The Na?/H? exchanger (NHE1) as a novel co-adjuvant target in paclitaxel therapy of triple-negative breast cancer cells

Dysregulation of Na?/H? exchanger isoform one (NHE1) activity is a hallmark of cells undergoing tumorigenesis and metastasis, the leading cause of patient mortality. The acidic tumor microenvironment is thought to facilitate the development of resistance to chemotherapy drugs and to promote extracellular matrix remodeling leading to metastasis. Here, we investigated NHE1 as a co-adjuvant target in paclitaxel chemotherapy of metastatic breast cancer. We generated a stable NHE1-knockout of the highly invasive, triple-negative, MDA-MB-231 breast cancer cells. The NHE1-knockout cells proliferated comparably to parental cells, but had markedly lower rates of migration and invasion in vitro. In vivo xenograft tumor growth in athymic nude mice was also dramatically decreased compared to parental MDA-MB-231 cells. Loss of NHE1 expression also increased the susceptibility of knockout cells to paclitaxel-mediated cell death. NHE1 inhibition, in combination with paclitaxel, resulted in a dramatic decrease in viability, and migratory and invasive potential of triple-negative breast cancer cells, but not in hormone receptor-positive, luminal MCF7 cells. Our data suggest that NHE1 is critical in triple-negative breast cancer metastasis, and its chemical inhibition boosts the efficacy of paclitaxel in vitro, highlighting NHE1 as a novel, potential co-adjuvant target in breast cancer chemotherapy

Na+ regulation in the intraerythrocytic malaria parasite

The maintenance of a low intracellular [Na⁺] ([Na⁺]i) is a crucial aspect of cellular physiology. In mammalian cells this is achieved through the extrusion of Na⁺ via the well-characterised Na⁺/K⁺-ATPase. Approximately 12 hr after invasion of the human erythrocyte by the malaria parasite there is a profound increase in the permeability of the erythrocyte membrane to a wide range of solutes, including Na⁺. Na⁺ enters the infected erythrocyte via parasite-induced 'New Permeability Pathways' and there is, as a result, an increase in [Na⁺] in the erythrocyte compartment, with [Na⁺]i eventually reaching levels similar to those in the extra erythrocytic plasma (~130 mM). The parasite itself maintains a low [Na⁺]i. The resulting large inwardly-directed electrochemical Na⁺ gradient across the parasite plasma membrane energises the accumulation within the parasite of at least one essential nutrient (inorganic phosphate). The aim of this thesis was to characterise the mechanisms involved in Na⁺ regulation in the mature asexual 'trophozoite' stage of the human malaria parasite Plasmodium falciparum. The Na⁺-sensitive, fluorescent dye Sodium-binding BenzoFuran Isophthalate (SBFI) was used to measure [Na⁺]i in parasites functionally isolated from their host cells by saponin-permeabilisation of the host erythrocyte membrane. Under physiologically relevant conditions the resting [Na⁺]i in isolated trophozoites was estimated to be ~11 mM. Maintenance of [Na⁺]i was sensitive to the P-type ATPase inhibitor orthovanadate, consistent with Na⁺ extrusion being via a P-type Na⁺-ATPase, similar to the ENA (exitus natru; exit of sodium)-type ATPases that operate in some other protozoa, fungi and lower plants. ENA ATPases have been predicted to antiport H⁺ and the data obtained here are consistent with this being true of the P. falciparum Na⁺ extrusion system. The P. falciparum genome encodes a number of putative P-type ATPases; one of these, PfATP4, was found to share significant sequence similarities to ENA ATPases of other protozoa. A recent study showed that mutations in PfATP4 confer resistance to a newly-described class of antimalarials, the spiroindolones. The effect of the spiroindolones on ion regulation was therefore investigated. Several spiroindolones were shown to cause a profound disruption of [Na⁺]i regulation. In parasites with mutant PfATP4 there was both an impairment of Na⁺ regulation and a decrease in the spiroindolone-sensitivity of Na⁺ regulation. These results are consistent with PfATP4 being a Na⁺-ATPase and the target of the spiroindolones. The physiological role of another putative Na⁺ transporter, the Na⁺/H⁺-exchanger PfNHE was also investigated, as previous studies on its contribution to regulation of [Na⁺]i and intracellular pH (pHi) have been controversial. On the basis of a bioinformatics analysis it was predicted that the protein functions as an amiloride-insensitive, plasma membrane Na⁺-extruder, like its closely related plant homologues. However physiological studies revealed no significant role for such an NHE in either pHi or [Na⁺]i regulation in the P. falciparum trophozoite. This study constitutes a significant advance in our understanding of fundamental aspects of the cell physiology of the intraerythrocytic parasite, as well as shedding light on the mode of action of what promises to be an important new class of antimalarials, the spiroindolones

Transcompartmental Sodium Imaging in Brain Cancer

According to the World Health Organization (WHO), cancers are the second-leading cause of death in the United States, next to cardiovascular disease. Although treatments of some solid tumors with high mortality are showing promise, there is one exception: glioblastoma multiforme (GBM). Even with advanced therapies, median survival rate for GBM patients is dismal because most are not diagnosed early enough. A hallmark of normal physiology is maintenance of large ion gradients across the cell membrane, mainly for sodium (Na + ) and potassium (K + ) ions. Under normal conditions, high and low Na + in extracellular (Na + ) and intracellular (Na + ) milieu, respectively (and e i the opposite is true for K + ), produce strong transmembrane and weak transendothelial Na + gradients, which contribute to the cell membrane potential (+ \u3c ) and blood-brain-barrier (BBB) integrity, respectively. The Na + /K + -ATPase in the cell membrane plays a crucial role in transporting Na + and K + against their respective electrochemical gradients by consuming ATP. A universal cancer hallmark is reduced oxidation due to downregulation of Na + /K + -ATPase, a consequence of inefficient metabolism which aids tumor survival. Electrolyte balance in the body is crucial for proper functioning of processes like action potential propagation, muscle movement, and maintaining cell volume, but electrolyte imbalances can lead to pathophysiologies like cancer. Translational magnetic resonance imaging (MRI) and spectroscopic imaging (MRSI) methods are an integral part of diagnosing and tracking cancer. Clinical MRI is largely based on detection of the 1 H nuclei in water molecules in soft tissues, where intrinsic 1 H-MRI contrasts provide anatomical separation between healthy tissue and lesion. But paramagnetic lanthanide(III) ions (Ln 3+ ), specifically gadolinium (Gd 3+ ), conjugated with a chelating molecule consisting of electron donors, provide superior 1 H-MRI contrast as the agent (e.g., Dotarem) extravasates into the lesion through leaky blood vessels to enhance the lesion’s appearance. Other nuclei are rarely considered for clinical applications, but these so-called “X-nuclei” (e.g., 23 Na, 31 P, or 17 O) can offer illuminating insights into the physiological processes. 23 Na-MRI has the potential to be a helpful screen for early cancer detection. However, normal 23 Na-MRI cannot separate the overlapping signals between Na + from different compartments like the blood vessel (Na + ), extracellular space (Na + ), and intracellular b e space (Na + ). In this thesis, I have developed a rigorous model which can be applied in i vivo to separate these individual signals by introducing small amounts of a paramagnetic contrast agent based on Ln 3+ metal ions. The model predicts that the induced 23 Na chemical shift and line broadening are monotonically increasing functions of both the agent’s concentration and negative charge, which was validated on a set of nine agents. This established model was then considered for in vivo applications to compartmentalize the 23 Na-MRSI signals in rat models. Since these agents extravasate from blood vessels, but are too negatively charged to enter cells, this method is able to separate and readout the 23 Na signals from Na + , Na + , and Na + with high fidelity by inducing chemical shift differences. b e i Examination of several GBM models in rodent brain, shows that tumors redistribute Na + from the extracellular milieu to dramatically weaken the transmembrane Na + gradient (compared to normal tissue) and concomitantly strengthen the transendothelial Na + gradient. This is a significant finding because the lower level of Na + imply that the e membrane of cancer cells is depolarized (i.e., and thus are they are non-excitable). Others have shown that high + \u3c is common to cells in a proliferative state. Since even immune cells can sense the level of Na + , it is evident the role of compartmental Na + in the tumor e niche will be important to study in vivo. I also report novel findings regarding variations in induced 23 Na line broadening and electrical activity between tumors and healthy tissue which have considerable oncologic significance. Despite the clinical popularity of 1 1 H-MRI, H-MRSI, and general imaging-based approaches, this project demonstrates the vast potential of 23 Na spectroscopic methods to allow novel explorations of the tumor habitat for early diagnosis and tracking treatments

Modulation de l'échangeur Na+/H+ de type 1 (NHE1) par le canal sodique dépendant du voltage Nav1.5 (implication dans l'invasivité de cellules cancéreuses mammaires humaines)

Les cellules cancéreuses mammaires invasives expriment des canaux sodiques NaV1.5 dont l activité semble être associée au développement métastatique. L activité de ce canal dans les cellules MDA-MB-231 conduit à une acidification péricellulaire favorable à l activité des cathepsines à cystéine B et S extracellulaires et à la dégradation de la matrice extracellulaire. Au cours de cette thèse, nous avons montré que l échangeur NHE1 est le principal régulateur du pH des cellules MDA-MB-231 et que l activité du canal NaV1.5 augmente l activité d efflux de protons par NHE1 vraisemblablement par modulation allostérique. NaV1.5 et NHE1 sont co-localisés dans des radeaux lipidiques et plus particulièrement dans les invadopodes des cellules MDA-MB-231. Les activités de NHE1 et NaV1.5 stimulent l activité protéolytique des invadopodes. Enfin, l activité du canal NaV1.5 semble moduler le cytosquelette et la morphologie des cellules cancéreuses MDA-MB-231 pour leur donner un phénotype invasif. En conclusion, NaV1.5 augmente l activité de NHE1 dans les invadopodes stimulant ainsi l invasivité des cellules cancéreuses mammaires.Invasive breast cancer cells express NaV1.5 sodium channels which activity seems to be associated with metastatic progression. The activity of the channel in MDA-MB-231 cells leads to a pericellular acidification favourable for the activity of extracellular cysteine cathepsins B and S and for extracellular matrix degradation. During this thesis, we have shown that NHE1 exchanger is the main pH regulator in MDA-MB-231 cells and that the activity of NaV1.5 channels increases protons efflux activity of NHE1 possibly through allosteric modulation. NaV1.5 and NHE1 are co-localised in lipid rafts and in invadopodia of MDA-MB-231 cells. The activity of NHE1 and NaV1.5 promotes the proteolytic activity of invadopodia. Finally, the activity of NaV1.5 channels seems to modulate cytoskeleton and morphology of MDA-MB-231 cancer cells to promote the acquisition of a proinvasive phenotype. In conclusion NaV1.5 increases NHE1 activity in invadopodia to stimulate breast cancer cells invasiveness.TOURS-Bibl.électronique (372610011) / SudocSudocFranceF

Rational design of novel antibody decorated nanoparticles targeting breast cancer cells

Every year there are over 50,000 new cases of breast cancer in UK, which represents the most common UK cancer (CR-UK). Despite significant advances in anti-breast cancer treatments, there is still a need for improved therapeutics to overcome drug resistance. HER2 (Human Epidermal Growth Factor Receptor 2) is a transmembrane oncoprotein encoded by the HER2/neu gene and overexpressed in approximately 20 to 30% of invasive breast cancers. Tumours overexpressing HER2 are more aggressive and carry a poor prognosis; thus, the receptor is a priority therapeutic target. One targeting entity is Trastuzumab (Tz), a monoclonal antibody recognized as one of the most effective agents against HER2+ breast cancer and has also been attached to chemotherapeutics to form antibody-drug conjugate (ADC). These ADCs, such as Kadcyla®, require cell binding to HER2 and access to the cell interior by endocytosis to release the payload. HER2 is, however, commonly termed the "endocytosis deficient' member of the HER family of receptors, thus challenging attempts to design ADCs that need access to lysosomes for drug release and activity. Previous studies in the laboratory showed that HER2 endocytosis was significantly promoted with concomitant lysosomal delivery and degradation via Tz-mediated crosslinking, and this presented work lies under the hypothesis that nanoparticles (NPs) decorated with sufficient numbers of Tz could also cause HER2 cross-linking, endocytosis, and HER2 degradation. Later data showed that HER2 crosslinking induced a form of endocytosis termed macropinocytosis to drive cell entry. The work presented initially investigated macropinocytosis as a process in different cell types and ways to inhibit this process using inhibitors targeting the sodium proton exchanger (NHE1) as a regulator of intracellular pH and this endocytic process. EIPA (5-(N-Ethyl-N-isopropyl) amiloride) as a macropinocytosis inhibitor, surprisingly significantly increased the internalisation of HER2; a result not observed with other NHE1 inhibitors amiloride and the more selective NHE1 inhibitor cariporide. EIPA was also shown to increase the uptake of the fluid phase and macropinocytosis marker dextran but had no effect on endocytosis of transferrin via clathrin-coated vesicles. The results suggest that EIPA targets need further analysis as modulators of HER2 internalisation and targets for breast cancer therapy. Fluorescently labelled Tz- decorated Poly (lactic glycolic acid) NPs were then generated and found to be highly selective for HER2 expressing breast cancer cells over controls. Upon incubation with cells, the decorated NPs rapidly accumulated on the cell surface and also appeared as large intracellular structures suggestive of macropinosomes. Rhodamine and Doxorubicin encapsulated Tz-PLGA NPs were synthesised, showing their capacity to drive internalisation of the fluorophore and cytotoxic drug into vesicular structures, with the later formulation enhancing the cytotoxicity of the drug over its soluble counterpart