Metastatic cells are preferentially vulnerable tolysosomal inhibitionMichael J. Morgana,b,1,2, Brent E. Fitzwaltera, Charles R. Owensb, Rani K. Powersa,c, Joseph L. Sottnikb, Graciela Gameza,James C. Costelloa,b,c, Dan Theodorescua,b,1,3, and Andrew Thorburna,b

aDepartment of Pharmacology, University of Colorado School of Medicine, Aurora, CO 80045; bUniversity of Colorado Comprehensive Cancer Center,Aurora, CO 80045; and cComputational Bioscience Program, University of Colorado School of Medicine, Aurora, CO 80045

Edited by Eileen P. White, The Cancer Institute of New Jersey, New Brunswick, NJ, and accepted by Editorial Board Member Rakesh K. Jain July 18, 2018(received for review April 19, 2017)

Molecular alterations that confer phenotypic advantages to tumorscan also expose specific therapeutic vulnerabilities. To search forpotential treatments that would selectively affect metastatic cells, weexamined the sensitivity of lineage-related human bladder cancer celllines with different lung colonization abilities to chloroquine (CQ) orbafilomycin A1, which are inhibitors of lysosome function and auto-phagy. Both CQ and bafilomycin A1 were more cytotoxic in vitro tohighly metastatic cells compared with their less metastatic counter-parts. Genetic inactivation of macroautophagy regulators and lyso-somal proteins indicated that this was due to greater reliance on thelysosome but not upon macroautophagy. To identify the mechanismunderlying these effects, we generated cells resistant to CQ in vitro.Surprisingly, selection for in vitro CQ resistance was sufficient to altergene expression patterns such that unsupervised cluster analysis ofwhole-transcriptome data indicated that selection for CQ resistancealone created tumor cells that were more similar to the poorly meta-static parental cells from which the metastatic cells were derived; im-portantly, these tumor cells also had diminished metastatic ability invivo. These effects were mediated in part by differential expression ofthe transcriptional regulator ID4 (inhibitor of DNA binding 4); depletionof ID4 both promoted in vitro CQ sensitivity and restored lung coloni-zation and metastasis of CQ-resistant cells. These data demonstratethat selection for metastasis ability confers selective vulnerability tolysosomal inhibitors and identify ID4 as a potential biomarker for theuse of lysosomal inhibitors to reduce metastasis in patients.

metastasis | chloroquine | autophagy | lysosome | ID4

Metastatic disease is the primary cause of death in cancer(1). Metastatic cancer cells colonize and survive environ-

mental stresses at distant sites because of molecular changes ac-quired during tumor evolution (1–3). For example, metastatic cellsare more resistant to cell death and have increased prosurvivalsignaling (4). Autophagy is a prosurvival process that facilitatescancer cell survival under metabolic and environmental stressesand has been implicated in multiple cellular functions importantfor metastasis, suggesting that autophagy inhibition might be auseful strategy for treating metastatic disease (5, 6).Two inhibitors of autophagy, chloroquine (CQ), a well-

tolerated Food and Drug Administration-approved antimalarialdrug (7), and its derivative, hydroxychloroquine (HCQ) (8), havebeen evaluated in patients with advanced cancer and are the onlyautophagy inhibitors currently available for use in people (9, 10).Chloroquine is a weak base with hydrophobic characteristics thatdiffuses into lysosomes, where it becomes protonated and trap-ped, thus leading to a rise in lysosomal pH. These lysosomes can nolonger fuse with autophagosomes, thus blocking autophagy (11).Other, more potent lysosomal inhibitors (12, 13) are also underdevelopment. Initial phase I trials suggest that CQ/HCQ therapy canblock autophagy in human tumors and may have clinical benefit (14–19). However, since CQ and HCQ can have autophagy-independentantitumor effects (7, 20, 21), it is unclear whether their clinicalbenefit is primarily due to their ability to inhibit autophagy, moregeneral lysosomal function, or lysosome-independent effects. Forexample, Piao et al. (22) reported that two genes, LDH1A1 and

HLTF, can regulate HCQ sensitivity and resistance, respectively,without directly affecting either lysosome function or autophagy.To evaluate the importance of autophagy in driving coloni-

zation of the lung (3), we used a well-established model oflineage-related human bladder cancer cell lines with differingmetastatic propensity (23, 24). We found that cells with greatermetastatic propensity displayed more in vitro cytotoxicity to twomechanistically different lysosomal inhibitors, CQ and bafilo-mycin A1 (BafA1), and to knockdown of the lysosomal proteinLAMP2. However, the metastatic and nonmetastatic cells wereequally sensitive to depletion of different autophagy regulators.Together, these data suggest that metastatic cells are more de-pendent on lysosomal function but not autophagy itself. To test thishypothesis, we generated CQ-resistant metastatic cells in vitro andfound that these cells overexpress the ID4 (inhibitor of DNAbinding 4, a dominant-negative helix–loop–helix protein) gene.Conversely, depletion of ID4 in poorly metastatic cells was sufficientto promote resistance to CQ and BafA1. Moreover, CQ-resistantcells have reduced metastatic ability, and this can be largely restoredby ID4 depletion. These data suggest that there is an intimate re-ciprocal relationship between metastatic ability and lysosome function

Significance

We show that there is a functional reciprocal relationship be-tween lysosome activity and metastasis that allows chloro-quine (CQ) and other inhibitors of lysosome function, such asbafilomycin A1, to preferentially kill human metastatic bladdercancer cells by targeting autophagy-independent lysosomefunctions. In addition, CQ treatment of bladder cancer cells andsubsequent acquisition of resistance to this therapy lead toaltered gene expression programs that drive a less aggressiveand metastatic phenotype via up-regulation of ID4 (inhibitor ofDNA binding 4). Clinically, this work provides a conceptualfoundation for using ID4 expression as a predictive and prog-nostic biomarker of CQ sensitivity and metastasis in patientswith bladder cancer.

Author contributions: M.J.M., C.R.O., J.L.S., J.C.C., D.T., and A.T. designed research;M.J.M., B.E.F., C.R.O., and G.G. performed research; J.L.S., J.C.C., and D.T. contributednew reagents/analytic tools; M.J.M., B.E.F., C.R.O., R.K.P., J.L.S., and J.C.C. analyzed data;and M.J.M., D.T., and A.T. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission. E.P.W. is a guest editor invited by theEditorial Board.

Published under the PNAS license.1To whom correspondence may be addressed. Email: morgan83@nsuok.edu or dan.theodorescu@cshs.org.

2Present address: Department of Natural Sciences, Northeastern State University,Tahlequah, OK 74464.

3Present address: Samuel Oschin Comprehensive Cancer Institute, Cedars-Sinai, LosAngeles, CA 90048.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1706526115/-/DCSupplemental.

Published online August 20, 2018.

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and that targeting the lysosome itself, rather than autophagy specif-ically, may provide an effective therapy in patients with metastasis.

ResultsMetastatic Bladder Cancer Cells Are More Sensitive to Chloroquineand Bafilomycin. Since autophagy helps cells survive environmentalstress, we reasoned that metastatic cells might rely on autophagyfor growth and survival at distant sites. To test this, we used twowell-established lineage-related human bladder cancer cell linemodels derived from T24t cells (25, 26). The FL and SLT serieswere derived by serial passaging of T24t to the lung and the liver,respectively, to generate metastatic cell lines with progressivelyincreased metastatic potential (SI Appendix, Fig. S1) (23, 24).We treated FL3 and T24t cells with chloroquine or bafilo-

mycin A1, which inhibit late stages of autophagy by preventinglysosome acidification. We then assessed loss of cell viability bypropidium iodide (PI) permeability over 48 h using INCUCYTEimaging. The more metastatic FL3 cells had substantially decreasedcell viability in response to these inhibitors compared with theT24t parental cells (Fig. 1A and Movies S1–S3). Additionalviability and cell-death assays [MTS tetrazolium compoundreduction and lactate dehydrogenase (LDH) release assays,

respectively] confirmed that FL3 cells were more sensitive tothe lysosomal inhibitors than T24t (Fig. 1 B–E). We then testedthe entire metastatic series and found each independently de-rived, more metastatic cell line was more sensitive to theseinhibitors than the poorly metastatic T24t cells from which theywere derived (SI Appendix, Fig. S2). Thus, in these closelymatched cells, in vitro cytotoxicity to CQ and BafA1 correlateswith increased metastatic potential.

Autophagic Flux Does Not Correlate with Sensitivity to CQ or BafA1.We next compared autophagic flux in the various cell lines (27).Sensitivity to lysosomal inhibitors was not correlated with theamount of autophagic flux as measured by LC3 Western assaysand tandem-mCherry-EGFP-LC3 flux measurements (SI Ap-pendix, Fig. S3). To test if the lysosomal inhibitors were capableof inhibiting a well-established autophagy-specific function, T24tand FL3 cells were put under starvation stress in Earle’s bal-anced salt solution (EBSS). BafA1 sensitized T24t and FL3 cellsequally to cell death. In contrast, more cytotoxicity was observedin FL3 compared with T24t cells in normal growth mediumconditions (Fig. 1 F and G). This suggests that both metastatic andpoorly metastatic cells have a similar requirement for autophagy

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Fig. 1. Differences in chloroquine and bafilomycin A1 toxicity in lowmetastatic T24t and its high metastatic FL3 derivative. (A) Phase-contrast images overlappedwith propidium iodide fluorescence (red), with PI uptake as a measure of loss of viability. T24t and FL3 cells are shown after treatment with or without 30 μM CQor 5 nM BafA1 for 48 h (photos represent the last frames in Movies S1–S3). (B–E) T24t and FL3 cells were treated with the indicated concentrations of CQ (B and C)or BafA1 (D and E) for 48 h; cell viability was analyzed by MTS (B and D) and cell death was analyzed by LDH release (C and E). n = 10 for CQ experiments and n =7 for BafA1 experiments. (F and G) mCherry-GFP-LC3 T24t and FL3 cells (as shown in SI Appendix, Fig. S2B) were plated in a six-well plate. The next day, cells weretreated with 5 nM BafA1 in the presence or absence of starvation media (EBSS). Cells were washed and grown in full media for 6 d, and then stained with crystalviolet (F) and quantitated (G). n = 3. [All bars indicate mean ± SEM; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001; n.s., nonsignificant (P > 0.2).]

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and harbor functional lysosomes under stress conditions but thatlysosomal inhibition selectively causes increased cytotoxicity in themetastatic cells under nutrient-replete conditions.

Lysosomal, Rather than Autophagy, Dependency Is Correlated withMetastatic Potential. We next tested whether differential sensi-tivity to lysosome inhibitors reflected an enhanced requirementfor macroautophagy in metastatic cells. Using an inducibledominant-negative ATG4 mutant construct (ATG4DN) (28), weeliminated production of LC3II under basal conditions or in thepresence of CQ (SI Appendix, Fig. S4A) in nutrient-replete orstarvation conditions (SI Appendix, Fig. S4 B and C). However,this had no effect on survival and clonogenic growth of eitherT24t or FL3 cells (SI Appendix, Fig. S4D). To test the re-quirement for autophagy, we used shRNAs that target VPS34(PIK3C3), ATG5, and ATG7, which are all required for auto-phagosome formation, as well as LAMP2, which is a lysosomalprotein needed for autophagosome–lysosome fusion (Fig. 2A).Knockdown of VPS34, ATG5, or ATG7 in T24t and FL3 cellsled to a reduction in cell number and increased cell death, butthe magnitude of these effects was similar across the lines (Fig. 2B–G and SI Appendix, Fig. S5A). In contrast, knockdown of

LAMP2 (with pan-LAMP2 shRNA) demonstrated preferentialcytotoxicity in FL3 cells as measured by both MTS and LDHassays (Fig. 3 A and B). Importantly, a LAMP2 isoform A-specific shRNA had no effect on viability of either cell type.Since isoform A of LAMP2, but not other splice variants, isnecessary for chaperone-mediated autophagy (CMA), this indi-cates CMA is not responsible for these effects (Fig. 3 A and B).To confirm these results, we monitored long-term cell viability

of individual cells in a population of GFP-NLS–tagged T24t andFL3 cells using propidium iodide staining during INCUCYTEimaging. Consistent with the MTS assays, all of the autophagy-targeted shRNAs reduced proliferation of both T24t andFL3 cells. ATG5 or ATG7 shRNAs caused minimal cytotoxicityin either cell line and, while VPS34 shRNA caused more toxicity,this was equivalent in both T24t and FL3 cells (Fig. 3C). Incontrast, LAMP2 depletion led to preferential reduction in cellviability of metastatic cells compared with the poorly metastaticcells (Fig. 3C). Together, these data indicate that although themetastatic and poorly metastatic cells are equally sensitive tomacroautophagy inhibition per se, metastasis propensity is as-sociated with more sensitivity to blockade of lysosomal function.

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Fig. 2. T24t and FL3 cells do not differ in their response to inhibition of autophagy with ATG5, ATG7, or VPS34 knockdown. (A) Western blotting showingknockdown of ATG5, ATG7, VPS34, or LAMP2 in T24t or FL3 cells. (B–G) T24t or FL3 cells in 48-well plates were infected with two different concentrations oflentivirus (200- or 400-μL aliquots) encoding nonsilencing (NS), ATG5, ATG7, or VPS34 shRNAs and allowed to grow for 6 d; cell viability was analyzed by MTS(B, D, and F) and cell death was analyzed by LDH release (C, E, and G). n = 4 for all experiments. [All bars indicate mean ± SEM; n.s., nonsignificant (P > 0.2).]ACTB, actin B protein.

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ID4 Expression Is Associated with CQ Resistance in Bladder CancerCells. To understand the molecular basis of the dependence ofmetastatic cells on lysosome function, we derived CQ-resistantversions of the metastatic cells by growing FL3 cells in pro-gressively higher concentrations and exposure times to CQ overseveral months (Fig. 4A). Three CQ-resistant FL3 derivatives,C1AZ, D1AZ, and D1BZ1, were developed. All three lines werealso resistant to BafA1 (Fig. 4 B–E) compared with the FL3 cells,and their level of resistance was similar to that of the parental,poorly metastatic T24t cells from which FL3 cells were derived.However, the CQ-resistant derivatives were not generally re-sistant to other cell-death inducers such as etoposide (Fig. 4 Fand G) or cell detachment (SI Appendix, Fig. S5B).Using microarrays, we examined differentially expressed genes

in the CQ-resistant lines relative to FL3 cells. A Venn diagramand accompanying gene lists indicate significant overlap acrosscell lines in genes whose expression is higher in the derivativescompared with that in FL3 cells (SI Appendix, Fig. S6). Geneswhose expression is lower than in FL3 cells are shown in SIAppendix, Fig. S7. The top 10 statistically significant up-regulatedand down-regulated genes in the CQ-resistant cells ranked interms of high fold changes across multiple cell lines are shown inSI Appendix, Tables S1 and S2, respectively. Interestingly, theexpression of LDH1A1 and HLTF, reported to mediate HCQsensitivity and resistance in some other cancer cell lines (22), wasnot significantly different across our cell lines that were all de-rived from the same parental line but selected for differences inmetastasis and then separately selected for resistance to CQ.Next, we examined the correlation between the expression levelsof the top 20 genes with CQ and BafA1 sensitivity using the NCI-60 cancer cell line panel (https://discover.nci.nih.gov/cellminer)(29, 30) (SI Appendix, Fig. S8 and Tables S1 and S2 and DatasetsS1 and S2). Among our top candidate genes, we found thatID4 was highly expressed in all CQ-resistant cells compared withthe CQ-sensitive FL3 cells (a 4- to 15-fold increase), and was alsoreduced in the metastatic derivative cells compared with T24t (SIAppendix, Fig. S9). ID4 expression was also significantly nega-tively correlated with sensitivity to both CQ and BafA1 in theNCI-60 cancer cell line panel (Pearson’s correlation coefficient−0.48 for CQ and −0.40 for BafA1; P < 0.05; population size47/60 for CQ and 60/60 for BafA1). Because no other candidategenes were significantly correlated with both CQ and BafA1sensitivity, we focused on ID4 for further study. Finally, it isnotable that since there are no bladder cancer cell lines in theNCI-60 panel, this finding suggests that ID4 expression is asso-ciated with CQ resistance across cancer types.

ID4 Expression Promotes Resistance to CQ and BafA1. To determineif ID4 expression regulates CQ sensitivity, we depleted ID4 inthree cell lines that were less sensitive to CQ/BafA1: the parentalT24t cells and the CQ-resistant derived C1AZ and D1BZ1 (SI Ap-pendix, Fig. S10A). Reduction in ID4 expression increased the cyto-toxicity of CQ and BafA1 in all three lines (Fig. 5 A–F and SIAppendix, Fig. S10 B–G) but had no direct effect on the autophagy/lysosome pathway, as we observed no change in basal or starvation-induced autophagy in the T24t cells with ID4 knockdown (SI Ap-pendix, Fig. S11). This suggests that ID4 does not directly controllysosomal processes but is involved in repressing the increased needof metastatic cells for lysosomal function.

ID4 Amplification/Expression Is Associated with Better Prognosis inBladder Cancer Patients.We next examined data from The CancerGenome Atlas (TCGA) to determine which tumor characteris-tics determine ID4 expression in human cancers. We foundseveral types of cancer had homozygous or heterozygous deletionof ID4 while in others it was amplified (SI Appendix, Fig. S12 A andB). Interestingly, amplification of ID4 in bladder urothelial carci-noma (BLCA) tumors occurred at a rate of 39.7% (162/408 tu-mors), while the average copy-number gain for another gene onchromosome 6 was only 16.2% (a significance of P = 0.03; SIAppendix, Fig. S12C). Amplification of ID4 was associated with

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Fig. 3. LAMP2 knockdown leads to differential cytotoxicity in T24t and FL3,while knockdown of the LAMP2A isoform involved in chaperone-mediatedautophagy has no cytotoxicity in either cell line. (A and B) T24t or FL3 cells(1,000) in 48-well plates were infected with two different concentrations oflentivirus (200- or 400-μL aliquots) encoding nonsilencing pan-LAMP2 or LAMP2AshRNAs and allowed to grow for 6 d; cell viability was analyzed by MTS (A) andcell death was analyzed by LDH release (B). n = 5. (C) T24t and FL3 GFP-NLS cellsin 48-well plates were infected with lentivirus encoding nonsilencing ATG5,ATG7, VPS34, or LAMP2 (pan-variant) shRNAs and followed by INCUCYTE im-aging of phase, green, and red channels in the presence of propidium iodide.Cell death of the knockdown T24t and FL3 GFP-NLS cells in the above INCUCYTEexperiments is shown as measured at the 140-h time point by PI permeability.Data are shown as the number of PI-positive cells per the total number of greencells. Data for each bar represent four technical replicates. [All bars indicatemean±SEM; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001; n.s., nonsignificant (P > 0.15).]

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improved patient survival in TCGA database (Fig. 5G; P = 0.012),and this was also true in ovarian cancers and uveal melanomas inTCGA datasets (SI Appendix, Fig. S12 D and E). ID4 mRNA cor-related with ID4 gain or loss in bladder cancer (Fig. 5H), and whenstratifying patients by both ID4 gain and mRNA expression, highexpression was associated with better survival (P = 0.033; Fig. 5I).High ID4 expression (as measured by RNA sequencing; RNA-seq)was also associated with better survival of bladder cancer patientsin two additional patient cohorts (SI Appendix, Fig. S13). Together,the cell line and human clinical studies presented here stronglysuggest that more aggressive cancers have reduced expression ofID4 (SI Appendix, Fig. S8C). These data also led us to hypothesizethat reduced ID4 expression confers both metastatic competenceand increased sensitivity to CQ, BafA1, and lysosomal inhibitors.

Selection for CQ Resistance Leads to Reversal of Gene ExpressionChanges and a Reduction in Metastatic Potential. ID4 expressionis low in metastatic cells, but not in the poorly metastatic T24t orin the CQ-resistant C1AZ, D1AZ, and D1BZ1 cell lines (SIAppendix, Fig. S9). To determine if this was part of more ex-tensive overall changes in gene expression patterns related to CQsensitivity, we evaluated the transcriptome of all of the cells.Analysis of these data indicated that acquisition of CQ resistanceby metastatic cells was associated with an overall change in geneexpression pattern so that they were closer to the original poorlymetastatic T24t cells from which FL3 was derived by in vivoselection for metastatic ability, rather than to the FL3 cellsthemselves from which they were directly derived (Fig. 6A).Principal component analysis further confirmed this relationship (SIAppendix, Fig. S14A). This suggests that there is a causal reciprocalrelationship between CQ sensitivity and metastatic propensity.

To test this hypothesis, we injected FL3 cells, or the CQ-resistant FL3 derivatives D1AZ, C1AZ, and D1BZ1, into the tailveins of female athymic NCr nu/nu mice and examined meta-static colonization of the lungs over 2 mo. Human tumor burdenin the lung was assessed by quantitative real-time PCR with ahuman-specific 12p primer set. This revealed that all three CQ-resistant cell lines were less metastatic than the parental FL3 cellline (Fig. 6B). Moreover, the D1AZ and D1BZ1 lines were as-sociated with better overall survival [death end points, defined byour Institutional Animal Care and Use Committee (IACUC);P = 0.029; Fig. 6C], while the C1AZ line had a strong trendtoward better overall survival (P = 0.12; Fig. 6C). In addition, ontermination of the experiment, we noticed two FL3-injected micehad large nonlung tumors but did not yet have signs of distress,suggesting the overall survival difference would have been evenmore profound if we had continued the experiment. Only one ofthe mice injected with CQ-resistant derivatives, a C1AZ mouse,had to be killed due to signs of distress. We conclude from thesedata that selection for CQ resistance selects for gene expressionpatterns that favor less aggressive tumors and that selection forCQ resistance also reverses the metastatic phenotype of bladdercancer cells.

ID4 Expression Inhibits the Metastatic Potential of CQ-Resistant CellLines. To test if these effects were due to ID4, we designed an invivo model to test the relative metastatic fitness in the CQ-resistant cells when ID4 expression was reduced (Fig. 7A). Tosimulate the heterogeneity present in tumors, we used a pooledpolyclonal population of the parental and three CQ-resistant celllines. Nonsilencing shRNA or ID4 shRNA were introduced intoeach of the four lines represented in the polyclonal population,

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creating a mix of eight different groups within the tumor cellpopulation (Fig. 7A, Left and SI Appendix, Fig. S14B). EachshRNA was labeled with a barcode to allow genetic identificationof the relative levels of each cell type (SI Appendix, Fig. S14 Cand D), and the parental FL3 cells, which have minimal ex-pression of ID4, were labeled with distinct barcodes for non-silencing and ID4 shRNAs. Equal numbers of cells from the fourgroups (parental FL3 with nonsilencing shRNA, parentalFL3 with ID4 shRNA, CQ-resistant pool with nonsilencingshRNA, and CQ-resistant pool with ID4 shRNA) were pooledand injected into the tail veins of nude mice. After 90 d, micewere killed; autopsies were carried out, and complete lungs andany visual extrapulmonary metastatic tumors were isolated.shRNA vector DNA sequences were amplified in all tissuescollected, and next-generation sequencing was used to identify

the relative amount (“count”) of each shRNA vector. A similarsequencing analysis was carried out on the preinjection pool ofcells (Fig. 7B). Determination of the percentage change in eachshRNA vector read count from the original preinjection poolcompared with that in the lungs was used as a measure of therelative metastatic fitness. As expected, the representation of themetastatic parental FL3 cells (irrespective of whether they hadnonsilencing or ID4 shRNA) increased in the lungs of most(70%) mice (Fig. 7C), while representation of the CQ-resistant,nonsilencing pool was increased in the lungs of only a singlemouse. In stark contrast, ID4 knockdown in the CQ-resistantpool led to increased representation of these tumor cells in thelungs in over one-third of mice (Fig. 7B). Additionally, whenexamining the nine nonpulmonary metastatic tumors, two tu-mors were composed predominantly of CQ-resistant cells with

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Fig. 5. Decreased ID4 expression leads to sensitivity to lysosomal inhibitors CQ and BafA1, while increased ID4 expression leads to less aggressive bladdercancer. (A–F) T24t- and FL3-derived CQ-resistant cell lines D1BZ1 and C1AZ were infected with lentivirus encoding ID4 or nonsilencing shRNA. Cells were thenplated in 48-well plates and treated with the indicated concentrations of CQ (A, C, and E) for 48 h or BafA1 (B, D, and F) for 72 h and cell death was analyzedby LDH release. n = 4 for CQ and BafA1 experiments. (All bars indicate mean ± SEM; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.) (G) Kaplan–Meier survival curve forTCGA BLCA patients. Overall survival in days is shown for patients with a copy-number gain of ID4 (red) or patients diploid for ID4 (black). (H) RSEM nor-malized ID4 mRNA expression values from TCGA BLCA patients (muscle invasive) subset into groups according to both copy number and expression. TheID4 loss group was defined as a GISTIC score less than 0 and ID4 mRNA expression in the lower quantile (<25%) of all ID4 expression values. The ID4 wild-typegroup was defined as a GISTIC score of 0 and ID4 mRNA expression between the middle quantiles (25 to 75%) of expression values. The ID4 gain group wasdefined as a GISTIC score greater than 0 and ID4 mRNA expression in the upper quantile (>75%) of all ID4 expression values. (I) Kaplan–Meier survival curvefor TCGA BLCA patients (muscle invasive) with both a copy-number gain of ID4 and high expression of ID4 (red) or patients diploid for ID4 with wild-typeID4 expression levels (black).

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ID4 knockdown, while no tumors were isolated from CQ-resistant cells harboring nonsilencing shRNA (Fig. 7D). Thisexperiment extends and confirms the previous experimentshowing that selection for CQ resistance reduces metastatic fit-ness of the FL3 cells and demonstrates that ID4 knockdownalone is sufficient to reverse this phenotype and thus increasemetastatic fitness. This demonstrates a cause-and-effect re-

lationship between ID4 expression, CQ resistance, and lungmetastasis. Taken together, these data indicate that ID4 is ametastasis suppressor in human bladder cancer cells and that itsincreased expression in CQ-resistant cells contributes to makingthese cells less metastatic (Fig. 6 B and C).

DiscussionWe have previously shown that some cancer cells are particularlydependent on autophagy for their growth and viability evenunder unstressed conditions, while others have no enhancedrequirement for autophagy (31, 32). Autophagy has also beenshown to affect multiple cellular processes important for me-tastasis. For example, it promotes resistance to anoikis, modu-lates epithelial-to-mesenchymal transition, promotes tumor cellmotility and subsequent invasion, influences cancer stem cellrenewal/differentiation, and maintains tumor cell dormancy (6).However, while previous studies have measured the impact ofblocking autophagy on metastasis, there are no studies that haveexamined if cancer cells that have already gained metastaticpotential differ in their autophagy dependence compared withnonmetastatic counterparts. Here we evaluated this relationship.Due to multiple influences of autophagy on metastatic pro-

cesses, we hypothesized that as a cancer cell becomes moremetastatic, it would more likely be dependent on autophagy forgrowth and survival. However, this hypothesis was incorrect; boththe high metastatic and the low metastatic cells had equivalentsensitivity to knockdown of essential autophagy regulators, in-dicating that autophagy was similarly required for growth/survivalof these cell lines. Our model is a late-stage model that prefer-entially reflects the ability of cells to survive, seed, and grow intarget organs after direct injection into the bloodstream. Thesemodels were used because there are no human bladder cancer cellmodels that can be used to study spontaneous metastasis to thelungs. Therefore, it is possible that differences in autophagy de-pendence might be observed in cells with alterations in earliersteps in the metastatic process such as invasion or extravasation.The more important conclusion of our work is that although

autophagy itself was similarly important, there was a substantialdifference in the sensitivity to inhibition of lysosome functionwhen highly metastatic cells were compared with poorly meta-static cells. Moreover, we identified an inverse reciprocal relation-ship between gene expression patterns promoting metastasis andthose promoting CQ/BafA1 resistance such that selection for me-tastasis bestows sensitivity to CQ/BafA1, and then selection for CQ/BafA1 resistance in the metastatic cells returns the cells to a poorlymetastatic phenotype. Inhibitor of DNA binding 4 is expressed inCQ-resistant/poorly metastatic cells and is highly down-regulated inmetastatic/CQ-sensitive cells. Moreover, knockdown of ID4 impartssensitivity to CQ and BafA1 and also confers increased metastaticcapability in the CQ-resistant cells, indicating that it is sufficient toexplain a large part of the phenotypic differences observed betweenhighly metastatic and poorly metastatic bladder cancer cells.ID4 is proposed to act as a dominant-negative transcription

factor (i.e., it binds to basic helix–loop–helix transcription factorsbut lacks a DNA-binding motif, and thus prevents transcriptionmediated by these factors); it likely acts by regulating tran-scription of a number of other genes in conferring lysosomaldependency and metastatic capability. ID4 promoter methylationis associated with unfavorable recurrence-free survival and anincreased risk of lymph node metastasis in breast cancer patients(33, 34). Consistent with this, we found ID4 expression is asso-ciated with less aggressive bladder cancers in human patientsand, importantly, reduction in ID4 expression led to increasedmetastatic potential in our metastatic fitness model. The thera-peutic implication of this association is that more aggressive andmetastatic cancers, which have lost ID4 expression, may be moreamenable to therapeutic treatment with CQ. The use of CQ andHCQ as autophagy inhibitors is being investigated in manyclinical trials (9, 10). Although some of the clinical studies in-volve patients selected for particular mutations such as theBRAF mutation in melanoma (e.g., NCT02257424), most of

A

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B

*

Fig. 6. Selection for CQ resistance leads to a reversal of the metastaticphenotype. (A) Dendrogram analysis of cell lines hierarchically clustered byexpression of all microarray probes. (B) Nine weeks after tail vein injection ofequal amounts of the FL3 cell line or its CQ-resistant derivatives D1AZ, C1AZ,or D1BZ1, mouse lungs were harvested and genomic DNA was isolated. Thegraph shows the amount of metastatic human tumor burden in the lung asassessed by qPCR with a human-specific primer. (C) As a measure of cell lineaggressiveness, the graph shows the overall survival of mice after tail veininjection of equal amounts of the FL3 cell line or its CQ-resistant derivativesD1AZ, C1AZ, or D1BZ1. (§P = 0.12, *P = 0.029.)

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Fig. 7. Knockdown of ID4 leads to greater metastatic fitness in CQ-resistant cell lines. (A) Schematic of the experiment to address metastatic fitness upon ID4modulation in parental FL3 cells and CQ-resistant cell lines. Lentiviral vectors encoding ID4 and nonsilencing shRNAs were modified with different barcodes so as toidentify the parental or resistant cells. Cells were pooled with 250,000 cells from each NSsh- and ID4sh-expressing cell line. Pooled cells were injected into the tail veinof nude mice (four separate experiments of 10 mice were injected; n = 4). After 90 d, or as required, mice were killed and the lungs of mice and visible nonpulmonarymetastatic tumors were isolated. Genomic DNA was purified from each lung or tumor sample and the vector sequences were amplified with unique secondarybarcodes for each lung or tumor. Next-generation sequencing was performed to identify the percentage of total reads that corresponded to each barcode as ameasurement of the relative quantity of each cell line within a given lung or tumor. This percentage was compared with the percentage of reads in the originalpreinjection cell pool to determine whether the relative percentage increased. (B) Each mouse was scored as to whether a given cell population increased its overallpercentage of reads compared with the preinjection cell pool as a measure of higher metastatic fitness. For each cell type (indicated by different colors), filled circles inthis panel indicate when the relative percentage of cells (measured by the overall NGS reads) increased in the lungs of a given mouse with respect to its originalpercentage of the initial preinjection population. (C) The graph shows the relative metastatic fitness for each cell type as denoted by the average percentage of micethat scored positively (ones that increased their representation as scored in B) for that cell type over the four injection groups (n = 4). [All bars indicate mean ± SEM;*P = 0.018; n.s., nonsignificant (P = 0.56); P value determined by Wilcoxon rank-sum test.] (D) The figure shows the anatomic position of nonpulmonary metastatictumors. Filled circles in this panel indicate the cell line dominating each tumor. (Pink, parental FL3 with nonsilencing shRNA; red, parental FL3 with ID4 shRNA; blue,CQ-resistant cell line with ID4 shRNA. There were no metastatic tumors dominated by a CQ-resistant cell line with nonsilencing shRNA.)

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these studies do not attempt to select patient-based tumorcharacteristics that would be expected to confer sensitivity toCQ, HCQ, and/or autophagy inhibition. Our work suggests thatin bladder cancer, and perhaps other tumor types as well,ID4 expression might provide such a marker. Our work also hasimplications for targeting autophagy and suggests that doing soby targeting the lysosome may have additional benefits comparedwith targeting earlier steps in the autophagy process—by tar-geting both autophagy-dependent tumor cell survival/growth, aswe saw in all of the cells tested here, while also targeting thelysosome dependency that we see only in more metastatic cells.Additionally, the reciprocal relationship that we identified whenwe selected for resistance to CQ implies that tumor cells thatfind a way to circumvent this dependency may consequentlybecome less aggressive by gaining additional ID4 expression.It is not clear what non–macroautophagy-dependent lysosomal

functions confer lysosome dependency on the metastatic cells.We ruled out chaperone-mediated autophagy (CMA) sinceLAMP2A is required for CMA (35), and knockdown of theLAMP2A splice variant alone has no effect on the growth andviability of the metastatic cells (Fig. 3 A and B). It is known thatupon oncogenic transformation, cancer cells undergo changes intheir lysosomes, including their content and activity, and differ-ences in the membrane composition of lysosomes and in sub-cellular localization (36, 37). Invasive and metastatic cells haveparticularly large numbers of lysosomes located at the peripheryof the cell instead of in their typical juxtanuclear region. Theseperipheral lysosomes have recently been implicated in the reg-ulation and degradation of focal adhesion proteins, the degra-dation of Rho proteins, including RhoA, and the degradation ofextracellular matrix upon exocytosis; these processes regulatecellular migration and invasion of metastatic cells (37, 38). It maybe that these processes are involved in the connection betweenmetastasis and additional sensitivity to lysosomal inhibitors.Some of the observed lysosome-dependency phenotype couldpossibly be linked to vulnerability of the lysosomal membrane tocationic amphiphilic drugs (39) and altered lysosomal sphingo-myelinase activity (37, 39). Further determination of the mech-anism by which metastatic cells like those tested here becomeparticularly dependent on the lysosome may identify other waysto capitalize on this susceptibility that could be used togetherwith drugs like chloroquine to target metastatic cells.

MethodsReagents. Chemicals, antibodies, and other reagents were obtained fromsources as detailed in SI Appendix, Methods. The ATG4B doxycycline-inducibledominant-negative construct (iA4DN) was cloned into the acceptor lentiviralplasmid pCW57.1 Tet RE inducible system as indicated in SI Appendix, Methods.

Cell Culture. T24t and the derived lung and liver metastatic series havepreviously been described (23, 24). All cell lines were authenticated by shorttandem repeat profiling as matching T24. All cells were grown in DMEM/F12 medium (Gibco) supplemented with 5% FBS and penicillin-streptomycin.

Real-Time PCR. Quantitative PCR reactions were performed using standardmethodologies and normalized to SDHA as housekeeping gene control. Fordetails, see SI Appendix, Methods.

Western Blots. Cells were lysed with modified RIPA buffer (150 mM NaCl, 1%Nonidet P-40, 0.5% sodium deoxycholate, 0.2% SDS, 50 mM Tris·HCl, 5 mMEDTA, 500 mM NaF) containing protease inhibitor mixture (Roche). Proteinlysates were sonicated briefly, resolved by SDS/PAGE, analyzed by Westernblot, and visualized by enhanced chemiluminescence.

MTS and LDH Release Viability/Cytotoxicity Assays. Cell proliferation/viabilitywasmeasured using theMTS assay (Promega kit), while cell death/cytotoxicitywasmeasuredby LDH release (G-Biosciences). For details, see SIAppendix,Methods.

Clonogenic Assays. For clonogenic assays, small numbers of cells (typically10,000) were plated in six-well plates. Cells were treated for 24 to 48 h withCQ or bafilomycin A1 and then rinsed and allowed to recover and grow for anumber of days (typically 6 d); cells were then fixed (10% methanol, 10% acetic

acid) and stained with crystal violet (BD). Plates were scanned and stain wassolubilized with 30% acetic acid, and absorbance was measured at 540 nm.Measurement of cell growth/viability was normalized to untreated control cells.

Microarray Analysis. cDNA probes made from RNA from T24t, FL3, C1AZ,D1AZ, and D1BZ1 cell lines were hybridized to the Affymetrix GeneChipPrimeView Human Gene Expression Array using standard Affymetrix pro-tocols and analyzed by Partek. For details, see SI Appendix, Methods.

Anoikis Experiments.Cells were plated on poly-HEMA–coated plates and cell deathwas analyzed by PI uptake/exclusion. For details, see SI Appendix, Methods.

Derivation of CQ-Resistant FL3 Cells. FL3 cells were selected for CQ resistanceby growing for several months in varying doses of CQ (lowest concentration,30 μM; highest concentration, 100 μM) for varying times (shortest time, 48 h;longest time, 72 h) as illustrated in Fig. 4A. For details, see SI Appendix,Methods.

INCUCYTE Imaging. Quantitative live-cell imaging to assess growth and via-bility was performed using an IncuCyte Zoom imaging system (Essen Bio-Science). For details, see SI Appendix, Methods.

TCGA Bladder Urothelial Carcinoma Data. Gene expression and copy-numberdata were downloaded from TCGA data portal (April 2016) for 408 bladderurothelial tumors. Copy-number data were downloaded from TCGA in theform of GISTIC (40) scores for each gene, discretized to values of −2/−1/0/1/2to represent homozygous loss, heterozygous loss, diploid, heterozygousgain, and homozygous gain, respectively. Overall survival data for TCGABLCA dataset were downloaded from cBioPortal (41, 42) (April 2016). Geneexpression data were downloaded from TCGA in the form of gene-levelRSEM (43) normalized RNA-seq counts.

Survival Analysis. Kaplan–Meier curves for Fig. 5 G and I and SI Appendix, Fig.S12 D and E were generated with copy-number and gene expression datadownloaded from TCGA. We used a GISTIC (40) score of greater than zero toidentify samples which had an ID4 copy-number gain and a score of zero toidentify samples diploid for ID4. Kaplan–Meier curves were generated in R(44) with the “survival” package (45, 46).

Kaplan–Meier curves for SI Appendix, Fig. S13 were generated from GeneExpression Omnibus datasets GSE32894 and GSE13507 (both represent datafrom Illumina expression bead chip arrays). The curves were generated usingthe R2 Genomics Analysis and Visualization Platform software hosted by theAcademic Medical Center in Amsterdam (r2.amc.nl). The Illumina Probe IDused for ID4 is ILMN_ 1721758. The expression cutoff modus in both datasetswas set at the median for the given dataset.

Cluster Dendrogram Analysis. Affymetrix CEL files from the GeneChip HumanPrimeView arrays for each cell line were normalized with robust multiarrayaverage (RMA) (47) in the “affy” package (48), and hierarchical clusteringwas performed with the “stats” package (44) using all 49,495 probes.

Copy-Number Frequency Simulations. To determine the significance ofID4 copy-number gain or loss frequency in each cancer type, we calculated thefrequency of copy-number gain or loss for all other genes on chromosome 6.We compared the frequencies of gain or loss of ID4 with the distribution offrequencies obtained for other chromosome 6 genes to obtain a P value.

Statistics. Unless otherwise indicated, statistical analysis was performed usingthe unpaired two-tailed Student t test. Statistical analysis of metastatic fit-ness was performed using a Wilcoxon rank-sum test. Hazard analysis of Pvalues for the Kaplan–Meier curve in Fig. 6C was done using a log-rank test.

shRNA Lentiviral Transduction. shRNA pLKO.1 vectors were acquired from theFunctional Genomics Facility at the University of Colorado Cancer Center.Lentiviruses were prepared largely according to protocols published by theRNAi Consortium (https://portals.broadinstitute.org/gpp/public/resources/protocols).For details, see SI Appendix, Methods.

Measurement of Autophagic Flux by Ratiometric Flow Cytometry. Cells stablyexpressing mCherry-GFP-LC3 were used for flow cytometric analysis as pre-viously described (49). For details, see SI Appendix, Methods.

Experimental Metastasis Assay. Five-week-old female athymic NCr nu/nu mice(Charles River), maintained in accordance with the University of Colorado Denverand IACUC guidelines, were inoculated with FL3 cells or chloroquine-resistant

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FL3 derivatives D1AZ, C1AZ, and D1BZ1. Mice were injected with 1 × 106

cells via the tail vein, with 10 mice per group. Nine weeks after injection(or as necessary under protocol guidelines due to animal distress or morbidity),mice were killed and human tumor burden in the lung was assessed byquantitative real-time PCR with a human-specific 12p TaqMan probe andprimer set using 0.5 μg of genomic DNA isolated from both lungs (50).

Metastatic Fitness Assay.Multiplex shRNA analysis of pooled populations. To deconvolute pooled pop-ulations of cells treated with the same shRNA (shNS and shID4; see SI Ap-pendix, Methods), we removed the endogenous canonical loop sequence(CTCGAG) and replaced it with eight unique 8-bp barcodes that could beread out using next-generation sequencing (NGS) using annealing oligomermixtures (SI Appendix, Table S3). For details, see SI Appendix, Methods.Metastatic competition of chloroquine-resistant cell lines. Four different experi-mental groups of 5-wk-old female athymic NCr nu/nu mice (Charles River),maintained in accordance with the University of Colorado Denver and IACUCguidelines, were inoculated with FL3, D1AZ, C1AZ, and D1BZ1 cells stablyexpressing barcoded shNS or shID4 constructs. All cell lines were trypsinized,counted, and pooled at an equal number of cells. Mice were injected with2 × 106 cells via the tail vein (i.v.). Mice were euthanized when they met

IACUC criteria for euthanasia or a terminal time point of 90 d post tumorchallenge. Lungs and grossly visible ectopic tumors were isolated and flash-frozen in liquid nitrogen.NGS library preparation and sequencing. Genomic DNA was isolated from eachsample (i.e., lungs or ectopic tumor) using the Gentra Puregene Kit (Qiagen).Sequences were then amplified with custom dual-indexed primers (51), andeach sample was quantified by Qubit dsDNA HS Assay Kit (Invitrogen) andKAPA Library Quant Kit for Illumina-based sequencers (Kapa Biosystems)before equimolar pooling for NGS. For details, see SI Appendix, Methods.

ACKNOWLEDGMENTS. We thank Anna Maria Cuervo (Albert Einstein) forthe LAMP2A-specific shRNA lentiviral vector. We also thank Tzu Lip Phang(Department of Medicine, UC Denver) for help with bioinformatical analysis.This work was supported in part by NIH Grants CA150925 and CA190170 (toA.T.), NIH Grants CA075115 and CA143971 (to D.T.), and shared resources (FlowCytometry; Functional Genomics; Genomics and Microarray Core; and ProteinProduction, Monoclonal Antibody, and Tissue Culture shared resources) fromUniversity of Colorado Cancer Center Support Grant P30CA046934. In addition,this work was supported by a Cancer League of Colorado Cancer Research Grant(to M.J.M.) from the Cancer League of Colorado in association with theUniversity of Colorado Cancer Center.

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