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Drug Sequestration in Lysosomes as One of the Mechanisms of Chemoresistance of Cancer Cells and the Possibilities of Its Inhibition
review-article
Author(s):
Jan Hraběta
1 ,
Marie Belhajová
1 ,
Hana Šubrtová
2 ,
Miguel Angel Merlos Rodrigo
2
,
3 ,
Zbyněk Heger
2
,
3 ,
Tomáš Eckschlager
1
,
*
Publication date (Electronic):
20 June 2020
Publisher:
MDPI
There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.
Abstract
Resistance to chemotherapeutics and targeted drugs is one of the main problems in successful cancer therapy. Various mechanisms have been identified to contribute to drug resistance. One of those mechanisms is lysosome-mediated drug resistance. Lysosomes have been shown to trap certain hydrophobic weak base chemotherapeutics, as well as some tyrosine kinase inhibitors, thereby being sequestered away from their intracellular target site. Lysosomal sequestration is in most cases followed by the release of their content from the cell by exocytosis. Lysosomal accumulation of anticancer drugs is caused mainly by ion-trapping, but active transport of certain drugs into lysosomes was also described. Lysosomal low pH, which is necessary for ion-trapping is achieved by the activity of the V-ATPase. This sequestration can be successfully inhibited by lysosomotropic agents and V-ATPase inhibitors in experimental conditions. Clinical trials have been performed only with lysosomotropic drug chloroquine and their results were less successful. The aim of this review is to give an overview of lysosomal sequestration and expression of acidifying enzymes as yet not well known mechanism of cancer cell chemoresistance and about possibilities how to overcome this form of resistance.
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Most cited references85
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A phase I/II trial of hydroxychloroquine in conjunction with radiation therapy and concurrent and adjuvant temozolomide in patients with newly diagnosed glioblastoma multiforme
Myrna R Rosenfeld, Xiaobu Ye, Jeffrey Supko … (2014)
Preclinical studies indicate autophagy inhibition with hydroxychloroquine (HCQ) can augment the efficacy of DNA-damaging therapy. The primary objective of this trial was to determine the maximum tolerated dose (MTD) and efficacy of HCQ in combination with radiation therapy (RT) and temozolomide (TMZ) for newly diagnosed glioblastoma (GB). A 3 + 3 phase I trial design followed by a noncomparative phase II study was conducted in GB patients after initial resection. Patients received HCQ (200 to 800 mg oral daily) with RT and concurrent and adjuvant TMZ. Quantitative electron microscopy and immunoblotting were used to assess changes in autophagic vacuoles (AVs) in peripheral blood mononuclear cells (PBMC). Population pharmacokinetic (PK) modeling enabled PK-pharmacodynamic correlations. Sixteen phase I subjects were evaluable for dose-limiting toxicities. At 800 mg HCQ/d, 3/3 subjects experienced Grade 3 and 4 neutropenia and thrombocytopenia, 1 with sepsis. HCQ 600 mg/d was found to be the MTD in this combination. The phase II cohort (n = 76) had a median survival of 15.6 mos with survival rates at 12, 18, and 24 mo of 70%, 36%, and 25%. PK analysis indicated dose-proportional exposure for HCQ. Significant therapy-associated increases in AV and LC3-II were observed in PBMC and correlated with higher HCQ exposure. These data establish that autophagy inhibition is achievable with HCQ, but dose-limiting toxicity prevented escalation to higher doses of HCQ. At HCQ 600 mg/d, autophagy inhibition was not consistently achieved in patients treated with this regimen, and no significant improvement in overall survival was observed. Therefore, a definitive test of the role of autophagy inhibition in the adjuvant setting for glioma patients awaits the development of lower-toxicity compounds that can achieve more consistent inhibition of autophagy than HCQ.
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Abnormal lysosomal trafficking and enhanced exosomal export of cisplatin in drug-resistant human ovarian carcinoma cells.
Roohangiz Safaei, Barrett Larson, Timothy C. Cheng … (2005)
Previous work has shown that cisplatin (CDDP) becomes concentrated in lysosomes, and that acquired resistance to CDDP is associated with abnormalities of protein trafficking and secretion. The lysosomal compartment in CDDP-sensitive 2008 human ovarian carcinoma cells was compared with that in CDDP-resistant 2008/C13*5.25 subline using deconvoluting imaging and specific dyes and antibodies. The lysosomal compartment in CDDP-resistant cells was reduced to just 40% of that in the parental CDDP-sensitive cells (P<0.002). This was accompanied by a reduced expression of the lysosome-associated proteins 1 and 2 (LAMP1 and LAMP2) as determined by both microscopy and Western blot analysis. The CDDP-resistant cells released more protein as exosomes and Western blot analysis revealed that these exosomes contained substantially more LAMP1 than those released by the CDDP-sensitive cells. Following loading of the whole cell with CDDP, the exosomes released from 2008/C13*5.25 cells contained 2.6-fold more platinum than those released from sensitive cells. Enhanced exosomal export was accompanied by higher exosomal levels of the putative CDDP export transporters MRP2, ATP7A, and ATP7B. Expression profiling identified significant increases in the expression of several genes whose products function in membrane fusion and vesicle trafficking. This study shows that the lysosomal compartment of human ovarian carcinoma cells selected for stable resistance to CDDP is markedly reduced in size, and that these cells abnormally sort some lysosomal proteins and the putative CDDP transporters into an exosomal pathway that also exports CDDP.
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Regulation of TFEB and V-ATPases by mTORC1
Introduction Target of rapamycin (TOR) complex 1 (TORC1) is a pivotal regulator of cell growth conserved from yeast to humans and implicated in cancer (Wullschleger et al, 2006; Abraham and Eng, 2008). Mammalian TORC1 (mTORC1) includes the atypical serine/threonine kinase mTOR and an adaptor protein, regulatory-associated protein of mTOR (Raptor) (Hara et al, 2002; Kim et al, 2002; Loewith et al, 2002). mTORC1 regulates cell growth, at least in part, by promoting cap-dependent translation and its best-characterized substrates are the eukaryotic initiation factor 4E binding protein-1 (4E-BP1) (Sonenberg and Hinnebusch, 2009) and S6 kinase 1 (S6K1) (Fingar and Blenis, 2004). mTORC1 is specifically inhibited by rapamycin (also called sirolimus) (Jacinto et al, 2004; Sarbassov et al, 2004) and rapamycin analogues are used clinically for many applications including cancer treatment (Abraham and Eng, 2008). Indeed, two rapamycin analogues, temsirolimus (Hudes et al, 2007) and everolimus (Motzer et al, 2008), are approved by the FDA for the treatment of advanced RCC (renal cell carcinoma). mTORC1 is regulated by a complex formed by the proteins tuberous sclerosis complex 1 (TSC1) and 2 (TSC2), which is essential for the relay of signals from oxygen (Brugarolas et al, 2004; Connolly et al, 2006; Kaper et al, 2006; Liu et al, 2006; DeYoung et al, 2008), energy stores (Inoki et al, 2003b; Corradetti et al, 2004; Shaw et al, 2004) and growth factors (Jaeschke et al, 2002; Kwiatkowski et al, 2002; Zhang et al, 2003a). mTORC1 regulation by hypoxia involves the protein regulated in development and DNA damage response 1 (REDD1), a conserved protein with a novel fold (Vega-Rubin-de-Celis et al, 2010), which, when overexpressed is sufficient to inhibit mTORC1 in a TSC1/TSC2-dependent manner (Brugarolas et al, 2004). Interestingly, in some settings, hypoxia signals are transduced via AMP-activated protein kinase (Liu et al, 2006; Wolff et al, 2011), which phosphorylates TSC2 and is normally involved in the relay of energy signals (Inoki et al, 2003b; Gwinn et al, 2008). mTORC1 regulation by growth factors involves TSC2 phosphorylation by Akt (Dan et al, 2002; Inoki et al, 2002; Manning et al, 2002; Potter et al, 2002), extracellular signal-regulated kinase (Erk) (Ma et al, 2005) and ribosomal S6 kinase (Rsk) (Roux et al, 2004). TSC2 functions as a GTPase-activating protein towards Ras homologue enriched in brain (Rheb) (Castro et al, 2003; Garami et al, 2003; Tee et al, 2003; Inoki et al, 2003a; Zhang et al, 2003b), a small GTPase that directly interacts with and activates mTORC1 (Sancak et al, 2007; Avruch et al, 2009). mTORC1 regulation by nutrients is independent of TSC1/TSC2 (Zhang et al, 2003a; Smith et al, 2005; Roccio et al, 2006) and involves its localization to a cellular compartment where it becomes receptive to activation by Rheb (Sancak et al, 2008, 2010). Amino-acid stimulation drives mTORC1 to the surface of the late endosome/lysosome in a manner that depends on a Rag GTPase heterodimer (RagA [or B] bound to RagC [or D]) and a multimeric complex termed the Ragulator (Kim et al, 2008; Sancak et al, 2010). Importantly, constitutive mTORC1 targeting to this compartment renders mTORC1 insensitive to amino-acid withdrawal (Sancak et al, 2010). The significance of the late endosome/lysosome in mTORC1 activation has been recently established in experiments manipulating endosome maturation (Flinn et al, 2010; Li et al, 2010). Disrupting endosome maturation with a constitutively active Rab5 or through depletion of hVps39 inhibited the activation of mTORC1 by growth factors and amino acids (Flinn et al, 2010; Li et al, 2010). A critical component of the endosome required for its acidification and maturation is the vacuolar H+-ATPase (V-ATPase) (Marshansky and Futai, 2008). V-ATPases are multisubunit complexes formed by a membrane-embedded V0 domain (a, d, e, c, c′ and c″ subunits) responsible for proton translocation, and a cytosolic V1 domain (A–H subunits), which provides energy through ATP hydrolysis (Forgac, 2007). V-ATPases are present in virtually every eukaryotic cell and V-ATPase function is essential for survival under a variety of conditions (Beyenbach and Wieczorek, 2006). However, little is known about the regulation of V-ATPases and in particular about how their expression is controlled. Here, we have uncovered a regulatory network linking V-ATPases and endocytosis to mTORC1 that involves the transcription factor EB (TFEB). TFEB is a basic helix-loop-helix (bHLH) leucine zipper transcription factor of the Myc family, microphthalmia transcription factor subfamily (Steingrimsson et al, 2004), that is translocated in a subset of renal tumours (Davis et al, 2003; Kuiper et al, 2003; Srigley and Delahunt, 2009). Results V-ATPase enrichment among mTORC1-regulated genes We identified V-ATPase genes in an unbiased screen for genes whose expression was tightly regulated by mTORC1. We used a robust experimental paradigm involving the combination of two interventions, one genetic and one pharmacologic. In Tsc2 +/+ immortalized mouse embryo fibroblasts (MEFs), but not Tsc2 −/−, mTORC1 is inhibited by serum deprivation (0.1% fetal bovine serum, FBS) (Figure 1A). In Tsc2 −/− cells, abnormally increased mTORC1 activity can be corrected by treatment with rapamycin (Figure 1B). By contrast, rapamycin has little effect on mTORC1 in Tsc2 +/+ cells in which mTORC1 is already inhibited by low serum (Figure 1B). Thus, under serum-deprived conditions, mTORC1 activity is low in Tsc2 +/+ cells (untreated or rapamycin treated), high in Tsc2 −/− cells, but lowered by rapamycin; a pattern referred to as a ‘low/low/high/low' or ‘LLHL' (Figure 1B). This experimental setup creates an ‘asymmetric' response to rapamycin (in Tsc2 −/− versus Tsc2 +/+ cells), which, as illustrated hereafter, can be harnessed to great advantage. Using an Affymetrix MOE430A array platform and duplicate samples, genes were identified with an LLHL expression pattern (upregulated in untreated Tsc2 −/− cells compared with a baseline formed by Tsc2 +/+ cells, untreated or rapamycin treated, and Tsc2 −/− rapamycin-treated cells). With a false discovery rate-corrected P-value (FDR Q) cutoff of 0.05 and a threshold of 1.5-fold, we identified 78 probe sets (henceforth referred to as probes). They corresponded to 75 genes (Figure 1C and D). We hypothesized that if the association of probes with the LLHL pattern was random, there would be similar numbers of probes with the three alternate patterns (HLLL, LHLL and LLLH). Strikingly, however, no probes fitted an HLLL or LHLL pattern, and only 22 probes conformed to an LLLH pattern (Figure 1C). Given these results and assuming a binomial distribution with equal probabilities for all patterns, the probability of finding 78 probes with an LLHL pattern by chance alone was below 1.6 × 10−28. To validate the microarray findings, qRT–PCR was performed on a subset of genes with the weakest LLHL association (highest P-values). In every instance, the LLHL pattern was confirmed and the fold change was even greater than expected (Figure 1E). Among the LLHL probes, we found six for V-ATPase genes (Figure 1D; Supplementary Table S1). The probability that there would be six V-ATPase probes among the 78 probes identified by chance alone was 20% reduction in mean expression levels in Tsc2 −/− MEFs by Tfeb knockdown), including several V-ATPases (Supplementary Table S3). LLHL ‘flattening' was confirmed for several V-ATPase genes by qRT–PCR (Figure 4E). Interestingly, the decrease in the number of genes with an LLHL pattern after Tfeb knockdown was specific for the LLHL pattern. In fact, Tfeb knockdown resulted in an increase in the number of genes with a LLLH pattern (35 versus 323; Figure 4C; Supplementary Table S4). These results suggest that Tfeb is an important effector downstream of mTORC1 involved in the regulation of a substantial number of genes. Regulation of endocytosis by V-ATPases, TFEB and mTORC1 Having determined that mTORC1 regulated V-ATPase expression in cells in culture and in mice and having established that TFEB was required for mTORC1-induced V-ATPase expression, we sought to examine the functional significance of V-ATPase regulation by mTORC1. Because V-ATPases are involved in vesicle trafficking (Marshansky and Futai, 2008), we evaluated the effects of mTORC1 on endocytosis. For these experiments, we used albumin, the most abundant protein in plasma and a protein that is taken up by macrophages and hydrolyzed to amino acids, which are then released in the circulation and are utilized by different cells (Guyton and Hall, 1996). While albumin uptake occurs through different pathways in different cell types (Mayor and Pagano, 2007), albumin is taken up by MEFs through a caveolin-dependent mechanism (Razani et al, 2001). FITC-conjugated bovine serum albumin (FITC-BSA) was efficiently taken up by MEFs, where it was found in punctate structures reminiscent of vesicles, which also contained V-ATPases (Figure 5A). In keeping with the idea that mTORC1 regulates endocytosis, FACS analyses showed higher levels of albumin uptake in Tsc2 −/− than Tsc2 +/+ cells (Figure 5B and C). In addition, treatment of Tsc2 −/− cells with rapamycin downregulated albumin uptake (Figure 5C). Albumin uptake required V-ATPases as determined by experiments using the V-ATPase inhibitor bafilomycin A1 (Bowman and Bowman, 2002) (Figure 5B and C). In addition, albumin uptake was downregulated by siRNA-mediated depletion of essential components of either the V0 or the V1 domain (alone or in combination) in highly transfectable HeLa cervical carcinoma cells (Figure 5D and E). Next, we tested whether Tfeb may be similarly involved in endocytosis. Indeed, Tfeb knockdown resulted in a statistically significant decrease in albumin uptake (Figure 5F). The effects of Tfeb knockdown on albumin uptake in Tsc2 −/− cells were similar to those achieved by treatment with rapamycin (Figure 5F). Taken together, these data show that mTORC1 promotes endocytosis in a Tfeb- and V-ATPase-dependent manner. Regulation of Tfeb phosphorylation and subcellular localization by the Tsc1/Tsc2 complex and rapamycin Having established the importance of Tfeb downstream of mTORC1, we explored the mechanism whereby mTORC1 regulated Tfeb. We observed that not only the expression levels of Tfeb appeared to be upregulated by mTORC1, but also that mTORC1 affected Tfeb electrophoretic mobility on SDS–PAGE (Figures 4B and 6A). In Tsc2 −/− cells, Tfeb was prominently found in a fast-migrating form(s) (Figures 4B and 6A). Treatment of Tsc2 −/− cells with rapamycin shifted Tfeb up giving rise to a pattern similar to that observed in wild-type cells (Figure 6A). Similar results were observed in Tsc1 +/+ and Tsc1 −/− cells (Figure 6B). To determine how quickly Tfeb mobility was affected by rapamycin, a time course was performed. As shown in Figure 6C, rapamycin treatment for 1 h was sufficient to change the distribution of Tfeb. Changes in Tfeb migration may reflect changes in phosphorylation, and phosphoproteomic studies have shown TFEB to be phosphorylated in >10 sites (Dephoure et al, 2008; Mayya et al, 2009). To determine whether Tfeb migration was affected by phosphorylation, we evaluated the effects of λ phosphatase treatment. As shown in Figure 6D, the migration of Tfeb was accelerated by treatment of cell lysates with λ phosphatase. Parenthetically, by comparison to similarly processed samples incubated without λ phosphatase for the same amount of time, phosphatase exposure not only accelerated Tfeb migration, but also led to a downregulation of total Tfeb protein levels (Figure 6D). Notably, even fast-migrating Tfeb in Tsc2 −/− cells was subject to a downshift following phosphatase treatment (Figure 6D). These results indicate that even in Tsc2 −/− cells, Tfeb retained some level of phosphorylation. We considered that the changes in Tfeb phosphorylation may be linked to changes in its subcellular localization and performed subcellular fractionation experiments. Whereas in Tsc2 +/+ cells, nuclear Tfeb was undetectable, Tfeb was found in nuclear fractions of Tsc2 −/− cells (Figure 6E). In the nucleus, Tfeb appeared to be present almost exclusively in a fast-migrating form (Figure 6E). To determine whether nuclear Tfeb retained some level of phosphorylation, nuclear extracts were treated with λ phosphatase. As shown in Figure 6F, this resulted in a further shift, indicating that nuclear Tfeb was also phosphorylated. Since phosphatase treatment collapsed both nuclear and cytosolic Tfeb onto an undistinguishable band, the differences in migration likely reflect differences in phosphorylation. Next, we sought to determine how rapamycin affected nuclear Tfeb in Tsc2 −/− cells. Rapamycin treatment caused a shift up in nuclear Tfeb mobility and a progressive decline in nuclear Tfeb (Figure 6G). Taken together, these data show that the migration and nuclear localization of Tfeb are coordinately regulated by Tsc1/Tsc2 and rapamycin and suggest that mTORC1 controls the phosphorylation state and nuclear localization of Tfeb. Regulation of TFEB phosphorylation and nuclear localization by mTORC1 To extend these studies, we examined the regulation of TFEB by mTORC1 in HeLa cells. HeLa cells were transduced with lentiviruses encoding a TSC2 shRNA or a scrambled control and, as for MEFs, cultured for 24 h in 0.1% FBS with or without rapamycin. The results were remarkably similar to those observed in MEFs. TSC2 inactivation led to a downshift in TFEB and this was reverted by rapamycin (Figure 7A and B). As in MEFs, phosphatase treatment accelerated TFEB migration even in cells depleted of TSC2 (Figure 7C). Subcellular fractionation studies showed the presence of fast-migrating TFEB in nuclear fractions of cells depleted of TSC2, but not in controls (Figure 7D) and nuclear TFEB retained some level of phosphorylation (Figure 7E). Thus, the regulation of TFEB phosphorylation and nuclear localization by TSC1/TSC2 and rapamycin was conserved across cell types. To unequivocally establish that TFEB regulation by TSC1/TSC2 and rapamycin was indeed mTORC1 dependent, we inactivated mTORC1 by knocking-down Raptor. HeLa cells were transduced with a Raptor shRNA (or a scrambled control) and, following selection, were subjected to TSC2 depletion using siRNA. As shown in Figure 7F, in cells depleted of Raptor, TSC2 RNAi failed to accelerate TFEB mobility. In a complementary set of experiments, Raptor siRNA (like rapamycin) retarded TFEB mobility in HeLa cells depleted of TSC2 (Figure 7G). Regardless of the means to deplete Raptor, Raptor inactivation prevented the accumulation of faster-migrating TFEB (Figure 7F and G). Taken together, our data indicate that mTORC1 coordinately regulates TFEB phosphorylation and nuclear localization in multiple cell types. mTORC1-dependent nuclear localization of TFEB requires C-terminal serine-rich motif To dissect TFEB regulation by mTORC1, we first identified conditions in which ectopically expressed TFEB behaved like endogenous TFEB. Epitope-tagged TFEB, when expressed at low levels in HeLa cells, preserved its regulation by mTORC1; it was enriched in the nuclei of TSC2-depleted cells and the levels were decreased by rapamycin (Figure 8A). To determine what regions were important for TFEB regulation by mTORC1, we examined a series of deletions (Figure 8B). TFEB233–476 and TFEB290–476 behaved like full-length TFEB, indicating that the N-terminal half of TFEB was dispensable for mTORC1-dependent nuclear localization (Figure 8C). Within this region, mutation of a serine residue (S142) proposed to be phosphorylated by ERK2 and to regulate TFEB nuclear localization (Settembre et al, 2011) had no appreciable effect (Figure 8D and E). Deletion of residues 1–320 resulted in a protein that was enriched in nuclei (Figure 8C). Interestingly, deletion of just 15 amino acids from the C-terminus abrogated mTORC1-dependent nuclear localization (Figure 8C). By contrast, deletion of seven amino acids had no effect (Figure 8C). These data indicated that there were critical residues between 462–469. This corresponded to a serine-rich region (462SSRRSSFS469). To further evaluate, this serine-rich motif, we mutated all S to A (5 × SA). Interestingly, TFEB5 × SA was largely cytoplasmic and failed to be driven into the nucleus in cells with active mTORC1 (Figure 8D and E). Thus, a serine(s) within this motif is necessary for mTORC1-dependent nuclear TFEB localization. Conversely, mutation of the same residues to aspartic acid resulted in an enrichment of TFEB in the nucleus (Figure 8D and E). TFEB5 × SD localized to the nucleus even in cells with inactive mTORC1. Interestingly, the percentage of cells with TFEB5 × SD in the nucleus was the same as for wild-type TFEB in mTORC1-active cells (Figure 8E; see also Figure 8A). These data show that the presence of phosphomimetic residues in the serine-rich motif obviates the mTORC1 requirement for TFEB nuclear localization. The simplest explanation for these data is that a serine(s) in the C-terminal serine-rich motif is phosphorylated in an mTORC1-dependent manner and that phosphorylation is both necessary and sufficient to drive TFEB into the nucleus (see model in Figure 8F). Complex TFEB regulation beyond mTORC1 by multiple signals Next, we examined TFEB in wild-type MEFs and HeLa cells grown under standard conditions (10% FBS). TFEB was found in both a slow and fast-migrating form(s) in MEFs, but predominantly in a slow migrating form(s) in HeLa cells (Figure 9A). We evaluated the effects of multiple interventions on TFEB mobility. Glucose withdrawal resulted in a downshift in TFEB in both MEFs and HeLa cells (Figure 9A). Amino acid withdrawal induced acute changes in TFEB migration in HeLa cells and subsequently led to the accumulation of slow migrating TFEB, which was also observed in MEFs (Figure 9A). The mobility of TFEB was also affected by serum deprivation in both MEFs and HeLa cells (Figure 9A). Finally, changes in TFEB mobility were also induced by treatment with a reducing agent, DTT. DTT led to a progressive shift in TFEB to a fast-migrating form(s) in both HeLa and MEFs (Figure 9A). Overall, these data show that TFEB is extensively and dynamically regulated by multiple stimuli. While post-translational modifications other than phosphorylation may be involved in the changes observed in TFEB migration, TFEB could be down-shifted by phosphatase treatment in every instance (data not shown). The dynamic changes in TFEB mobility induced by the various interventions could not be explained by their expected effects on mTORC1 suggesting that TFEB regulation involves multiple pathways and is likely to be rather complex. This is consistent with the finding that TFEB is phosphorylated in >10 sites (Dephoure et al, 2008; Mayya et al, 2009; Yu et al, 2011). Finally, since TFEB is an oncogene (Davis et al, 2003; Kuiper et al, 2003; Srigley and Delahunt, 2009), we tested whether TFEB was implicated in the regulation of cell proliferation. As shown in Figure 9B, stable depletion of TFEB in both MEFs and HeLa cells lowered the rates of cell expansion. Discussion Using a robust experimental paradigm, we uncovered a novel regulatory network linking endocytosis to TFEB and mTORC1. An unbiased gene expression analysis coupled with a design amenable to refined statistics led us to identify a connection between mTORC1 and V-ATPase genes. Publicly available data sets confirmed the link and experiments in mice showed that mTORC1 regulated V-ATPase expression also in vivo. A recent study had established that TFEB regulated lysosomal biogenesis (Sardiello et al, 2009) and among the genes regulated by TFEB we found several V-ATPases. These results led us to hypothesize that V-ATPase regulation by mTORC1 may be mediated by TFEB. Indeed, TFEB was necessary for V-ATPase upregulation by mTORC1. mTORC1 regulated TFEB phosphorylation and nuclear localization and a C-terminal serine-rich motif was identified that is essential for mTORC1-dependent TFEB nuclear localization. Our data suggest that mTORC1 induces the phosphorylation of a serine(s) within this motif driving thereby TFEB to the nucleus. TFEB regulates the expression of V-ATPases and other lysosomal genes and TFEB was required for mTORC1-induced endocytosis. To our knowledge this is the first study to show that endogenous TFEB is regulated at the subcellular localization level and that mTORC1 promotes TFEB nuclear localization. These data link an oncogenic transcription factor that is a master regulator of lysosomal biogenesis, TFEB, to mTORC1 and endocytosis. An increasing amount of evidence implicates TORC1 in endocytosis. mTOR was identified in an RNAi screen for kinases involved in clathrin-mediated endocytosis (Pelkmans et al, 2005) and Tsc2-deficient cells were previously shown to have increased fluid-phase endocytosis (Xiao et al, 1997). In Drosophila melanogaster, a sensitized screen for genes that modified a tissue-specific dTOR overexpression phenotype identified an important regulator of endocytosis, Hsc70-4, a clathrin uncoating factor, and the authors showed that dTOR was involved in fat body endocytosis (Hennig et al, 2006). Interestingly, besides V-ATPases, mTORC1 regulated the expression of multiple lysosomal genes (Figure 1D; see also Supplementary Table S5). These effects may be mediated, at least in part, by TFEB, which regulates lysosome biogenesis (Sardiello et al, 2009). Thus, one mechanism whereby mTORC1 and TFEB may affect endocytosis is by regulating V-ATPase levels and lysosomes. The lysosome is the final destination not only for endosomal cargo but also for autophagosomes. In response to starvation, mTORC1 is inhibited leading to the formation of autophagosomes that engulf intracellular components for recycling and sustenance (He and Klionsky, 2009; Kroemer et al, 2010). Autophagosomes and their content are delivered to lysosomes, which accumulate in the perinuclear area (Korolchuk et al, 2011). Autophagosomes fuse with lysosomes to generate autolysosomes, a process that rapidly depletes the lysosomal pool (Yu et al, 2010). The degradation of macromolecules and release of their constituents into the cytosol reactivates mTORC1 (Yu et al, 2010). mTORC1 reactivation terminates autophagy and through a poorly understood mechanism repletes the lysosome pool (Yu et al, 2010). However, how the pool of lysosomes is repleted is unknown. Our results suggest that lysosomal reformation in response to autophagy, which is mTORC1 dependent (Yu et al, 2010), may be mediated, at least in part, by TFEB. mTORC1 coordinately regulates TFEB phosphorylation and nuclear localization. mTORC1 promoted TFEB nuclear localization and this process required a C-terminal serine-rich motif. Serine substitution to non-phosphorylatable residues abrogated TFEB regulation by mTORC1. More importantly, mutation to phosphomimetic amino acids was sufficient to reproduce the effect of mTORC1 on TFEB nuclear localization obviating the need for mTORC1. These data support a model in which mTORC1 (or an effector kinase) directly phosphorylates a serine(s) within the C-terminal serine-rich motif driving thereby TFEB to the nucleus. Consistent with this notion, nuclear TFEB was phosphorylated. This model may seem at odds with the observation that nuclear TFEB is fast-migrating and possibly hypo-phosphorylated by comparison to cytoplasmic (presumably cytosolic) TFEB. However, as our data suggest, nuclear TFEB may be phosphorylated on sites that are not phosphorylated on cytosolic TFEB, and thus no inferences can be drawn about the degree of phosphorylation based on SDS–PAGE migration. Furthermore, while we have identified a critical motif involved in mTORC1 regulation, other sites in TFEB appear to be regulated by mTORC1 (Yu et al, 2011). Our results further emphasize the link between mTORC1 and the late endosome/lysosome and provide evidence for the existence of bidirectional regulatory loops. mTORC1 localizes to the surface of the late endosome/lysosome in a highly choreographed manner (Korolchuk et al, 2011; Narita et al, 2011). The late endosome/lysosome is necessary for mTORC1 activation (Flinn et al, 2010; Li et al, 2010; Sancak et al, 2010) and mTORC1 is reactivated under conditions of persistent starvation following macromolecule degradation in autolysosomes (Yu et al, 2010). However, not only is mTORC1 downstream of the lysosome, but, inasmuch as mTORC1 regulates TFEB and V-ATPases, mTORC1 is also upstream. mTORC1 reactivation by autolysosomes requires Spinster, a lysosomal efflux sugar transporter. Spinster has been proposed to act by regulating lysosomal pH (Rong et al, 2011), which is controlled primarily by the V-ATPase. Interestingly, not only has luminal pH been implicated in mTORC1 regulation, but changes in cytosolic pH occur in response to alterations in nutrient conditions (Korolchuk et al, 2011) and have been implicated in the regulation of lysosome topology and mTORC1 (Korolchuk et al, 2011). Recently, TFEB was found to regulate autophagy gene expression (Settembre et al, 2011). Starvation led to ERK2 inactivation, which in turn, decreased TFEB phosphorylation and increased its nuclear localization. This model is in principle compatible with our results. TFEB is phosphorylated in over 10 sites (Dephoure et al, 2008; Mayya et al, 2009; Yu et al, 2011), and mechanisms likely exist that drive TFEB into the nucleus independently of the activation state of mTORC1. However, the studies do differ in one aspect. Whereas mutation of the putative ERK2 phosphorylation site, S142, to A is reported to be sufficient to drive TFEB into the nucleus (Settembre et al, 2011), we find that, at least under low serum conditions, the subcellular localization of TFEB is not appreciably altered by a S142A mutation. The experimental conditions studied by Settembre et al (2011), which involve evaluating ectopically expressed TFEB in cells in the absence of glucose, amino acids and serum, are quite different from ours. Individual deprivation of glucose, amino acids or serum has profound, dynamic and diverse effects on TFEB (see Figure 9A). Given these observations and the complexity of TFEB phosphorylation (Dephoure et al, 2008; Mayya et al, 2009; Yu et al, 2011), how starvation of all three sources will affect TFEB is unclear. Consistent with this complexity, the changes in TFEB migration observed following deprivation of individual factors could not be explained by the predicted changes on mTORC1 activity. In fact, we speculate that the discovery that mTORC1 regulates TFEB may have been possible only because of the tight experimental system we used. Besides V-ATPases and lysosomal genes, many other genes downstream of mTORC1 were regulated in a Tfeb-dependent manner. Approximately 25% of genes induced by mTORC1 required Tfeb. Experiments are ongoing to determine how many of these genes are directly regulated by Tfeb. A similar experimental setup previously identified Hif-1α as an mTORC1 effector (Brugarolas et al, 2003), and Hif-1α is an important mediator of a metabolic gene expression programme (Duvel et al, 2010). Along with sterol regulatory element-binding protein-1 (Porstmann et al, 2008), a transcription factor whose nuclear localization is regulated by mTORC1, and Hif-1α, TFEB is an important mediator of mTORC1 effects on gene expression. The discovery that mTORC1 regulates TFEB has clinical implications. It may explain the expression of melanocyte lineage markers in tumours of patients with TSC (Martignoni et al, 2008), a syndrome arising from mutations in either the TSC1 or TSC2 genes, resulting in constitutive mTORC1 activation (El-Hashemite et al, 2003; Kenerson et al, 2007). Patients with TSC are predisposed to develop renal angiomyolipomas, which express melanocytic markers such as HMB45 and Melan-A (Martignoni et al, 2008), and since expression of these proteins in melanomas is thought to be driven by TFEB (or related family members with whom TFEB can heterodimerize) (Steingrimsson et al, 2004), these markers may be similarly driven by TFEB in renal angiomyolipomas. Interestingly, TFEB functions as an oncogene in the kidney. The TFEB gene is translocated in a subset of renal tumours where it is subjected to a robust heterologous promoter (Davis et al, 2003; Kuiper et al, 2003). Translocation carcinomas tend also to aberrantly express melanocytic markers (Srigley and Delahunt, 2009), which may be similarly driven by TFEB. TFEB activation may also account for the expression of cathepsin-K, a lysosomal protease that has been recently proposed as a marker for this tumour type (Martignoni et al, 2009). Interestingly, translocation carcinomas are often recognized by immunohistochemical studies showing high levels of nuclear TFEB (Srigley and Delahunt, 2009). Because TFEB nuclear localization is regulated by mTORC1 and as we show, sirolimus excludes TFEB from the nucleus, sirolimus, or its derivatives, temsirolimus and everolimus, may be effective against this tumour type. While this study focused on a small subset of genes, many genes were identified whose expression was regulated by mTORC1 not only positively but also negatively (Supplementary Figures S8–S10; Supplementary Table S1). Moreover, genes were also found whose expression was regulated by Tsc2 independently of mTORC1 (LLHH and HHLL patterns). As previously reported (Brugarolas et al, 2003), this would point towards mTORC1-independent functions of the Tsc1/Tsc2 complex (Supplementary Figure S8; Supplementary Table S1). In addition, the expression of some genes appeared to be regulated by rapamycin to a significantly greater extent than by Tsc2 (HLHL and LHLH patterns) (Supplementary Figure S8; Supplementary Table S1). Importantly, in all these settings, analyses of alternative patterns showed the associations to be statistically significant (Supplementary Figure S8). Incidentally, among the genes with an HLHL pattern, there was enrichment for genes implicated in glycolysis, pentose phosphate pathway and fatty acid biosynthesis (Supplementary Table S5). These pathways were recently reported to be regulated by mTORC1 (Duvel et al, 2010) and our results suggest that they are affected by rapamycin to a greater extent than by Tsc1/Tsc2. In summary, our work uncovered a novel regulatory network connecting mTORC1 to a master regulator of lysosome biogenesis, TFEB, which is essential for mTORC1-induced endocytosis. Materials and methods Cell culture, drug and hypoxia treatments MEFs Tsc2 +/+; p53 −/− (Tsc2 +/+) and Tsc2 −/−; p53 −/− (Tsc2 −/−) were a gift of DJ Kwiatkowski (Brigham and Women's Hospital, Harvard Medical School, Boston, MA). Cells were grown in high glucose Dulbecco's modified Eagle's medium (Sigma) supplemented with 10% FBS (HyClone) and 1% (v/v) penicillin/streptomycin (P/S) (Gibco) in a humidified incubator at 37°C and 5% CO2. For experiments, cells were plated to achieve ∼70% confluency at the time of treatment. For low-serum experiments, cells were washed twice in PBS and medium was changed to the same base medium but supplemented with 0.1% FBS and cells were harvested after 24 h. Where indicated, rapamycin (LC Laboratories) was used at 25 nM in methanol and methanol was used as vehicle. Where indicated, cells were treated with actinomycin D (Sigma) at 10 μg/ml, bafilomycin A1 (LC Laboratories) at 100 nM, or DTT (10 mM). For growth curves, cells were plated on day 0, maintained in 10% FBS and triplicates were counted every 24 h. For hypoxia experiments, cells were exposed to 1% O2 and 5% CO2 at 37°C in a hypoxia chamber (Coy Laboratory Products). For starvation time courses, cells were washed with PBS twice and starved of amino acids (amino acid-free media), glucose (glucose-free media) or serum (0.1% FBS) for different amounts of time. Hif-1α, Tfeb, Raptor and TSC2 shRNAs Recombinant lentiviruses were generated in HEK293T cells by transient transfection using calcium phosphate with the envelope plasmid pMD2.G (Database ID p486), packaging plasmid psPAX2 (p485) and the specific plasmid of interest. pMD2.G, psPAX2 and pLKO.1 or pGIPZ vectors were transfected at a ratio of 3:8:10. Supernatants were collected for 2 days, passed through a 0.45-μm filter, and used to infect MEFs or HeLa cells for 24 h in medium containing 8 μg/ml polybrene (Sigma). MEFs and HeLa cells were selected with 2 μg/ml puromycin for at least 1 week and HeLa cells were further enriched by FACS. The following vectors were used: pLKO.1 vectors with a Hif-1α shRNA (5′-AGAGGTGGATATGTCTGGG-3′) (p481) generously provided by LB Gardner (Nemetski and Gardner, 2007), a mouse Tfeb shRNA from Open Biosystems (sequence A, TRCN0000085548, 5′-CGGCAGTACTATGACTATGAT-3′) (p633) or a designed Tfeb shRNA (sequence B, 5′-GGCAGTACTATGACTATGATG-3′) (p652), a human TFEB shRNAs from Open Biosystems (sequence 110, TRCN0000013110, 5′-GAGACGAAGGTTCAACATCAA-3′) (p700) or (sequence 111, TRCN0000013111, 5′-GAACAAGTTTGCTGCCCACAT-3′) (p701), a raptor shRNA from Addgene (Addgene plasmid 1858) (p741) or a scrambled sequence (5′-GGGTCTGTATAGGTGGAGA-3′) (p480). pGIPZ vectors containing a TSC2 (p496) or a scrambled (p483) shRNA were from Open Biosystems. Western blotting and antibodies Cell lysates and western blotting was performed as previously described (Vega-Rubin-de-Celis et al, 2010), using antibodies from the following sources: S6K1, phospho-S6K1 (T389), S6, phospho-S6 (S235/236), total 4E-BP1, phospho-4E-BP1 (T37/46), phospho-4E-BP1 (S65) from Cell Signaling; HIF-1α, TSC1 and Menin from Bethyl laboratories; TSC2, ATF4 and c-Myc from Santa Cruz; Raptor from Millipore; Erk1/2, phospho-Erk1/2 (T183/Y185) and Tubulin from Sigma; Cyclophilin B from Abcam; GLUT-1 from Novus Biologicals; and Grp78 from BD Biosciences. Atp6v1a and Atp6v1b2 antibodies were a generous gift from D Brown (Massachusetts General Hospital, Boston, MA); and Atp6v0a1 and Atp6v0a3 antibodies were kindly provided by V Marshansky (Massachusetts General Hospital, Boston, MA). Tfeb antibodies were developed in collaboration with Bethyl Laboratories. For the remaining Materials and methods, please see the Supplementary data section. Accession numbers Microarrays were deposited in GEO (http://www.ncbi.nlm.nih.gov/geo/) under accession numbers GSE27982 and GSE28021. Supplementary Material Supplementary Data Supplementary Table S1 Supplementary Table S3 Supplementary Table S4 Review Process File
Author and article information
Journal
Journal ID (nlm-ta): Int J Mol Sci
Journal ID (iso-abbrev): Int J Mol Sci
Journal ID (publisher-id): ijms
Title:
International Journal of Molecular Sciences
Publisher:
MDPI
ISSN
(Electronic):
1422-0067
Publication date
(Electronic):
20
June
2020
Publication date Collection:
June
2020
Volume: 21
Issue: 12
Electronic Location Identifier: 4392
Affiliations
[1
]Department of Paediatric Haematology and Oncology, 2nd Faculty of Medicine, Charles
University and Motol University Hospital, CZ-150 06 Prague, Czech Republic;
janhrabeta@
123456gmail.com
(J.H.);
mariebelhajova@
123456gmail.com
(M.B.)
[2
]Department of Chemistry and Biochemistry, Mendel University in Brno, CZ-613 00 Brno,
Czech Republic;
hanasub7@
123456gmail.com
(H.Š.);
merlos19792003@
123456gmail.com
(M.A.M.R.);
zbynek.heger@
123456mendelu.cz
(Z.H.)
Author notes
Author information
Hana Šubrtová
https://orcid.org/0000-0001-9268-4275
Miguel Angel Merlos Rodrigo
https://orcid.org/0000-0002-1920-0948
Zbyněk Heger
https://orcid.org/0000-0002-3915-7270
Article
Publisher ID: ijms-21-04392DOI: 10.3390/ijms21124392
PMC ID: 7352242
PubMed ID: 32575682
SO-VID: 47ef0fd7-48f5-4a6e-93a8-d8f33d89ace0
Copyright © © 2020 by the authors.
License:
Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( http://creativecommons.org/licenses/by/4.0/).
History
Date
received
: 26
May
2020
Date
accepted
: 18
June
2020
Categories
Subject:
Review
ScienceOpen disciplines: Molecular biology
Data availability:
ScienceOpen disciplines: Molecular biology