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Transcriptional control of autophagy–lysosome function drives pancreatic cancer metabolism

Abstract

Activation of cellular stress response pathways to maintain metabolic homeostasis is emerging as a critical growth and survival mechanism in many cancers1. The pathogenesis of pancreatic ductal adenocarcinoma (PDA) requires high levels of autophagy2,3,4, a conserved self-degradative process5. However, the regulatory circuits that activate autophagy and reprogram PDA cell metabolism are unknown. Here we show that autophagy induction in PDA occurs as part of a broader transcriptional program that coordinates activation of lysosome biogenesis and function, and nutrient scavenging, mediated by the MiT/TFE family of transcription factors. In human PDA cells, the MiT/TFE proteins6—MITF, TFE3 and TFEB—are decoupled from regulatory mechanisms that control their cytoplasmic retention. Increased nuclear import in turn drives the expression of a coherent network of genes that induce high levels of lysosomal catabolic function essential for PDA growth. Unbiased global metabolite profiling reveals that MiT/TFE-dependent autophagy–lysosome activation is specifically required to maintain intracellular amino acid pools. These results identify the MiT/TFE proteins as master regulators of metabolic reprogramming in pancreatic cancer and demonstrate that transcriptional activation of clearance pathways converging on the lysosome is a novel hallmark of aggressive malignancy.

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Figure 1: Coordinate induction of an autophagy–lysosome gene program in PDA by MiT/TFE proteins.
Figure 2: Constitutive nuclear import of MiT/TFE factors controls autophagy–lysosome function in PDA.
Figure 3: MiT/TFE proteins maintain autolysosome-derived pools of amino acids.
Figure 4: MiT/TFE proteins are required for PDA growth.

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Accession codes

Primary accessions

Gene Expression Omnibus

Data deposits

RNA-sequencing data have been deposited in the Gene Expression Omnibus under accession number GSE62077.

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Acknowledgements

We would like to thank A. Kimmelman, L. Ellisen, W. Kim and R. Mostoslavsky for advice and helpful comments on the manuscript, S. Gygi for access to proteomics data analysis software, F. Kottakis, Y. Mizukami and M. Leisa for technical support, and C. Ivan for bioinformatics support. This work was supported by grants from the National Institutes of Health (P50CA1270003, P01 CA117969-07, R01 CA133557-05) and the Linda J. Verville Cancer Research Foundation to N.B. N.B. holds the Gallagher Endowed Chair in Gastrointestinal Cancer Research. R.M.P. holds a Hirshberg Foundation for Pancreatic Cancer seed grant. N.B. and R.M.P. are members of the Andrew Warshaw Institute for Pancreatic Cancer Research.

Author information

Authors and Affiliations

Authors

Contributions

R.M.P. and N.B. conceived and designed the study. R.M.P. and S.S. performed all experiments involving PDA cells and mouse models. R.M.P. and B.N.N. performed the metabolite measurements. J.F. and J.L. performed immunohistochemistry on human tissue sections. M.B. and W.H. performed quantitative proteomics measurements and analysis. K.N.R. and S.R. performed computational analysis. V.D. and M.K.S. performed pathology assessment and electron microscopy analysis. C.R.F. provided essential reagents. N.J.D. and G.S. supervised the metabolite analysis. R.Z. and J.S. contributed to the study design. R.M.P. and N.B. wrote the manuscript with feedback from all authors.

Corresponding author

Correspondence to Nabeel Bardeesy.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Tumour-specific expression and constitutive activation of MiT/TFE proteins in PDA.

a, Immunofluorescence staining of autophagosomes (LC3) and lysosomes (LAMP2) showing extensive overlap of these organelles and increased LC3 immunofluorescence in PDA cell lines (graph, right; error bars indicate mean ± s.d. for N = 3 independent experiments with at least 130 cells scored. **P < 0.001. Significance was analysed using two-tailed Student’s t-test). Scale bar, 11 μm. b, GSEA of different human PDA data sets for enrichment of the autophagy–lysosome gene signature in tumour versus normal tissue (Gene Expression Omnibus (GEO) accession numbers indicated). c, Mean expression (reads per kilobase per million (RPKM)) of TFE3, MITF and TFEB individually or cumulatively as a meta-gene formed by the mean of the three transcription factors (3TF) in primary tumour specimens from the indicated malignancies (The Cancer Genome Atlas (TCGA) data set). d, TFE3 and MITF antibody validation by western blotting in cells treated with the indicated siRNA. TFE3 antibody immunohistochemistry (IHC) validation using alveolar soft part sarcoma (ASPS) tissue. This antibody (MRQ-37, Cell Marque) is used as a clinical diagnostic for ASPS. Scale bars: 100 μm (top); 20 μm (bottom). e, Gene expression analysis showing upregulation of MiT/TFE genes in subsets of PDA relative to normal pancreatic ductal tissue. SAGE data50 from normal microdissected pancreatic ductal cells (normal microdissected control, black box; HPDE, grey box), cultured PDA cells (cell line), and PDA xenografts and primary tumour tissues (tumour). A.U., arbitrary units. f, Quantitative reverse transcription–polymerase chain reaction (qRT–PCR) analysis of MiT/TFE expression levels in a panel of human PDA cell lines. Note that PL18 and HupT3 preferentially express high levels of MITF, while 8988T, PSN1 and Panc1 express TFE3 at higher levels. The highest levels of TFEB were detected in 8902 and A13A cells. g, RT–PCR analysis reveals that PDA cells express distinct MITF isoforms. Note the complete absence of all MITF isoforms in normal HPDE cells. PDA cells lack the melanoma-specific M isoform (detected in M14 melanoma cells, lane 2). h, MITF, TFE3 and TFEB protein levels in a panel of non-PDA cell lines, patient-derived PDA cultures and PDA cell lines. i, GSEA analysis showing correlation between expression of MiT/TFE factors and autophagy–lysosome gene set in primary human PDA specimens (TCGA data set). j, GSEA analysis showing correlation between cumulative expression of MiT/TFE factors (see Methods) and the autophagy–lysosome gene set in human PDA cell lines (Cancer Cell Line Encyclopedia (CCLE) data set). k, Database for Annotation, Visualization and Integrated Discovery (DAVID) analysis of gene sets correlating with increasing expression of TFE3 (left) or MITF (right) in human PDA cell lines (CCLE data set).

Extended Data Figure 2 MiT/TFE-dependent regulation of autophagy–lysosome gene expression in PDA cell lines.

a, ChIP analysis of Flag–MITF (left) and Flag–TFE3 (right) binding to autophagy–lysosome genes in 8902 and Panc1 cells, respectively. Histograms show the amount of immunoprecipitated DNA detected by qPCR normalized to input and plotted as relative enrichment over mock control. Error bars indicate mean ± s.e.m. for N = 3 independent experiments. *P < 0.05. EV, empty vector. b, siRNA-mediated knockdown of MITF in HupT3 and PL18 cells causes a decrease in autophagy–lysosome gene expression, assayed 48 h after siRNA transfection. *P < 0.05. c, Knockdown of TFE3 in PSN1, Panc1 and 8988T cells, or of TFEB in 8902 cells, causes a decrease in autophagy–lysosome gene expression. *P < 0.05. d, HPDE, HPNE and QGP1 control cells show minimal changes in autophagy–lysosome gene expression upon knockdown of the MiT/TFE genes. e, Decreased autophagy–lysosome gene expression after MITF knockdown (left; PL18 cells; *P < 0.05) or TFE3 knockdown (right; 8988T cells; *P < 0.01) is rescued by transient ectopic expression of MITF or TFE3. Cells were transfected with expression constructs for MITF or TFE3 24 h post-siRNA transfection. After 48 h, gene expression was assayed. f, Expression of dominant-negative MITF (MITF-DN) in HupT3 cells causes a decrease in autophagy–lysosome gene expression compared with control cells (left; *P < 0.02). Similar results are seen in PSN1 cells expressing doxycycline (Dox)-inducible MITF-DN upon addition of 1 μg ml−1 of Dox for 48 h (right; *P < 0.05). For all graphs error bars indicate mean ± s.d. for N = 3 independent experiments. Significance was analysed using two-tailed Student’s t-test.

Extended Data Figure 3 MiT/TFE transcription factors escape mTOR-mediated cytoplasmic retention in PDA.

a, Subcellular localization of ectopically expressed TFEB-GFP in HPDE cells under full nutrient and starvation conditions (3 h HBSS) (left) and Torin-1-dependent nuclear localization of ectopically expressed Flag–MITF (left) or Flag–TFE3 (right) in HPNE cells (right). b, Subcellular localization of ectopically expressed GFP–MITF (left) or GFP–TFE3 (right) in HupT3 and 8988T cells, respectively, under full nutrient and starvation conditions (3 h HBSS). c, d, Subcellular fractionation studies showing that endogenous TFE3 (c) and MITF (d) are constitutively nuclear localized in PDA cell lines. e, Immunofluorescence staining of endogenous TFEB in A13A and 8902 PDA cells. Note the predominant nuclear localization under both full nutrient (fed) and starved conditions. f, Subcellular fractionation of PL18 and HupT3 PDA cells under full nutrient, amino acid (AA) starved and amino acid re-fed conditions shows constitutive nuclear residence of endogenous MITF regardless of the nutrient status of the cells. Lamin = nuclear fraction; GAPDH = cytoplasmic fraction. g, Subcellular fractionation of Panc1 cells and a primary patient-derived culture (PDAC1) showing constitutive nuclear localization of TFE3 (in Panc1) and MITF (in PDAC1) independent of Torin 1 treatment. h, Immunoblot for p-ERK1/2 in the indicated cell lines treated with vehicle or with the MEK inhibitor, AZD6244 (AZD). i, Neither AZD6244 nor Torin 1 affect MITF localization (PL18 cells) or TFE3 localization (8988T cells). j, Immunoblot showing readily detectable p-p70S6K in the indicated non-PDA and PDA cell lines, and extinction of phosphorylation upon Torin 1 treatment. k, Immunofluorescence showing that amino acid re-feeding of starved 8988T and Panc1 PDA cells results in mTOR (green) translocation from a diffuse cytoplasmic distribution to the lysosome (LAMP2; red), as indicated by co-localization with LAMP2. Scale bar, 20 μm. l, The indicated non-PDA (top) and PDA cell lines (bottom) stably expressing Flag-tagged TFE3 (F-TFE3) or MITF (F-MITF) were treated with vehicle or Torin 1. Cells were then lysed, subjected to Flag immunoprecipitation and immunoblotted for Flag and 14-3-3. Note that in all cell lines, 14-3-3 is detected in the anti-Flag immunoprecipitates and binding is lost upon Torin 1 treatment. All data shown are representative of at least N = 3 independent experiments.

Extended Data Figure 4 IPO8 drives increased MiT/TFE nuclear import in PDA cells.

a, Identification of IPO8 as a PDA-specific binding partner of TFE3. HPDE (left), PSN1 (middle) and 8988T cells (right) stably expressing Flag–TFE3 or control vector were subjected to affinity purification, followed by multiplexed quantitative proteomics analysis using tandem-mass tag (TMT) reagents. The graphs show normalized protein intensities in the Flag–TFE3 and control samples. Note the specific enrichment of IPO8 in the Flag–TFE3-expressing PDA cell lines. In HPDE cells, IPO8 did not score as a significantly enriched interactor (mean log2 ratio Flag–TFE3/control = −0.14 (N = 3), P = 0.30 (paired two-tailed t-test), while in PSN1 and 8988T IPO8 was significantly enriched in TFE3 immunoprecipitates (mean log2 ratio Flag–TFE3/control = 4.35 (N = 3), P = 0.015 (PSN1) and mean log2 ratio Flag–TFE3/control = 1.70 (N = 3), P = 0.002 (8988T)). b, Immunoprecipitation of endogenous IPO8 with Flag–TFE3 in a PDA cell line (PSN1) and primary PDA culture (PDAC3). c, qRT–PCR showing increased expression of IPO8 in PDAC cell lines and primary patient-derived cultures (red bars) compared to control pancreatic ductal cells (HPDE and HPNE, black bars). d, Quantification of IPO8 immunohistochemistry staining intensity (0 = no staining to 3 = high staining) in normal (N = 11) and PDA (N = 110) patient samples. e, Subcellular fractionation and immunoblot analyses of the indicated cell lines transfected with control siRNA (siCTRL) or siIPO8. Note that siIPO8 leads to a marked decrease in nuclear TFE3 in PDA cells (PSN1 and Panc1) (left) and in whole cell lysates (right). f, Immunoblot of whole cell lysates showing that IPO8+IPO7 knockdown decreases the levels of MITF and TFEB in PL18 and 8902 cells, respectively. g, Knockdown of IPO8 has no effect on total TFE3 protein (left) or mRNA (right) levels in HPDE and HPNE cells. Error bars indicate mean ± s.d. for N = 3 independent experiments. h, Torin-1-induced TFE3 nuclear localization in HPNE cells is unaffected by knockdown of IPO8. i, qRT–PCR showing that siRNA-mediated knockdown of IPO8, or of both IPO8 and IPO7, effectively reduces target expression without significantly affecting the expression of TFE3, TFEB or MITF mRNA levels. Error bars indicate mean ± s.d. for N = 3 independent experiments. j, Immunoblot of 8988T cells transfected with control siRNA (siCTRL) or siIPO8 and treated with cycloheximide (CHX) for the indicated time points shows a decrease in steady-state levels and stability of TFE3 upon loss of IPO8. Data are representative of N = 3 independent experiments.

Extended Data Figure 5 Altered lysosome morphology and function following loss of MiT/TFE proteins.

a, RNAi-mediated knockdown of MITF in PL18 (right; N = 259 siCTRL, N = 263 siMITF) or TFEB in PaTu8902 cells (left; N = 273 siCTRL, N = 147 siTFEB) causes aberrant lysosome morphology and an increase in lysosome diameter as visualized by immunofluorescence staining for LAMP2. **P < 0.001; bar, mean. b, Lysosome size in HPDE cells is not affected by knockdown of MITF (N = 156) and TFE3 (N = 81), and is only slightly increased by TFEB knockdown (N = 198) relative to siCTRL (N = 296). NS, not significant; *P < 0.05; bar, mean. Scale bar, 7.5 μm. c, Electron microscopy of 8988T PDA cells transfected with siCTRL (panels 1–5) or siTFE3 (panels 6–10). Note that TFE3 loss causes an accumulation of undigested material shown by asterisks indicating a defect in clearance (graph indicates percentage lysosomes filled with cargo; N = 80 lysosomes for siCTRL and N = 153 lysosomes for siTFE3) and an increase in average lysosome diameter (quantified in graph on the right; N = 63 lysosomes in siCTRL and N = 68 lysosomes in siTFE3). Scale bar, 1 μm. **P < 0.001. d, Immunofluorescence staining with LC3 (green) and LAMP2 (red) in 8988T cells after siRNA-mediated knockdown of TFE3, shows similar accumulation of undigested LC3-positive aggregates encapsulated within enlarged LAMP2 positive lysosomes (bottom) compared with control cells (top). Magnifications of the boxed regions are shown (right). Scale bar, 7.5 μm. Error bars represent mean ± s.d. Data are representative of at least N = 3 independent experiments. Significance was analysed using two-tailed Student’s t-test.

Extended Data Figure 6 Ectopic MiT/TFE expression in PDA cell lines causes an increase in autophagy–lysosome function.

a, b, Ectopic expression of MITF induces autophagy–lysosome genes in HPDE cells (a), and causes an increased abundance of LC3 puncta (b) as measured by immunoflourescence staining of endogenous LC3 (red) and quantified in the graph on the right. *P < 0.01, **P < 0.001. Scale bar, 7.5 μm. c, Ectopic expression of Flag-tagged MITF or TFE3 in HPDE cells (left) or HPNE cells (right) causes an increase in autophagic flux as measured by the increase in LC3-II versus LC3-I, following treatment with 25 μM chloroquine (CQ) for 18 h. MITF and TFE3 protein expression is represented by immunoblot for Flag. d, Expression of MITF in 8902 PDA cells (which lack MITF expression) or in MiaPaca cells (which express low levels of all MiT/TFE members) causes an increase in autophagy–lysosome gene expression. *P < 0.05. e, Dox-inducible expression of MITF in MiaPaca cells causes an increase in endogenous LC3-positive puncta, as measured in the graph on the right, indicating increased autophagy induction. N = 68 cells, −Dox; N = 100 cells, +Dox; **P < 0.001. Error bars represent mean ± s.d. Data are representative of at least N = 3 independent experiments. Significance was analysed using two-tailed Student’s t-test. Scale bar, 15 μm.

Extended Data Figure 7 Role of lysosome in maintaining amino acid levels in PDA.

a, Amino acid uptake was measured as fold change in extracellular amino acids in 8988T cells after transfection with two siRNAs against TFE3 relative to siCTRL. Media was changed 1 h before media samples were harvested for analysis. Data are matched to results presented in Fig. 3b. b–d, Effect of BafA1 (b), siTFE3 (c), and siATG5 (d) on intracellular amino acid levels in the indicate cell lines. Error bars represent mean ± s.d. for N = 3 independent experiments. *P < 0.05. Significance was analysed using two-tailed Student’s t-test.

Extended Data Figure 8 MiT/TFE proteins couple amino acid metabolism to energy homeostasis in PDA.

a, Knockdown of TFE3 in PSN1 and 8988T cells causes an increase in p-ACC (Ser 79) and p-AMPK (Thr 172) levels. b, Knockdown of TFE3 (in 8988T and PSN1 cells) or MITF (in HupT3 cells) causes a decrease in cellular ATP levels. N = 3 independent experiments; *P < 0.05, **P < 0.001. c, BafA1 treatment (150 nM) for 18 h induces p-ACC and p-AMPK in PDA cells but not in HPDE, HPNE or QGP1 cells. d, Forced expression of MITF or TFE3 in HPDE cells causes a decrease in p-ACC and p-AMPK levels. Error bars represent mean ± s.d. Data are representative of at least 3 independent experiments. Significance was analysed using two-tailed Student’s t-test.

Extended Data Figure 9 Regulation of in vitro and in vivo growth by MiT/TFE proteins.

a, A panel of human PDA cell lines were infected with shRNAs targeting MITF, TFE3 or TFEB. Growth relative to cells infected with shGFP control was assayed 8–10 days after infection. Note that sensitivity to individual knockdown correlates with the relative expression of each protein (see Extended Data Fig. 1f). Coloured bars indicate knockdown condition, which leads to the greatest growth impairment. b, Selective sensitivity of PDA cells compared with non-PDA cells (QGP1, HPNE and the non-small-cell lung cancer cell line H460) to treatment with 50 μM chloroquine (CQ) for 4 days. Error bars indicate mean ± s.e.m. *P < 0.05. c, Immunoblot showing robust detection of LC3-II across a panel of primary patient-derived cultures (PDAC 1–6) and PDA cell lines. d, Subcellular fractionation showing that PDA patient cultures have constitutively nuclear MITF and TFE3. e, qRT–PCR showing that MITF (left) or TFE3 (right) knockdown suppressed multiple autophagy–lysosome genes in patient-derived PDA cells. *P < 0.01. f, Immunofluorescence staining for LAMP2 showing that MITF (N = 212 siCTRL, N = 220 siMITF), TFE3 (N = 228 siCTRL, N = 244 siTFE3) and TFEB (N = 229 siCTRL, N = 271 siTFEB) knockdown results in enlarged, dysmorphic lysosomes in patient-derived PDA cultures and quantified in the graph on the right. Scale bar, 7.5 μm. **P < 0.0001. g, Knockdown of the indicated MiT/TFE factors inhibits colony formation in a series of primary PDA cultures. h, 8988T cells infected in vitro with shGFP or shTFE3 (left) and PL18 cells infected with shGFP or two hairpins targeting MITF (shMITF_1 and shMITF_2; right) were implanted subcutaneously on both flanks of SCID mice (N = 4 mice per group). Tumour xenograft growth was monitored over the course of 70 (8988T) and 50 (PL18) days. Error bars indicate mean ± s.e.m. i, Forced expression of MITF in KrasG12D mouse PanIN cells causes an increase in autophagy–lysosome gene expression relative to control cells in vitro. *P < 0.005. Data are representative of N = 3 independent experiments. Significance was analysed using two-tailed Student’s t-test.

Supplementary information

Supplementary Table 1

This file contains the Autophagy-Lysosome gene set. A list of genes associated with autophagy and lysosome function and biogenesis were curated as described in materials and methods. (XLS 44 kb)

Supplementary Table 2

This file shows enrichment of autophagy-lysosome gene set in primary human PDA versus matched normal tissue. A list of autophagy-lysosome genes (symbol, rank in list, rank metric score, running enrichment score and core enrichment) corresponding to heat map in Figure 1c. (XLS 90 kb)

Supplementary Table 3

This file shows statistically significant metabolites changes in PDA cells following TFE3 knockdown. List of metabolites showing statistically significant change in abundance following knockdown of TFE3 with 2 siRNA in 2 PDA cell lines (8988T and PSN1). Cells were transfected with siRNA and metabolite extracts were prepared 48 hrs post transfection and analyzed by GC/MS and LC/MS by Metabolon Inc. Red = metabolites which show a decrease in abundance, Blue = metabolites which show an increase in abundance. (XLSX 70 kb)

Supplementary Table 4

This table contains PDA Patient Information. All cases in the TMAs were obtained from resected PDA biopsies. Clinical information was available for 18/31 normal tissue controls and 153/354 PDA samples. Primary PDA cultures were established from malignant ascites. (XLS 38 kb)

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Perera, R., Stoykova, S., Nicolay, B. et al. Transcriptional control of autophagy–lysosome function drives pancreatic cancer metabolism. Nature 524, 361–365 (2015). https://doi.org/10.1038/nature14587

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