification induced low grade blastomere cytofragmentation (P < 0.05) and low chromatin damage only in embryos at the morula stage (P < 0.001). Mitochondrial (mt) distribution pattern was affected by vitrification only in early embryos (P < 0.001). Mitochondrial activity did not change upon vitrification in morula-stage embryos but it was reduced in blastocyst-stage embryos (P < 0.05). Intracellular ROS levels significantly increased in embryos at the morula and blastocyst stages (P < 0.001). Colocalization of active mitochondria and ROS increased only in vitrified blastocysts. CONCLUSIONS: In conclusion, this study elucidates the developmentally-related and mild effects of vitrification on morphology, nuclear and bioenergy/oxidative parameters of mouse embryos and demonstrates that vitrification is a suitable method for preserving predictive parameters of embryo ability to induce a full-term pregnancy.