Metastatic invasion is the major cause of cancer-related deaths. In this study, we introduce two-pore channels (TPC), a recently described class of NAADP- and PI(3,5)P2–sensitive Ca2+-permeable cation channels in the endolysosomal system of cells, as candidate targets for the treatment of invasive cancers. Inhibition of the channel abrogated migration of metastatic cancer cells in vitro. Silencing or pharmacologic inhibition of the two-pore channel TPC2 reduced lung metastasis of mammary mouse cancer cells. Disrupting TPC function halted trafficking of β1-integrin, leading to its accumulation in EEA1-positive early endosomes. As a consequence, invasive cancer cells were no longer able to form leading edges, which are required for adequate migration. Our findings link TPC to cancer cell migration and provide a preclinical proof of concept for their candidacy as targets to treat metastatic cancers. Cancer Res; 77(6); 1427–38. ©2017 AACR.

Metastasis is the major cause of cancer-related deaths. The formation of secondary, metastatic growth includes proliferation and extensive vascularization of the primary tumor, detachment and invasion of tumor cells, circulation of single tumor cells, arrest in distant sites, extravasation, and, finally, proliferation within the organ (1). Extensive studies in the last decades contributed to a better understanding of these processes. However, still about 90% of cancer-associated mortality is due to metastasis. Therefore, new strategies to prevent metastasis are urgently needed (1, 2).

To successfully colonize secondary sites, cancer cells gained the ability to migrate (2, 3). A crucial step in migration is the binding to ligands of the extracellular matrix (ECM), which is mediated by integrins. These cell surface receptors display a diverse family of glycoproteins consisting of 18 α-subunits and 8 β-subunits. Besides mediating cell attachment to the ECM, integrins are linked to the cytoskeleton through the formation of clusters with actin-associated proteins such as vinculin. Furthermore, ligation of integrins induces a network of intracellular signaling pathways including the activation of focal adhesion kinase (FAK) and Src, altogether regulating cell migration (3).

To fulfill the function as migration-promoting receptors, integrins have to be trafficked to the front of the cell for assembly of focal adhesions. These adhesions act as traction sites for the movement and are afterwards disassembled at the rear of the cell for detachment. Hence, integrins need to be dynamically turned over, which is achieved by endocytic trafficking (4, 5). After endocytosis, integrins are carried to early endosomes where sorting into recycling and degradative pathways takes place. Under normal conditions, the larger portion recycles back to the plasma membrane. Disturbing this process can alter the composition of focal adhesions, thereby influencing cell migration (4).

There is increasing evidence that two-pore channels (TPC) are key players in the regulation of endocytic transport (6–8). It has been postulated that inhibiting these channels alters Ca2+ signaling during endolysosomal fusion events, resulting in defects in vesicle trafficking (9–11). TPCs are assigned to the superfamily of voltage-gated ion channels (8). There are two different TPC subtypes present in primates, TPC1 and TPC2. TPC1 is primarily expressed on endosomal membranes while TPC2 dominates on lysosomal membranes (8, 10, 12). Activation of these channels is presumably triggered by the second messenger nicotinic acid adenine dinucleotide phosphate (NAADP) and by phosphatidylinositol 3,5-bisphosphate [PI(3,5)P2; refs. 8, 13–15]. Distinct experiments suggested TPCs as Ca2+-permeable channels indicating that TPCs might be involved in Ca2+ release from endo- and lysosomal stores (7–9, 13, 16).

This work unveils that TPCs play a crucial role in the formation of metastasis as silencing TPC1 and TPC2 reduced the adhesion and migration of invasive tumor cells in vitro. Similar results were achieved with Ned-19, an antagonist of NAADP (17), and tetrandrine, recently shown to inhibit TPC1 and TPC2 (18). Remarkably, both silencing of TPC2 with siRNA or by pharmacologic inhibition showed efficacy in a metastatic in vivo model. Inhibition of TPCs led to the accumulation of integrins in endocytic vesicles and impaired formation of leading edges, indicating that TPCs are significantly involved in integrin recycling.

Cell lines and reagents

The cancer cell line T24 was obtained from Dr. B. Mayer (Surgical Clinic, LMU, Munich, Germany) and recently authenticated by the DSMZ. HUH7 were purchased from Japanese Collection of Research Bioresources and 4T1-Luc (4T1) from PerkinElmer. Method of cell authentication was short tandem repeat (STR) DNA typing by using 8 different and highly polymorphic STR loci. T24 cells were grown in McCoy, HUH7 cells in DMEM high glucose, and 4T1 cells in RPMI1640 medium. All media were supplemented with 10% FCS. trans-Ned-19 (Ned-19) was purchased from Tocris Bioscience, and tetrandrine from Santa Cruz Biotechnology.

qRT-PCR

qRT-PCR of TPC1 and TPC2 was performed as described previously (19). Following primers were used: TPC2 forward (fw): 5′-GTA CCC CTC TTG TGT GGA CG-3; reverse (rv), 5′-GGC CCT GAC AGT GAC AAC TT-3′; TPC1 fw, 5′-GGA GCC CTT CTA TTT CAT CGT-3′; rv, 5′-CGG TAG CGC TCC TTC AAC T-3′; actin was used as housekeeping gene. The following primers were used: fw, 5′-CCA ACC GCG AGA AGA TGA-3′; rv, 5′-CCA GAG GCG TAC AGG GAT AG-3′.

Whole-endolysosomal patch clamp

Whole-endolysosomal patch-clamp recordings were performed by modified conventional patch-clamp. T24 cells were treated with 1 μmol/L vacuolin-1 for at least 4 hours. Currents were recorded using an EPC-10 patch-clamp amplifier and PatchMaster acquisition software (HEKA). Data were digitized at 40 kHz and filtered at 2.8 kHz. Recording pipettes had a resistance of 8–10 MΩ. Liquid junction potential was corrected. Pipette solution (corresponding to luminal endolysosomal solution) contained 140 mmol/L sodium methanesulfonate, 5 mmol/L potassium methanesulfonate (KMSA), 2 mmol/L calcium methanesulfonate, 1 mmol/L CaCl2, 10 mmol/L HEPES and 10 mmol/L MES, pH 4.6. Bath solution (corresponding to cytosolic solution) contained 140 mmol/L KMSA, 5 mmol/L KOH, 4 mmol/L NaCl, 0.39 mmol/L CaCl2, 1 mmol/L EGTA, and 10 mmol/L HEPES, pH 7.2. PI(3,5)P2 was used in a water-soluble diC8 form (A.G. Scientific). All compounds including PI(3,5)P2 were prepared as high-concentration stock solutions, added to the bath solutions to match the final concentration indicated. pH of bath solution and pipette solution were adjusted with KOH and MSA, respectively. All recordings were obtained at 21°C–23 °C and were analyzed using PatchMaster and Origin 6.1 (OriginLab) software.

Tissue staining

Two-micron thick tissue sections were used. Antigen retrieval was done with Target Retrieval Solution from Dako and the sections were heated for 30 minutes in the microwave. Blocking of endogenous peroxidase was done in 7.5% hydrogen peroxide for 10 minutes. Anti-TPC2 (atlas antibodies, diluted 1:60) was applied as primary antibody for 1 hour at room temperature. An unspecific control antibody (rabbit IgG isotype, diluted 1:60) was used under the same conditions as for anti-TPC2 to detect any background staining of the primary antibody. For antibody detection, ImmPress reagent anti-rabbit Ig was utilized according to the manual and DAB+ for 3 minutes at room temperature was used as a chromogen. Slides were counterstained with hematoxylin for 20 seconds and finally embedded in mounting medium and covered with glass coverslips.

Cell transfection

A total of 2 × 105 T24 cells were transfected using the ScreenFect A Transfection Kit (Genaxxon bioscience). TPC1 and TPC2 were silenced using siRNA from Santa Cruz Biotechnology. Nontargeting siRNA was used as a control.

Boyden chamber assay

A total of 1 × 105 treated or silenced cells were placed on top of the Transwell chamber (Corning) in media without FCS. Twenty-four–well plate was filled with medium with 10% FCS. Transwell chambers were placed in it and incubated for 16 hours. Migrated cells were fixed and stained with crystal violet/methanol. The top of the Transwell chamber was cleaned and pictures were taken. Migrated cells were counted with ImageJ (NIH, Bethesda, MD).

Endolysosomal preparation

Isolation of endolysosomal proteins was conducted as described before (20). In short, three 6-well plates of T24 cells transfected with siTPC1, siTPC2, or siNT were washed with PBS. Five-hundred microliters of homogenization buffer was added and the cells were detached with a cell scraper. Homogenization was performed with a Potter S Homogenizer (B. Braun) at 12 × 900 rpm. Homogenized cells were collected into a microcentrifuge tube and were centrifuged at 14,000 × g, 4°C for 15 minutes. Supernatant was collected into a 1.5-mL ultracentrifuge tube (Beckman), one volume of 16 mmol/L CaCl2 was added and the preparation was mixed by shaking at 4°C for 5 minutes. Ultracentrifugation was performed at 18,000 × g, 4°C for 15 minutes. Supernatant was removed and the formed pellet was resuspended in 250 μL wash buffer. After ultracentrifugation at 25,000 ×g, 4°C for 15 minutes, supernatant was removed and pellet was resuspended in 50 μL wash buffer. Protein concentration was determined and 20 μg of endolysosomal preparation was used for Western blot analysis.

Adhesion assay

A total of 5 × 104 silenced or stimulated T24 cells were seeded onto μ-slides 8-well ibidiTreat and allowed to attach for 1 hour. Subsequently, cells were fixed with 4% paraformaldehyde (PFA) and stained with rhodamine-phalloidin (Molecular Probes) and Hoechst 33342 (Hoechst; Sigma) for 30 minutes. Cells were mounted with FluorSave Reagent mounting medium (Merck) and analyzed with a Zeiss LSM 510 Meta confocal microscope (Jena).

Chemotaxis

A total of 6 × 104 T24 cells were seeded on μ-Slide Chemotaxis3D (IBIDI) and allowed to attach for 3 hours. Chemotaxis experiment (20 hours) was performed as described previously (21). Cell tracking was performed with ImageJ plugin Manual tracking (Fabrice Cordelières, Institut Curie, Orsay, France). Thirty cells were tracked per experiment. Data analysis was done with Chemotaxis and Migration Tool (IBIDI). Rayleigh test was used to describe uniformity of distribution of cell endpoints. With P > 0.05, uniformity is rejected (22).

Measurement of cell death

Flow cytometry analysis was performed as described previously (19). Specific cell death was calculated as follows: specific cell death = [(x – Co) / (100 – Co)] × 100

Membrane staining

A total of 2 × 104 HUH7 cells were seeded onto μ-slides 8-well ibidiTreat. For membrane labeling, the PKH 26 red fluorescent cell linker mini kit was used (Sigma). After silencing or pharmacologic treatment, cells were incubated with the dye according to the manufacturer's instructions for 2 minutes. After removal, cells were incubated with pure FCS for 1 minutes followed by an incubation of 2 hours in medium. Cells were then fixed with PFA and stained with Hoechst for 10 minutes. Washed cells were mounted in FluorSave Reagent mounting medium and the cells were analyzed by confocal microscopy.

Lysotracker staining

A total of 2 × 104 T24 cells were seeded onto μ-slides 8-well ibidiTreat and were silenced or stimulated. Thereafter, the cells were incubated for 30 minutes with 1 μmol/L Lysotracker Red DND-99 (Molecular Probes) and Hoechst in PBS. After removal, pictures were taken immediately with a Zeiss LSM 510 Meta confocal microscope.

β1-integrin internalization

A total of 1 × 104 T24 cells were seeded onto μ-slides 8-well ibidiTreat and were silenced or stimulated. Cells were starved for 90 minutes and stained with β1-integrin (TS2/16; Santa Cruz Biotechnology) in cold 0.01% BSA/DMEM without FCS for 45 minutes at 4°C. Washed cells were incubated with 0.01% BSA/DMEM with FCS for 1 hour at 37°C. Next, 1 μmol/L phorbol 12-myristate 13-acetate (Merck) in 0.01% BSA/DMEM with FCS was added for 30 minutes at 37°C. After washing and fixing cells with PFA, 0.1% Triton-X100 was added. Alexa Fluor 488-goat-anti-mouse (Molecular Probes) and Hoechst were used for 1 hour to stain cells. Washed cells were embedded in FluorSave Reagent mounting medium and analyzed by confocal microscopy.

Immunocytochemistry

A total of 1 × 104 T24 cells were seeded onto μ-slides 8-well ibidiTreat (IBIDI). Silenced or treated cells were fixed with PFA prior to permeabilization with 0.2% Triton X-100/PBS. Hereafter, unspecific binding sites were blocked with 1% BSA/PBS containing 0.1% Triton X-100. Antibodies were diluted in blocking solution. Used antibodies are given as follows: β1-integrin (TS2/16), EEA1, LAMP3 (Santa Cruz Biotechnology). Nuclei were stained with Hoechst. Cells were embedded in FluorSave Reagent mounting medium and analyzed by confocal microscopy.

Staining of migrating cells

A confluent T24 cell layer was scratched with a pipette tip, after silencing or treatment. Cells were allowed to migrate for 5 hours and subsequently fixed with PFA. Samples were stained as described under the section “Immunocytochemistry.” Antibodies given below were used: β1-integrin (TS2/16), vinculin (Santa Cruz Biotechnology), pFAKSr732, pSrcpY418 (Invitrogen), Alexa Fluor 680-goat-anti-rabbit and Alexa Fluor 643-goat-anti-mouse (Molecular Probes). Rhodamine-phalloidin was used to stain actin, Hoechst for nuclei.

Western blot analysis

Western blot analysis was performed as described previously (19). Following antibodies were used: TPC2 (Alomone), LAMP3, β1-integrin (TS2/16), FAK (Santa Cruz Biotechnology), pFAKSr732, pSrcpY418 (Molecular Probes), Src (L4A1; Cell Signaling Technology), actin (Millipore), HRP-goat-anti-rabbit (Bio-Rad), and HRP-goat-anti-mouse (Santa Cruz Biotechnology).

In vivo mouse model

4T1-Luc cells were pretreated with 150 μmol/L Ned-19, 10 μmol/L tetrandrine or DMSO for 24 hours. Alternatively, cells were silenced with siTPC2 or siNT for 72 hours. Female BALB/cOlaHsd mice (Envigo) were inoculated with 1 × 105 4T1-Luc cells via the tail vein. After five days, imaging of the mice was performed using the IVIS Lumina system with Living Image software 4.4 (Caliper Life Sciences) immediately after intraperitoneal injection of 6 mg luciferin (potassium salt, PerkinElmer). The total signal per defined region of interest was calculated as photons/second/cm2 (total flux/area). Animal experiments were approved by the District Government of Upper Bavaria in accordance with the German Animal Welfare and Institutional guidelines.

Statistical analysis

GraphPad Prism Software was used for all statistical analyses. In some experiments, data were normalized to control or as indicated.

TPCs in cancer cells

TPCs have been described in different cell types (9, 18, 23); however, not in cancer cells so far. TPC2 mRNA is distinctly expressed in T24 (bladder cancer), Jurkat (leukemia), and HUH7 (HCC) cells (Fig. 1A). mRNA levels of TPC1 were particularly high in T24 and HUH7 cells (Fig. 1B). Tumor tissue sections of HCC patients showed a highly positive staining for TPC2 (Fig. 1C). Together, these data suggest the presence of TPCs in cancer cells. To verify TPC functionality in T24 cancer cells, we isolated lysosomes and performed endolysosomal patch-clamp experiments. The applied whole-endolysosmal patch-clamp method is illustrated in Fig. 1D. PI(3,5)P2-elicited currents were strongly reduced after applying tetrandrine (Fig. 1E and F), recently shown to inhibit TPC1 and TPC2 (18).

Figure 1.

TPCs in cancer cells. A and B, mRNA levels of TPC2 and TPC1 in different cancer cell lines were assessed by qRT-PCR using the SYBR Green PCR Master Mix. Expression level of MDA-MB-231 cells was set at 1. Bars, SEM of three independent experiments performed in duplicate. C, Human liver sections (HCC and surrounding tissue) were stained with an antibody against TPC2 or with an unspecific isotype control antibody (Co-IgG). Counterstaining was done with hematoxylin. One representative specimen is shown. D, Cartoon illustrating whole-endolysosomal patch-clamp method. E, Representative current-voltage relation recorded from a vacuolin-enlarged endolysosomal vesicle manually isolated from cultured T24 cells. Shown are the effect of 1 μmol/L PI(3,5)P2 and the blocking effect of 500 nmol/L tetrandrine. F, Population data for current amplitudes at −100 mV obtained from experiments as shown in E. *, P < 0.05, Student t test; data are shown as mean ± SEM.

Figure 1.

TPCs in cancer cells. A and B, mRNA levels of TPC2 and TPC1 in different cancer cell lines were assessed by qRT-PCR using the SYBR Green PCR Master Mix. Expression level of MDA-MB-231 cells was set at 1. Bars, SEM of three independent experiments performed in duplicate. C, Human liver sections (HCC and surrounding tissue) were stained with an antibody against TPC2 or with an unspecific isotype control antibody (Co-IgG). Counterstaining was done with hematoxylin. One representative specimen is shown. D, Cartoon illustrating whole-endolysosomal patch-clamp method. E, Representative current-voltage relation recorded from a vacuolin-enlarged endolysosomal vesicle manually isolated from cultured T24 cells. Shown are the effect of 1 μmol/L PI(3,5)P2 and the blocking effect of 500 nmol/L tetrandrine. F, Population data for current amplitudes at −100 mV obtained from experiments as shown in E. *, P < 0.05, Student t test; data are shown as mean ± SEM.

Close modal

TPCs affect cancer cell migration and adhesion

Previous studies in TPC2-deficient MEF cells showed defects in the endolysosmal degradation pathway (9, 11). As alterations in this pathway through inhibition of another endolysosome-associated protein, the vacuolar ATPase (V-ATPase), have been described to reduce the ability of invasive cancer cells to migrate (21), we hypothesized that TPCs affect cancer cell migration. Silencing TPC1 and TPC2 by siRNA in T24 cells resulted in significantly reduced migration through pores in Boyden chamber assays (Fig. 2A). Knockdown efficiency is shown in Fig. 2B and C. Moreover, these cells showed diminished adhesion after 1 hour of seeding (Fig. 2D). To get more information about the migratory defect, movement of the silenced cells exposed to a diffusive gradient of FCS (0%–10%) was analyzed. Figure 2E shows control cells moving clearly toward the highest FCS concentrations, whereas knockdown cells displayed reduced directional migration, which is expressed as P value in the Rayleigh test. Not only directed migration but also cell mobility seems to be affected as velocity was significantly reduced in silenced cells. Thus, TPC1 and TPC2 seem to play a pivotal role in two major steps of metastasis formation: adhesion and migration.

Figure 2.

Silencing of TPC1 and TPC2 leads to reduced migration and adhesion in cancer cells. For all experiments, T24 cells were silenced with siRNA against TPC1 and TPC2 for 72 hours. A, siRNA-treated cells were allowed to migrate in Transwell chambers for 16 hours. B, qPCR analysis of TPC1 and TPC2 mRNA levels were assessed with the SYBR Green PCR Master Mix. Bars, SEM of three independent experiments performed in duplicate. C, Western blot analysis of TPC1 and TPC2 protein levels in endolysosomal preparations. LAMP3 served as loading control and marker for lysosomes. Bars, SEM of three independent experiments, *, P < 0.05 (t test). D, After silencing, cells were allowed to adhere for 1 hour, fixed, and stained (rhodamine-phalloidin, Hoechst). Cells were analyzed by confocal microscopy. Scale bars, 150 μm. Bars in A and D are the SEM of three independent experiments, *, P < 0.05 (one-way ANOVA, Dunnett multiple comparison test). E, Movement of silenced T24 cells along a FCS gradient was monitored (20 hours) by live cell imaging and analyzed for directed migration (P value) and velocity (ImageJ, IBIDI software). Dashed line, P = 0.05. Bars are the SEM of three independent experiments. *, P < 0.05 (t test).

Figure 2.

Silencing of TPC1 and TPC2 leads to reduced migration and adhesion in cancer cells. For all experiments, T24 cells were silenced with siRNA against TPC1 and TPC2 for 72 hours. A, siRNA-treated cells were allowed to migrate in Transwell chambers for 16 hours. B, qPCR analysis of TPC1 and TPC2 mRNA levels were assessed with the SYBR Green PCR Master Mix. Bars, SEM of three independent experiments performed in duplicate. C, Western blot analysis of TPC1 and TPC2 protein levels in endolysosomal preparations. LAMP3 served as loading control and marker for lysosomes. Bars, SEM of three independent experiments, *, P < 0.05 (t test). D, After silencing, cells were allowed to adhere for 1 hour, fixed, and stained (rhodamine-phalloidin, Hoechst). Cells were analyzed by confocal microscopy. Scale bars, 150 μm. Bars in A and D are the SEM of three independent experiments, *, P < 0.05 (one-way ANOVA, Dunnett multiple comparison test). E, Movement of silenced T24 cells along a FCS gradient was monitored (20 hours) by live cell imaging and analyzed for directed migration (P value) and velocity (ImageJ, IBIDI software). Dashed line, P = 0.05. Bars are the SEM of three independent experiments. *, P < 0.05 (t test).

Close modal

Pharmacologic inhibition of TPC1 and TPC2 reduces cancer cell migration and adhesion

To further investigate the potential of TPCs as novel targets for cancer therapy, two pharmacologic inhibitors of TPC1 and TPC2 were used. The NAADP antagonist Ned-19 (17) showed significant reduction of migration through pores in T24 and HUH7 cells (Fig. 3A). Along this line, treatment with tetrandrine abrogated migration in Boyden chambers in T24, HUH7, and 4T1 cells (Fig. 3B). Both TPC inhibitors further impaired adhesion in T24 cells as displayed in Fig. 3C. Importantly, Ned-19 and tetrandrine had no effect on cell survival in all tested concentrations and at all tested time points (Fig. 3D). These results strengthen our finding that TPC1 and TPC2 are required for migration of cancer cells.

Figure 3.

Ned-19 and tetrandrine inhibit cancer cell migration and adhesion. A and B, T24, HUH7, and 4T1 cells were pretreated with Ned-19 and tetrandrine for 8 hours (T24: 250 μmol/L Ned-19, 15 μmol/L Tet; HUH7: 150 μmol/L Ned-19, 2,5 μmol/L Tet; 4T1: 150 μmol/L Ned-19, 10 μmol/L Tet). Cells were allowed to migrate in Transwell chambers for 16 hours in the presence of the inhibitor. C, After 24 hours of treatment with inhibitors used in A and B, T24 cells were allowed to adhere for 1 hour, fixed, and stained (rhodamine-phalloidin, Hoechst). Cells were analyzed by confocal microscopy. Scale bars, 150 μm. Bars in AC are the SEM of three independent experiments, *, P < 0.05 (t test). D, Cell death was assessed after 24 hours of treatment with inhibitors used in A and B by flow cytometry analysis. Data represent means of three independent experiments performed in triplicates ± SEM.

Figure 3.

Ned-19 and tetrandrine inhibit cancer cell migration and adhesion. A and B, T24, HUH7, and 4T1 cells were pretreated with Ned-19 and tetrandrine for 8 hours (T24: 250 μmol/L Ned-19, 15 μmol/L Tet; HUH7: 150 μmol/L Ned-19, 2,5 μmol/L Tet; 4T1: 150 μmol/L Ned-19, 10 μmol/L Tet). Cells were allowed to migrate in Transwell chambers for 16 hours in the presence of the inhibitor. C, After 24 hours of treatment with inhibitors used in A and B, T24 cells were allowed to adhere for 1 hour, fixed, and stained (rhodamine-phalloidin, Hoechst). Cells were analyzed by confocal microscopy. Scale bars, 150 μm. Bars in AC are the SEM of three independent experiments, *, P < 0.05 (t test). D, Cell death was assessed after 24 hours of treatment with inhibitors used in A and B by flow cytometry analysis. Data represent means of three independent experiments performed in triplicates ± SEM.

Close modal

Accumulation of enlarged acidic vesicles after pharmacologic inhibition and TPC silencing

As TPC1 and TPC2 are located in the endolysosomal system (8, 10, 12) of cells, investigating this process after inhibition of TPCs seemed mandatory. First, membranes of cells were stained with PKH26 fluorescent dye and subsequently allowed to recycle. As Fig. 4A indicates, cells treated with Ned-19 and tetrandrine as well as cells silenced with siTPC1 and siTPC2 accumulated up to 2-fold enlarged vesicles compared with control cells, indicating that general recycling was impaired after TPC inhibition. Staining with Lysotracker Red DND-99 showed that the accumulated enlarged vesicles are acidic compartments. This experiment further indicates that the acidification of endocytic vesicles was neither affected after pharmacologic inhibition nor after siRNA silencing (Fig. 4B). To elucidate the connection between disturbed recycling and inhibited cell migration, we selected key proteins of migration that are predominantly trafficked via the endolysosomal system, such as integrins.

Figure 4.

Inhibition of TPCs disrupts endocytic recycling. A, Membranes were stained with PKH 26 red in HUH7 cells after 24 hours of Ned-19 (150 μmol/L) and tetrandrine (2.5 μmol/L) treatment or after 72 hours of TPC1 and TPC2 silencing. Cells were allowed to recycle again for 2 hours. Fixed cells were analyzed by confocal microscopy. B, Ned-19 (250 μmol/L, 24 hours), tetrandrine (15 μmol/L, 24 hours)-treated or siRNA-silenced (72 hours) T24 cells were stained with Lysotracker Red DND-99 and analyzed by confocal microscopy. Nuclei in A and B were stained with Hoechst. Vesicles were analyzed with ImageJ. Bars, SEM of three independent experiments, *, P < 0.05 (t test). Scale bars, 20 μm.

Figure 4.

Inhibition of TPCs disrupts endocytic recycling. A, Membranes were stained with PKH 26 red in HUH7 cells after 24 hours of Ned-19 (150 μmol/L) and tetrandrine (2.5 μmol/L) treatment or after 72 hours of TPC1 and TPC2 silencing. Cells were allowed to recycle again for 2 hours. Fixed cells were analyzed by confocal microscopy. B, Ned-19 (250 μmol/L, 24 hours), tetrandrine (15 μmol/L, 24 hours)-treated or siRNA-silenced (72 hours) T24 cells were stained with Lysotracker Red DND-99 and analyzed by confocal microscopy. Nuclei in A and B were stained with Hoechst. Vesicles were analyzed with ImageJ. Bars, SEM of three independent experiments, *, P < 0.05 (t test). Scale bars, 20 μm.

Close modal

Disturbed β1-integrin recycling through inhibition of TPCs

Integrins feature prominently in cancer cell adhesion and migration. Under normal conditions, these transmembrane receptors get rapidly recycled through the endolysosomal system (4). As inhibition of TPCs hampered endocytosis, we checked whether recycling of β1-integrin is hindered as well. Internalization assays of β1-integrin in T24 cells showed an up to 2-fold enlargement of β1-integrin–positive vesicles after treatment with Ned-19 or tetrandrine compared with control (Fig. 5A, left). As shown in the right panel of Fig. 5A, comparable results were achieved when TPC1 or TPC2 was silenced in the cells. Moreover, staining of β1-integrin–positive vesicles with EEA1 and LAMP3 displayed a higher overlap of β1-integrin vesicles with EEA1, indicating an accumulation in early endosomes rather than in lysosomes (Fig. 5B). In summary, these findings illustrate that TPC inhibition or silencing evokes disturbances in β1-integrin recycling.

Figure 5.

β1-Integrin recycling is hindered after TPC inhibition. A, Internalized β1-integrin was stained in T24 cells after 24 hours of Ned-19 (250 μmol/L) and tetrandrine (15 μmol/L) treatment or after 72 hours of siRNA silencing. Scale bars, 20 μm (top pictures) and 10 μm (bottom pictures). Vesicles were analyzed with ImageJ. Bars, SEM of three independent experiments, *, P < 0.05 (t test). B, Ned-19 (250 μmol/L, 24 hours) and tetrandrine (15 μmol/L, 24 hours)-treated or siRNA-silenced (72 hours) T24 cells were stained for β1-integrin (green), EEA1, LAMP3 (red), and nuclei with Hoechst (blue). Scale bars, 10 μm. Pictures of cells in A and B were taken with a confocal microscope.

Figure 5.

β1-Integrin recycling is hindered after TPC inhibition. A, Internalized β1-integrin was stained in T24 cells after 24 hours of Ned-19 (250 μmol/L) and tetrandrine (15 μmol/L) treatment or after 72 hours of siRNA silencing. Scale bars, 20 μm (top pictures) and 10 μm (bottom pictures). Vesicles were analyzed with ImageJ. Bars, SEM of three independent experiments, *, P < 0.05 (t test). B, Ned-19 (250 μmol/L, 24 hours) and tetrandrine (15 μmol/L, 24 hours)-treated or siRNA-silenced (72 hours) T24 cells were stained for β1-integrin (green), EEA1, LAMP3 (red), and nuclei with Hoechst (blue). Scale bars, 10 μm. Pictures of cells in A and B were taken with a confocal microscope.

Close modal

Polarization and protrusion of the cell are essential for accurate cell migration. Protrusions in the direction of migration are stabilized through integrins linked to the actin cytoskeleton. Therefore, integrins have to be recycled to the leading edges of migrating cells (4, 5). To explore the ability to form leading edges, scratch assays were performed. Under these conditions, Ned-19 as well as tetrandrine inhibited the formation of wound-directed, β1-integrin-, pSrc-, pFAK-, and vinculin-positive polarized lamellipodia (Fig. 6A, top). Accordingly, silencing of TPC1 or TPC2 resulted in the same effects (Fig. 6A, bottom). These findings can be easily accounted for by disturbed β1-integrin trafficking to the front of the cell. Consistently, total protein levels of β1-integrin, FAK, pFAK, Src, and pSrc were not significantly altered after Ned-19 treatment (Fig. 6B), further corroborating an effect on β1-integrin recycling.

Figure 6.

TPC function is required for the formation of leading edges. A, T24 cells were scratched and let migrate for 5 hours after a pretreatment with 250 μmol/L Ned-19 and 15 μmol/L tetrandrine for 16 hours or after siRNA silencing for 72 hours. Fixed cells were stained for actin, β1-integrin, pSrc, pFAK, and vinculin (white). Nuclei were stained with Hoechst. One representative picture out of three independent experiments is shown. Scale bars, 20 μm. White arrows, leading edges. B, Total protein amounts of β1-integrin, pSrc, Src, pFAK, and FAK were detected by Western blot analysis in T24 cells after treatment with 250 μmol/L Ned-19 for 24 hours. One representative blot is shown. Quantification was done with ImageJ. Bars, SEM of three independent experiments.

Figure 6.

TPC function is required for the formation of leading edges. A, T24 cells were scratched and let migrate for 5 hours after a pretreatment with 250 μmol/L Ned-19 and 15 μmol/L tetrandrine for 16 hours or after siRNA silencing for 72 hours. Fixed cells were stained for actin, β1-integrin, pSrc, pFAK, and vinculin (white). Nuclei were stained with Hoechst. One representative picture out of three independent experiments is shown. Scale bars, 20 μm. White arrows, leading edges. B, Total protein amounts of β1-integrin, pSrc, Src, pFAK, and FAK were detected by Western blot analysis in T24 cells after treatment with 250 μmol/L Ned-19 for 24 hours. One representative blot is shown. Quantification was done with ImageJ. Bars, SEM of three independent experiments.

Close modal

Reduced formation of lung metastasis in vivo by pharmacologic TPC inhibition and siRNA silencing of TPC2

To extend the relevance of TPC inhibition on chemotactic migration and invasion, we investigated whether tetrandrine was effective in abrogating cancer cell dissemination in a mouse model. The 4T1-Luc syngeneic metastatic mouse mammary cancer model was used for this purpose (24–26). 4T1-Luc cells express a luciferase reporter, which enables for live imaging. After intravenous injection, 4T1-Luc cells disseminate to distant organs homing preferably to lungs. Along this line, control animals showed easily detectable lung metastasis after five days of 4T1-Luc cell injection. Remarkably, both pretreatment with Ned-19 or tetrandrine significantly diminished formation of lung metastasis (Fig. 7A and B). The data were supported by a subsequent in vivo experiment using TPC2-silenced 4T1-Luc cells, which also formed significantly fewer lung tumors compared with control transfected cells (Fig. 7C). These findings strongly support the hypothesis that targeting TPCs might be a very promising and viable strategy for metastatic cancer therapy.

Figure 7.

Inhibition of TPC function in 4T1-Luc cells reduces the formation of lung metastasis in vivo. A and B, 4T1-Luc cells were pretreated with 150 μmol/L Ned-19, 10 μmol/L tetrandrine, or DMSO for 24 hours. C, 4T1-Luc cells were silenced with siTPC2 or siNT for 72 hours. AC, 1 × 105 cells were injected intravenously into BALB/cOlaHsd mice. On day five after cell inoculation, bioluminescence signals were measured by imaging the mice in ventrodorsal position. The total signal per defined region of interest was calculated as photons/second/cm2 (total flux/area). Bars, SEM of 7 animals. *, P < 0.05 (t test).

Figure 7.

Inhibition of TPC function in 4T1-Luc cells reduces the formation of lung metastasis in vivo. A and B, 4T1-Luc cells were pretreated with 150 μmol/L Ned-19, 10 μmol/L tetrandrine, or DMSO for 24 hours. C, 4T1-Luc cells were silenced with siTPC2 or siNT for 72 hours. AC, 1 × 105 cells were injected intravenously into BALB/cOlaHsd mice. On day five after cell inoculation, bioluminescence signals were measured by imaging the mice in ventrodorsal position. The total signal per defined region of interest was calculated as photons/second/cm2 (total flux/area). Bars, SEM of 7 animals. *, P < 0.05 (t test).

Close modal

This study designates TPCs as promising targets for the treatment of invasive cancers. Disruption of TPC function abrogates cancer cell migration in vitro and in vivo, resulting from disturbed integrin trafficking in the endolysosomal system.

TPCs are Ca2+-permeable cation channels located in the membrane of endosomes and lysosomes (7–10, 12, 13, 16). In recent years, evidence has accumulated that TPCs are substantially implicated in the regulation of endolysosomal trafficking processes. Thus, a lack of TPC2 in mouse embryonic fibroblasts resulted in an accumulation of LDL and EGF/EGFR in intracellular vesicles (9) and led to delayed PDGFRβ degradation (11). Pharmacologic inhibition of TPCs resulted in accumulation of cholera toxin within endolysosomes, which is normally delivered to the Golgi, and enlarged lysosomes dramatically (10). Disrupting TPC function also reduced Ebola virus trafficking through endosomal vesicles, preventing infection (18). Consistently, we observed an accumulation of integrins in early endosomes after TPC inhibition in invasive cancer cells.

When looking at general endocytic trafficking and receptor recycling, another protein located in the endolysosmal membrane of cells comes into focus, the V-ATPase. V-ATPases are multiunit proton pumps, which actively transport protons from the cytoplasm into intracellular compartments thereby regulating its pH (27). In several reports, it has been observed that altering pH by inhibition of V-ATPase results in impaired trafficking and recycling of signaling molecules. Hence, disturbed V-ATPase function led to the accumulation of Notch in the endolysosomal system (28) and of cholesterol in intracellular compartments (29). In cancer cells, inhibition of V-ATPase affected EGF receptor (21) and transferrin receptor internalization, the latter resulting in apoptosis (19).

Taken together, targeting the endolysosmal system either by TPC or V-ATPase inhibition can apparently both impair adequate endocytic trafficking. As it is quite evident for V-ATPase inhibition that this is due to altered pH, there is still an ongoing discussion regarding TPCs. Impaired Ca2+ signaling may be one reason; however, it has also been postulated that alkalinizing of the lysosomal pH is responsible for the inhibition of autophagosomal-lysosomal fusion (30). In our study, we observed an accumulation of enlarged acidic vesicles after TPC inhibition, hence no alkalinizing of pH. Other recent publications have also found no evidence for changes in endolysosomal pH under basal conditions in TPC-deficient cells (9, 11, 31).

Alterations in Ca2+ signaling are widely accepted to lead to impaired trafficking and fusion of endocytic vesicles (9, 10). This hypothesis is further supported by the finding that Ca2+ chelators, BAPTA and EGTA-AM, are able to inhibit fusion of late endosomes and lysosomes (32). In this context, it is interesting to note that another Ca2+-permeable endolysosomal channel, TRPML1, has been proposed to be required for fusion between late endosomes and lysosomes in Drosophila (33). In humans, loss or mutation of this channel leads to the lysosomal storage disorder mucolipidosis type IV (34–36), further supporting the importance of Ca2+ signaling.

Regardless of what the main cause of this impairment is, inhibition of TPC function led to a clear accumulation of β1-integrins in early endosomes. Integrins link the cell to the ECM enabling adhesion and migration (37). To migrate, the cell must be polarized meaning that different molecular processes occur at the front and back of a moving cell (5). Therefore, several proteins are trafficked towards the front of the cell. For example, growth factor receptors and chemokine receptors are recycled to specific sites of the leading edge to mediate promigratory signals. Importantly, adhesive contacts are regulated by the recycling and degradation of integrins (4). Hence, the turnover of integrins is crucial for migration. We could observe in our studies, in addition to an accumulation of β1-integrin in enlarged vesicles, reduced β1-integrin localization at the leading edge of migrating cells after TPC inhibition or silencing. These findings suggest that disrupted TPC function alters β1-integrin trafficking, resulting in reduced adhesion to ECM and insufficient polarization of the cell.

Besides mediating adhesion to ECM, integrins are also essential for cell migration as they regulate promigratory signaling pathways. Thus, integrin ligation induces clustering, resulting in the activation of FAK. Active FAK recruits Src family kinases to focal adhesions, altogether promoting cell migration and invasion. In addition, focal contacts contain different actin-associated proteins, such as vinculin, which links integrin to the cytoskeleton (3). In our study, inhibition of TPC function or expression clearly diminished the accumulation of integrin, pFAK, pSrc, and vinculin at the leading edge of migrating cells. This suggests that adequate β1-integrin trafficking is crucial for the initiation of promigratory mechanisms.

To fulfill these functions, integrins form heterodimeric receptors. Pairing of α and β subunits decides for the specific binding of certain matrix ligands. Previous investigations on the β1-integrin subunit revealed a central role in adhesion, extravasation, and migration in T24 cancer cells (38). Moreover, reports show that β1-integrin and FAK signaling is implicated in the initial proliferation of cancer cells disseminated into the lungs (39). Our own experiments are consistent with these notions. Further research over the last several years led to the development of integrin-targeted therapeutics, which are now tested in clinical studies against cancer and other diseases (3). The mAb volociximab is the first α5β1 integrin antagonist in clinical trials against metastatic clear cell renal cell carcinoma, metastatic melanoma, non–small cell lung cancer, and peritoneal cancer among others (40, 41). In addition, several therapeutic antibodies targeting αvβ3 integrin are under development or in clinical phases as antitumor agents such as Vitaxin and CNTO 95 (40). Taken together, targeting integrins is a highly promising and viable strategy for the treatment of metastatic cancers.

In summary, our study reveals a potential novel role for TPCs in the formation of metastasis. Impaired TPC function reduced adhesion and migration of cancer cells in vitro and diminished formation of metastasis in vivo. Most likely, this is due to disturbed trafficking of integrins known to act promigratory. Here we link TPCs to fundamental processes in cancer cell migration, rendering them new and attractive targets for the treatment of invasive carcinomas.

No potential conflicts of interest were disclosed.

Conception and design: C. Grimm, L.S. Schneider, C. Wahl-Schott, M. Biel, A.M. Vollmar

Development of methodology: O.N.P. Nguyen, C. Grimm, L.S. Schneider, M. Ulrich, C. Wahl-Schott, M. Biel, A.M. Vollmar

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): C. Grimm, C. Atzberger, K. Bartel, A. Watermann, M. Ulrich, D. Mayr

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): O.N.P. Nguyen, C. Grimm, L.S. Schneider, Y.-K. Chao, C. Atzberger, K. Bartel, D. Mayr, C. Wahl-Schott, M. Biel

Writing, review, and/or revision of the manuscript: O.N.P. Nguyen, C. Grimm, L.S. Schneider, C. Atzberger, C. Wahl-Schott, M. Biel, A.M. Vollmar

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): Y.-K. Chao, K. Bartel

Study supervision: O.N.P. Nguyen, C. Grimm, C. Wahl-Schott, A.M. Vollmar

We thank Cheng-Chang Chen for the outstanding instructions and supervision of the lysosomal patch-clamp recordings. We also thank Kerstin Loske for the great support during the animal experiments.

This work was supported by funding from the German Research Foundation (SFB/TRR152 TP04 to C. Grimm, TP06 to C. Wahl-Schott, TP12 to M. Biel, and FOR1406 to A.M. Vollmar).

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.
Fidler
IJ
. 
Timeline: the pathogenesis of cancer metastasis: the ‘seed and soil’ hypothesis revisited
.
Nat Rev Cancer
2003
;
3
:
453
58
.
2.
Chaffer
CL
,
Weinberg
RA
. 
A perspective on cancer cell metastasis
.
Science
2011
;
331
:
1559
64
.
3.
Hood
JD
,
Cheresh
DA
. 
Role of integrins in cell invasion and migration
.
Nat Rev Cancer
2002
;
2
:
91
100
.
4.
Maritzen
T
,
Schachtner
H
,
Legler
DF
. 
On the move: endocytic trafficking in cell migration
.
Cell Mol Life Sci
2015
;
72
:
2119
34
.
5.
Ridley
AJ
. 
Cell migration: integrating signals from front to back
.
Science
2003
;
302
:
1704
09
.
6.
Brailoiu
E
,
Churamani
D
,
Cai
X
,
Schrlau
MG
,
Brailoiu
GC
,
Gao
X
, et al
Essential requirement for two-pore channel 1 in NAADP-mediated calcium signaling
.
J Cell Biol
2009
;
186
:
201
09
.
7.
Zong
X
,
Schieder
M
,
Cuny
H
,
Fenske
S
,
Gruner
C
,
Rötzer
K
, et al
The two-pore channel TPCN2 mediates NAADP-dependent Ca2+-release from lysosomal stores
.
Pfluegers Arch/Eur J Physiol
2009
;
458
:
891
99
.
8.
Calcraft
PJ
,
Ruas
M
,
Pan
Z
,
Cheng
X
,
Arredouani
A
,
Hao
X
, et al
NAADP mobilizes calcium from acidic organelles through two-pore channels
.
Nature
2009
;
459
:
596
600
.
9.
Grimm
C
,
Holdt
LM
,
Chen
C-C
,
Hassan
S
,
Müller
C
,
Jörs
S
, et al
High susceptibility to fatty liver disease in two-pore channel 2-deficient mice
.
Nat Commun
2014
;
5
:
4699
.
10.
Ruas
M
,
Rietdorf
K
,
Arredouani
A
,
Davis
LC
,
Lloyd-Evans
E
,
Koegel
H
, et al
Purified TPC isoforms form NAADP receptors with distinct roles for Ca2+ signaling and endolysosomal trafficking
.
Curr Biol
2010
;
20
:
703
09
.
11.
Ruas
M
,
Chuang
KT
,
Davis
LC
,
Al-Douri
A
,
Tynan
PW
,
Tunn
R
, et al
TPC1 has two variant isoforms, and their removal has different effects on endo-lysosomal functions compared to loss of TPC2
.
Mol Cell Biol
2014
;
34
:
3981
92
.
12.
Rietdorf
K
,
Funnell
TM
,
Ruas
M
,
Heinemann
J
,
Parrington
J
,
Galione
A
. 
Two-pore channels form homo- and heterodimers
.
J Biol Chem
2011
;
286
:
37058
62
.
13.
Schieder
M
,
Rotzer
K
,
Bruggemann
A
,
Biel
M
,
Wahl-Schott
CA
. 
Characterization of two-pore channel 2 (TPCN2)-mediated Ca2+ currents in isolated lysosomes
.
J Biol Chem
2010
;
285
:
21219
22
.
14.
Wang
X
,
Zhang
X
,
Dong
X-p
,
Samie
M
,
Li
X
,
Cheng
X
, et al
TPC proteins are phosphoinositide- activated sodium-selective ion channels in endosomes and lysosomes
.
Cell
2012
;
151
:
372
83
.
15.
Jha
A
,
Ahuja
M
,
Patel
S
,
Brailoiu
E
,
Muallem
S
. 
Convergent regulation of the lysosomal two-pore channel-2 by Mg2+, NAADP, PI(3,5)P2 and multiple protein kinases
.
EMBO J
2014
;
33
:
501
11
.
16.
Ruas
M
,
Davis
LC
,
Chen
CC
,
Morgan
AJ
,
Chuang
KT
,
Walseth
TF
, et al
Expression of Ca2+-permeable two-pore channels rescues NAADP signalling in TPC-deficient cells
.
EMBO J
2015
;
34
:
1743
58
.
17.
Naylor
E
,
Arredouani
A
,
Vasudevan
SR
,
Lewis
AM
,
Parkesh
R
,
Mizote
A
, et al
Identification of a chemical probe for NAADP by virtual screening
.
Nat Chem Biol
2009
;
5
:
220
26
.
18.
Sakurai
Y
,
Kolokoltsov
AA
,
Chen
CC
,
Tidwell
MW
,
Bauta
WE
,
Klugbauer
N
, et al
Two-pore channels control Ebola virus host cell entry and are drug targets for disease treatment
.
Science
2015
;
347
:
995
98
.
19.
Schneider
LS
,
von Schwarzenberg
K
,
Lehr
T
,
Ulrich
M
,
Kubisch-Dohmen
R
,
Liebl
J
, et al
Vacuolar-ATPase inhibition blocks iron metabolism to mediate therapeutic effects in breast cancer
.
Cancer Res
2015
;
75
:
2863
74
.
20.
Schieder
M
,
Rotzer
K
,
Bruggemann
A
,
Biel
M
,
Wahl-Schott
C
. 
Planar patch clamp approach to characterize ionic currents from intact lysosomes
.
Sci Signal
2010
;
3
:
pl3
.
21.
Wiedmann
RM
,
von Schwarzenberg
K
,
Palamidessi
A
,
Schreiner
L
,
Kubisch
R
,
Liebl
J
, et al
The V-ATPase-inhibitor archazolid abrogates tumor metastasis via inhibition of endocytic activation of the Rho-GTPase Rac1
.
Cancer Res
2012
;
72
:
5976
87
.
22.
Zengel
P
,
Nguyen-Hoang
A
,
Schildhammer
C
,
Zantl
R
,
Kahl
V
,
Horn
E
. 
μ-Slide Chemotaxis: a new chamber for long-term chemotaxis studies
.
BMC Cell Biol
2011
;
12
:
21
.
23.
Favia
A
,
Desideri
M
,
Gambara
G
,
D'Alessio
A
,
Ruas
M
,
Esposito
B
, et al
VEGF-induced neoangiogenesis is mediated by NAADP and two-pore channel-2-dependent Ca2+ signaling
.
Proc Natl Acad Sci U S A
2014
;
111
:
E4706
E15
.
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.
Tao
K
,
Fang
M
,
Alroy
J
,
Sahagian
GG
. 
Imagable 4T1 model for the study of late stage breast cancer
.
BMC Cancer
2008
;
8
:
228
.
27.
Forgac
M
. 
Vacuolar ATPases: rotary proton pumps in physiology and pathophysiology
.
Nat Rev Mol Cell Biol
2007
;
8
:
917
29
.
28.
Kobia
F
,
Duchi
S
,
Deflorian
G
,
Vaccari
T
. 
Pharmacologic inhibition of vacuolar H+ ATPase reduces physiologic and oncogenic Notch signaling
.
Mol Oncol
2014
;
8
:
207
20
.
29.
Kozik
P
,
Hodson
NA
,
Sahlender
DA
,
Simecek
N
,
Soromani
C
,
Wu
J
, et al
A human genome-wide screen for regulators of clathrin-coated vesicle formation reveals an unexpected role for the V-ATPase
.
Nat Cell Biol
2012
;
15
:
50
60
.
30.
Lu
Y
,
Hao
BX
,
Graeff
R
,
Wong
CWM
,
Wu
WT
,
Yue
J
. 
Two Pore Channel 2 (TPC2) inhibits autophagosomal-lysosomal fusion by alkalinizing lysosomal pH
.
J Biol Chem
2013
;
288
:
24247
63
.
31.
Cang
C
,
Zhou
Y
,
Navarro
B
,
Seo
Y-j
,
Aranda
K
,
Shi
L
, et al
mTOR regulates lysosomal ATP-Sensitive Two-Pore Na+ channels to adapt to metabolic state
.
Cell
2013
;
152
:
778
90
.
32.
Pryor
PR
,
Mullock
BM
,
Bright
NA
,
Gray
SR
,
Luzio
JP
. 
The role of intraorganellar Ca(2+) in late endosome-lysosome heterotypic fusion and in the reformation of lysosomes from hybrid organelles
.
J Cell Biol
2000
;
149
:
1053
62
.
33.
Wong
C-O
,
Li
R
,
Montell
C
,
Venkatachalam
K
. 
Drosophila TRPML is required for TORC1 activation
.
Curr Biol
2012
;
22
:
1616
21
.
34.
Bargal
R
,
Avidan
N
,
Ben-Asher
E
,
Olender
Z
,
Zeigler
M
,
Frumkin
A
, et al
Identification of the gene causing mucolipidosis type IV
.
Nat Genet
2000
;
26
:
118
23
.
35.
Slaugenhaupt
SA
,
Acierno
JS
 Jr
,
Helbling
LA
,
Bove
C
,
Goldin
E
,
Bach
G
, et al
Mapping of the mucolipidosis Type IV gene to chromosome 19p and definition of founder haplotypes
.
Am J Hum Genet
1999
;
65
:
773
78
.
36.
Chen
C-C
,
Keller
M
,
Hess
M
,
Schiffmann
R
,
Urban
N
,
Wolfgardt
A
, et al
A small molecule restores function to TRPML1 mutant isoforms responsible for mucolipidosis type IV
.
Nat Commun
2014
;
5
:
4681
.
37.
Huttenlocher
A
,
Horwitz
AR
. 
Integrins in Cell Migration
.
Cold Spring Harbor Perspect Biol
2011
;
3
:
a005074
a74
.
38.
Heyder
C
,
Gloria-Maercker
E
,
Hatzmann
W
,
Niggemann
B
,
Zänker
KS
,
Dittmar
T
. 
Role of the β1-integrin subunit in the adhesion, extravasation and migration of T24 human bladder carcinoma cells
.
Clin Exp Metastasis
2005
;
22
:
99
106
.
39.
Shibue
T
,
Weinberg
RA
. 
Integrin 1-focal adhesion kinase signaling directs the proliferation of metastatic cancer cells disseminated in the lungs
.
Proc Natl Acad Sci U S A
2009
;
106
:
10290
95
.
40.
Millard
M
,
Odde
S
,
Neamati
N
. 
Integrin targeted therapeutics
.
Theranostics
2011
;
1
:
154
88
.
41.
Almokadem
S
,
Belani
CP
. 
Volociximab in cancer
.
Expert Opin Biol Ther
2012
;
12
:
251
57
.