Impact of Oncogenic Changes in p53 and KRAS on Macropinocytosis and Ferroptosis in Colon Cancer Cells and Anticancer Efficacy of Niclosamide with Differential Effects on These Two Processes.

Mutations in p53 and KRAS are seen in most cases of colon cancer. The impact of these mutations on signaling pathways related to cancer growth has been studied in depth, but relatively less is known on their effects on amino acid transporters in cancer cells. This represents a significant knowledge gap because amino acid nutrition in cancer cells profoundly influences macropinocytosis and ferroptosis, two processes with opposing effects on tumor growth. Here, we used isogenic colon cancer cell lines to investigate the effects of p53 deletion and KRAS activation on two amino acid transporters relevant to macropinocytosis (SLC38A5) and ferroptosis (SLC7A11). Our studies show that the predominant effect of p53 deletion is to induce SLC7A11 with the resultant potentiation of antioxidant machinery and protection of cancer cells from ferroptosis, whereas KRAS activation induces not only SLC7A11 but also SLC38A5, thus offering protection from ferroptosis as well as improving amino acid nutrition in cancer cells via accelerated macropinocytosis. Niclosamide, an FDA-approved anti-helminthic, blocks the functions of SLC7A11 and SLC38A5, thus inducing ferroptosis and suppressing macropinocytosis, with the resultant effective reversal of tumor-promoting actions of oncogenic changes in p53 and KRAS. These findings underscore the potential of this drug in colon cancer treatment.

Induction of Oxidative Stress and Ferroptosis in Triple-Negative Breast Cancer Cells by Niclosamide via Blockade of the Function and Expression of SLC38A5 and SLC7A11.

The amino acid transporters SLC38A5 and SLC7A11 are upregulated in triple-negative breast cancer (TNBC). SLC38A5 transports glutamine, methionine, glycine and serine, and therefore activates mTOR signaling and induces epigenetic modifications. SLC7A11 transports cystine and increases the cellular levels of glutathione, which protects against oxidative stress and lipid peroxidation via glutathione peroxidase, a seleno (Se)-enzyme. The primary source of Se is dietary Se-methionine (Se-Met). Since SLC38A5 transports methionine, we examined its role in Se-Met uptake in TNBC cells. We found that SLC38A5 interacts with methionine and Se-Met with comparable affinity. We also examined the influence of Se-Met on Nrf2 in TNBC cells. Se-Met activated Nrf2 and induced the expression of Nrf2-target genes, including SLC7A11. Our previous work discovered niclosamide, an antiparasitic drug, as a potent inhibitor of SLC38A5. Here, we found SLC7A11 to be inhibited by niclosamide with an IC50 value in the range of 0.1-0.2 µM. In addition to the direct inhibition of SLC38A5 and SLC7A11, the pretreatment of TNBC cells with niclosamide reduced the expression of both transporters. Niclosamide decreased the glutathione levels, inhibited proliferation, suppressed GPX4 expression, increased lipid peroxidation, and induced ferroptosis in TNBC cells. It also significantly reduced the growth of the TNBC cell line MB231 in mouse xenografts.

Amino acid transporter SLC38A5 is a tumor promoter and a novel therapeutic target for pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) cells have a great demand for nutrients in the form of sugars, amino acids, and lipids. Particularly, amino acids are critical for cancer growth and, as intermediates, connect glucose, lipid and nucleotide metabolism. PDAC cells meet these requirements by upregulating selective amino acid transporters. Here we show that SLC38A5 (SN2/SNAT5), a neutral amino acid transporter is highly upregulated and functional in PDAC cells. Using CRISPR/Cas9-mediated knockout of SLC38A5, we show its tumor promoting role in an in vitro cell line model as well as in a subcutaneous xenograft mouse model. Using metabolomics and RNA sequencing, we show significant reduction in many amino acid substrates of SLC38A5 as well as OXPHOS inactivation in response to SLC38A5 deletion. Experimental validation demonstrates inhibition of mTORC1, glycolysis and mitochondrial respiration in KO cells, suggesting a serious metabolic crisis associated with SLC38A5 deletion. Since many SLC38A5 substrates are activators of mTORC1 as well as TCA cycle intermediates/precursors, we speculate amino acid insufficiency as a possible link between SLC38A5 deletion and inactivation of mTORC1, glycolysis and mitochondrial respiration, and the underlying mechanism for PDAC attenuation. Overall, we show that SLC38A5 promotes PDAC, thereby identifying a novel, hitherto unknown, therapeutic target for PDAC.

Potent Inhibition of Macropinocytosis by Niclosamide in Cancer Cells: A Novel Mechanism for the Anticancer Efficacy for the Antihelminthic.

Niclosamide, a drug used to treat tapeworm infection, possesses anticancer effects by interfering with multiple signaling pathways. Niclosamide also causes intracellular acidification. We have recently discovered that the amino acid transporter SLC38A5, an amino acid-dependent Na+/H+ exchanger, activates macropinocytosis in cancer cells via amino acid-induced intracellular alkalinization. Therefore, we asked whether niclosamide will block basal and SLC38A5-mediated macropinocytosis via intracellular acidification. We monitored macropinocytosis in pancreatic and breast cancer cells using TMR-dextran and the function of SLC38A5 by measuring Li+-stimulated serine uptake. The peptide transporter activity was measured by the uptake of glycylsarcosine. Treatment of the cancer cells with niclosamide caused intracellular acidification. The drug blocked basal and serine-induced macropinocytosis with differential potency, with an EC50 of ~5 µM for the former and ~0.4 µM for the latter. The increased potency for amino acid-mediated macropinocytosis is due to direct inhibition of SLC38A5 by niclosamide in addition to the ability of the drug to cause intracellular acidification. The drug also inhibited the activity of the H+-coupled peptide transporter. We conclude that niclosamide induces nutrient starvation in cancer cells by blocking macropinocytosis, SLC38A5 and the peptide transporter. These studies uncover novel, hitherto unknown, mechanisms for the anticancer efficacy of this antihelminthic.

Dishevelled-1 DIX and PDZ domain lysine residues regulate oncogenic Wnt signaling.

DVL proteins are central mediators of the Wnt pathway and relay complex input signals into different branches of the Wnt signaling network. However, molecular mechanism(s) that regulate DVL-mediated relay of Wnt signals still remains unclear. Here, for the first time, we elucidate the functional significance of three DVL-1 lysines (K/Lys) which are subject to post-translational acetylation. We demonstrate that K34 Lys residue in the DIX domain regulates subcellular localization of ß-catenin, thereby influencing downstream Wnt target gene expression. Additionally, we show that K69 (DIX domain) and K285 (PDZ domain) regulate binding of DVL-1 to Wnt target gene promoters and modulate expression of Wnt target genes including CMYC, OCT4, NANOG, and CCND1, in cell line models and xenograft tumors. Finally, we report that conserved DVL-1 lysines modulate various oncogenic functions such as cell migration, proliferation, cell-cycle progression, 3D-spheroid formation and in-vivo tumor growth in breast cancer models. Collectively, these findings highlight the importance of DVL-1 domain-specific lysines which were recently shown to be acetylated and characterize their influence on Wnt signaling. These site-specific modifications may be subject to regulation by therapeutics already in clinical use (lysine deacetylase inhibitors such as Panobinostat and Vorinostat) or may possibly have prognostic utility in translational efforts that seek to modulate dysfunctional Wnt signaling.

A Proton-Coupled Transport System for ß-Hydroxy-ß-Methylbutyrate (HMB) in Blood-Brain Barrier Endothelial Cell Line hCMEC/D3.

ß-Hydroxy-ß-methylbutyrate (HMB), a leucine metabolite, is used as a nutritional ingredient to improve skeletal muscle health. Preclinical studies indicate that this supplement also elicits significant benefits in the brain; it promotes neurite outgrowth and prevents age-related reductions in neuronal dendrites and cognitive performance. As orally administered HMB elicits these effects in the brain, we infer that HMB crosses the blood-brain barrier (BBB). However, there have been no reports detailing the transport mechanism for HMB in BBB. Here we show that HMB is taken up in the human BBB endothelial cell line hCMEC/D3 via H+-coupled monocarboxylate transporters that also transport lactate and ß-hydroxybutyrate. MCT1 (monocarboxylate transporter 1) and MCT4 (monocarboxylate transporter 4) belonging to the solute carrier gene family SLC16 (solute carrier, gene family 16) are involved, but additional transporters also contribute to the process. HMB uptake in BBB endothelial cells results in intracellular acidification, demonstrating cotransport with H+. Since HMB is known to activate mTOR with potential to elicit transcriptomic changes, we examined the influence of HMB on the expression of selective transporters. We found no change in MCT1 and MCT4 expression. Interestingly, the expression of LAT1 (system L amino acid transporter 1), a high-affinity transporter for branched-chain amino acids relevant to neurological disorders such as autism, is induced. This effect is dependent on mTOR (mechanistic target of rapamycine) activation by HMB with no involvement of histone deacetylases. These studies show that HMB in systemic circulation can cross the BBB via carrier-mediated processes, and that it also has a positive influence on the expression of LAT1, an important amino acid transporter in the BBB.

The Wnt non-canonical signaling modulates cabazitaxel sensitivity in prostate cancer cells.

BACKGROUND: Despite new drugs, metastatic prostate cancer remains fatal. Growing interest in the latest approved cabazitaxel taxane drug has markedly increased due to the survival benefits conferred when used at an earlier stage of the disease, its promising new therapeutic combination and formulation, and its differential toxicity. Still cabazitaxel's mechanisms of resistance are poorly characterized. The goal of this study was thus to generate a new model of acquired resistance against cabazitaxel in order to unravel cabazitaxel's resistance mechanisms. METHODS: Du145 cells were cultured with increasing concentrations of cabazitaxel, docetaxel/ taxane control or placebo/age-matched control. Once resistance was reached, Epithelial-to-Mesenchymal Translation (EMT) was tested by cell morphology, cell migration, and E/M markers expression profile. Cell transcriptomics were determined by RNA sequencing; related pathways were identified using IPA, PANTHER or KEGG software. The Wnt pathway was analyzed by western blotting, pharmacological and knock-down studies. RESULTS: While age-matched Du145 cells were sensitive to both taxane drugs, docetaxel-resistant cells were only resistant to docetaxel and cabazitaxel-resistant cells showed a partial cross-resistance to both drugs concomitant to EMT. Using RNA-sequencing, the Wnt non-canonical pathway was identified as exclusively activated in cabazitaxel resistant cells while the Wnt canonical pathway was restricted to docetaxel-resistant cells. Cabazitaxel-resistant cells showed a minimal crossover in the Wnt-pathway-related genes linked to docetaxel resistance validating our unique model of acquired resistance to cabazitaxel. Pharmacological and western blot studies confirmed these findings and suggest the implication of the Tyrosine kinase Ror2 receptor in cabazitaxel resistant cells. Variation in Ror2 expression level altered the sensitivity of prostate cancer cells to both drugs identifying a possible new target for taxane resistance. CONCLUSION: Our study represents the first demonstration that while Wnt pathway seems to play an important role in taxanes resistance, Wnt effectors responsible for taxane specificity remain un-identified prompting the need for more studies.

Aromatase Acetylation Patterns and Altered Activity in Response to Sirtuin Inhibition.

Aromatase, a cytochrome P450 member, is a key enzyme involved in estrogen biosynthesis and is dysregulated in the majority of breast cancers. Studies have shown that lysine deacetylase inhibitors (KDI) decrease aromatase expression in cancer cells, yet many unknowns remain regarding the mechanism by which this occurs. However, advances have been made to clarify factors involved in the transcriptional regulation of the aromatase gene (CYP19A1). Yet, despite aromatase being a primary target for breast cancer therapy, its posttranslational regulation has been virtually unexplored. Acetylation is a posttranslational modification (PTM) known to alter the activity and stability of many oncoproteins, and given the role of KDIs in regulating aromatase expression, we postulate that aromatase acetylation acts as a novel posttranslational regulatory mechanism that impacts aromatase expression and/or activity in breast cancer. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis revealed that aromatase is basally acetylated on several lysine residues (108, 169, 242, 262, 334, 352, and 354) in MCF-7 cells, and treatment with a SIRT-1 inhibitor induced additional acetylation (376, 390, 440, and 448). These acetylated lysine residues are in regions critical for aromatase activity. Site-directed mutagenesis and overexpression studies demonstrated that K108R/Q or K440R/Q mutations significantly altered aromatase activity in breast cancer cells without altering its subcellular localization.Implications: These findings demonstrate a novel posttranslational regulation of aromatase and uncover novel anticancer effects of deacetylase inhibitors, thus providing new insight for ongoing development of deacetylase inhibitors as cancer therapeutics. Mol Cancer Res; 16(10); 1530-42. ©2018 AACR.

Sm-p80-based vaccine trial in baboons: efficacy when mimicking natural conditions of chronic disease, praziquantel therapy, immunization, and Schistosoma mansoni re-encounter.

Sm-p80-based vaccine efficacy for Schistosoma mansoni was evaluated in a baboon model of infection and disease. The study was designed to replicate a human vaccine implementation scenario for endemic regions in which vaccine would be administered following drug treatment of infected individuals. In our study, the Sm-p80-based vaccine reduced principal pathology producing hepatic egg burdens by 38.0% and egg load in small and large intestines by 72.2% and 49.4%, respectively, in baboons. Notably, hatching rates of eggs recovered from liver and small and large intestine of vaccinated animals were significantly reduced, by 60.4%, 48.6%, and 82.3%, respectively. Observed reduction in egg maturation/hatching rates was supported by immunofluorescence and confocal microscopy showing unique differences in Sm-p80 expression in worms of both sexes and matured eggs. Vaccinated baboons had a 64.5% reduction in urine schistosome circulating anodic antigen, a parameter that reflects worm numbers/health status in infected hosts. Preliminary analyses of RNA sequencing revealed unique genes and canonical pathways associated with establishment of chronic disease, praziquantel-mediated parasite killing, and Sm-p80-mediated protection in vaccinated baboons. Overall, our study demonstrated efficacy of the Sm-p80 vaccine and provides insight into some of the epistatic interactions associated with protection.

Schistosoma egg-induced liver pathology resolution by Sm-p80-based schistosomiasis vaccine in baboons.

Schistosomiasis remains a serious chronic debilitating hepato-intestinal disease. Current control measures based on mass drug administration are inadequate due to sustained re-infection rates, low treatment coverage and emergence of drug resistance. Hence, there is an urgent need for a schistosomiasis vaccine for disease control. In this study, we assessed the anti-pathology efficacy of Schistosoma mansoni large subunit of calpain (Sm-p80)-based vaccine against schistosomiasis caused by infections with Schistosoma mansoni in baboons. We also evaluated the disease transmission-blocking potential of Sm-p80 vaccine. Immunisations with Sm-p80-based vaccine resulted in significant reduction of hepatic egg load in vaccinated baboons (67.7% reduction, p = 0.0032) when compared to the control animals, indicative of reduction in pathology. There was also a significant reduction in sizes of egg-induced granulomas in baboons immunised with Sm-p80 vaccine compared to their control counterparts. Egg hatching rate analysis revealed an overall 85.6% reduction (p = 0.0018) in vaccinated animals compared to the controls, highlighting the potential role of Sm-p80 vaccine in disease transmission. The findings on anti-pathology efficacy and transmission-blocking potential presented in this study have formed the basis for a large-scale double-blinded baboon experiment that is currently underway.

Cabazitaxel regimens inhibit the growth of prostate cancer cells and enhances the anti-tumor properties of PEDF with various efficacy and toxicity.

BACKGROUND: Taxanes chemotherapies represent the major therapeutic alternative for symptomatic mCRPC. While docetaxel is the most commonly prescribed Taxane for mCRPC; cabazitaxel has been approved for patients unresponsive to docetaxel. Still mCRPC remains incurable and patients often experience severe side effects. Recently, the FIRSTANA trial first demonstrated the absence of superiority in overall survival between cabazitaxel and docetaxel in mCRPC patients. Inversely, different toxicity were reported suggesting that cabazitaxel may provide a first line treatment option for some patients urging for a deeper characterization of cabazitaxel mechanisms of action as well as a re-evaluation of cabazitaxel conventional dose and schedule. In this study, our goal was therefore to evaluate the anti-tumor efficacy of various cabazitaxel regimens delivered as monotherapy or in combination with PEDF, a known anti-angiogenic and anti-neoplastic agent. METHODS: CRPC cells undergoing Taxane treatment were evaluated for cell proliferation, migration and death, and apoptosis using crystal violet staining, chemotaxis, cell cycle, and TUNEL assays. In vitro data were corroborated in CL1 CRPC xenografts where mice received intermittent or metronomic low-doses cabazitaxel ± PEDF. RESULTS: We found that cabazitaxel inhibits the proliferation of CRPC cells with a higher efficacy than docetaxel in vitro. As expected, high-doses of Taxanes blocked the cells in mitosis. Surprisingly, low-doses of cabazitaxel induced more cell death than docetaxel mainly through apoptosis. In vivo, intermittent cabazitaxel lead to disease stabilization when combined with PEDF. Unexpectedly, low-doses of cabazitaxel delayed tumor growth with severe toxicity for some of the doses tested. Other results showed that PEDF and low-doses of cabazitaxel combination inhibited the migration of tumor cell and increased the tumoricidal activity of macrophages toward prostate tumor cells. CONCLUSIONS: Our findings highlight the great promise of cabazitaxel drug and predict a possible move of cabazitaxel forward within the therapeutic sequence of prostate cancer.

Cross-species prophylactic efficacy of Sm-p80-based vaccine and intracellular localization of Sm-p80/Sm-p80 ortholog proteins during development in Schistosoma mansoni, Schistosoma japonicum, and Schistosoma haematobium.

Schistosomiasis remains a major global health problem. Despite large-scale schistosomiasis control efforts, clear limitations such as possible emergence of drug resistance and reinfection rates highlight the need for an effective schistosomiasis vaccine. Schistosoma mansoni large subunit of calpain (Sm-p80)-based vaccine formulations have shown remarkable efficacy in protecting against S. mansoni challenge infections in mice and baboons. In this study, we evaluated the cross-species protective efficacy of Sm-p80 vaccine against S. japonicum and S. haematobium challenge infections in rodent models. We also elucidated the expression of Sm-p80 and Sm-p80 ortholog proteins in different developmental stages of S. mansoni, S. haematobium, and S. japonicum. Immunization with Sm-p80 vaccine reduced worm burden by 46.75% against S. japonicum challenge infection in mice. DNA prime/protein boost (1 + 1 dose administered on a single day) resulted in 26.95% reduction in worm burden in S. haematobium-hamster infection/challenge model. A balanced Th1 (IFN-γ, TNF-α, IL-2, and IL-12) and Th2 (IL-4, IgG1) type of responses were observed following vaccination in both S. japonicum and S. haematobium challenge trials and these are associated with the prophylactic efficacy of Sm-p80 vaccine. Immunohistochemistry demonstrated that Sm-p80/Sm-p80 ortholog proteins are expressed in different life cycle stages of the three major human species of schistosomes studied. The data presented in this study reinforce the potential of Sm-p80-based vaccine for both hepatic/intestinal and urogenital schistosomiasis occurring in different geographical areas of the world. Differential expression of Sm-p80/Sm-p80 protein orthologs in different life cycle makes this vaccine potentially useful in targeting different levels of infection, disease, and transmission.

Novel Treatment of Melanoma: Combined Parasite-Derived Peptide GK-1 and Anti-Programmed Death Ligand 1 Therapy.

Recent successes in the development of new therapies for metastatic melanoma, such as mitogen-activated protein kinase pathway inhibitors, anticytotoxic T lymphocyte-associated antigen-4, and programmed cell death protein 1/programmed cell death ligand 1 (PD-L1) pathway-blocking antibodies, as well as combination strategies, all yielded promising results, changing the continually evolving landscape of therapeutic options for patients with melanoma. One promising new treatment modality is based on the use of immunomodulatory monoclonal antibodies that enhance the function of components of the antitumor immune response such as T cells or block immunologic checkpoints that restrain effective antitumor immunity. Program death-1 receptor and its ligand, PD-L1, is a major mechanism by which a tumor suppresses T cell-mediated antitumor immune responses. Studies in mice have shown that GK-1, an 18 amino acid peptide from Taenia crassiceps cisticerci, has the potential to be used as a primary or adjuvant component for the treatment of cancers by stimulating proinflammatory cytokines. The authors hypothesized that treatment with GK-1 in combination with anti-PD-L1 will increase survival in mice bearing melanoma tumors. C57BL/6 mice were injected with B16-F10-luc2 cells and separated into four groups: control, GK-1, anti-PD-L1, and GK-1/anti-PD-L1. The tumor sizes were measured and monitored using calipers and bioluminescence. The GK-1 peptide in combination with anti-PD-L1 showed significantly longer survival (34 days) compared with the other groups (23-27 days). This means an increase; survival increased 47.82% in the mice treated with GK-1+anti-PD-L1, 21.7% in mice treated with GK-1 alone, and 6.08% in those mice treated with anti-PD-L1 only. Blood samples were collected at days 0, 14, and at euthanization or end of the experiment and monitored for cytokines using mouse-specific V-PLEX Proinflammatory Panel. A decrease in TNF-α, IL-4, IL-5, IL-6, and IL-10 serum levels was observed in the GK-1/anti-PD-L1 combination group that may explain the beneficial effects of the combination treatment in prolonging the life of mice bearing melanoma. The data indicate that GK-1/anti-PD-L1 combined therapy affectively increases survival and warrants further clinical investigations.

Vacuolar H+-ATPase in the nuclear membranes regulates nucleo-cytosolic proton gradients.

The regulation of the luminal pH of each organelle is crucial for its function and must be controlled tightly. Nevertheless, it has been assumed that the nuclear pH is regulated by the cytoplasmic proton transporters via the diffusion of H+ across the nuclear pores because of their large diameter. However, it has been demonstrated that ion gradients exist between cytosol and nucleus, suggesting that the permeability of ions across the nuclear pores is restricted. Vacuolar H+-ATPase (V-H+-ATPase) is responsible for the creation and maintenance of trans-membrane electrochemical gradient. We hypothesize that V-H+-ATPase located in the nuclear membranes functions as the primary mechanism to regulate nuclear pH and generate H+ gradients across the nuclear envelope. We studied the subcellular heterogeneity of H+ concentration in the nucleus and cytosol using ratio imaging microscopy and SNARF-1, a pH indicator, in prostate cells. Our results indicate that there are proton gradients across the nuclear membranes that are generated by V-H+-ATPase located in the outer and inner nuclear membranes. We demonstrated that these gradients are mostly dissipated by inhibiting V-H+-ATPase. Immunoblots and V-H+-ATPase activity corroborated the existence of V-H+-ATPase in the nuclear membranes. This study demonstrates that V-H+-ATPase is functionally expressed in nuclear membranes and is responsible for nuclear H+ gradients that may promote not only the coupled transport of substrates, but also most electrochemically driven events across the nuclear membranes. This study represents a paradigm shift that the nucleus can regulate its own pH microenvironment, providing new insights into nuclear ion homeostasis and signaling.

Porphyrin Immobilized Nanographene Oxide for Enhanced and Targeted Photothermal Therapy of Brain Cancer.

Brain cancer is a fatal disease that is difficult to treat because of poor targeting and low permeability of chemotherapeutic drugs through the blood brain barrier. In a comparison to current treatments, such as surgery followed by chemotherapy and/or radiotherapy, photothermal therapy is a remarkable noninvasive therapy developed in recent years. In this work, porphyrin immobilized nanographene oxide (PNG) was synthesized and bioconjugated with a peptide to achieve enhanced and targeted photothermal therapy for brain cancer. PNG was dispersed into the agar based artificial tissue model and demonstrated a photo-to-thermal conversion efficiency of 19.93% at a PNG concentration of only 0.5 wt %, with a heating rate of 0.6 °C/s at the beginning of irradiation. In comparison, 0.5 wt % graphene oxide (GO) indicated a photo-to-thermal conversion efficiency of 12.20% and a heating rate of 0.3 °C/s. To actively target brain tumor cells without harming healthy cells and tissues surrounding the laser path, a tripeptide l-arginyl-glycyl-l-aspartic (RGD) was further grafted to PNG. The photothermal therapy effects of PNG-RGD completely eliminated the tumor in vivo, indicating its excellent therapeutic effect for the treatment of brain cancer.

Regulation of retinoid mediated cholesterol efflux involves liver X receptor activation in mouse macrophages.

Removal of cholesterol from macrophage-derived foam cells is a critical step to the prevention of atherosclerotic lesions. We have recently demonstrated the functional importance of retinoids in the regulation of the steroidogenic acute regulatory (StAR) protein that predominantly mediates the intramitochondrial transport of cholesterol in target tissues. In the present study, treatment of mouse macrophages with retinoids, particularly all-trans retinoic acid (atRA) and 9-cis RA, resulted in increases in cholesterol efflux to apolipoprotein AI (Apo-A1). Activation of the PKA pathway by a cAMP analog, (Bu)2cAMP, markedly augmented retinoid mediated cholesterol efflux. Macrophages overexpressing hormone-sensitive lipase increased the hydrolysis of cholesteryl esters and concomitantly enhanced the efficacy of retinoic acid receptor and liver X receptor (LXR) ligands on StAR and ATP-binding cassette transporter A1 (ABCA1) protein levels. RAs elevated StAR promoter activity in macrophages, and an increase in StAR levels augmented cholesterol efflux to Apo-A1, suggesting retinoid-mediated efflux of cholesterol involves enhanced oxysterol production. Further studies revealed that retinoids activate the LXR regulated genes, sterol receptor-element binding protein-1c and ABCA1. These findings provide insights into the regulatory events in which retinoid signaling effectively enhances macrophage cholesterol efflux and indicate that retinoid therapy may have important implications in limiting and/or regressing atherosclerotic cardiovascular disease.

Vacuolar H(+)-ATPase signaling pathway in cancer.

Up-regulated aerobic glycolysis is a hallmark of malignant cancers. Little is understood about the reasons why malignant tumors up-regulate glycolysis and acidify their microenvironment. Signaling pathways involved in glucose changes are numerous. However, the identity of the internal glucose signal remains obscure. In this review we address the question of the significance of vacuolar proton ATPase (V-ATPase) and its relationship to up-regulated glycolysis in tumors. We know that glycolysis is extremely sensitive to changes in pH. Importantly, the V-ATPase activity is sensitive to glucose availability. Therefore, we propose that pH acts as the glucose signal via the V-ATPase that responds to changes in intracellular pH and acts as a sensor. We hypothesize that the increase in glycolysis leads to intracellular acidification and activates the V-ATPase to maintain a more alkaline intracellular pH in tumors by up-regulating glycolysis. This review attempts to provide a comprehensive description of the current knowledge about the role of V-ATPase in cancer, highlighting its role as a key player in the pH signaling pathway.

Function of a subunit isoforms of the V-ATPase in pH homeostasis and in vitro invasion of MDA-MB231 human breast cancer cells.

It has previously been shown that highly invasive MDA-MB231 human breast cancer cells express vacuolar proton-translocating ATPase (V-ATPases) at the cell surface, whereas the poorly invasive MCF7 cell line does not. Bafilomycin, a specific V-ATPase inhibitor, reduces the in vitro invasion of MB231 cells but not MCF7 cells. Targeting of V-ATPases to different cellular membranes is controlled by isoforms of subunit a. mRNA levels for a subunit isoforms were measured in MB231 and MCF7 cells using quantitative reverse transcription-PCR. The results show that although all four isoforms are detectable in both cell types, levels of a3 and a4 are much higher in MB231 than in MCF7 cells. Isoform-specific small interfering RNAs (siRNA) were employed to selectively reduce mRNA levels for each isoform in MB231 cells. V-ATPase function was assessed using the fluorescent indicators SNARF-1 and pyranine to monitor the pH of the cytosol and endosomal/lysosomal compartments, respectively. Cytosolic pH was decreased only on knockdown of a3, whereas endosome/lysosome pH was increased on knockdown of a1, a2, and a3. Treatment of cells with siRNA to a4 did not affect either cytosolic or endosome/lysosome pH. Measurement of invasion using an in vitro transwell assay revealed that siRNAs to both a3 and a4 significantly inhibited invasion of MB231 cells. Immunofluorescence staining of MB231 cells for V-ATPase distribution revealed extensive intracellular staining, with plasma membrane staining observed in approximately 18% of cells. Knockdown of a4 had the greatest effect on plasma membrane staining, leading to a 32% reduction. These results suggest that the a4 isoform may be responsible for targeting V-ATPases to the plasma membrane of MB231 cells and that cell surface V-ATPases play a significant role in invasion. However, other V-ATPases affecting the pH of the cytosol and intracellular compartments, particularly those containing a3, are also involved in invasion.

Plasmalemmal vacuolar H+-ATPases in angiogenesis, diabetes and cancer.

Angiogenesis, i.e., new blood vessel formation, is required in normal and pathological states. A dysfunction in the microvascular endothelium occurs in diabetes, leading to decreased blood flow and limb amputation. In cancer, angiogenesis is increased to allow for growth, invasion, and metastasis of tumor cells. Better understanding of the molecular events that cause or are associated with either of these diseases is needed to develop therapies. The tumor and angiogenic cells micro-environment is acidic and not permissive for growth. We have shown that to survive this environment, highly metastatic and angiogenic cells employ vacuolar H+-ATPase at their plasma membranes (pmV-ATPases) to maintain an alkaline pHcyt. However, in lowly metastatic and in microvascular endothelial cells from diabetic model, the density of pmV-ATPase and the cell invasiveness are decreased. Therefore, the overexpression of the pmV-ATPase is important for cell invasion, and essential for tumor progression, angiogenesis and metastasis. Both, cancer and diabetes are heterogenous diseases that involve many different proteins and signaling pathways. Changes in pHcyt have been associated with the regulation of a myriad of proteins, signaling molecules and pathways affecting many if not all cellular functions. Since changes in pHcyt are pleiotropic, we hypothesize that alteration in a single protein, pmV-ATPase, that can regulate pHcyt may explain the dysfunction of many proteins and cellular pathways in diabetes and cancer. Our long term goal is to determine the molecular mechanisms by which pmV-ATPase expression regulates tumor angiogenesis and metastasis. Such knowledge would be useful to identify targets for cancer therapy.

Plasmalemmal vacuolar-type H+-ATPase in cancer biology.

Vacuolar-type H+-adenosine triphosphatase (V-ATPase) is one of the most fundamental enzymes in nature. V-ATPases are responsible for the regulation of proton concentration in the intracellular acidic compartments. It has similar structure with the mitochondrial F0F1-ATP synthase (F-ATPase). dagger The V-ATPases are composed of multiple subunits and have various physiological functions, including membrane and organelle protein sorting, neurotransmitter uptake, cellular degradative processes, and cytosolic pH regulation. The V-ATPases have been involved in multidrug resistance. Recently, plasma membrane V-ATPases have been involved in regulation of extracellular acidity, essential for cellular invasiveness and proliferation in tumor metastasis. The current knowledge regarding the structure and function of V-ATPase and its role in cancer biology is discussed.