The abundance of the multimeric vacuolar ATP-dependent proton pump, V-ATPase, on the plasma membrane of tumor cells correlates with the invasiveness of the tumor cell, suggesting the involvement of V-ATPase in tumor metastasis. V-ATPase is hypothesized to create a proton efflux leading to an acidic pericellular microenvironment that promotes the activity of proinvasive proteases. An alternative, not yet explored possibility is that V-ATPase regulates the signaling machinery responsible for tumor cell migration. Here, we show that pharmacologic or genetic reduction of V-ATPase activity significantly reduces migration of invasive tumor cells in vitro. Importantly, the V-ATPase inhibitor archazolid abrogates tumor dissemination in a syngeneic mouse 4T1 breast tumor metastasis model. Pretreatment of cancer cells with archazolid impairs directional motility by preventing spatially restricted, leading edge localization of epidermal growth factor receptor (EGFR) as well as of phosphorylated Akt. Archazolid treatment or silencing of V-ATPase inhibited Rac1 activation, as well as Rac1-dependent dorsal and peripheral ruffles by inhibiting Rab5-mediated endocytotic/exocytotic trafficking of Rac1. The results indicate that archazolid effectively decreases metastatic dissemination of breast tumors by impairing the trafficking and spatially restricted activation of EGFR and Rho-GTPase Rac1, which are pivotal for directed movement of cells. Thus, our data reveals a novel mechanism underlying the role of V-ATPase in tumor dissemination. Cancer Res; 72(22); 5976–87. ©2012 AACR.

Cancer is a major cause of mortality primarily because of tumor metastasis (1). The vacuolar (V)-ATPase is emerging as a prominent player in the invasiveness of cancer cells (2, 3). Consistently, pharmacologic inhibition of V-ATPase has been proposed as a strategy to oppose metastatic processes (3–5).

V-ATPases are ATP-dependent proton pumps ubiquitously expressed and thought to regulate the pH in endomembrane systems. V-ATPase comprises 2 major domains: the cytoplasmic V1 domain, which consists of 8 subunits (A–H), mediates ATP hydrolysis; the membrane-bound V0 subunit is composed by subunits a, d, e, and the proteolipids c, c′, and c″. The c subunits form an H+-binding rotor ring that transports protons from the cytoplasm to the endosomal/lysosomal lumen or the extracellular space (6, 7).

Few specific small molecule inhibitors of V-ATPase have been developed and their binding properties have been extensively studied (8). The archazolids are a novel group of V-ATPase inhibitors, first isolated from cultivated myxobacteria Archangium gephyra (9). Like the known V-ATPase inhibitors bafilomycin A1 or concanamycin, archazolids bind to the V0 subunit c of V-ATPase. However, the molecular mechanism of their inhibitory action is different. Archazolids do not penetrate in between 2 c subunits, but most likely bind to the membrane facing side of a single subunit c (10). Archazolids potently inhibit V-ATPase activity (IC50: nanomolar range) exerting little or no effects on Na+/K+-ATPases and mitochondrial F-ATPases (8). To date, archazolid A, B, and F have been isolated from their natural producer and archazolid A and B have also been successfully chemically synthesized (9, 11–13).

It has been observed that the abundance of V-ATPase on the plasma membrane correlates with an invasive phenotype of tumor cells (3, 4). In addition, cell surface V-ATPase activity has been postulated to create an acidic extracellular microenvironment, which is required for the activation of proteases known to be important for tumor cell invasion (14). Consistent with this, V-ATPase blockade reduces the activity of matrix metalloproteinase MMP-9, albeit it increases MMP-2 in vitro (4). Besides plasma membrane-localized V-ATPase, also intracellular V-ATPase may be involved in proteolytic activation of proteases acting within the lysosome or facilitating the traffic of secretory vesicles containing MMPs or cathepsins to the cell surface (2, 4, 15). Most of the work on V-ATPase in tumor cell invasion has focused on the impact of this proton pump on matrix degradation, whereas little has been done to assess whether V-ATPase may also influence the signaling leading to tumor cell motility and migration.

Cell migration is the result of a complex interplay of signaling machineries. Among these signaling axes, those emanating from growth factor receptors, such as EGFR, are important for directed migration, whereas Rho-GTPases, a subgroup of the Ras superfamily of GTPases, regulate cell morphology, and actin cytoskeleton, and are, therefore, indispensable for controlling the machinery of cell migration (16–18).

Here, we report that the V-ATPase inhibitor archazolid hinders the migration of invasive cancer cells in vitro and in vivo. We suggest that V-ATPase contributes to spatially restricted activation of migratory transducers such as EGFR and Rac1, which is required for the acquisition of polarized and directed tumor cell migration.

Compounds

Archazolid A was purified and isolated as described previously (9), archazolid B was synthesized chemically as described (ref. 12; Supplementary Fig. S1A). Both compounds were dissolved in dimethyl sulfoxide (DMSO) and show similar bioactivity (Supplementary Fig. S1B). The following antibodies were used: actin, cortactin and Rab5A (Santa Cruz), anti-pAkt/Akt, pEGFR/EGFR (cell signaling technologies), anti-Rac1 (Millipore), goat–anti–rabbit-488, goat–anti-mouse-488 and goat–anti-mouse-633 (AlexaFluor, Life technologies), goat–anti-rabbit (Dianova), goat–anti-mouse IgG2b (Biozol). Phalloidin rhodamine was purchased from Sigma.

Cell culture

The human breast cancer cell line SKBR3 was provided in 2009 by Dr. B. Mayer (University of Munich, Munich, Germany). Cells were tested every 6 months for their estrogen receptor, PgR, Her2, and EGFR status. Hela cells were from American Type Culture Collection and routinely tested for Mycoplasma contamination with MycoAlert Mycoplasma Detection Kit (LONZA) and cell cross-contamination with StemElite ID System (Promega). SKBR3 were maintained in McCoy's (PAA) medium supplemented with 10% heat-inactivated fetal calf serum (FCS, Biochrom AG) and 1% glutamine. Hela cells were grown in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% South American serum (EuroClone) and 1% L-glutamine. 4T1-Luc cells were recently (< 6 months) purchased from Caliper (Alamenda, CA) and cultivated in RPMI with 10% FCS.

Immunocytochemistry/flow cytometry

SKBR3 cells were seeded on IBIDI-μ-slides (IBIDI, Martinsried, Germany), fixed with 4% formaldehyde (15 minutes, RT) and permeabilized with 0.2% Triton X-100/PBS. Antibodies were diluted in 1% bovine serum albumin/PBS containing 0.1% Triton. For uptake of EGF-rhodamine (Invitrogen) archazolid treated as well as control, cells were starved for 2 hours and incubated for 5 to 15 minutes with EGF-rhodamine and analyzed by confocal microscopy (LSM 510 Meta; Zeiss, Oberkochen, Germany).

For analysis of EGFR and Her2 on the plasma membrane SKBR3 cells were treated with archazolid (10 nmol/L, 24 hours), trypsinized, washed with ice cold HEPES buffered saline, labeled anti–EGFR (Fig. 2) antibody and AlexaFluor488-trastuzumab, respectively (Supplementary Fig. S3) and analyzed with FACScan flow cytometer.

Rac1 activation assay

Rac1 was induced in cells treated with archazolid (24 hours, DMEM media) by adding epidermal growth factor (EGF 100 ng/mL) for 2 minutes followed by a pull-down assay according to the manufacturer's instructions (Thermo Fisher Scientific).

Western blot

Proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes using tank blotting. For the detection of protein levels, the ECL detection system (Amershan Pharmacia Biotech) or the Odyssey Infrared Imaging system version 2.1 (LI-COR Biosciences) was used.

Quantitative real-time PCR

The ABI 7300 RealTime PCR system with the TaqMan Universal PCR Mastermix (Life Technologies Corporation) was used. Probe and primers for the V-ATPase subunit c (ATP6L) and GAPDH (housekeeping gene) were supplied as mix (Life Technologies Corporation). Fluorescence increase was analyzed using the ABI 7300 system software. Calculation of relative mRNA was done according to Pfaffl (19).

Measurement of apoptosis

Apoptotic cell death was quantified as described (20) permeabilizing cells, staining with propidiumiodide (50 μg/μL) and analysis by flow cytometry (FACSCalibur, Becton Dickinson).

siRNA transfection

1 × 106 SKBR3 cells were transfected with control siRNA or siRNA targeting ATP6L (Thermo Scientific Dharmacon) using the Amaxa system. Downregulation of ATP6L was proved by real-time PCR.

Liposome-based cell transfection (Fugene)

The Fugene transfection reagent (Roche) was used to transfect plasmids: pCMV-BamHI, pCMV-Rab5, pGFP-Rac1-C1, 1014, pRaichu-CRIB (CFP-CRIB-YFP/PAK-CRIB), pCDNA3-Rac1-HA [wild-type Rac1 (RacWT)], and pCDNA3-RacQ61L-HA [constitutive active Rac1 (RacQL)].

Boyden chamber assay

Cells were treated with archazolid at a confluence of 70% for 24/16 hours. 1 × 105 cells were suspended in media without FCS and added on top of the Boyden chamber membrane (Corning). Culture medium with 10% FCS was added on the bottom of the membrane. Chambers were incubated at 37°C, 5% CO2 for 4 hours. Migrated cells were fixed and stained with crystal violet/methanol. Cells not migrated were removed with a q-tip. Pictures (×10 magnifications) were taken and migrated cells were counted and normalized to control cells (100% migration). For control purpose, cells were not exposed to FCS gradient (−Co, 0% migration).

Chemotaxis

SKBR3 cells (5 × 106) were seeded with media containing 2% FCS on IBIDI chemotaxis μ-slides and allowed to attach for 16 hours. FCS gradient and chemotaxis analysis was conducted as described before (21).

Cell adhesion/xCelligence

4 × 104 SKBR3 cells/well were allowed to adhere on fibronectin coated IBIDI μ-slides (30 minutes, 37°C) and fixed with 4% paraformaldehyde. Pictures were taken using the Axiovert, Zeiss, and number of adhered cells was counted.

For impedance/adherence time measurement 5 × 103 cells/well were seeded in the respective electrode plate (xCelligence, Roche, E-Plate 16). Cell impedance was monitored and adhesion time analyzed using the manufacturer's software (Software RTCA 1.2.1).

In vivo experiments

Twenty female BALB/cByJRj mice (Janvier) were housed in individually ventilated cages with a 12 hours day/night cycle and food and water ad libitum. Mice were inoculated with 1 × 105 4T1-Luc cells via the tail vein. Twenty-four and 4 hours before tumor cell injection the treatment group (n = 10) was medicated with 1 mg/kg archazolid (5% DMSO/PBS) intravenously. On day 8, after tumor cell inoculation bioluminescence imaging of the mice was conducted under anesthesia (2% isoflurane in oxygen) using the IVIS Lumina system with Living Image software 3.2 (Caliper Life Sciences) 15 minutes after intraperitoneal injection of 6 mg Na-luciferin (Promega). Mice were sacrificed through cervical dislocation; lungs were harvested, weighed, and imaged. Regions of Interest (ROI) were defined and the total signal per ROI was calculated as photons/second/cm2 (total flux/area). All animal experiments were conducted according to the guidelines of the German law for protection of animal life and approved by the local ethics committee.

Statistical analysis

All experiments were conducted at least 3 times in duplicates/triplicates. Results are expressed as mean value ± SEM. One-way ANOVA/Dunnett and individual students t tests were conducted using GraphPadPrism. P values less than 0.05 were considered as significant.

Archazolid inhibits cell migration

Treatment with archazolid (Supplementary Fig. S1A) inhibits migration of the breast cancer cell lines SKBR3 and MDA-MB 231 as well of the pancreatic cancer cell line L3.6pl using either Boyden chamber (Fig. 1A, Supplementary Fig. S2A) or wound healing-type of assays (Supplementary Fig. S2B). Importantly, archazolid, at all the concentrations and timepoints tested, had no effects on cell survival (Fig. 1A, Supplementary S2A bottom right). Archazolid affects chemotaxis of SKBR3 cells exposed to a diffusive gradient of FCS (0%–10%; Fig. 1B). Whereas control cells move toward the highest FCS concentration, cells treated with archazolid clearly display reduced directional migration (expressed as y-forward migration index). Only slight alterations in the cumulative distance and in the velocity of cell movement are observed suggesting that directed migration rather than cell mobility is mainly affected by archazolid.

Figure 1.

Archazolid inhibits cancer cell migration. A, SKBR3 cells treated with archazolid (24 hours) were allowed to migrate in Boyden chambers (4 hours). Bars (left) indicate percentage of migration [cells stimulated with FCS (+Co) are set as 100%; −Co, no FCS gradient applied] and are the mean ± SEM of 3 independent experiments conducted in duplicates; *, P < 0.05. Right panel shows that inhibition of migration by archazolid is not because of apoptosis induction. Bars represent the mean ± SEM of 3 independent experiments conducted in triplicates; *, P < 0.05. B, movement of SKBR3 cells treated with archazolid (16 hours) along a FCS gradient was monitored (24 hours) by live cell imaging and analyzed [Image-J (NIH); IBIDI software]. Bars represent the y-forward migration index (control cells are set as 1) as well as velocity and accumulative distance and are the mean ± SEM of 3 independent experiments; *, P < 0.05 (t test).

Figure 1.

Archazolid inhibits cancer cell migration. A, SKBR3 cells treated with archazolid (24 hours) were allowed to migrate in Boyden chambers (4 hours). Bars (left) indicate percentage of migration [cells stimulated with FCS (+Co) are set as 100%; −Co, no FCS gradient applied] and are the mean ± SEM of 3 independent experiments conducted in duplicates; *, P < 0.05. Right panel shows that inhibition of migration by archazolid is not because of apoptosis induction. Bars represent the mean ± SEM of 3 independent experiments conducted in triplicates; *, P < 0.05. B, movement of SKBR3 cells treated with archazolid (16 hours) along a FCS gradient was monitored (24 hours) by live cell imaging and analyzed [Image-J (NIH); IBIDI software]. Bars represent the y-forward migration index (control cells are set as 1) as well as velocity and accumulative distance and are the mean ± SEM of 3 independent experiments; *, P < 0.05 (t test).

Close modal

Archazolid affects EGFR cellular distribution and cell polarization

Growth factor receptors, including the epidermal growth factor receptor (EGFR), play a crucial role in directed cell migration as confirmed by using an EGFR inhibitor (Supplementary Fig. S4). Whereas the amount of EGFR protein on the cell surface increased upon archazolid treatment (24 hours; Fig. 2A left, Supplementary Fig. S3), the total amount of EGFR was unchanged (Fig. 2A right) and was still able to be activated as indicated by phosphorylation at Tyr 1068, although the extent and kinetic of activation were slightly decreased (Fig. 2A bottom). Conversely, we observed dramatic changes of EGFR surface distribution after archazolid treatment by confocal microscopy analysis of notpermeabilized cells. In control cells, EGFR localizes and is enriched at the leading edge of migrating cells. In archazolid-treated cells, in contrast, EGFR was distributed all over the cell surface and failed to accumulate along the leading edge extending in the direction of migration. This altered cellular distribution of EGFR likely reflects archazolid inhibition of lamellipodia extension as revealed by antiactin staining (Fig. 2C left). Furthermore, the localization of the microtubule-organizing center (MTOC) was affected. Whereas in control cells the MTOC is localized at the very front of the nucleus in the direction of migration, the MTOC of archazolid treated cells was randomly positioned with respect to the nucleus and the front of migration (Fig. 2C right).

Figure 2.

Archazolid affects EGFR localization. A, EGFR on the plasma membrane was analyzed by FACS (left) and total EGFR by Western blot after 24 hours of archazolid treatment (right). EGFR phosphorylation (Tyr1068) upon EGF stimulation was reduced in archazolid treated cells (bottom). Representative experiments out of 3 independent experiments are shown. B, cells treated (archazolid, 24 hours) or untreated (control) were scratched, fixed, and stained for EGFR without permeabilization (controlled by negative staining for actin) after 5 hours of migration time. C, archazolid inhibits lamellipodia extension as observed by actin staining in permeabilized cells (left). The localization of the centrosome (MTOC; stained for γ-tubulin) is altered by archazolid (right, arrows). Representative images out of 3 independent experiments are shown.

Figure 2.

Archazolid affects EGFR localization. A, EGFR on the plasma membrane was analyzed by FACS (left) and total EGFR by Western blot after 24 hours of archazolid treatment (right). EGFR phosphorylation (Tyr1068) upon EGF stimulation was reduced in archazolid treated cells (bottom). Representative experiments out of 3 independent experiments are shown. B, cells treated (archazolid, 24 hours) or untreated (control) were scratched, fixed, and stained for EGFR without permeabilization (controlled by negative staining for actin) after 5 hours of migration time. C, archazolid inhibits lamellipodia extension as observed by actin staining in permeabilized cells (left). The localization of the centrosome (MTOC; stained for γ-tubulin) is altered by archazolid (right, arrows). Representative images out of 3 independent experiments are shown.

Close modal

We tested if archazolid affects EGFR internalization and trafficking, which may in turn control EGFR signaling to actin remodeling. EGFR is rapidly internalized upon ligand binding. However, in archazolid treated cells EGFR internalization was significantly delayed (Fig. 3A) paralleled by a delay in maximal activation and a significant reduction in the duration of Akt signaling in archazolid treated cells (Fig. 3B). Likewise for EGFR, the polarized distribution of activated Akt in migrating cells was significantly impaired (Fig. 3C).

Figure 3.

Archazolid affects EGF internalization and Akt. A, SKBR3 cells treated or untreated and starved for 2 hours were exposed to EGF-rhodamine for 5 and 15 minutes and analyzed by confocal microscopy. B, phosphorylated Akt (Ser473) was detected by Western blot upon exposure to EGF (100 ng/mL). C, localization of pAkt was analyzed by confocal microscopy in control and archazolid treated cells. A–C, representative experiments out of 3 independent experiments are shown.

Figure 3.

Archazolid affects EGF internalization and Akt. A, SKBR3 cells treated or untreated and starved for 2 hours were exposed to EGF-rhodamine for 5 and 15 minutes and analyzed by confocal microscopy. B, phosphorylated Akt (Ser473) was detected by Western blot upon exposure to EGF (100 ng/mL). C, localization of pAkt was analyzed by confocal microscopy in control and archazolid treated cells. A–C, representative experiments out of 3 independent experiments are shown.

Close modal

Archazolid inhibits ruffle formation by preventing Rac1 activation

Cells that attach and spread on ECM substrates are known to activate signaling pathways and undergo morphologic changes in their shape and remodeling of the actin cytoskeleton that recapitulate events typical of migratory cells (22). Thus, we tested the effect of archazolid on cell spreading onto fibronectin-coated plates. Archazolid treatment did not significantly alter the number of adhered cells, suggesting that adhesion receptors responsible for fibronectin interaction are functional (Fig. 4A left). Conversely, archazolid-treated, freshly adherent SKBR3 cells underwent prominent morphologic changes that were monitored in real time both by changes in impedance, which is directly proportional to the area of cells in contact with the substrate (xCelligence, Roche, Fig. 4A right) and by determination of ruffle formation (Fig. 4B left). In contrast to control, archazolid-treated cells were significantly less efficient in spreading and failed to form prominent actin rich peripheral lamellipodia and ruffles.

Figure 4.

Archazolid affects localization and activation of Rac1. A, archazolid treated SKBR3 cells (24 hours) were seeded on fibronectin (30 minutes), fixed, and counted. Bars represent percentage of adhered cells (control cells, 100%; left). Kinetics of cell spreading (control cells, 100%) was analyzed by impedance measurement (xCELLigence RTCA, Roche) as shown by a representative graph (right). B, the formation of ruffles (left) and cellular localization of Rac1 (right) in freshly adhered cells untreated or treated with archazolid was analyzed by confocal microscopy. C, a Rac1 pull-down assay was conducted in control and archazolid-treated cells upon a 2-minute EGF stimulation. A representative experiment out of 3 independent experiments is shown. D, SKBR3 treated with archazolid (24 hours) were engaged in a scratch assay and stained for Rac1, cortactin, and F-actin (left). Representative images out of 3 independent experiments are shown. E, overexpression of cells with a RacWT or a constitutive active Rac1 mutant (RacQL) was able to abrogate inhibition of migration by archazolid (Boyden chamber setting). Representative images out of 3 independent experiments are shown. A, B, and E, bars always represent the mean ± SEM of 3 independent experiments conducted in triplicates. *, P < 0.05 (t test).

Figure 4.

Archazolid affects localization and activation of Rac1. A, archazolid treated SKBR3 cells (24 hours) were seeded on fibronectin (30 minutes), fixed, and counted. Bars represent percentage of adhered cells (control cells, 100%; left). Kinetics of cell spreading (control cells, 100%) was analyzed by impedance measurement (xCELLigence RTCA, Roche) as shown by a representative graph (right). B, the formation of ruffles (left) and cellular localization of Rac1 (right) in freshly adhered cells untreated or treated with archazolid was analyzed by confocal microscopy. C, a Rac1 pull-down assay was conducted in control and archazolid-treated cells upon a 2-minute EGF stimulation. A representative experiment out of 3 independent experiments is shown. D, SKBR3 treated with archazolid (24 hours) were engaged in a scratch assay and stained for Rac1, cortactin, and F-actin (left). Representative images out of 3 independent experiments are shown. E, overexpression of cells with a RacWT or a constitutive active Rac1 mutant (RacQL) was able to abrogate inhibition of migration by archazolid (Boyden chamber setting). Representative images out of 3 independent experiments are shown. A, B, and E, bars always represent the mean ± SEM of 3 independent experiments conducted in triplicates. *, P < 0.05 (t test).

Close modal

Lamellipodia extension and ruffle formation are primarily controlled by the Rho GTPase Rac1, which localizes to leading edges to promote these migratory structures. Thus, we tested whether archazolid may affect either the localization or activation (or both) of Rac1. Confocal analysis of freshly adherent control cells showed a distinct localization of Rac1, which, as expected, accumulated at the cell periphery where actin-rich lamellipodial sheets are localized. Archazolid completely abrogated the distribution of Rac1 to the plasma membrane most likely because the peripheral enrichment of filamentous actin (ruffles) also disappears upon archazolid treatment (Fig. 4B, right). A similar picture was observed in migrating cells in a scratch assay. Under these conditions, archazolid inhibited the formation of wound–directed, Rac1-, cortactin-, and F-actin–positive polarized lamellipodia (Fig. 4D). All these morphologic phenotypes can be easily accounted by a direct inhibitory effect of archazolid on Rac1 activation. Consistent with this hypothesis, EGF–induced Rac-GTP levels were significantly reduced by archazolid treatment (Fig. 4C), suggesting that pathways leading to the activation of this GTPase are a primary target of archazolid action. This is further supported by overexpression of either RacWT or RacQL, which could both rescue the inhibition of migration caused by archazolid (Fig. 4F).

Downregulation of V-ATPase subunit c inhibits migration and Rac1 activation

Archazolid binds to the V-ATPase subunit c. To unequivocally establish that the effects of archazolid are because of the inhibition of V-ATPase subunit c, we genetically silenced the V0 subunit c (ATP6L) using siRNA. Downregulation of ATP6L impaired both cell migration and polarized lamellipodia and ruffle formation observed in a scratch wound healing assay (Fig. 5A and B). This effect is not cell type specific, as pancreatic cancer cells L3.6pl were equally inhibited by silencing of ATP6L (Supplementary Fig. S5). Notably and similar to archazolid, silencing of ATP6L inhibited both the activation of Rac1 (Fig. 5C) and its accumulation at the leading edge of migrating SKBR3 cells because of loss of ruffles (Fig. 5B, right). These results support the hypothesis that the V-ATPase subunit c plays a crucial role during the migration process by critically participating in the activation of Rac1.

Figure 5.

Downregulation of the V-ATPase subunit c abrogates cell migration. A, cells were transfected with siRNA either nontargeted (NT) or targeted against the subunit c (ATP6L) for 24 hours, followed by a Boyden chamber assay and quantification of migrated cells (left). Downregulation of ATP6L was confirmed by real-time PCR (middle) and no apoptotic cell death is induced by siRNA transfection (right). B, cells were transfected with siRNA-ATP6L and freshly seeded. Cells with ruffles were counted and expressed as percentage of total adhered cells (left panel). siRNA-ATP6L treated cells were used in a scratch assay and stained for ATP6L, Rac1, and F-actin (phalloidin) Representative images out of 3 independent experiments are shown (right). C, Rac1 pull-down assay was conducted in ATP6L-silenced cells. One representative experiment out of 3 independent experiments is shown (left). Downregulation of ATP6L was proved for each single experiment by real-time PCR (right). A–C, bars represent always the mean ± SEM of 3 independent experiments; *, P < 0.05 (t test).

Figure 5.

Downregulation of the V-ATPase subunit c abrogates cell migration. A, cells were transfected with siRNA either nontargeted (NT) or targeted against the subunit c (ATP6L) for 24 hours, followed by a Boyden chamber assay and quantification of migrated cells (left). Downregulation of ATP6L was confirmed by real-time PCR (middle) and no apoptotic cell death is induced by siRNA transfection (right). B, cells were transfected with siRNA-ATP6L and freshly seeded. Cells with ruffles were counted and expressed as percentage of total adhered cells (left panel). siRNA-ATP6L treated cells were used in a scratch assay and stained for ATP6L, Rac1, and F-actin (phalloidin) Representative images out of 3 independent experiments are shown (right). C, Rac1 pull-down assay was conducted in ATP6L-silenced cells. One representative experiment out of 3 independent experiments is shown (left). Downregulation of ATP6L was proved for each single experiment by real-time PCR (right). A–C, bars represent always the mean ± SEM of 3 independent experiments; *, P < 0.05 (t test).

Close modal

V-ATPase-inhibition affects Rab5-induced Rac1 activation

Recently, the recycling molecule Rab5 has been reported to play an important role in the activation of the Rac1–mediated actin remodeling and cell migration (23). In keeping with these observations, we show that Rab5 is sufficient to induce the formation of Rac1-dependent actin-rich migratory structures, including circular dorsal ruffles and peripheral ruffles (Fig. 6A). Thus, we tested whether archazolid interfered also with Rab5-evoked morphologic changes. Both, peripheral and circular dorsal ruffle formation was significantly reduced in Rab5-expressing Hela cells after 24 hours of archazolid treatment (Fig. 6A). Similarly, Rab5-mediated activation of Rac1 (Fig. 6B left) was significantly inhibited by archazolid treatment (Fig. 6B right).

Figure 6.

Rab5-dependent Rac activation is inhibited by archazolid. A, Hela cells were transfected with Rab5WT and treated with archazolid (10 nmol/L, 24 hours). Ruffle formation was induced with 10% FCS and 10 ng/mL HGF for 5 minutes. Samples were stained for actin using phalloidin-FITC. Number of ruffles was counted (PR, peripheral ruffles; CDR, circular dorsal ruffles). Bars represent ruffling index and are the mean ± SEM of 3 independent experiments conducted in triplicates. *, P < 0.05 (t test). B, Rab5 expression induces Rac1 activation (left) similar to EGF. Rab5-dependent Rac activation is reduced after archazolid treatment (right). SKBR3 cells were transfected with pcMV-Rab5 followed by a Rac1 pull-down assay. Representative experiments out of 3 independent experiments are shown. C, Rab5 expression leads to enlarged early endosomes containing Rac1 (arrows, left). Rab5 and Rac-GFP were expressed in Hela cells being stained for EEA1. Localization of active Rac1 was analyzed in archazolid-treated (24 hours) and untreated cells expressing Rab5, Rac1-HA, and CRIB-YFP. Arrows show active PAK-CRIB at the enlarged endosomes (right). Representative images out of 3 independent experiments are shown.

Figure 6.

Rab5-dependent Rac activation is inhibited by archazolid. A, Hela cells were transfected with Rab5WT and treated with archazolid (10 nmol/L, 24 hours). Ruffle formation was induced with 10% FCS and 10 ng/mL HGF for 5 minutes. Samples were stained for actin using phalloidin-FITC. Number of ruffles was counted (PR, peripheral ruffles; CDR, circular dorsal ruffles). Bars represent ruffling index and are the mean ± SEM of 3 independent experiments conducted in triplicates. *, P < 0.05 (t test). B, Rab5 expression induces Rac1 activation (left) similar to EGF. Rab5-dependent Rac activation is reduced after archazolid treatment (right). SKBR3 cells were transfected with pcMV-Rab5 followed by a Rac1 pull-down assay. Representative experiments out of 3 independent experiments are shown. C, Rab5 expression leads to enlarged early endosomes containing Rac1 (arrows, left). Rab5 and Rac-GFP were expressed in Hela cells being stained for EEA1. Localization of active Rac1 was analyzed in archazolid-treated (24 hours) and untreated cells expressing Rab5, Rac1-HA, and CRIB-YFP. Arrows show active PAK-CRIB at the enlarged endosomes (right). Representative images out of 3 independent experiments are shown.

Close modal

In Rab5-expressing, untreated cells, Rac1 can, as previously reported (23), localize to enlarged early endosomes [revealed by the presence of the early endosome antigen-1 (EEA1)] (Fig. 6C left). Treatment with archazolid, however, prevented the accumulation of Rac1 on Rab5 vesicles, as revealed both by directly monitoring Rac1 cellular distribution (Supplementary Fig. S6) or using a Rac1 biosensor (PAK-CRIB) that specifically binds to activated Rac1 (Fig. 6C right).

Archazolid inhibits metastasis in vivo

To extend the relevance of the inhibitory effects of archazolid on chemotactic migration and cell invasion, we next tested whether this drug was effective in impairing cancer dissemination in xenograft mouse models. To this end, we used the 4T1-Luc syngeneic metastatic mouse breast cancer model (24–27). This model is based on the 4T1-Luc mouse mammary tumor cell line, which when injected intravenously disseminates to distant organs, homing to lungs, liver, bone, and brain over a short period of time. These cells are engineered to express a luciferase reporter to enable real time monitoring of developing tumor by live imaging. Under these conditions, mock treated, 4T1-injected animals formed easily detectable lung metastases, which were drastically inhibited by intravenous injection of archazolid, which at the concentrations tested (1 mg/kg i.v.) did not cause any obvious signs of toxicity (Fig. 7A and B and Supplementary Fig. S7A and S7B).

Figure 7.

Metastasis of 4T1 Luc cells is decreased by archazolid in vivo. Archazolid reduces the amount of metastasis in the lung of BALB/cByJRj mice. 1 × 105 4T1-Luc cells were injected intravenously into pretreated (1 mg/kg archazolid) and untreated mice. A, on day 8 after cell inoculation bioluminescence signals were recorded by imaging the mice dorsoventral and ventrodorsal. Color bar scales were equalized. B, day 8, mice were sacrificed, lungs were harvested, and imaged separately (Supplementary Fig.S7). ROIs were defined and the total signal per ROI was calculated as photons/second/cm2 (total flux/area). Bars represent the mean ± SEM. *, P < 0.05 (t test).

Figure 7.

Metastasis of 4T1 Luc cells is decreased by archazolid in vivo. Archazolid reduces the amount of metastasis in the lung of BALB/cByJRj mice. 1 × 105 4T1-Luc cells were injected intravenously into pretreated (1 mg/kg archazolid) and untreated mice. A, on day 8 after cell inoculation bioluminescence signals were recorded by imaging the mice dorsoventral and ventrodorsal. Color bar scales were equalized. B, day 8, mice were sacrificed, lungs were harvested, and imaged separately (Supplementary Fig.S7). ROIs were defined and the total signal per ROI was calculated as photons/second/cm2 (total flux/area). Bars represent the mean ± SEM. *, P < 0.05 (t test).

Close modal

This study gives evidence for a role of V-ATPase in tumor metastasis and reveals novel molecular mechanisms of its action, which are depicted in a cartoon (Supplementary Fig. S8)

It has been reported for breast and pancreatic cancer cells that the abundance of V-ATPase on the plasma membrane correlates with an invasive phenotype (3, 28–31). Our observation that V-ATPase is localized on the plasma membrane of the invasive cancer cell line SKBR3, but not in the nontumor mammary epithelial cells MCF-10A (Supplementary Fig. S9) is consistent with this notion. Genetic knock down of ATP6L leads both to the disappearance of V-ATPase at the leading edge of breast cancer cells as well as to a significant decrease of their migratory activity extending and corroborating data by Lu and colleagues using hepatocellular carcinoma cells (32).

With respect to the mode of action of V-ATPase in cancer metastasis, one currently accepted model invokes that V-ATPase, highly concentrated on the cell surface, may provide a localized proton efflux, thus generating an acidic extracellular microenvironment, which is, in turn, favorable for the activation of a variety of proinvasive proteases (4, 33).

Our work unveils an alternative mode of action for V-ATPase. We propose that V-ATPase regulates the signaling machinery responsible for tumor cell migration. More specifically, we showed that V-ATPase inhibition by archazolid impairs spatial restriction of migratory signaling molecules such as Rac1 and EGFR, which is pivotal for directed and polarized cell movement.

Spatial restriction of signaling, achieved by redistributing signaling molecules to confined areas of the plasma membrane (e.g., leading edge) upon chemotactic stimuli, has emerged as essential for directed cell migration (17). Within this context, endocytosis has been recently proposed to serve as an integrated trafficking network to direct where signaling molecules are activated and is thus important for virtually all polarized functions, first and foremost cell migration (34). Consistently endo/exocytic cycles of receptor tyrosine kinases, such as EGFR, are essential for guided motility (35, 36). EGFR is also a potent guidance receptor, which induces actin remodeling leading to the formation of various migratory protrusions such lamellipodia (peripheral ruffles) or dorsal surface circular ruffles, an obligatory step for 2-dimensional cell motility (16). Own experiments using an EGFR inhibitor support this notion (Supplementary Fig. S4).

V-ATPase inhibition by archazolid in migrating tumor cells alters EGFR localization, reduces its internalization, and impairs EGFR downstream signaling by ablating Akt activation and preventing its accumulation at the leading edge. These combined effects, ultimately, impact on the ability of cells to polarize. Not surprisingly, the PI3K/Akt pathway plays a major role in the migratory programs of cells (17).

Notably, a general influence of V-ATPase inhibition on internalization as well as recycling processes has been reported before (37, 38). This finding was also confirmed for archazolid, which inhibits uptake of dextran, but not of transferrin/transferrin-R after short time of treatment, however, abrogates transferrin/transferrin-R uptake after prolonged exposure (Supplementary Fig. S10A and S10B).

About mechanisms underlying V-ATPase action on internalization/recycling, it is important to know that endosomes and lysosomes, where recycling and degradation takes place, are acidified by V-ATPases (39). The acidic pH is pivotal for the dissociation of ligands from its receptors and degradation/recycling processes (35). Increase of pH induced by bafilomycin, for example, has been shown to affect the delivery of endocytosed molecules to late endosomes or lysosomes in some, but not all cell lines tested (37, 40, 41). We analyzed SKBR3 cells by transmission electron microscopy (Supplementary Fig. S10C) and found considerably more and enlarged multivesicular bodies (MVB) in cells after archazolid treatment. MVBs are degradative endosomal organelles, mediating trafficking between early and late endosomes (40, 42). Interestingly, this phenomenon was also described in a study focusing on the involvement of V-ATPase activity in Notch Signaling (42). As an even more acidic pH is necessary for lysosomal degradation process, V-ATPase inhibition by archazolid might inhibit the final fusion with lysosomes, halting the degradative, and probably also recycling routes, leading to enlargement of MVBs. Likely these “high pH” MVB are inefficient degradative factory that may trap cargos and endosomal associated molecules that would otherwise be routed to recycling pathways for subsequent round of signaling. Ultimately, this may result in an impaired delivery of internalized molecules, such as EGFR, to the proper location on the plasma membrane preventing their proper and sustained activation. Similar alteration in trafficking of endosomal membrane associated molecules recycling back to the plasma membrane may also occur for impaired activation of Rac1, which after archazolid treatment, is no longer able to enter the Rab5-mediated endosomal routes for subsequent delivery back to the plasma membrane. How changes in endosomal pH affect Rac1 recruitment to these organelles is unclear at this stage. One possibility is that local changes of the pH at the plasma membrane, where Rac1 or its GEFs are primarily localized and activated, are required for Rac1 (or its activators) to subsequently enter endocytic organelles. Indeed similar reduction of Rac1 in endosomes could be observed after interference with clathrin (23) suggesting that plasma membrane localized Rac may follow a canonical endocytic routes that may require local changes of pH to be effective. Whether this is indeed the case remains, however, to be tested in future investigations.

Whatever the case, impaired endocytic traffic induced by V-ATPase is hypothesized to affect the spatially restricted activation of Rac1, a central regulator of actin remodeling and cell migration (17, 43). Consistently, archazolid treatment or silencing the V0 subunit c of the V-ATPase, the binding site of archazolid, abrogate Rac1-dependent F-actin remodeling, F-actin–mediated formation of ruffles or lamellipodia as well as the recruitment of active Rac1 to these migratory structures. These results also show that archazolid exerts its effect by specifically targeting V-ATPase, or more precisely of the V0 subunit c.

Spatial restriction of Rac1 signaling has been shown to depend on Rab5 endo/exocitic cycles (23). Rab5, in addition to be a master regulator of endocytosis (44) by controlling the biogenesis and fate of early endosomes, was also shown to be sufficient to promote, through Rac1, the formation of a diverse set of migratory protrusions, including lamellipodia and circular dorsal ruffles (23). Notably, archazolid robustly inhibits all these structures. These effects are accompanied by loss of Rac1 from endosomal membrane, which results in reduced delivery of this GTPase to cell protrusion, and impaired directional migration. Thus, archazolid appears to interfere both with endocytic trafficking of Rac1 as well as EGFR. Whether EGFR needs to be internalized and recycled to trigger Rac1 internalization and activation or the 2 events are occurring at the same time independently of each other remains to be addressed in future work.

Finally, as in vivo data on V-ATPase inhibitors are very limited, we like to point to the fact that effects of archazolid in cell culture are recapitulated in a mouse model of tumor dissemination. This indicates that pharmacologic interference with V-ATPase represents a viable strategy both to decipher functions of this enzyme in cancer progression as well as to prevent the acquisition of migratory and invasive properties, ultimately leading to metastasis.

No potential conflicts of interest were disclosed.

Conception and design: S. Zahler, R. Müller, G. Scita, A.M. Vollmar

Development of methodology: A. Palamidessi, D. Trauner, A.M. Vollmar

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): R.M. Wiedmann, L. Schreiner, R. Kubisch, G. Vereb, E. Wagner, A.M. Vollmar

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): K.v. Schwarzenberg, A. Palamidessi, J. Liebl, C. Schempp, G. Vereb, S. Zahler, E. Wagner, A.M. Vollmar

Writing, review, and/or revision of the manuscript: K.v. Schwarzenberg, J. Liebl, D. Trauner, S. Zahler, E. Wagner, R. Müller, G. Scita, A.M. Vollmar

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): K.v. Schwarzenberg, E. Wagner, A.M. Vollmar

Study supervision: E. Wagner, A.M. Vollmar

Performed experiments: C. Schempp

The authors thank Prof. Dr. Wanner and Lina Schneider for their excellent work.

This work was supported by The German Research Foundation (DFG), FOR 1406, Vo 376-14/15 (A.M. Vollmar), MU 1254/14-1 (R. Müller), and WA 1648/3-1 (E. Wagner); and Cluster of Excellence Nanosystems Initiative Munich (E. Wagner).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1.
Chaffer
CL
,
Weinberg
RA
. 
A perspective on cancer cell metastasis
.
Science
2011
;
331
:
1559
64
.
2.
Fais
S
,
De Milito
A
,
You
H
,
Qin
W
. 
Targeting vacuolar H+-ATPases as a new strategy against cancer
.
Cancer Res
2007
;
67
:
10627
30
.
3.
Sennoune
SR
,
Bakunts
K
,
Martinez
GM
,
Chua-Tuan
JL
,
Kebir
Y
,
Attaya
MN
, et al
Vacuolar H+-ATPase in human breast cancer cells with distinct metastatic potential: distribution and functional activity
.
Am J Physiol Cell physiology
2004
;
286
:
C1443
52
.
4.
Chung
C
,
Mader
CC
,
Schmitz
JC
,
Atladottir
J
,
Fitchev
P
,
Cornwell
ML
, et al
The vacuolar-ATPase modulates matrix metalloproteinase isoforms in human pancreatic cancer
.
Lab Invest
2011
;
91
:
732
43
.
5.
Supino
R
,
Petrangolini
G
,
Pratesi
G
,
Tortoreto
M
,
Favini
E
,
Bo
LD
, et al
Antimetastatic effect of a small-molecule vacuolar H+-ATPase inhibitor in in vitro and in vivo preclinical studies
.
J Pharmacol Exp Ther
2008
;
324
:
15
22
.
6.
Forgac
M
. 
Vacuolar ATPases: rotary proton pumps in physiology and pathophysiology
.
Nat Rev Mol Cell Biol
2007
;
8
:
917
29
.
7.
Saroussi
S
,
Nelson
N
. 
The little we know on the structure and machinery of V-ATPase
.
J Exp Biol
2009
;
212
:
1604
10
.
8.
Huss
M
,
Sasse
F
,
Kunze
B
,
Jansen
R
,
Steinmetz
H
,
Ingenhorst
G
, et al
Archazolid and apicularen: novel specific V-ATPase inhibitors
.
BMC Biochemistry
2005
;
6
:
13
.
9.
Sasse
F
,
Steinmetz
H
,
Hofle
G
,
Reichenbach
H
. 
Archazolids, new cytotoxic macrolactones from Archangium gephyra (Myxobacteria). Production, isolation, physico-chemical and biological properties
.
J Antibiot
2003
;
56
:
520
5
.
10.
Bockelmann
S
,
Menche
D
,
Rudolph
S
,
Bender
T
,
Grond
S
,
von Zezschwitz
P
, et al
Archazolid A binds to the equatorial region of the c-ring of the vacuolar H+-ATPase
.
J Biol Chem
2010
;
285
:
38304
14
.
11.
Menche
D
,
Hassfeld
J
,
Li
J
,
Rudolph
S
. 
Total synthesis of archazolid A
.
J Am Chem Soc
2007
;
129
:
6100
1
.
12.
Roethle
PA
,
Chen
IT
,
Trauner
D
. 
Total synthesis of (-)-archazolid B
.
J Am Chem Soc
2007
;
129
:
8960
1
.
13.
Horstmann
N
,
Essig
S
,
Bockelmann
S
,
Wieczorek
H
,
Huss
M
,
Sasse
F
, et al
Archazolid A-15-O-beta-D-glucopyranoside and iso-archazolid B: potent V-ATPase inhibitory polyketides from the myxobacteria Cystobacter violaceus and Archangium gephyra
.
J Nat Prod
2011
;
74
:
1100
5
.
14.
Mason
SD
,
Joyce
JA
. 
Proteolytic networks in cancer
.
Trends Cell Biol
2011
;
21
:
228
37
.
15.
McHenry
P
,
Wang
WL
,
Devitt
E
,
Kluesner
N
,
Davisson
VJ
,
McKee
E
, et al
Iejimalides A and B inhibit lysosomal vacuolar H+-ATPase (V-ATPase) activity and induce S-phase arrest and apoptosis in MCF-7 cells
.
J Cell Biochem
2010
;
109
:
634
42
.
16.
Buccione
R
,
Orth
JD
,
McNiven
MA
. 
Foot and mouth: podosomes, invadopodia and circular dorsal ruffles
.
Nat Rev Mol Cell Biol
2004
;
5
:
647
57
.
17.
Jiang
P
,
Enomoto
A
,
Takahashi
M
. 
Cell biology of the movement of breast cancer cells: intracellular signalling and the actin cytoskeleton
.
Cancer Lett
2009
;
284
:
122
30
.
18.
Sahai
E
,
Marshall
CJ
. 
RHO-GTPases and cancer
.
Nat Rev Cancer
2002
;
2
:
133
42
.
19.
Pfaffl
MW
. 
A new mathematical model for relative quantification in real-time RT-PCR
.
Nucleic Acids Res
2001
;
29
:
e45
.
20.
Nicoletti
I
,
Migliorati
G
,
Pagliacci
MC
,
Grignani
F
,
Riccardi
C
. 
A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry
.
J Immunol Methods
1991
;
139
:
271
9
.
21.
Rothmeier
AS
,
Ischenko
I
,
Joore
J
,
Garczarczyk
D
,
Furst
R
,
Bruns
CJ
, et al
Investigation of the marine compound spongistatin 1 links the inhibition of PKCalpha translocation to nonmitotic effects of tubulin antagonism in angiogenesis
.
FASEB J
2009
;
23
:
1127
37
.
22.
Applewhite
DA
,
Barzik
M
,
Kojima
S
,
Svitkina
TM
,
Gertler
FB
,
Borisy
GG
. 
Ena/VASP proteins have an anti-capping independent function in filopodia formation
.
Mol Biol Cell
2007
;
18
:
2579
91
.
23.
Palamidessi
A
,
Frittoli
E
,
Garre
M
,
Faretta
M
,
Mione
M
,
Testa
I
, et al
Endocytic trafficking of Rac is required for the spatial restriction of signaling in cell migration
.
Cell
2008
;
134
:
135
47
.
24.
Aslakson
CJ
,
Miller
FR
. 
Selective events in the metastatic process defined by analysis of the sequential dissemination of subpopulations of a mouse mammary tumor
.
Cancer Res
1992
;
52
:
1399
405
.
25.
Yang
J
,
Mani
SA
,
Donaher
JL
,
Ramaswamy
S
,
Itzykson
RA
,
Come
C
, et al
Twist, a master regulator of morphogenesis, plays an essential role in tumor metastasis
.
Cell
2004
;
117
:
927
39
.
26.
Li
J
,
Sahagian
GG
. 
Demonstration of tumor suppression by mannose 6-phosphate/insulin-like growth factor 2 receptor
.
Oncogene
2004
;
23
:
9359
68
.
27.
Tao
K
,
Fang
M
,
Alroy
J
,
Sahagian
GG
. 
Imagable 4T1 model for the study of late stage breast cancer
.
BMC Cancer
2008
;
8
:
228
.
28.
Sennoune
SR
,
Luo
D
,
Martinez-Zaguilan
R
. 
Plasmalemmal vacuolar-type H+-ATPase in cancer biology
.
Cell Biochem Biophys
2004
;
40
:
185
206
.
29.
Hinton
A
,
Sennoune
SR
,
Bond
S
,
Fang
M
,
Reuveni
M
,
Sahagian
GG
, et al
Function of a subunit isoforms of the V-ATPase in pH homeostasis and in vitro invasion of MDA-MB231 human breast cancer cells
.
J Biol Chem
2009
;
284
:
16400
8
.
30.
Hinton
A
,
Bond
S
,
Forgac
M
. 
V-ATPase functions in normal and disease processes
.
Pflugers Arch
2009
;
457
:
589
98
.
31.
Torigoe
T
,
Izumi
H
,
Ise
T
,
Murakami
T
,
Uramoto
H
,
Ishiguchi
H
, et al
Vacuolar H(+)-ATPase: functional mechanisms and potential as a target for cancer chemotherapy
.
Anti-cancer Drugs
2002
;
13
:
237
43
.
32.
Lu
X
,
Qin
W
,
Li
J
,
Tan
N
,
Pan
D
,
Zhang
H
, et al
The growth and metastasis of human hepatocellular carcinoma xenografts are inhibited by small interfering RNA targeting to the subunit ATP6L of proton pump
.
Cancer Res
2005
;
65
:
6843
9
.
33.
Kubota
S
,
Seyama
Y
. 
Overexpression of vacuolar ATPase 16-kDa subunit in 10T1/2 fibroblasts enhances invasion with concomitant induction of matrix metalloproteinase-2
.
Biochem Biophys Res Commun
2000
;
278
:
390
4
.
34.
Sigismund
S
,
Confalonieri
S
,
Ciliberto
A
,
Polo
S
,
Scita
G
,
Di Fiore
PP
. 
Endocytosis and signaling: cell logistics shape the eukaryotic cell plan
.
Physiol Rev
2012
;
92
:
273
366
.
35.
Scita
G
,
Di Fiore
PP
. 
The endocytic matrix
.
Nature
2010
;
463
:
464
73
.
36.
Jones
MC
,
Caswell
PT
,
Norman
JC
. 
Endocytic recycling pathways: emerging regulators of cell migration
.
Curr Opin Cell Biol
2006
;
18
:
549
57
.
37.
Baravalle
G
,
Schober
D
,
Huber
M
,
Bayer
N
,
Murphy
RF
,
Fuchs
R
. 
Transferrin recycling and dextran transport to lysosomes is differentially affected by bafilomycin, nocodazole, and low temperature
.
Cell Tissue Res
2005
;
320
:
99
113
.
38.
Tawfeek
HA
,
Abou-Samra
AB
. 
Important role for the V-type H(+)-ATPase and the Golgi apparatus in the recycling of PTH/PTHrP receptor
.
Am J Physiol Endocrinol Metab
2004
;
286
:
E704
10
.
39.
Nishi
T
,
Forgac
M
. 
The vacuolar (H+)-ATPases–nature's most versatile proton pumps
.
Nat Rev Mol Cell Biol
2002
;
3
:
94
103
.
40.
Clague
MJ
,
Urbe
S
,
Aniento
F
,
Gruenberg
J
. 
Vacuolar ATPase activity is required for endosomal carrier vesicle formation
.
J Biol Chem
1994
;
269
:
21
4
.
41.
van Weert
AW
,
Dunn
KW
,
Gueze
HJ
,
Maxfield
FR
,
Stoorvogel
W
. 
Transport from late endosomes to lysosomes, but not sorting of integral membrane proteins in endosomes, depends on the vacuolar proton pump
.
J Biol Chem
1995
;
130
:
821
34
.
42.
Vaccari
T
,
Duchi
S
,
Cortese
K
,
Tacchetti
C
,
Bilder
D
. 
The vacuolar ATPase is required for physiological as well as pathological activation of the Notch receptor
.
Development
2010
;
137
:
1825
32
.
43.
Wertheimer
E
,
Gutierrez-Uzquiza
A
,
Rosemblit
C
,
Lopez-Haber
C
,
Sosa
MS
,
Kazanietz
MG
. 
Rac signaling in breast cancer: a tale of GEFs and GAPs
.
Cell Signal
2012
;
24
:
353
62
.
44.
Zerial
M
,
McBride
H
. 
Rab proteins as membrane organizers
.
Nat Rev Mol Cell Biol
2001
;
2
:
107
17
.