Transcription factor EB (TFEB) is a master regulator of lysosomal biogenesis and autophagy with critical roles in several cancers. Lysosomal autophagy promotes cancer survival through the degradation of toxic molecules and the maintenance of adequate nutrient supply. Doxorubicin (DOX) is the standard of care treatment for triple-negative breast cancer (TNBC); however, chemoresistance at lower doses and toxicity at higher doses limit its usefulness. By targeting pathways of survival, DOX can become an effective antitumor agent. In this study, we examined the role of TFEB in TNBC and its relationship with autophagy and DNA damage induced by DOX. In TNBC cells, TFEB was hypo-phosphorylated and localized to the nucleus upon DOX treatment. TFEB knockdown decreased the viability of TNBC cells while increasing caspase-3 dependent apoptosis. Additionally, inhibition of the TFEB-phosphatase calcineurin sensitized cells to DOX-induced apoptosis in a TFEB dependent fashion. Regulation of apoptosis by TFEB was not a consequence of altered lysosomal function, as TFEB continued to protect against apoptosis in the presence of lysosomal inhibitors. RNA-Seq analysis of MDA-MB-231 cells with TFEB silencing identified a down-regulation in cell cycle and homologous recombination genes while interferon-γ and death receptor signaling genes were up-regulated. In consequence, TFEB knockdown disrupted DNA repair following DOX, as evidenced by persistent γH2A.X detection. Together, these findings describe in TNBC a novel lysosomal independent function for TFEB in responding to DNA damage.
Breast cancer is a leading cause of death among women. Notably, five-year survival rates have increased from 75% in 1975 to 91% in 2015 [1–3], partly due to the development of targeted therapies such as estrogen receptor and human epidermal growth factor receptor 2 inhibitors . However, there remains a significant percentage of breast cancers, namely triple-negative breast cancers (TNBC), which cannot be treated by targeted therapies and only respond to cytotoxic chemotherapy, typically either taxanes or doxorubicin (DOX) [4–6]. DOX, an anthracycline antibiotic, induces cellular apoptosis through intercalation and topoisomerase II inhibition, resulting in DNA double-strand breaks, with concomitant generation of mitochondrial reactive oxygen species (mtROS) [7–9]. Limitations to using DOX are that remission is achieved in only 30% of patients, and high cumulative doses of DOX cause cardiotoxicity [5,10,11]. Therefore, it is crucial to understand mechanisms through which breast cancer cells survive DOX-induced cell stress and exploit therapeutic targets for lowering the effective dose of DOX. Acquired resistance to chemotherapeutic agents in breast cancer patients is demonstrated to be an outcome of altered drug transporter expression and efflux, lysosomal trapping of the drug and adaptive activation of proliferative and survival signaling such as lysosomal autophagy which enables cancer cells to evade the toxicity of chemotherapeutics [12–15].
Autophagy is a catabolic process crucial for the maintenance of cellular homeostasis . Autophagy is considered both a tumor suppressor during the early stages of neoplasia as well as a contributor to cancer growth and proliferation . During nutrient insufficiency, autophagy sustains growth and supports cancer progression by recycling cellular and extracellular macronutrients. Autophagy also prevents proteotoxicity and oxidative stress through degrading damaged cellular organelles and molecules [13,16]. Furthermore, the autophagy-lysosome system is implicated in cancer cell resistance to cytotoxic chemotherapy treatment through sequestration of basic molecules . Earlier studies have found that anthracycline treatment results in stimulation of lysosomal autophagy, specifically in cancer cells, as an adaptive survival mechanism [18–22]. The strategy of targeting autophagy is clinically tested in multiple cancer types, including breast cancer; using the lysomotropic agent chloroquine (CQ) , while pre-clinical animal and in vitro models suggest that autophagy inhibition is efficacious in sensitizing breast cancer cells to anthracycline-based chemotherapy [18,19,22,23]. Despite the evidence that autophagy is activated by and promotes resistance to DNA damaging agents, little is known about how the DNA damage response regulates autophagy or if autophagy promotes DNA repair.
Due to the physiological importance of the autophagy-lysosome system, it is subject to stringent regulation to prevent uncontrolled catabolism. The primary promoters of lysosomal biogenesis and function are the microphthalmia (MiT/TFE) family of transcription factors . The MiT/TFE protein family of basic helix–loop–helix transcription factors consists of MITF, TFE3, TFEC, and TFEB, of which TFEB is best characterized for its role in the regulation of lysosomal function . Upon mTOR phosphorylation, TFEB is restricted to the cytosol through binding with 14–3–3, however, in the absence of mTOR repression, TFEB translocates to the nucleus . TFEB binds to CLEAR (Co-ordinated lysosomal enhancement and regulation) promoters, which are upstream of several lysosomal and autophagy genes . The MiT/TFE family of proteins are frequently overexpressed in several cancers, including pancreatic ductal adenocarcinoma, melanoma, and renal cell carcinoma [27–29]. Recent studies have also determined that TFEB protein overexpression in breast tumors is associated with increased mortality, while expression of a constitutively active TFEB isoform in breast cancer cells promotes tumor growth in mouse xenografts [30,31]. It remains to be examined if or how the molecular subtype of breast cancer alters reliance on TFEB for survival. Two prior studies have found that TFEB is activated by DOX in LoVo colorectal cancer cells and MCF7 breast cancer cells [17,32]. Additionally, one study showed that TFEB function promotes resistance to DOX in LoVo colorectal cancer cells . Further research is warranted to examine if in TNBC cells, TFEB is activated by DOX and whether TFEB counters DOX-induced DNA damage through regulation of lysosomal function or a non-lysosomal pathway.
In the current study, we characterized the role of TFEB in TNBC during DOX treatment and explored the link between TFEB, autophagy, and the DNA damage response. Our results demonstrated that TFEB is activated and hypo-phosphorylated in MDA-MB-231 and BT549 cells upon treatment with DOX. TFEB knockdown is sufficient to exacerbate DOX-induced apoptosis in MDA-MB-231, BT549, and SUM159 cell lines while simultaneously decreasing cell viability. We find that inhibition of the TFEB phosphatase calcineurin increases Dox-induced apoptosis while overexpression of constitutively active TFEBS211A rescues the effect. Our data show that this phenotype is not dependent on lysosomal function as TFEB mediated protection from apoptosis persists in the presence of lysosomal inhibitors. Using transcriptome analysis in breast cancer cells, we uncovered the role of TFEB in controlling genes related to homologous recombination, cell cycle, and interferon-γ signaling pathways. In accordance, knockdown of TFEB leads to increased DNA damage, as measured by γH2A.X, and decreased repair of DNA damage following DOX treatment. Together these results describe a novel transcription network in TNBC cells regulated by TFEB, which promotes expression of DNA repair and cell survival genes in response to genotoxic stress.
Materials and methods
Cell lines, chemicals, and antibodies
MCF10A cells were obtained from American type culture collection (ATCC, CRL-10317) and grown in DMEM/F12 1:1 (Hyclone) supplemented with 5% horse serum, l-glutamine (2 mM), sodium pyruvate (1 mM), insulin (10 µg/ml), epidermal growth factor (20 ng/ml), hydrocortisone (500 ng/ml), and cholera toxin (100 ng/ml). MDA-MB-231 cells were a gift from Dr. G. Robichaud (Université de Moncton) and grown in DMEM high glucose + 9% fetal bovine serum. BT-549 cells were obtained from ATCC (HTB-122) and cultured in RPMI 1640 supplemented with 9% fetal bovine serum and 0.8 µg/ml Insulin. SUM159 cells were grown in Ham's F12 + 5% FBS, 5 µg/ml Insulin, 1 µg/ml Hydrocortisone, and 10 mM HEPES. The cell lines have not been genetically authenticated. Primary and secondary antibodies are listed in Supplementary Table S1.
Expression of TFEB-Ha (SKU#: ADV-225358), mutant TFEBS211A-Flag, and TFEB shRNA (shADV-225358) was accomplished using adenoviral vectors obtained from Vector Biolabs. The control vectors, Ad-GFP, Ad-mCherry, and scrambled shRNA GFP, were also obtained from Vector Biolabs (Cat#: 1060, 1767, and 1122, respectively). The CLEAR luciferase construct was a gift from Dr. A. Ballabio (TIGEM). SiRNA knockdown of TFEB was performed using Ambion silencer select siRNA oligonucleotides (Thermo-Fisher Scientific Cat# 4392420). The siRNAs used in this paper were siTFEB#1: # s15495, siTFEB#2: # s15496, siRNA negative control Cat# 4390844. Transfection was achieved using Lipofectamine RNAiMAX following the manufacturer's instructions with a concentration of 10 nM of siRNA per plate.
Cell culture and preparation of lysates, and immunoblotting
All cell culture experiments described were conducted on cells between passage number 3 and 20. Adenoviral infection of cells was done 24 h post-plating, and the multiplicity of infection (MOI) was kept constant between the control and experimental constructs. All experiments were conducted within 72 h post-infection, except viability (120 hours), and colony formation assays (10–15 days). Cell lysate generation and immunoblotting was performed as previously described . Protein levels were quantified by densitometry using Image Lab (Bio-rad), and corrected to the protein stain. Immunoblots and quantification represent three independent experiments.
Immunofluorescence and fluorescence microscopy
Cells were plated on glass coverslips in 35 mm dishes and allowed to settle for 24 h. Subsequently, cells were washed three times in warm PBS before being fixed with pre-warmed 4% formaldehyde in PBS for 5 min. Cells were blocked and permeabilized with 3% bovine serum albumin (BSA) and 0.1% Triton-X-100 in PBS for 30 min before incubation with the primary antibody for 1 h followed by incubation with fluorophore-conjugated secondary antibodies for 1 h.
Cellular proteolytic activity was assayed using DQ-BSA Red by incubating cells with 100 µg/ml DQ-BSA in serum-free media for 5 h after which cells were washed in PBS and fixed with 4% formaldehyde. Lysosomes were labeled using LysoTracker Red DND-99 or LysoTracker Green DND-26 by incubating cells with 250 nM of the dye in media for 15 min before being washed in PBS and fixed with 4% formaldehyde.
Coverslips were mounted onto glass slides containing Vectashield with DAPI (Vector Laboratories) and imaged with a Zeiss Axio Observer Z1 equipped with an Apotome.2 structural illumination unit using a 63× Plan-Apochromat objective (NA: 1.4, oil) or 20× LD A-Plan objective (NA: 0.35, air). Images were processed for analysis in Zen (Carl Zeiss), and image processing was identical for each image set. Images were analyzed in Cell Profiler, and data are represented as the mean per cell unless otherwise noted [35,36]. Experiments with data displayed from one independent experiment have been repeated at least once.
Cell viability and colony formation assay
Cell viability was assessed using Presto Blue (Thermo-Fisher Scientific). Briefly, cells were seeded in 96-well plates and following 18 h of DOX treatment, the cells were washed once with PBS and incubated for 48 h in drug-free media. Following 48 h, Presto Blue was added to the media, the plate was incubated for 3 h at 37°C, and fluorescent intensity was read on a microplate fluorometer (Synergy H4).
For colony formation assays, cells were transfected with siRNAs and incubated for 2 days. Cells were then trypsinized and re-plated at a density of 500 cells per well. Cells were allowed to adhere for 24 h before being treated. After treatment, the media was changed every 3 days for 10–13 days, then colonies were fixed with 4% formaldehyde and stained with 0.5% Crystal Violet and counted manually. Mean values represent six treatments from two independent experiments.
Cell permeability assay
The microtitre plate cell permeability apoptosis assay was conducted using SyTOX Blue and Hoechst 33342 (Thermo-Fisher Scientific). Cells were seeded in a 96 well plate and incubated for 1 h at room temperature to reduce edge effects. Cells were incubated with SyTOX Blue and Hoechst 33342 for 30 min before each well of the plate was imaged once with a Zeiss Axio Observer Z1 at 5× magnification. Images were processed in ImageJ and the number of SyTOX Blue and Hoechst 33342 cells counted with Cell Profiler. The fraction of SyTOX Blue to Hoechst 33342 positive cells was calculated for each well and data is displayed as a mean of at least five wells per treatment.
Luciferase reporter assay
CLEAR promoter activity was assayed using a firefly luciferase reporter gene driven by two CLEAR sequences, as described previously . Cells were transduced with adenoviruses containing the CLEAR-luciferase construct and luciferase activity was measured 72 h after adenoviral transduction and following 18 h of DOX treatment. Protein lysates were collected by adding passive lysis buffer (Biotium) to each plate before 30 min of shaking, after which lysates were clarified by centrifugation. Luciferase activity was measured according to the manufacturer's instructions with a Synergy H4 plate reader, and luminescence was normalized to protein concentration. Values are represented as fold change to vehicle-treated cells.
RNA-seq analysis and bioinformatics
MDA-MB-231 cells were transfected with either of two siRNA's targeting TFEB or a non-targeting control and cultured for 48 h before cells were harvested and RNA extracted using the Qiagen RNeasy mini kit according to the manufacturer's instructions. RNA-Seq was conducted by McGill University and the Genome Quebec Innovation Center (Montreal, Canada) with the Illumina NovaSeq 6000 S2 PE100 — 50 M platform. Paired-end reads were pseudoaligned to the Homo sapiens GRCh38 transcriptome and quantified using kallisto . Transcripts were summarized to gene-level counts with the R package tximport and testing for differential gene expression was completed with DESeq2 [39,40]. Testing for differential expression was conducted only on genes with an average estimated count of greater than 0.3. Genes were considered significantly differentially expressed if the adjusted P-value was less than 0.01 for both siTFEB#1 and siTFEB#2 groups, and the fold change was occurring in the same direction. GO and KEGG term enrichment was conducted with DAVID and EnrichR [41,42]. Geneset enrichment analysis was accomplished with GSEA 3.0 (Broad Institute) using the parameters: Number of permutations = 1000 and Permutation type = geneset, and the gene-level count for siTFEB#1 and siTFEB#2 was averaged together to create the ranked list [43–46]. Heatmap visualizations were created with Morpheus: (https://software.broadinstitute.org/morpheus). RNA-Seq data is desposited in NCBI's Gene Expression Omnibus under the assecession number GSE139203 .
Statistical analysis was performed with Graph Pad Prism 6. Data sets with three or more groups were analyzed using one-way, or two-way analysis of variance (ANOVA) as appropriate and significant differences between individual groups were assessed with Tukey's post-hoc test. Data sets with two groups were analyzed using a two-tailed student's t-test. Statistical significance was attributed if the P-value was less than 0.05. Data are displayed with a as mean +/− standard error of the mean (SEM) unless otherwise noted.
DOX activates TFEB in breast cancer cell lines
Prior studies have shown that DOX induces autophagy in TNBC and prostate cancer, while TFEB nuclear translocation and mTOR deactivation in response to DOX has been found in MCF7 and LoVo breast and colorectal cancer cells [17,19,21,32]. We sought to confirm the induction of this pathway in TNBC cells. We first investigated whether TFEB protein levels are altered by DOX treatment in MDA-MB-231 and BT549 claudin-low breast cancer cells, a subtype of TNBC, as well as in MCF7 luminal A breast cancer cells and MCF10A non-cancerous breast cells. TFEB protein expression was up-regulated upon 1 µM DOX treatment in MCF10A, MCF7, and MDA-MB-231 cells but not in BT549 cells (Figure 1A,B). Following DOX treatment all cell lines displayed a distinct molecular mass decrease of ∼5 kDa for the 70 kDa TFEB protein suggesting a posttranslational modification. Although prior studies have found TFEB nuclear translocation in response to DOX, it was unknown if altered phosphorylation of TFEB contributes to its altered localization. Indeed, DOX treatment decreased phosphorylation of TFEB at serine 211 in all four cell lines tested as evident from bands of phosphorylated TFEB at 70 and 50 kDa, which represent two TFEB splice isoforms  (Figure 1A,B). We next ascertained if DOX modulates upstream regulators of TFEB. Activated mTORC1 phosphorylates TFEB at serine 211 [49–51], however, DOX treatment had no significant effect on S6K phosphorylation, a downstream signaling readout of mTOR activity, in MCF10A, MDA-MB-231, and BT549 cells however a marginal but significant decrease was observed in MCF7 cells (Figure 1A,B). We also found that DOX treatment increased the phosphorylated to total ratio of S6 at threonine 240/244 in MDA-MB-231 cells, and was unaltered in the other cell lines tested (Figure 1A,B). Taken together, these results show that TFEB dephosphorylation occurs independently of mTORC1 activity in MDA-MB-231 and BT549 cells.
DOX treatment activates TFEB in breast cancer cells.
We next questioned if TFEB activation in MDA-MB-231 cells following incubation with DOX caused TFEB nuclear translocation. DOX-treated MDA-MB-231 cells displayed increased nuclear staining compared with vehicle control, as determined by immunofluorescence microscopy (Figure 1C,D). Using a luciferase gene reporter assay in which firefly luciferase is driven by two CLEAR sequences  we further confirmed that DOX treatment activated TFEB. In MDA-MB-231 cells, DOX caused a significant increase in luciferase activity compared with vehicle, indicating increased TFEB transcriptional activity (Figure 1E). These results suggest that DOX is sufficient to activate TFEB in breast cancer cell lines.
Loss of TFEB reduces MDA-MB-231 cell viability alone and in combination with DOX
We hypothesized that TFEB activation by DOX was a cytoprotective response. To study this, we incubated MDA-MB-231 cells with DOX for 18 h followed by a chase in drug-free media for 48 h, then estimated viability using the reduction in resazurin (a proxy for metabolic activity) through the addition of the Presto Blue reagent. In MDA-MB-231 cells, knockdown of TFEB with two siRNAs (Supplementary Fig. S1A) resulted in a significant reduction in metabolic activity, which was further decreased by DOX (Figure 1F). Also, knockdown of TFEB with two different siRNAs reduced colony-forming ability of MDA-MB-231 cells by between 50 and 20% (Figure 1G,H). To ascertain whether decreased viability was an outcome of augmented cell death, we assayed the rate of cell death in TFEB knockdown cells through measurement of membrane permeability. We observed a 7.5–10-fold more permeable cells following TFEB knockdown when compared with control, which indicates a significant increase in cell death for cells lacking TFEB (Figure 1I). These results show that TFEB knockdown leads to loss of viability and increased cell death.
TFEB knockdown reduces the viability of BT549 and SUM159 cells
We next explored whether the phenotype caused by TFEB knockdown was specific to MDA-MB-231 cells or observed in other claudin-low models such as BT549 and SUM159 cells. Knockdown of TFEB in BT549 cells with two different siRNAs (Supplementary Fig. S1B) reduced metabolic activity by 50 and 25%, and this effect was additive when combined with DOX (Figure 2A). TFEB knockdown in SUM159 cells resulted in no significant change to metabolic activity; however, we find that TFEB knockdown led to a two-fold higher sensitivity to DOX at concentrations of 0.5 and 1 µM (Figure 2B,C). Similarly, TFEB knockdown (Supplementary Fig. S1C) did not reduce the colony-forming ability of SUM159 cells; however, colonies from TFEB siRNA treated cells were significantly smaller, as measured by the percent area covered by cells (Figure 2D–F). The colony-forming ability of SUM159 cells remained unaffected by DOX treatment; however, when TFEB knockdown is combined with DOX, viability is reduced by 50% (Figure 2G). These results show that TFEB knockdown significantly reduces the viability of three different claudin-low breast cancer cell lines.
TFEB knockdown reduces the viability of BT549 and SUM159 cells.
TFEB regulates Caspase-3 dependent cell death
Since TFEB knockdown caused loss of viability in TNBC cell lines, we examined whether this corresponded with increased apoptosis signaling and changes in autophagy proteins. Adenoviral delivery of short-hairpin RNA (shRNA) targeting TFEB in MDA-MB-231 cells resulted in a substantial reduction in TFEB protein levels, and impaired autophagy (Figure 3A,B). The extent of autophagic activation was ascertained by examining protein levels of non-lipidated LC3-I and lipidated LC3-II, respectively. DOX treatment of MDA-MB-231 cells elicited a significant increase in both LC3-I and LC3-II, whereas knockdown of TFEB attenuated DOX-induced amplification of LC3-I and II independent of statistically significant changes in autophagy cargo receptor, SQSTM1 (Figure 3A,B). These results suggest that DOX-induced TFEB activation up-regulates protein levels of LC3B. Knockdown of TFEB in MDA-MB-231 cells also resulted in a two-fold increase in protein levels of cleaved caspase-3, an executor of apoptosis, in response to 1 µM DOX when compared with control (Figure 3A,B). In BT549 cells, TFEB knockdown in combination with DOX treatment resulted in a significant increase in caspase-3 cleavage (Figure 3C,D) when compared with control. Surprisingly, shRNA targeting of TFEB in BT549 cells did not lead to significant changes in levels of the autophagy-related proteins SQSTM1 and LC3-I (Figure3C,D). Similar to MDA-MB-231 cells, DOX-induced an increase in LC3-II content in BT549 cells, which was not observed with TFEB knockdown (Figure 3C,D).
TFEB regulates Caspase-3 activation.
To further test if the pro-survival role of TFEB was specific to DOX-treated cancer cells, TFEB was overexpressed in non-cancerous MCF10A cells. Overexpression of TFEB in MCF10A cells caused a two-fold increase in LC3-I with a corresponding increase in LC3-II (Figure 3E,F). MCF10A cells treated with 1 µM DOX showed a significant increase in cleaved caspase-3 protein levels; however, TFEB overexpression blunted this increase. (Figure 3E,F). Surprisingly, overexpression of TFEB did not rescue metabolic activity in response to DOX in MCF10A cells (Figure 3G). These results show that TFEB functions to prevent caspase activation in multiple cell types in response to DOX and create a pro-survival milieu within the cell.
Calcineurin inhibition sensitizes breast cancer cells to DOX
TFEB is dephosphorylated by the phosphatase Calcineurin (CaN), while CaN agonists and antagonists are capable of modulating TFEB activity [53,54]. We investigated whether DOX treatment in breast cancer cells alters CaN protein expression resulting in increased TFEB dephosphorylation. MDA-MB-231 cells treated with DOX for 18 h exhibited a significant decrease in CaN protein levels while there was no change in the other cell lines studied, effects that are inconsistent with TFEB dephosphorylation (Supplementary Fig. S2). To investigate whether CaN activity was altered by DOX, leading to increased TFEB activation, BT549 cells were treated CaN chemical inhibitor cyclosporine A and its ability to phenocopy TFEB knockdown analyzed. Indeed, BT549 cells co-treated with 10 µM cyclosporine A (CsA) and 1 µM DOX led to a significant increase in cleaved caspase-3 and cleaved PARP compared with DOX treatment alone (Figure 4A,B). To test whether TFEB inhibition precipitated the pro-apoptotic effect of CsA, BT549 cells were transduced with either mutant TFEBS211A or wildtype TFEB. Both TFEB and TFEBS211A overexpression were sufficient to rescue DOX-induced apoptosis, as shown by reduced levels of cleaved PARP and cleaved caspase-3; however, only TFEBS211A expression was sufficient to rescue increased apoptosis caused by the combination of CsA and DOX (Figure 4A–D). Even though mutant TFEBS211A rescued the induction of apoptosis by CsA and DOX, CsA treatment did not change the phosphorylation status of TFEB at serine 211 in the presence of DOX; however, this phosphorylation was significantly increased by CsA in VEH treated cells (Figure 4A,B). Together, these findings suggest that phosphatase activity is necessary for the pro-survival function of TFEB.
Calcineurin inhibition enhances doxorubicin-induced apoptosis in a TFEB dependent manner.
DOX augments lysosomal function in a TFEB-independent manner
Since a primary outcome of TFEB activity is lysosomal biogenesis and activation, we hypothesized that lysosomal function would be comprised by TFEB knockdown, leading to cell death and decreased viability. First, we assayed whether DOX treatment in MDA-MB-231 cells was associated with increased lysosomal proteolysis by performing the DQ-BSA Red (double quenched bovine serum albumin) assay. DQ-BSA Red is taken up by cells and subsequently degraded in the lysosome, eliminating self-quenching and resulting in fluorescence. We observed that treatment with 1 µM DOX results in a two-fold increase in DQ-BSA fluorescence compared with the vehicle, indicating that DOX significantly increases lysosomal activity (Figure 5A,B). Likewise, MDA-MB-231 cells treated with DOX for 18 h depicted a significant increase in the number of lysosomes per cell after staining with the acidophilic dye LysoTracker Red DND-99 (Figure 5C,D). To test whether increased lysosomal biogenesis was caused by TFEB, we visualized lysosomes in DOX treated MDA-MB-231 cells with and without TFEB silencing. Surprisingly, TFEB knockdown in DOX treated MDA-MB-231 cells did not suppress lysosomal biogenesis as measured by fluorescence microscopy of LysoTracker stained cells (Figure 5C,D).
DOX induces lysosomal biogenesis independently of TFEB.
Since TFEB knockdown did not suppress lysosomal biogenesis induced by DOX, we sought to confirm whether individual autophagy-lysosome genes may be affected. Treatment with DOX resulted in a significant increase in the mRNA expression of TFEB, MCOLN1 (Mucolipin-1), ATP6V1H (ATPase H+ Transporting V1 Subunit H), HEXA (Hexosaminidase A), ATP6V1E1 (ATPase H+ Transporting V1 Subunit E1), and SGSH (N-Sulfoglucosamine Sulfohydrolase) (Figure 5E). Notably, only up-regulation of ATP6V1E1 and SGSH was suppressed by TFEB knockdown, while the other genes examined remained unaffected (Figure 5E). Knockdown of TFEB also significantly reduced mRNA expression of ZKSCAN3, a transcriptional repressor of autophagy , likely a compensatory response to decreased TFEB levels (Figure 5E). Altogether, these results suggest that TFEB is not necessary for the induction of the autophagy-lysosome pathway by DOX in MDA-MB-231 cells.
Anti-apoptotic functions of TFEB are independent of its lysosomal action
Prior studies have attributed the pro-survival function of TFEB to its role in regulating lysosomal biogenesis [27,32]. Since TFEB knockdown did not disrupt autophagy-related genes or lysosomal biogenesis in response to DOX equally in all cell lines, we questioned if the lysosomal function was necessary for the anti-apoptotic effects of TFEB expression in TNBC cells. To study this, we overexpressed a constitutively active form of TFEB with serine 211 mutated to alanine (S211A) in BT549 cells (Supplementary Fig. S1D) and co-treated the cells with DOX + 100 µM CQ or DOX + 25 nM BafA1. In both experiments, the expression of TFEBS211A was sufficient to reduce the cleavage of caspase-3 and PARP (Figure 6A,B and Supplementary Fig. S3). Incubation of BT549 cells with autophagy inhibitors CQ or BafA1 elicited accumulation of LC3-II (prominently in the TFEBS211A groups) and the loss of Cathepsin D maturation into the active 25 kDa isoform, which was associated with increased levels of cleaved caspase-3 and cleaved PARP (Figure 6A,B and Supplementary Fig. S3). Notably, despite the presence of lysosomal inhibitors, TFEBS211A expression remained sufficient to rescue levels of cleaved caspase-3 and PARP. The ability of TFEBS211A expression to reduce cellular apoptosis is not dependant on reversing the effect of lysosomal inhibitors since the level of active cathepsin D remains low and similar to the vector control (Figure 6A,B and Supplementary Fig. S3). Also, we observe that 18 h treatment with either CQ or BafA1 is sufficient to eliminate LysoTracker staining in BT549 cells, while expression of TFEBS211A could not rescue this effect (Supplementary Fig. S4A,B). This result shows that lysosomal function is not required for TFEB to inhibit apoptosis and reduce DNA damage in BT549 cells.
Inhibition of lysosomal acidification does not prevent the effects of TFEB overexpression.
RNA-Seq identifies TFEB-dependent regulation of the cell cycle and DNA repair genes in TNBC cells
To identify the pathways which are regulated by TFEB in TNBC cells, MDA-MB-231 cells were treated with either control siRNA or one of two siRNA's targeting TFEB exons 4 or 7 and subjected to RNA-Seq transcriptomic analysis. Principle component analysis (PCA) and distance clustering showed that the transcriptome of cells treated with siTFEB#1 and siTFEB#2 displayed little similarity (Figure 7A,B), thus we classified genes as differentially expressed compared with control if the adjusted P-value was below 0.01 for each siRNA individually and the fold change was occurring in the same direction. This method identified 1864 genes, which were differentially expressed by both TFEB siRNA's and over-represented Gene Ontology (GO), and KEGG terms were determined using DAVID (Figure 7C and Supplementary Fig. S5A). Numerous GO terms related to the cell cycle were enriched in the differentially expressed genes from TFEB knockdown cells, including ‘mitotic nuclear division’, ‘cell division’, and ‘G1/S transition of mitotic cell cycle’, while the KEGG term ‘Cell cycle’ was also enriched (Figure 7D, Supplementary Fig. S5A). Interestingly, genes with the associated GO term ‘DNA synthesis involved in DNA repair’, including RAD51, RAD51C, XRCC3, and EXO1, were found to be enriched in the list of genes differentially expressed by TFEB knockdown (Figure 7E). Gene set enrichment analysis (GSEA) was also conducted to evaluate enrichment in the up-regulated and down-regulated genes, which identified that numerous cell cycle and DNA recombination terms were over-represented the genes down-regulated by TFEB knockdown (Supplementary Fig. S5B). Likewise, GSEA identified that the KEGG pathway ‘Homologous Recombination’ is globally down-regulated by TFEB knockdown, which includes RAD51, RAD52, RAD54L/B, BRCA2, and three POLD subunits (Figure 7F,G). Together, this result shows that TFEB is necessary for the expression of homologous recombination genes breast cancer cells.
Transcriptomic analysis of MDA-MB-231 cells with TFEB knockdown.
To discover mechanisms by which TFEB knockdown induces cell death and decreases viability, we used GSEA to identify genesets that were significantly up-regulated by treatment with TFEB siRNA (Figure 8A). Notably, the Reactome geneset ‘Interferon γ signaling’ was found to be enriched in the group of genes up-regulated by TFEB knockdown, which includes Interferon-gamma receptors 1 and 2 (IFNGR1/2), IRF2, IRF7, and several major histocompatibility complex (MHC) class I and II genes (Figure 8B,C). Cross-talk between IFN-γ and the induction of death receptor signaling has been previously described, thus it is not surprising that several death receptor signaling genes were differentially regulated by TFEB knockdown [56–58]. Differentially expressed genes involved in death receptor signaling were found to be up-regulated after TFEB knockdown, including both TNF related apoptosis-inducing ligand (TRAIL/TNFSF10) and TNFRSF1A associated via death domain (TRADD), while apoptosis inhibiting proteins Baculoviral IAP Repeat Containing 2/3 (BIRC2/3) and BCL2 Associated Athanogene 4 (BAG4) were down-regulated (Figure 8D). These findings indicate that TFEB knockdown increases pro-apoptotic genes involved in IFN-γ and death receptor signaling.
Interferon signaling is up-regulated by TFEB knockdown.
TFEB augments DNA damage repair capacity of breast cancer cells
The primary mechanism of DOX-induced cell death in proliferating cells is the induction of DNA double-strand breaks through DNA intercalation and topoisomerase inhibition [59–61]. Prior reports suggest that tumors with the augmented capacity to undergo DNA damage repair offer resistance to genotoxic chemotherapy . Additionally, certain tumors can be sensitized to chemotherapeutic agents by co-treating them with DNA damage repair inhibitors [63,64]. Since DOX caused TFEB activation in breast cancer cells, and RNA-Seq identified a significant number of DNA repair genes which were down-regulated by TFEB knockdown, we questioned whether TFEB modulates DOX-induced DNA damage repair. As detected by immunofluorescence, knockdown of TFEB in untreated MDA-MB-231 cells did not cause a significant increase in the formation of γH2A.X foci (Figure 9A,B), a phosphorylated histone variant that marks the site of DNA damage . Likewise, following treatment with DOX, DNA damage levels as measured by γH2A.X were unaltered in TFEB knockdown cells compared with control (Figure 9A,B). We postulated that the kinetics of DNA damage repair might be altered by TFEB knockdown, and thus we examined DNA damage after 18 h of treatment followed by a 4 h chase in drug-free media. MDA-MB-231 cells deficient in TFEB exhibited two-fold greater DNA damage than control cells, indicating that TFEB is necessary for efficient DNA damage repair (Figure 9A,B). Furthermore, when MDA-MB-231 cells were treated with a 100 nM concentration of DOX, only a marginal increase in γH2A.X foci was found in control cells (Figure 9C,D). In contrast, 100 nM DOX treatment in TFEB knockdown cells caused a five-fold increase in the number of γH2A.X foci per nuclei, indicating a significant increase to the sensitivity to DOX-induced DNA damage, potentially mediated by a suppression of the DNA repair processes (Figure 9C,D).
TFEB knockdown delays DNA damage repair.
To confirm whether overexpression of TFEB could result in a reduction in DNA damage after DOX treatment, MDA-MB-231 cells were treated with control or TFEB overexpression adenoviruses and γH2A.X levels assessed by immunofluorescence. Although an equal proportion of DOX-treated cells were classified in γH2A.X positive (93% for both groups), overexpression of TFEB resulted in a slight but significant decrease in γH2A.X intensity (Figure 10A–C). Similarly, overexpression of TFEB in BT549 cells caused significantly decreased phosphorylation of H2A.X at serine 139 (i.e. γH2A.X) following 18 h of 1 µM DOX treatment compared to control (Figure 10D,E). We found that regulation of DNA repair by TFEB is not dependent on the lysosome. In BT549 cells treatment with lysosomal inhibitors CQ or Bafilomycin strongly increased DNA damage induced by DOX, however, overexpression of TFEBS211A was able to reverse this effect (Figure 11A,B). Likewise, expression of TFEBS211A was also sufficient to rescue γH2A.X increases caused by CsA treatment, but a similar effect was not observed with TFEB overexpression (Figure 11C,D). These findings, together with transcriptomics data, show that TFEB regulates DNA damage repair independently of its regulation of the lysosome.
TFEB overexpression reduces γH2A.X levels.
TFEB reduces H2A.X phosphorylation independently of the lysosome.
In summary, our results show that DOX activates TFEB in two claudin-low breast cancer cell lines and the suppression of TFEB by shRNA and siRNA knockdown sensitizes the cells to lower doses of DOX. Loss of TFEB disrupts DNA damage repair in breast cancer cell lines, which is associated with decreased expression of homologous recombination genes. The anti-apoptotic effect of TFEB is not the result of the increased lysosomal function and is dependent on the activity of calcineurin (Figure 12A,B).
Proposed mechanism for the role of TFEB in TNBC.
DOX and the related anthracycline class of chemotherapy agents are a commonly used treatment for TNBC and hormone receptor-positive cancers that are unresponsive to targeted therapy. TNBC is more responsive to cytotoxic chemotherapy, including DOX, than other subtypes of breast cancer. However, the therapeutic response rate remains low at ∼30% given that specific TNBC subtypes are inadequately susceptible to DOX [5,10,66]. Furthermore, TNBC is more prone to relapse than other types of breast cancer . The high rate of relapse, together with the frequent toxicity of DOX, when given in higher doses, has motivated several investigations into the mechanisms through which cancer cells resist chemotherapy. Several reports have identified that cytoprotective autophagy is activated in response to DOX and other chemotherapeutic agents in breast cancer; however, the regulators of this response remain uncharacterized. TFEB is a regulator of autophagy and is an emerging target for the treatment of cancer, given that several cancers rely on TFEB to survive . Indeed, TFEB and TFE3, both members of the MITF family with redundant functions, are distinctly involved in genetic rearrangements that drive the formation of renal cell carcinomas and sarcomas [29,68]. The goal of our study was to characterize the role of TFEB in TNBC and further studying the regulation of the pro-survival autophagy response in breast cancer induced by DNA damaging agents like doxorubicin.
This research has identified that the master regulator of lysosomal biogenesis, TFEB, is activated upon DOX treatment; however, knockdown of TFEB cannot completely suppress the activation by DOX of the autophagy-lysosome pathway in MDA-MB-231 cells. Our findings are in agreement with other studies that show that DOX increases autophagy in multiple cancer types; however, this activation cannot be explained by TFEB function in TNBC cells [15,18,19,69]. This result is puzzling given that TFEB is established as the master regulator of lysosomal biogenesis, however we propose that in the breast cancer cells the primary regulator of the autophagy-lysosome system is not TFEB and that this role is fulfilled by other established autophagy regulators, including the FOXO family or other MiT/TFE family members . Indeed, these results do not rule out the regulation of the autophagy-lysosome pathway by MiT/TFE family members in cancer, instead we suggest that these transcription factors are not interchangeable, and may have distinct tissue or context-specific functions. Future work should be dedicated to understanding how the MiT/TFE family members co-operate, and if the loss of multiple members has a different effect than the loss of a single-family member alone.
Our study demonstrated that DOX-induced activation of TFEB is independent of mTOR signaling. mTORC1 independent activation of TFEB has previously been reported in pancreatic ductal adenocarcinoma cells where evasion of MiT/TFE factors from mTORC1 inhibition facilitates anabolic maintenance pathways while concomitantly exploiting survival advantages from TFEB, TFE3, and MITF activity . In contrast, DOX stimulates TFEB in colorectal cancer cells and cervical cancer cells in association with decreased mTORC1 activity . Also, a recent report by Brady et al.  proposes that DNA damage induces p53 up-regulation resulting in mTORC1 inhibition and TFEB/TFE3 activation. Both MDA-MB-231 and BT549 cells have loss-of-function p53 mutations and, therefore, cannot activate TFEB in a p53-mTORC1 dependent manner. It likely that p53 mutant cancer cells develop an alternate mechanism of TFEB activation in response to DNA damage and future studies will be aimed at characterizing and targeting this pathway for cancer therapy.
The activation of TFEB and lysosomal activity by DOX seems to be specific to proliferating cells given that in cardiomyocytes and neurons, TFEB and autophagy are suppressed by DOX treatment [72,73]. The difference in response to DOX between cancerous and non-cancerous cells is not surprising because proliferating and senescent cells have different DNA damage repair requirements, such as the inhibition of homologous recombination in G0/G1 phase cells, or the need to avoid replicating excessively damaged DNA in proliferating cells [74,75]. In our study, the mechanism of TFEB activation in response to DOX remains enigmatic given mTORC1 activity, and calcineurin protein levels are inconsistent with TFEB activation. However, it is likely that calcineurin activity increases independent of its protein levels due to increased intracellular calcium, a potential consequence of endoplasmic reticulum dysfunction often precipitated by DOX [76,77]. Prior studies reported that downstream targets of calcineurin, including NFAT and NFκB, are activated by doxorubicin treatment, in both cancerous and cardiac cells [78–80]. It is therefore not surprising that combining DOX with the calcineurin inhibitor cyclosporine A (CsA) phenocopied knockdown of TFEB.
Our study identifies a novel role for TFEB in regulating apoptosis and the DNA damage response to DOX, which is independent of the lysosome. Transcriptomic analysis of TFEB knockdown MDA-MB-231 cells revealed that genes involved in homologous recombination (HR), a type of homology-directed repair, are down-regulated following TFEB knockdown, while genes involved in IFN-γ and death receptor signaling are up-regulated. Although direct regulation of HR by TFEB has not previously been described, others have shown that knockdown of MITF results in the down-regulation of BRCA1, RAD51L3, RAD54, and XRCC3 . Furthermore, a recent report found that TFEB and TFE3 double knock mouse embryonic fibroblasts displayed alterations in DNA damage repair genes, which the authors attribute to increased p53 stabilization and transcriptional activity . In our study, MDA-MB-231, BT549, and SUM159 cells have mutant p53, and therefore, the regulation of DNA repair is likely an alternate mechanism or direct effect. Additional studies are required to elucidate the exact mechanism by which TFEB knockdown leads to increased DNA damage both in the presence and absence of genotoxic agents. Based on our results, we postulate that in the absence of TFEB, DNA resection following DNA damage is not compromised, instead the process of strand invasion, homology search, and DNA synthesis is delayed. This mechanism could explain why double strand breaks, as detected by γH2A.X, persist for longer following DOX treatment in cells with knockdown of TFEB. One limitation to our study is that γH2A.X is an indirect measure of DNA damage, and changes in detection could also be caused by altered DNA damage signaling or increased γH2A.X foci elimination separate from repair. Thus, future research will be devoted to examining whether direct markers of DNA damage and repair are altered by TFEB activity and if homologous recombination is necessary for these processes.
Our data indicate that TFEB is required for efficient DNA repair in TNBC; however, it is less clear as to whether increased DNA damage in TFEB knockdown cells is responsible for the induction of cell death signaling. Prior studies have found that DNA damage induces IFN-α and IFN-γ gene expression through NFκB activation, while other models posit that persistent DNA damage leads to cytosolic double-stranded DNA which can be sensed by the cGAS (cGMP-AMP synthase) STING (Stimulator of Interferon Genes) pathway to induce an interferon response [82–84]. On the contrary, regulation of the IFN-extrinsic cell death axis may be due to a direct effect of TFEB, considering that when TFEB is overexpressed in BT549 cells, DOX-induced apoptosis is effectively inhibited while DNA damage is only partially rescued. Future studies will be focused on the regulation of the NFκB signaling pathway and the necessity of cGAS-STING for induction of apoptosis in TFEB knockdown cells.
In conclusion, our results highlight that TFEB is a therapeutically relevant target for sensitizing TNBC to lower doses of DOX. Additionally, our data provide novel evidence towards the involvement of TFEB in DNA damage repair and apoptosis regulation independent of its lysosomal effects.
analysis of variance
BCL2 Associated Athanogene 4
bovine serum albumin
co-ordinated lysosomal enhancement and regulation
gene set enrichment analysis
major histocompatibility complex
multiplicity of infection
principle component analysis
transcription factor EB
triple-negative breast cancer
TNFRSF1A associated via death domain
L.S. and T.P designed the research. L.S., D.B., and F.I. performed experiments. L.S. and T.P. analyzed the data. L.S and T.P. wrote the paper. Y.E. and P.C.K. provided intellectual inputs, technical assistance, reviewed and proof-read manuscript.
This work was funded by grants to T.P from the Natural Sciences and Engineering Research Council of Canada [RGPIN-2014-03687], Diabetes Canada [NOD_OG-3-15-5037-TP, NOD_SC-5-16-5054-TP], the Beatrice Hunter Cancer Research Institute and the New Brunswick Health Research Foundation. L.S. is a graduate trainee in the Cancer Research Training Program of the Beatrice Hunter Cancer Research Institute, with funds provided by the Terry Fox Research Institute (TFRI), Canadian Breast Cancer Foundation – Atlantic Region, and the New Brunswick Health Research Foundation. L.S. is also funded by a doctoral Alexander Graham Bell Canada Graduate Scholarship from NSERC. D.B. is funded by Postdoctoral fellowships from the New Brunswick Health Research Foundation and Dalhousie Medicine New Brunswick. T.P is a Diabetes Canada Scholar.
We thank the New Brunswick Health Research Foundation, Beatrice Hunter Cancer Research Institute and Dalhousie Medicine New Brunswick for granting a graduate studentship to L.S. We thank Dr. Ballabio for providing us with a CLEAR-PGC1α luciferase reporter construct.
The authors declare that there are no competing interests associated with the manuscript.
Mr. Logan Slade and Dr. Thomas Pulinilkunnil are the guarantors of this work, had full access to all the data, and take full responsibility for the integrity of data and the accuracy of data analysis.