Role of the V1G1 subunit of V-ATPase in breast cancer cell migration

V-ATPase is a large multi-subunit complex that regulates acidity of intracellular compartments and of extracellular environment. V-ATPase consists of several subunits that drive specific regulatory mechanisms. The V1G1 subunit, a component of the peripheral stalk of the pump, controls localization and activation of the pump on late endosomes and lysosomes by interacting with RILP and RAB7. Deregulation of some subunits of the pump has been related to tumor invasion and metastasis formation in breast cancer. We observed a decrease of V1G1 and RAB7 in highly invasive breast cancer cells, suggesting a key role of these proteins in controlling cancer progression. Moreover, in MDA-MB-231 cells, modulation of V1G1 affected cell migration and matrix metalloproteinase activation in vitro, processes important for tumor formation and dissemination. In these cells, characterized by high expression of EGFR, we demonstrated that V1G1 modulates EGFR stability and the EGFR downstream signaling pathways that control several factors required for cell motility, among which RAC1 and cofilin. In addition, we showed a key role of V1G1 in the biogenesis of endosomes and lysosomes. Altogether, our data describe a new molecular mechanism, controlled by V1G1, required for cell motility and that promotes breast cancer tumorigenesis.


V1G1 modulates invasive phenotype of breast cancer cells. Alterations of V-ATPase subunits have
been implicated in the invasive phenotype of breast cancer cells 1,2,36 . Therefore, we analyzed the abundance of some subunits of the pump in MCF7, poorly invasive breast cancer cells, and MDA-MB-231, highly invasive breast cancer cells. Western blot analysis showed an increase in V1C1 abundance in the MDA-MB-231 cell line as previously reported 38 , while we observed a decrease of V1G1 and V0D1 levels ( Fig. 1a) associated to a decrease of the V1G1 transcript (Fig. 1b). These data suggest that V1G1 levels could be related to the aggressiveness of breast cancer cells. The analysis of V1G1 expression from UALCAN platform 39 showed that while in less aggressive breast cancer subclasses V1G1 transcript levels are higher compared to normal breast tissues, in triple negative breast cancer they are reduced (Fig. 1c). Regarding protein levels, with the help of The Human Protein Atlas database 40 , we found that they are decreased in breast cancer compared to normal tissues (Supplementary Fig. 1 online). Therefore, we decided to analyze V1G1 protein levels in additional breast cancer cell lines of different subtypes: the luminal A (LA) subtype (MCF7, HCC1428, MD-MB-415, T47D and ZR75-1 cells), the HER2 positive (H) subtype (MDA-MB-453 cells), the triple negative A (TNA) subtype (MDA-MB-468 cells) and the triple negative B (TNB) subtype (MDA-MB-231, BT-549 and Hs578T cells) [41][42][43] . Western blot analysis showed a strong decrease of V1G1 protein abundance in TNB cell lines compared to other breast cancer cell lines of different subtypes (Fig. 1d). As V1G1 is a subunit of the proton pump that controls acidification of extracellular space required for cell migration, we analyzed cell migration in vitro using a wound healing assay on confluent monolayers of these different breast cancer cell lines. Cells were scratched and cells migrating toward the wound were imaged at T0 and 24 h after the scratch. We observed a strong increase in cell migration in TNB cell lines (BT-549, Hs578T and MDA-MB-231 cell lines) (Fig. 1e), as expected, associated to a strong decrease of V1G1 protein abundance compared to MCF7 cells (Fig. 1d), while the increase of V1G1 levels seems to be associated with a decrease in cell migration in HCC1428, T47D, ZR75-1 and MDA-MB-468 cells compared to MCF7 cells, although this reduction is not statistically significant (Fig. 1d, e).
Then, we modulated V1G1 expression in breast cancer cell lines to analyze cell migration and matrix metalloproteinases activation, two processes controlled by V-ATPase during tumor formation and dissemination 2,10 . We performed a wound-healing assay on confluent monolayers of V1G1-silenced MCF7 cells and HA-V1G1 overexpressing MDA-MB-231 cells. In wound-healing migration assay, cells migrating toward the wound were imaged at T0 and 8 h after scratch. Interestingly, we showed a correlation between V1G1 levels and cell migration. In fact, V1G1 depletion in MCF7 cells increased cell migration (Fig. 2a), while HA-V1G1 overexpression in MDA-MB-231 cells decreased cell migration (Fig. 2b). Interestingly, overexpression of V1G1 impaired cell migration in vitro also in Hs578T cells, an additional TNB cell line (Fig. 2c).
The activation of MMPs, and therefore the degradation of the molecular components of extracellular matrix, improves cell migration in vivo. A class of MMPs are the gelatinases that play a critical role in cancer progression and among which are included MMP-2 and MMP-9 37 . In order to measure the relative amounts of active enzymes secreted by MDA-MB-231 overexpressing HA-V1G1, we performed a gelatin zymography 44 . Gelatin zymography showed two strong bands for MDA-MB-231 cells, corresponding to the zone where the gelatin was digested by active MMP-2 and MMP-9, that became smaller when HA-V1G1 was overexpressed (Fig. 2d) suggesting a decrease in the activation of the proteinases in MDA-MB-231 after HA-V1G1 overexpression (Fig. 2d). In addition, we observed a decrease of the total amount of MMP-2 in MDA-MB231 cells overexpressing HA-V1G1 (Fig. 2e). These data suggest that overexpression of V1G1 affects abundance and activation of MMPs. V1G1 modulates membrane receptor stability and signaling. Breast cancer is a highly heterogeneous tumor, as breast cancer cell lines exhibit diverse gene expression profiling and clinical features 26,41 . MDA-MB-231 triple negative cells are featured also by high levels of EGFR expression compared to luminal A cell lines such as MCF7 26 . EGFR signaling regulates cell proliferation and survival, and the overactivation of the receptor promotes cancer development 22,25 . Endocytosis plays an important role in EGFR regulation. The internalization and the subsequent degradation into lysosomes control the activation of the receptor 21,22 . In order to establish if V1G1 modulates EGFR turnover, we measured EGFR degradation in HeLa cells overexpressing and silencing V1G1. To this purpose, cells were starved, incubated with cycloheximide to prevent synthesis of new EGFR and incubated with EGF to induce EGFR degradation. The amount of EGFR was detected by Western blot analysis. www.nature.com/scientificreports/ The analysis showed that about 80% of EGFR, in control cells, was degraded 3 h after the incubation with EGF, while V1G1 overexpression and silencing inhibited EGF-induced EGFR degradation (Fig. 3a, b), suggesting a critical role of V1G1 in the modulation of the receptor. MDA-MB-231 cells exhibit high expression of EGFR compared to the MCF7 cell line 26 . Therefore, to establish whether V1G1 affects EGFR in MDA-MB-231 cell lines, we overexpressed HA-V1G1 in these cells, where we observed lower level of V1G1 compared to MCF7 (Fig. 1a, d). Western Blot analysis showed a significant and TNB (triple negative B) groups, were analyzed by Western blot using specific anti-V1G1, anti-RAB7, and anti-tubulin antibodies. (e) Wound healing assay of different breast cancer cell lines. Cells were imaged at the initial time point (T0) and 24 h after the scratch. Cell migration was measured as the ratio between closed area of the wound in MCF7 cells (referring sample set to 1) and each cell line. Data in panels d and e represent the mean ± s.e.m of at least three experiments and statistical analysis was performed using one-way ANOVA followed by Dunnett's multiple comparisons test with control (MCF7 cells) as referring sample set to 1 (a.u. arbitrary unit). *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. www.nature.com/scientificreports/ reduction of EGFR protein amount in HA-V1G1 overexpressing MDA-MB-231 cells (Fig. 3c) associated also to a strong reduction of Low-Density Lipoprotein Receptor (LDLR) (Fig. 3c). Interestingly, also in Hs578T cells HA-V1G1 overexpression caused a reduction of EGFR protein amount (Fig. 3d). In addition, we showed that V1C1 levels are not affected by V1G1 overexpression in MDA-MB231 cells (Fig. 3c). EGFR stimulates several signaling pathways, such as phosphatidylinositol 3 kinase (PI3K/AKT) and Rasmitogen-activated protein kinase (MAPK/ERK) pathways, which promote cell survival and proliferation 45 . In order to evaluate the receptor signaling, we analyzed the activation of AKT and ERK in HA-V1G1 overexpressing MDA-MB-231 cells (Fig. 3e). We observed in these cells a significant reduction in the activation of both AKT and ERK while the total amount of these kinases was unchanged (Fig. 3e). Therefore, these data suggest that V1G1, regulating EGFR signaling, could control tumor cell survival and progression. To support this hypothesis, we inactivated AKT using the GDC-0068 inhibitor (1 µM) for 24 h, thus inhibiting EGFR signaling pathway (Fig. 3f). Interestingly, inactivation of AKT induced a decrease of cell migration in MDA-MB-231 cells 8 h after the scratch (Fig. 3g).

V1G1 affects cell migration through RAC1 dependent pathway. Cell migration is controlled
by the Rho GTPase RAC1 that, regulating actin cytoskeleton, promotes lamellipodia extension and ruffle formation 19,[28][29][30] . Therefore, we analyzed by western blotting RAC1 protein amount in MDA-MB-231 overexpressing HA-V1G1. The analysis showed a decrease in RAC1 abundance in cells overexpressing HA-V1G1 (Fig. 4a). Therefore, we decided to analyze the levels of cofilin, a downstream effector of RAC1 that controls actin dynamics 31 , and we found a decrease of cofilin protein amount in MDA-MB-231 cells upon overexpression of HA-V1G1 (Fig. 4a).
RAC1 regulates cell protrusions, cell-extracellular matrix interactions and generation of traction forces activating vinculin, a well-characterized focal adhesion protein that plays a key role in regulating cell motility [46][47][48] . Therefore, we analyzed cellular localization of vinculin in MDA-MB-231 overexpressing HA-V1G1. Confocal microscopy showed a distinct localization of vinculin at the cell cortex in control cells, as expected, while in HA-V1G1 overexpressing cells vinculin appeared delocalized (Fig. 4b). www.nature.com/scientificreports/ All together these data suggest that V1G1 is essential for the regulation of RAC1 and, consequently, for the activation of the downstream pathway that controls cell migration.

Role of RAB7 in breast cancer cells. V1G1 stability and localization on late endosomes and lysosomes
are strongly regulated by RILP and RAB7 7,9 . The functional interaction between V1G1 and RILP/RAB7 promotes the intravesicular acidification required for maturation of endosomes and lysosomes 2,3 . Interestingly, RILP controls cell migration in MDA-MB-231 cells, and, in these cells, its expression is lower compared with the www.nature.com/scientificreports/  www.nature.com/scientificreports/ less-invasive MCF7 cell line ( Fig. 5a and 49 ). Thus, having demonstrated a key role of V1G1 in controlling breast cancer cell migration in vitro, we decided to analyze the role of RAB7. First, we investigated abundance of RAB7 in these cells. The analysis showed a strong decrease of RAB7 levels in MDA-MB-231 compared to MCF7 cells (Fig. 5a), associated to a decrease of the RAB7 transcript (Fig. 1b) 50 .
In addition, also in other TNB cell lines (BT-549 and Hs578T) we observed a strong decrease of RAB7 protein abundance (Fig. 1d). These data suggest that the balance of these factors is strongly regulated, as possibly the functional interaction between V1G1, RILP and RAB7 is required to control cell migration, and therefore invasiveness of breast cancer cells.
In order to evaluate whether other RAB proteins are differentially expressed in breast cancer cells, we analyzed the protein amount of RAB5, a protein involved in early steps of endocytosis, and RAB6, a protein involved in the transport from the Golgi to the endoplasmic reticulum and in exocytosis 51,52 . In MDA-MB-231 cells the levels of RAB5 and RAB6 proteins decreased compared to MCF7 cells (Fig. 5a). These data suggest an alteration of trafficking in highly invasive compared to poorly invasive breast cancer cells.
As V1G1, similarly to RILP 49,53 , drives cell migration, we analyzed the role of RAB7 in breast cancer cell migration. The wound healing assay in MDA-MB-231 cells showed that overexpression of HA-RAB7 reduced cell migration in vitro (Fig. 5b). In addition, similarly to V1G1, overexpression of HA-RAB7 reduced the amount of EGFR (Fig. 5c).
Altogether, these data indicate that the complex V1G1/RILP/RAB7 is crucial for the regulation of cell motility in vitro in breast cancer cells, suggesting that downregulation of these three factors is required for cancer progression and, in particular, for cell migration in breast cancer. V1G1 controls lysosome biogenesis. V-ATPase controls maturation of late endosomes and lysosomes 3 .
In order to establish the role of V1G1 in this process, we monitored intracellular distribution of lysosomes in V1G1 depleted MCF7 cells using a specific vital dye, LysoTracker DND-99 that stains acidic organelles in live imaging microscopy. As shown in Fig. 6a, V1G1 depleted MCF7 displayed a significant increase of the dimension of acidic compartments stained with LysoTracker DND-99 (Fig. 6b) and intensity quantification revealed an increase of about twofold in V1G1 depleted compared to control cells (Fig. 6c). These data demonstrate that acidic compartments are affected by V1G1 depletion. Thus, we decided to analyze the protein abundance of TFE3, a master regulator of lysosome biogenesis 54 . Western blot analysis revealed a strong reduction of TFE3 expression in V1G1 depleted cells (Fig. 6d) suggesting that V1G1 plays a critical role in lysosomal biogenesis.

Discussion
V-ATPase is a multi-subunit complex composed by several subunits that control activation and regulation of the pump in different location [1][2][3] . Multiple isoforms exist for many subunits of the pump and these isoforms are ubiquitous or cell-or organelle-specific 3 . Notably, abnormal expression of specific isoforms of the pump correlates with cancer cell aggressiveness [1][2][3][11][12][13][14][15]55 . Here, we showed that expression of V1G1, a component of the peripheral stator of the pump, is strongly decreased in TNB breast cancer cells compared to breast cancer cells of other subtypes (Fig. 1d). This finding was confirmed by analysis of V1G1 expression using the UALCAN database (Fig. 1c). This downregulation, in TNB breast cancer cells, is associated to the previously shown increase in V1C1 protein amount (Fig. 1a) 38 , suggesting that V1G1 negatively regulates breast cancer migration in vitro. Indeed, we showed a correlation between V1G1 expression and cell migration in vitro as in TNB cell lines low levels of V1G1 are associated to an increase of cell migration compared to luminal A subtype MCF7 (Fig. 1d, e). As the G subunit of the V1 domain has three isoforms 3 , it will be interesting in the future to evaluate expression of the other subunits because in the G2-null mouse brain a switch between the different G isoforms has been observed as a compensatory mechanism 56 .
Interestingly, in TNB cell lines we observed also a decrease of RAB7 protein abundance (Figs. 1d, 5a). RAB7 is a key regulator of endocytosis that controls, together with RILP, protein stability of V1G1 required for the recruitment and activation of the pump on endolysosomal membranes 7,8 . Silencing of V1G1 in MCF7 cells affects lysosomal morphology as lysosomes looks larger (Fig. 6a, b) and, notably, increased size of lysosomes is often associated to pathological conditions 57 . Moreover, the decrease in the amount of TFE3 (Fig. 6d), a master regulator of lysosomal biogenesis 54 , suggests that V1G1 controls lysosome biogenesis. Accordingly, overexpression or silencing of V1G1 in HeLa cells affects cathepsin D maturation 7 and EGF-induced EGFR degradation (Fig. 3a, b).
Inappropriate activation of EGFR in cancer drives tumorigenesis and is associated to poor prognosis 22 . Increased expression and/or gene amplification of EGFR have been observed in many human cancers including triple negative breast cancer and, accordingly, MDA-MB-231 cells express high levels of EGFR compared to MCF7 cells that belong to the luminal A subtype (Fig. 3c) 26,58,59 . To mimic MCF7 condition, we overexpressed V1G1 or RAB7 in MDA-MB-231 and Hs578T cells and we observed a decrease of EGFR amount associated with a decrease in its activation (Figs. 3c, e, 5c). RAB7 and RILP control EGFR downregulation 6,60-63 , possibly also through the recruitment of the V-ATPase proton pump on the membranes of endosomes and lysosomes, mediated by the V1G1 subunit, required for their maturation 7 . Thus, we suppose that overexpression of V1G1 and RAB7, in MDA-MB-231, promotes recruitment of the pump on endocytic organelles negatively affecting the recruitment of the pump on the plasma membrane (Fig. 7). Also, upon overexpression of HA-V1G1 or HA-RAB7, TNB cells showed a decrease of EGFR and of LDLR (Figs. 3c, d, 5c) and a simultaneous decrease of cell migration (Figs. 2b, c, 5b), similarly to what happens upon RILP overexpression 49 . On the contrary, V1G1 depletion in MCF7 cells increased cell migration in vitro (Fig. 2a), as observed upon RILP depletion 49,53 . In light of these data, it will be interesting in the future to validate the role of V1G1 in tumor invasiveness in vivo.
The signaling of EGF activates RAC1 to control cytoskeletal reorganization, a crucial event in the metastatic cascade 19 . Overexpression of RAC1 correlates with poor prognosis in breast cancer cells 27  www.nature.com/scientificreports/ reduction or knockdown of V-ATPase activity significantly reduce migration of invasive tumor cells in vitro preventing activation of EGFR and RAC1 16 . In MDA-MB-231 cells we observed a decrease in RAB7 abundance, but we have previously associated RAB7 depletion to a decrease in cell migration combined to alterations in β1-integrin activation, distribution and trafficking and decreased RAC1 activation 64 . We suppose that, in MDA-MB-231 cells, overexpression of RAC1 could bypass the RAB7-dependent negative regulation of cell motility. In fact, in these cells overexpression of V1G1 reduces cell migration (Fig. 2b), RAC1 total protein amount (Fig. 4a) and, at the same time, we observed a decrease of cofilin, a key regulator of actin dynamics that promotes cell migration 31 (Fig. 4a). RAC1 regulates the activation of vinculin, a well-characterized focal adhesion protein involved in many steps of cell migration 46,48 . In triple negative breast cancer cells reduction of tumorigenic phenotypes is accompanied by reduced expression of RAC1, alpha-actinin, vinculin and FAK 65 and silencing of vinculin reduces cell viability www.nature.com/scientificreports/ and decreases tumor volume in animal models 66 . The decrease in vinculin assembly observed in MDA-MB-231 cells overexpressing V1G1 is associated to a decrease in RAC1 protein amount (Fig. 4a). Therefore, the decrease of cell migration observed in these cells (Fig. 2b) could be associated to a reduced assembly of vinculin at the leading edge (Fig. 4b). Altogether, these data showed that HA-V1G1 overexpression in MDA-MB-231 cells downregulates EGFR signaling that, through the activation of RAC1, controls cell migration (Fig. 7). The promoting effect of V-ATPase on cancer invasion is ensured also by maintaining acidic extracellular microenvironment required to increase secretion and activation of degradative enzymes such as matrix metalloproteinases that, by remodeling the extracellular environment, promote cell migration and invasion 37 . MMP-2 and MMP-9 are highly expressed in malignant tumors and positively correlate with an aggressive malignant phenotype and poor outcome in breast cancer patients 34,67 . Accordingly, we observed that V1G1 overexpression decreases the activation of secreted MMP-2 and MMP-9 (Fig. 2d, e), thus confirming a role of this subunit in processes related to cell migration.

Conclusion
Altogether these data indicate that V1G1 has a key role in the regulation of several processes that promote the invasive phenotype of breast cancer cells and may be a novel cell migration suppressor factor. In particular, V1G1, controlling EGFR stability, modulates EGFR downstream signaling pathways required for the cell migration (Fig. 7).  The specificity of PCR products was checked by performing a melting-curve test. The relative expression level was calculated using the comparative C T method and expressed as a "fold change", as described previously 60 .

Methods
Western blotting. Cells were lysed with RIPA or Laemmli buffer and processed for Western blot analysis as previously described 7,60 . Signal was captured on a film or using Bio-Rad ChemiDoc MP Imaging Systems (Hercules, CA, USA). Densitometric analysis was performed using NIH ImageJ (Bethesda, MD, USA) or Image Lab software (Bio-Rad). Rabbit anti-RILP antibody was previously described 5 . Commercial antibodies used are listed in Table 1. HRP-conjugated secondary antibodies were from Invitrogen or Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Wound-healing assay. The wound healing assay was performed as previously described 60,68 . Cells were imaged in at least 3 fields at the moment of the scratch (T0) and after 8 h or 24 h. We measured the wound area www.nature.com/scientificreports/ with the software ImageJ for each time point and we calculated the closed area of the wound for each sample as the difference between the wound area at T0 and at T8h or T24h. We set to 1 the closed area in the control cells and we measured cell migration as the ratio between the closed area of the wound in control cells and HA-V1G1 overexpressing or V1G1 silencing cells. When indicated, we performed the wound healing assay on confluent monolayer of MDA-MB-231 cells treated for 24 h with the inhibitor GDC-0068 (1 µM) or with DMSO as control. Cells, in medium supplemented with the inhibitor, were imaged at the moment of the scratch (T0) and after 8 h.
Gelatin zymography. Gelatin zymography was performed with 3ug of each sample, as previously described 60 . Briefly, secreted proteins were separated under denaturing but non reducing conditions through a polyacrylamide gel containing gelatin, a synthetic substrate of the gelatinases. Coomassie Blue staining allowed visualization of the clear region (the gelatin digested zone) on a uniform blue background of undegraded substrate 44 . Using the NIH ImageJ software, the clear region on a uniform blue background was quantify for each sample.
EGFR degradation assay. To measure EGFR degradation cells were treated and processed as previously described 60 .
Confocal immunofluorescence microscopy and live imaging. MDA-MB-231 cells were fixed for 15 min with 3% paraformaldehyde at room temperature, permeabilized for 5 min with 0.1% Triton X-100 and then processed as previously described 60 . Primary antibodies used are described in Table 1

Analysis of V1G1 expression level between breast cancer and normal tissues. To analyze TCGA
level RNA-seq of V1G1 in various breast cancer subclasses we used UALCAN (http://ualca n.path.uab.edu) 39 .
With the Human Protein Atlas (www.prote inatl as.org), we detected the V1G1 protein expression among normal and cancer tissues 40 .

Statistical analysis.
All experiments were conducted at least 3 times. Results are expressed as mean value ± SEM. Data were statistically analysed using Student's t-test. Comparisons between multiple groups were made by one-way ANOVA followed by Dunnett's multiple comparisons test using GraphPad Prism (*p ≤ 0.05, **p ≤ 0.01 and ***p ≤ 0.001).

Data availability
All data are available from the first author on request. www.nature.com/scientificreports/