Inhibitors of Vacuolar ATPase Proton Pumps Inhibit Human Prostate Cancer Cell Invasion and Prostate-Specific Antigen Expression and Secretion

Abstract

Vacuolar ATPases (V-ATPases) comprise specialized and ubiquitously distributed pumps that acidify intracellular compartments and energize membranes. To gain new insights into the roles of V-ATPases in prostate cancer (PCa) we studied the effects of inhibiting V-ATPase pumps in androgen-dependent (LNCaP) and androgen-independent (C4-2B) cells of a human PCa progression model. Treatment with nanomolar concentrations of the V-ATPase inhibitors bafilomycin A or concanamycin A reduced the in vitro invasion in both cell types by 80%, regardless that V-ATPase was prominent at the plasma membrane of C4-2B cells and only traces were detected in the low-metastatic LNCaP parental cells. In both cell types intracellular V-ATPase was excessive and co-localized with prostate-specific antigen (PSA) in the Golgi compartment. V-ATPase inhibitors reversibly excluded PSA from the Golgi and led to the accumulation of largely dispersed PSA-loaded vesicles of lysosomal composition. Inhibition of acridine orange staining and transferrin receptor recycling suggested defective endosomal and lysosomal acidification. The inhibitors, additionally, interfered with the AR-PSA axis under conditions that reduced invasion. Bafilomycin A significantly reduced steady-state and R1881-induced PSA mRNA expression and secretion in the LNCaP cells which are androgen-dependent, but not in the C4-2B cells which are androgen ablation-resistant. In the C4-2B cells, an increased susceptibility to V-ATPase inhibitors was detected after longer treatments, as proliferation was reduced and reversibility of bafilomycin-induced responses impaired. These findings make V-ATPases attractive targets against early and advanced PCa tumors.

INTRODUCTION

Vacuolar adenosine triphosphatases (V-ATPases) are protein complexes that use the energy of ATP hydrolysis to pump protons across endosomal and lysosomal membranes ¹. The luminal acidic pH generated by V-ATPases facilitates processes such as protein sorting and secretion, receptor recycling, endocytosis, and protein degradation ^2–6. Plasmalemmal V-ATPases present in cells specialized for active proton secretion, including cancer cells, pump protons to the extracellular space. They sustain the low pH that activates the extracellular metalloproteases, allowing for tumor cell motility, proliferation, and metastasis ^7–10. Accumulated evidence have shown V-ATPase involvement in tumor invasion and multi-drug resistance in breast cancer ^11–14, oral squamous cell carcinoma ^{15, 16}, hepatocellular carcinoma ⁸, melanoma ¹⁷, pancreatic cancer ⁹, and PCa ¹⁸. Additional information regarding the functions of V-ATPases in PCa is limited; Iejimalide B, an inhibitor of V-ATPases, induces cell cycle arrest and apoptosis in prostate cancer (PCa) cells ¹⁹.

PCa is the second most common cancer in men in the United States. PCa screening is performed by testing the serum levels of the biomarker prostate-specific antigen (PSA), an androgen-dependent ²⁰ serine protease secreted by both healthy and cancerous prostate tissue ²¹. PSA expression is under control of the androgen receptor (AR). The AR is activated by binding of dihydrotestosterone, which facilitates formation of dimers that are subsequently relocated to the nucleus ²². Nuclear AR is a transcription factor that binds androgen response elements in the promoter region of PSA and other target genes ²³. Increased PSA serum levels correlate to an extent with aggressiveness of the cancer and unfavorable outcome for PCa patients ^{20, 21, 24}. Localized (androgen-dependent) PCa is treated by combining androgen ablation and radiation therapies which initially successfully reduces tumor growth and progression. However, androgen ablation-resistant disease eventually develops, where cancer cells have adapted to low androgen levels and have elevated invasive capacity. This poses a major clinical challenge to reduce high lethality rates from metastatic tumors and cancer recurrence after prostatectomy ²⁵.

In this study, the effects of V-ATPase inhibitors on PCa invasion and PSA biology were investigated. We used the LNCaP → C4-2B cell model of PCa progression that mimics the advancing disease from androgen-dependency to androgen ablation-resistance ²⁶. We showed that PSA is down-regulated (reduced mRNA and secretion) by inhibiting V-ATPases in the LNCaP cells regardless of the presence of R1881, suggesting that V-ATPase inhibitors interfered with the AR-PSA axis. Unlike the LNCaP cells, PSA expression and secretion were not altered in the androgen-independent C4-2B cells in spite of a dramatic reduction of the in vitro invasion of both cell types. Both cell types show an extensive distribution of intracellular V-ATPase pumps, but a distinctive distribution at the plasma membrane. Plasma membrane V-ATPases were abundant in the C4-2B cells, which are also more susceptible to V-ATPase inhibitors. Together these findings make V-ATPase pumps attractive targets against early and advanced PCa tumors. Combined with other therapies, V-ATPase inhibitors could help prevent transformation into the castration-resistant phenotype.

MATERIALS AND METHODS

Cell culture

LNCaP and PC-3 cells (both from ATCC, Manassas, VA, USA) and C4-2B cells (kind gift from Prof. Dr. George N. Thalmann) were cultured in T-medium (DMEM, Sigma Aldrich, St. Louis, MO, USA; 20% F12 nutrient mixture, 5 µg/mL insulin, 25 µg/mL adenine hydrochloride, 10 µg/mL transferrin, 0.25 µg/mL biotin, 15 pg/mL trijodothyronine, 100 U/mL penicillin/streptomycin) supplemented with 10% FBS. Cells were maintained at 37°C and 5% CO₂ in a humidified atmosphere. Media was routinely changed every 2–3 days, and cells passaged at 80–90% confluency.

Antibody generation

The polyclonal antibody to V-ATPase was developed against the peptide N-I¹⁶²KHKIMLPPRNRGT¹⁷⁵-C of the subunit V₁A by BioGenes (Berlin, Germany). Single peptides were used for the immunization of 2 rabbits over 35 days. The animals were intramuscularly immunized using BioGenes' adjuvant mixed 2:1 with the V₁A antigen. Parts of the sera were affinity purified against the peptide that was used for immunization. Antibodies were tested for specificity by performing BLAST alignment searches and by Western blotting and immunocytochemistry experiments.

Immunocytochemistry

Cells were fixed with 4% paraformaldehyde for 10 min at RT and cell membranes permeabilized with 0.02% TritonX-100 in PBS. Cells were blocked with 5% GS-PBS for 30 min at RT. Incubation with primary antibodies was performed at 1:100 dilution in 5% GS-PBS for 1 h at RT (LAMP-1, LAMP-2, clathrin, Na⁺K⁺-ATPase, Giantin antibodies: Abcam, Cambridge, MA, USA; AR and transferrin receptor/TR antibodies: Invitrogen). Cells were washed with PBS and incubated for 30 min with the secondary fluorescent antibodies (AF488 and AF546, Invitrogen; 1:500 in 5% GS-PBS). Cells were washed with PBS and mounted onto microscope slides in mounting media. Primary and secondary antibody controls were included for all immunostaining experiments. For acridine orange staining, cells were incubated for 30 min at 37°C with 1 µM acridine orange diluted in cell culture medium and fixed with paraformaldehyde as described above. Slides were analyzed with the Zeiss LSM510 confocal system.Line profiles of fluorescent intensity were obtained using ZEN 2009 Light Edition © Carl Zeiss MicroImaging software.

Plasma membrane isolation

Plasma membrane fractions were obtained by Percoll density gradient centrifugation as described before ²⁷.

RNA isolation and cDNA synthesis

RNA was isolated from cells grown in multiwell plates with the RNeasy Mini kit (Qiagen, Germantown, MD, USA), following the manufacturer’s instructions using the QIAshredder (Qiagen) to homogenize cell lysates. Complimentary DNA was synthesized from 2,000 ng RNA with the RETROscript^® cDNA kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions.

Quantitative realtime PCR (qRT-PCR)

Specific primers were designed against V-ATPase subunits, PSA, and TATA-binding protein (TBP) as an internal standard; primer sequences are listed in Table S1. The SYBR Green I Mastermix (Roche, Indianapolis, IN, USA) was used for the following PCR conditions: denaturation at 95°C for 10 min, followed by 40 cycles of: 30 s at 95°C, 30 s at 65°C, and 20 s at 72°C. qRT-PCR was performed on the Roche LightCycler480 instrument.

Western blotting

Whole cell lysates were obtained by incubating cells for 10 min on ice with lysis buffer (25 mM Tris, 8 mM MgCl₂, 1 mM DTT, 15% glycerol, 1% TritonX-100, protease inhibitor cocktail). Insoluble cell debris was removed by centrifugation of lysates at 13,000 rpm for 10 min at 4°C. The protein concentration was determined by bicinchoninic acid assay (Pierce, Rockford, IL, USA) against a BSA standard curve. 80 µg total protein were analyzed by SDS-PAGE and immunoblotted with primary antibodies overnight, followed by incubation and detection with secondary HRP-conjugated antibodies (Invitrogen). Densitometry quantification of western blot bands was performed using ImageJ software.

Cell treatments and PSA secretion

Cells were incubated for 24 h with culture media containing 5% charcoal-treated FBS to deplete cells of androgens and then incubated for 24 h or 48 h with 10 nM bafilomycin A (Baf.A), 10 nM R1881, or both, in T-media supplemented with 1% charcoal-treated FBS. RNA isolation, RT-PCR, Western blotting and immunostaining were then performed as described above. PSA secreted into the cell culture media was quantified using an ELISA kallikrein 3/PSA kit (R&D Biosystems) according to the manufacturer’s instructions.

Invasion and proliferation assays

Invasion Matrigel invasion assays were performed according to the manufacture’s instructions (BD Biosciences, San Jose, CA, USA). LNCaP and C4-2B cells were seeded for ~40 hours before treatment. Cells in invasion chambers were treated for 24 h with 10 nM Baf.A, then excess cells were removed with a cotton swab from the top of the chamber, invaded cells were fixed in methanol for 2 min and stained with Toluidine Blue for 2 min. Inserts were excised, mounted in immersion oil, and counted under the microscope. Proliferation. LNCaP and C4-2B cells were treated with 10 nM Baf.A or Conc.A (24 h or 48 h) and cell proliferation measured with the MTT cell proliferation assay kit (ATCC) by reading absorbance at 570 nm.

Statistical analysis

Student’s t-tests were performed to determine statistically significant differences in V-ATPase subunit mRNA expression levels between cell lines, and in mRNA and secretion levels after treatments, using GraphPad Prism 4 software.

RESULTS

Two PCa epithelial cell models with different invasiveness potential were compared to study the effects of V-ATPase inhibitors on PCa cell invasion and PSA biology. We measured the in vitro invasion of the poorly invasive, LNCaP, and highly invasive, C4-2B, human PCa cells treated with bafilomycin A (Baf.A) and Concanamycin A (Conc.A), two highly potent (IC₅₀ 1–10 nM) and specific V-ATPase inhibitors ^{28, 29}. V-ATPase inhibitors are known to reduce the in vitro invasion of highly invasive human breast cancer cells, which express V-ATPases at the cell surface, but no the invasion of the poorly invasive cell lines, which do not express plasmmalemal V-ATPases ¹¹.

The V-ATPase inhibitors prevented in vitro invasion of both human PCa cells, LNCaP and C4-2B, by about 80 % (Fig. 1B,D) under conditions where proliferation was not compromised (10 nM Baf.A or Conc.A for 24 hours) (Fig. 1A,C). However, after 48 hours of treatment, proliferation of the highly invasive C4-2B cells was significantly reduced (Fig. 1A,C). Suppression of the in vitro invasion of both PCa cells could be explained if the poorly invasive and highly invasive cells express V-ATPases at the cell surface, as the hallmark of V-ATPase pumps in cancer biology is their specialized role at the plasma membrane where V-ATPases facilitate extracellular acidification and invasion ^{5, 9, 11, 12, 30, 31}.

Figure 1. Effect of V-ATPase inhibitors on cell proliferation and in vitro invasion of PCa cells.

Baf.A and Conc.A prevents invasion of LNCaP and C4-2B cells. Poorly invasive LNCaP (A, B) and highly invasive C4-2B (C, D) cells were treated with Baf.A or Conc.A (10 nM) for 24 hours or 48 hours. Proliferation (A, C) was measured with the MTT cell proliferation assay and invasion (B, D) with a Matrigel invasion assay. N.S., not significant; *, p<0.05; **, p<0.01; significance levels relative to untreated control.

We made an antibody against the V-ATPase subunit V₁A to study V-ATPase cellular distribution. Subunit V₁A does not have isoforms and is therefore a component of every V-ATPase complex assembled in the cell. Western blots of whole cell lysates revealed one major band that migrated at about 70-kDa, the molecular mass of subunit V₁A (Fig. 2A) ^{2, 7}. Treatment with the peptide antigen competed labeling, validating the antibody’s specificity for subunit V₁A. Plasma membrane fractions isolated from the poorly invasive LNCaP cells had only trace amounts of V-ATPase subunit V₁A, and V₁A was not detectable at the plasma membrane by immunofluorescence in these cells (Fig. 2C). Immunoblots showed V₁A present at the plasma membrane of the highly invasive C4-2B cells (Fig. 2B) but at lower levels than the plasma membrane protein Na⁺K⁺-ATPase. Thus, like other cancer cells, expression of V-ATPase pumps at the plasma membrane correlates with an increased invasive phenotype in PCa cells.

Figure 2. Cellular distribution of V-ATPase in PCa cells.

(A) Peptide antibody specificity for subunit V₁A. Western blot of whole cell lysates from the LNCaP cells were probed with anti-V₁A antibody in the presence and absence of its peptide antigen (10 µM). The same membrane was probed with β-actin (loading control). Antibody (6 ug) and peptide (60ug) were incubated 24 hours at 4°C (wells 4 &5) or 2 hours at RT (wells 5 &6). Lanes 1, 3, and 5 (20 µg protein); Lanes 2, 4, and 6 (50 µg protein). (B) Plasma membrane fractions from LNCaP and C4-2B cells. The same gel was proved with the plasma membrane protein Na⁺K⁺-ATPase. WCL=whole cell lysate; PM=plasma membrane. (C) V-ATPase is present in endosomal and lysosomal membranes of LNCaP cells. Cells were co-immunostained with antibodies against the V-ATPase subunit V₁A and the indicated marker proteins of the endocytic recycling pathway (transferring receptor, clathrin), secretory pathway (clathrin), lysosomes (LAMP-1), and plasma membrane (Na,K-ATPase). Proteins were labeled with AlexaFluor secondary antibodies, and co-localization analyzed using confocal microscopy determining a line profile of fluorescent intensity. (D) V-ATPase and PSA co-localize in the Golgi compartment of PCa cells. LNCaP and C-4-2B cells were co-immunostained with anti-V₁A, Golgi protein giantin, and PSA as described above. Scale bar =10 µm.

Suppression of invasion after treating LNCaP cells with Baf.A and Conc.A considering that have elfin amounts of plasmalemmal V-ATPases (Fig. 2B) suggests novel roles for intracellular V-ATPases that can influence the invasive phenotype of androgen-dependent PCa cells. Co-immunostaining with the anti-V₁A antibody and antibodies to subcellular markers showed the intracellular V-ATPase pumps distributed throughout the endomembrane system of LNCaP (Fig. 2C) and C4-2B cells (Fig. S1). V-ATPase signal was predominant in the Golgi compartment (Fig. 2D); the Golgi tether protein giantin completely co-localized with V-ATPase in both cell lines. PSA was abundant in the Golgi compartment and notably, we showed that PSA completely to co-localize with Golgi V-ATPase (Fig. 2D). V-ATPase in the Golgi and peri-Golgi regions was positive also for endosomal proteins such as clathrin and tranferrin receptor, and partly overlapped with the lysosomal marker LAMP-1 (Fig. 2C).

V-ATPase function is necessary for protein trafficking across endocytic and secretory pathways ^{6, 32}; thus, we anticipated PSA trafficking from the Golgi and its secretion to be V-ATPase dependent. Because PSA expression and secretion are induced by androgen ²², we measured PSA secretion from the androgen-dependent LNCaP cells after 24 and 48 hours exposure to R1881, a dihydrotestosterone analogue, which stimulates androgen receptor-mediated PSA secretion; and after exposure to Baf.A to block V-ATPase function. As expected, secretion of PSA was stimulated by a saturating concentration of R1881 (10 nM) (Fig. 3A), and both basal and R1881-induced PSA secretion were decreased in the presence of Baf.A.

Figure 3. Effect of V-ATPase inhibitors on R1881-induced PSA secretion and cellular distribution.

(A) Baf.A treatment reduces PSA secretion. The LNCaP cells were incubated with 10 nM Baf.A in the presence and absence of 10 nM R1881 (24 or 48 hours) or with 0.01% DMSO (Control). The PSA secreted was quantified by ELISA and normalized to cell number. (B) Baf.A treatment disturbs PSA recruitment in the Golgi. The LNCaP cells treated as described in A were immunostained with antibodies to V₁A and PSA and analyzed as described for Figure 2C. ***, p<0.001; **, p<0.01; significance levels relative to controls.

In addition to suppressing PSA secretion, V-ATPase inhibitors disturbed PSA recruitment in the Golgi compartment. Treatment with Baf.A (Figs. 3B, 4A,B) or Conc.A (Fig. S2) for 16 hours and up to 48 hours excluded PSA from the Golgi. PSA became dispersed in discrete vesicles throughout the cell, whereas V-ATPase remained in the Golgi (Fig.S3). This response was observed in the LNCaP and C2-4B cells alike (Fig. 4A,B), and therefore was independent of the invasive potential of the PCa cells. Removal of Baf.A from the media restored compartmentalization of PSA with V-ATPase in the Golgi of the LNCaP cells (Fig. 4A), in line with the notion that PSA recruitment into the Golgi required active intracellular V-ATPases. In the highly invasive C4-2B cells, reversibility was detected at 16 hours (Fig. 4B), but was impaired after 48 hours incubation with the V-ATPase inhibitor. Susceptibility of the C4-2B cells to Baf.A and Conc.A treatments was also evident in the proliferation assays after 48 hours (Fig. 1B).

Figure 4. Formation of lysosomal PSA-loaded vesicles by treatment with inhibitors of V-ATPase pumps.

(A) PSA vesicle formation is reversible in the LNCaP cells (16 – 48 hour), (B) but not in the C4-2B cells (48 hours) treated with Baf.A. The LNCaP (A, C) and C4-2B (B) cells were incubated with 10 nM Baf.A (16, 24, or 48 hours) or with 0.01% DMSO (Control). The V-ATPase inhibitor was removed by incubation in normal growth media for an additional 24 hours (Wash) to restore PSA compartmentalization with V-ATPase in the Golgi. (C) PSA-containing vesicles have lysosomal composition. The LNCaP cells were treated with 10 nM Baf.A (24 hours) or with 0.01% DMSO (Control). (A-C) Cells were immunostained with antibodies to V₁A, PSA, LAMP1, LAMP2, or LC-3 and analyzed as described for Figure 2C. Scale bar =10 µm.

The dispersion of PSA was detected after administrating Baf.A alone or in combination with R1881 (Fig. 3B). R1881 alone did not disturb PSA compartmentalization in the Golgi regardless that R1881 enhanced PSA secretion, indicating that Baf.A-induced formation of PSA-containing vesicles differed from normal PSA secretory events. Treatment with V-ATPase inhibitors likely targeted PSA for lysosomal degradation via autophagy-independent processes, because: 1) vesicles containing PSA were positive for the lysosomal markers LAMP1 and LAMP2 (Fig. 4C); 2) the autophagic LC-3-positive vesicles were observed as distinct punctae (Fig. 4C); 3) the ratio LC-3I/LC3II was unchanged, as assessed by Western blots (not shown); and 4) PSA-containing vesicles did not form under conditions that block autophagy (100 nM Baf.A, 2 hours) (Fig. S4) ³³.

The fact that V₁A is absent in the lysosomal PSA-containing vesicles suggests that the organelles were not acidified and degradation events impaired. Accordingly, V-ATPase inhibitors prevented vesicle staining with acridine-orange, therefore supporting the idea that endosomal and lysosomal acidification are defective (Fig. 5A). In addition, transferrin receptor recycling to the cell surface was blocked, as indicated by its retention in the Golgi with V-ATPase pumps in the Baf.A-treated cells (Fig. 5B), suggesting that cell-surface transport is disrupted upon V-ATPase inhibition. Finally, total intracellular PSA was significantly greater when R1881 was added in the presence of Baf. A (Fig. 5C), suggesting that PSA is not degraded when V-ATPase function is inhibited.

Figure 5. Effect of V-ATPase inhibitors on endosomal acification and trafficking.

(A) V-ATPase inhibitors disturb endosomal and lysosomal acidification. Cells were treated with 10 mnM Baf.A or Conc.A for 24 h, incubated with 1 µM acridine orange, and analyzed by confocal microscopy. (B) Transferrin Receptor recycling to the plasma membrane is reduced by Baf. A. LNCaP treated with 10 nM Baf.A or 0.01% DMSO (Control) for 24 hours were analyzed as described for Figure 2C. (C) Baf.A treatment cause intracellular accumulation of PSA. Cells were treated with R1881 and/or Baf.A as described for Figure 3A, whole cell lysates analyzed by immunoblots using antibodies against PSA and β-actin (loading control), and relative PSA protein levels quantified with Image J software (n=3–4). Scale bar =10 µm. **, p<0.01; significance levels relative to controls.

We measured PSA transcripts levels by qRT-PCR and determined that basal PSA was down-regulated at the transcriptional level in the poorly invasive androgen-dependent LNCaP cells treated with Baf.A. PSA mRNA decreased by 1.8- and 2.6-fold at 24 and 48 hours incubations with Baf.A (Fig. 6A). In addition to lowering PSA at steady-state, Baf.A dramatically suppressed R1881-induction of PSA, suggesting that V-ATPase inhibition interferes with AR transcriptional activity. A more modest decreased of AR protein was observed in the LNCaP cells after treatment with Baf.A for 48 hours in the presence (induced state) and absence (steady state) of R1881 (Fig.6B). PSA secretion, PSA expression, and AR protein were not reduced in the C4-2B cells by Baf.A after 24 hours (Fig. 6C), when invasion significantly declined (Fig. 1D). These results attribute increasingly important roles to plasma membrane-associated V-ATPase pumps in the highly invasive PCa cells, whereas intracellular V-ATPase pumps appear to be primarily responsible for defective PSA expression and secretion and reduction of invasion triggered by Baf.A and Conc.A in the LNCaP cells.

Figure 6. V-ATPase inhibition interfere with the AR-PSA axis in LNCaP, but not in the C4-2B cells.

(A) Baf.A treatment reduces PSA mRNA expression in LNCaP cells. Cells were treated with 10 nM Baf.A and/or R1881 and PSA mRNA was quantified by qRT-PCR using specific primers (Table S1) and expressed as a ratio relative to the internal control TATA-binding protein (TBP) (B) Baf.A treatment lower AR protein levels in the LNCaP cells. Immunoblots of whole cell lysates were analyzed using antibodies to the AR and β-actin (loading control) and relative AR protein quantified as described for Figure 5C. (C) PSA secretion, PSA expression (mRNA, protein), and AR protein levels are not altered by Baf.A in the C4-2B cells. Cells were treated with Baf.A and analyzed as described above for B (AR protein), A (PSA mRNA), and for Figures 5C (PSA protein) and 2A (Secreted PSA). Relative protein levels were quantified with Image J software (n=3–4). ***, p<0.001; significance levels relative to controls.

DISCUSSION

This study revealed important new roles for V-ATPase pumps in PCa cells. Pharmacological inactivation of V-ATPase interferes with the AR-PSA axis. It reduces PSA secretion and PSA mRNA expression in the androgen-dependent LNCaP cells. Together with a dramatic reduction (by 80%) of the in vitro invasion of LNCaP (poorly invasive) and C4-2B (highly invasive) cells, these findings make V-ATPase pumps attractive new targets against early and advanced PCa tumors. In this study, we used Baf.A at its IC₅₀ concentration (10 nM) to avoid any secondary and unspecific effects ³⁴ and reproduced our studies using Conc.A, another V-ATPase inhibitor for which V-ATPase is the only known target, arguing against a general toxic effect independent of V-ATPase inhibition.

The link of plasma membrane-associated V-ATPases and invasion of high-metastatic cells has been broadly disseminated ^{5, 9, 11–13}. Our findings indicate that intracellular V-ATPases also contribute to the invasive phenotype of PCa cells because the low-metastatic androgen-dependent LNCaP cells have only traces of V-ATPase pumps at the cell surface and excessive intracellular V-ATPase pumps. It has been shown that Golgi associated intracellular V-ATPases are necessary for Golgi functions ³⁵ and that V-ATPases in endosomal and lysosomal compartments support protein trafficking and degradation in all eukaryotic cells ^{2, 4, 5}, ⁶. In this context, PCa cells are not different. V-ATPase inhibitors decrease PSA secretion and recycling of the transferrin receptor to the plasma membrane. They also lead to accumulation of PSA-loaded vesicles of lysosomal nature (LAMP-1 and LAMP-2 positive), which could be degradative or secretory lysosomes. Since the catalytic subunit of the V-ATPase complex (V₁A) is absent from the PSA-loaded vesicles, we expected luminal acidification to be impaired. A defective endosomal and lysosomal acidification can help explain the membrane trafficking defects; and it is supported by absence of acridine orange staining in Baf.A and Con.A treated cells.

The scope of V-ATPase in PCa physiology extends beyond the boundaries of the organelles where they are present. V-ATPase inhibitors suppress androgen responses such as R1881 induction of PSA mRNA expression in the androgen-dependent LNCaP cells, but not in the C4-2B cells that have developed androgen resistance. One explanation to how V-ATPase inhibitors reduce PSA mRNA is that by excluding PSA from the Golgi and redistributing it into the lysosomal vesicles, V-ATPase inhibitors activate a negative feed-back loop and PSA expression is decreased. Time course analysis of PSA mRNA expression levels (not shown) support this model. PSA vesicle formation is detected at least at 16 hours treatment and precedes PSA mRNA down-regulation, which is detected only after 24 hours. It has been shown that V-ATPase inhibition lowers the cytosolic pH ¹¹. An aberrant cytosolic pH can interfere with the AR-PSA axis, and we will expect other cellular processes to be defective too. Any cytosolic pH defect will be more drastic in the C4-2B cells where the plasma membrane-associated V-ATPase pumps are more abundant.

The isogenic nature of the LNCaP and C4-2B cell lines and their resemblance to androgen-ablation resistant progressive disease ²⁶ allow us to compare cellular events associated with V-ATPase function in progressively invasive PCa cells. Our results revealed an increasing importance of V-ATPase in the metastatic C4-2B PCa cells. The C4-2B cells express significantly more V-ATPase pumps at the cell surface and many V_o subunit isoforms are upregulated (Table S2) relative to LNCaP (up to 9-fold) and to non-cancerous (up to 60-fold) prostate cells. Additionally, V-ATPase inhibitors suppress proliferation in the C4-2B cells (48 hours incubation) and Baf.A-induced responses become irreversible over time.

Future work will focus on identifying the V-ATPase subunit isoforms directly involved in PCa invasion, and suppression of PSA expression and secretion to selectively target prostate tumors in vivo. Baf.A and Conc.A do not discriminate between V-ATPase isoforms ^{28, 29}, although the V-ATPase complex comprises multiple subunit isoforms which combine to yield tissue- and organelle-specific V-ATPase pumps ⁶, ⁷. Our attempts at siRNA-mediated knockdown V₁A resulted in lower mRNA expression (up to 70%) but stable V₁A protein expression (not shown). Obviously, knockdown of subunit isoforms of large multisubunit complexes such as V-ATPase is not trivial; targeting of multiple subunits simultaneously may be necessary to reach inactivation levels comparable to the V-ATPase inhibitors.

The AR in LNCaP cells carries the mutation T877A in the ligand-binding domain ^{36, 37} which is equally distributed in those patients who have received hydroxyflutamide or bicalutamide therapy ³⁸. This clinically relevant mutation prevents a blockade of receptor function by most anti-androgen therapies in LNCaP cells ^{36, 37}. Therefore, V-ATPase inhibitors may result in important clinical benefits for patients expressing anti-androgen resistant mutations such as T877A. In the long-term, responsiveness of tumor cells to V-ATPase inhibitors might be used to control and prevent prostate tumor metastases. V-ATPase inhibitors may also help distinguish androgen-dependent and -independent tumor cells, as PSA secretion and mRNA expression are reduced only in the androgen-dependent cells (LNCaP cells). Combined with other therapies, V-ATPase inhibitors could additionally help prevent transformation into the castration-resistant phenotype.

Novelty and relation to the state of the field.

Down-regulation of PSA mRNA by V-ATPase proton pump inhibitors is novel. Reduction of the in vitro invasion of LNCaP and C4-2B cells alike by V-ATPase inhibitors, regardless that V-ATPase is prominent at the plasma membrane of C4-2B cells and only traces are detected in the low-metastatic LNCaP parental cells, is a new concept in the V-ATPase field. These findings make V-ATPase attractive new targets against early and advanced PCa tumors. They could pave the way for the investigation of new cellular processes that intertwine V-ATPase and intracellular pH homeostasis with the AR-PSA axis.

Abbreviations

AR

androgen receptor

Baf.A

bafilomycin A

Conc.A

concanamycin A

LAMP-1

lysosomal-associated membrane protein 1

LAMP-2

lysosomal-associated membrane protein 2

PCa

prostate cancer

PSA

prostate-specific antigen

TBP

TATA-binding protein

V-ATPase

vacuolar adenosine triphosphatase