Functional Role of Autophagy-Mediated Proteome Remodeling in Cell Survival Signaling and Innate...

Please cite this article in press as: Mathew et al., Functional Role of Autophagy-Mediated Proteome Remodeling in Cell Survival Signaling and InnateImmunity, Molecular Cell (2014), http://dx.doi.org/10.1016/j.molcel.2014.07.019

Molecular Cell

Article

Functional Role of Autophagy-MediatedProteome Remodeling in Cell SurvivalSignaling and Innate ImmunityRobin Mathew,1,2,6 Sinan Khor,1,2,6 Sean R. Hackett,3 Joshua D. Rabinowitz,3 David H. Perlman,4,5 and Eileen White1,2,*1Rutgers Cancer Institute of New Jersey, 195 Little Albany Street, New Brunswick, NJ 089032Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, NJ, 088543Department of Chemistry, and the Lewis-Sigler Institute for Integrative Genomics, Princeton University, Washington Road, Princeton,NJ 085444Department of Molecular Biology, the Lewis-Sigler Institute for Integrative Genomics, and the Princeton Proteomics andMass Spectrometry

Core, Princeton University, Washington Road, Princeton, NJ 085445Present address: Department of Chemistry, Princeton University, Washington Road, Princeton, NJ 085446Co-first author

*Correspondence: [email protected]

http://dx.doi.org/10.1016/j.molcel.2014.07.019

SUMMARY

Ras-driven cancer cells upregulate basal autophagythat degrades and recycles intracellular proteinsand organelles. Autophagy-mediated proteome de-gradation provides free amino acids to supportmetabolism and macromolecular synthesis, whichconfers a survival advantage in starvation and pro-motes tumorigenesis. While the degradation of iso-lated protein substrates by autophagy has beenimplicated in controlling cellular function, the extentand specificity by which autophagy remodels thecellular proteome and the underlying functional con-sequences were unknown. Here we compared theglobal proteome of autophagy-functional and -defi-cient Ras-driven cancer cells, finding that autophagyaffects the majority of the proteome yet is highlyselective. While levels of vesicle trafficking proteinsimportant for autophagy are preserved during star-vation-induced autophagy, deleterious inflammatoryresponse pathway components are eliminated evenunder basal conditions, preventing cytokine-inducedparacrine cell death. This reveals the global, func-tional impact of autophagy-mediated proteomeremodeling on cell survival and identifies criticalautophagy substrates that mediate this process.

INTRODUCTION

Macroautophagy (hereafter referred to as autophagy) is a stress-

induced, self-cannibalization mechanism that captures da-

maged proteins and organelles in autophagosomes, which

then fuse with lysosomes where their cargo is degraded. In

normal cells, basal autophagy occurs at low levels to prevent

chronic tissue damage and to maintain homeostasis (Mathew

et al., 2007a, 2007b). Autophagy substrates are degraded and

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recycled to provide carbon and nitrogen sources for metabolism

and biosynthesis of macromolecules and for production of

cofactors for redox balance and energy (Mathew and White,

2011; Rabinowitz and White, 2010). The specificity of cargo de-

livery is poorly understood but partly achieved by ubiquitin modi-

fication on cargo that is recognized by specific domains in the

cargo receptor proteins such as p62/SQSTM1 (p62) (Vadlamudi

and Shin, 1998). The captured cargo is then sequestered by au-

tophagosomes that assemble around them, through coordinate

action of the products of essential autophagy genes such as

Atg5 and Atg7, deletions of which abrogate autophagy (Mizush-

ima, 2007). While short-lived intracellular proteins are degraded

primarily by the ubiquitin proteasome system (UPS), autophagy

remains the only known mechanism by which cells eliminate

aggregated proteins and entire organelles.

Recent evidence suggests that autophagy is critical for cancer

cell metabolism, survival, and tumor maintenance (Guo et al.,

2011, 2013a; Rao et al., 2014; Rosenfeldt et al., 2013; Strohecker

et al., 2013). In contrast to normal cells, basal autophagy is

dramatically upregulated in many cancers, such as those driven

by activation of Ras oncogenes, where it is critical for survival

and for tumorigenesis (Guo et al., 2011, 2013b; Lock et al.,

2011; Yang et al., 2011). Metabolic stress due to a deficient

microenvironment and deregulated cell growth may contribute

to this autophagy addiction (Degenhardt et al., 2006), but the

mechanism for this autophagy-mediated survival and growth

of Ras-driven cancers is only beginning to emerge.

Autophagy degrades the proteome, a major amino acid and

energy source for metabolically stressed cancer cells, facilitating

both survival and proliferation. Oncogenic mutations in Ras sup-

press mitochondrial production of acetyl-coenzyme A and the

utilization of fatty acids via b-oxidation, presumably elevating

the demand for protein degradation by autophagy to support

mitochondrial function through anaplerosis (Mathew and White,

2011; White, 2013). Consistent with this, autophagy deficiency

in Ras-expressing tumors causes damaged proteins and defec-

tivemitochondria to accumulate, leading to impairment of energy

metabolism and reduced tumor growth. Although the exact

mechanism by which autophagy supports metabolism and

olecular Cell 55, 1–15, September 18, 2014 ª2014 Elsevier Inc. 1

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Figure 1. SILAC as a Means to Monitor Proteome Remodeling by Autophagy

(A) SILAC-based proteomics comparing autophagy-deficient and -competent cells. Autophagy-deficient cells were given medium containing 13C-Lysine and13C15N Arginine for at least eight doublings to fully label their proteome. Cells were then subjected to a starvation time course (0, 3, and 5 hr Hank’s balanced salt

solution [HBSS]), trypsin digested, and analyzed by ultraperformance liquid chromatography-mass spectrometry (UPLC-MS).

(B) Atg5- or Atg7-deficient cell lines do not have functional autophagy. Starved iBMK cells or TDCLs were lysed and immunoblotted for the indicated antibodies.

b-actin was used as a loading control.

(C) Autophagy-deficient cells have ER stress in starvation. Starved iBMK cells or TDCLs were lysed and immunoblotted for the indicated antibodies. b-actin was

used as a loading control.

(legend continued on next page)

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Autophagy Defects Prime the Interferon Response

2 Molecular Cell 55, 1–15, September 18, 2014 ª2014 Elsevier Inc.


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stress survival is unknown, defects in autophagy cause dere-

gulation of amino acid levels and depletion of key substrates in

mitochondrial metabolism, compromising mitochondrial respira-

tion (Guo et al., 2011, 2013a; Rao et al., 2014; Rosenfeldt et al.,

2013; Yang et al., 2011). Cancer cells are increasingly reliant on

amino acids such as glutamine for mitochondrial function, for

nucleotide and lipid synthesis, and for redox homeostasis (De-

Berardinis et al., 2007; Son et al., 2013). Despite this important

role for autophagy in cancer metabolism and stress survival, a

comprehensive understanding of autophagy substrates and the

specific mechanisms by which autophagy supports cancer cell

survival are currently lacking. Additionally, although autophagy

is selective to certain cellular substrates in specific contexts,

whether or not autophagy targets specific pathways to alter

cell function is not understood (Mizushima and Komatsu, 2011).

Using isogenic cell models comparing autophagy-intact and

-deficient cells (Atg5+/+ and Atg5�/�) and the technique of Stable

Isotope Labeling by Amino acids in Cell culture (SILAC) for proteo-

mic analysis, we examined the role of autophagy in remodeling the

proteome in basal and starvation conditions. We discovered that

autophagy-mediated proteome remodeling affects the majority

of the proteome and is selective, preserving cellular function of

Ras-driven cancer cells. Our results show that autophagy selec-

tively eliminates proteins (autophagy protein substrates) involved

in pathways that are nonessential or toxic for survival under stress,

whilepreferentially sparing those involved inpathwaysessential for

themaintenanceof functional autophagy and stress survival (auto-

phagy-resistant proteins). Specifically, we found that proteins

involved in the innate immune response, such as the retinoic-

acid-inducible gene-I (RIG-I) pathway, accumulated in auto-

phagy-deficient cells. Conversely, the soluble N-ethylmaleimide-

sensitive factor (NSF) attachment receptor (SNARE)-mediated

vesicle trafficking, lysosome, and endocytosis pathways essential

for intracellular protein trafficking between endoplasmic reticulum

(ER), Golgi apparatus, endosomes, and lysosomes escaped auto-

phagy-mediated elimination. These findings suggest that auto-

phagy extensively remodels the proteome and that defects in

autophagy may prime nonimmune cells for constitutive innate im-

mune and inflammatory signaling with profound implications for

cell survival, partly explaining the requirement for autophagy for

sustaining aggressive cancer growth.

RESULTS

Global Impact of Autophagy on the Cellular ProteomeTo assess the impact of autophagy on the overall cellular prote-

ome, we employed SILAC (Ong et al., 2002) to compare relative

protein levels in isogenic wild-type (WT) (Atg5+/+) and auto-

phagy-deficient (Atg5�/�) HRasG12V-transformed immortalized

baby mouse kidney epithelial (iBMK) cells (Guo et al., 2011)

in normal growth media and following 3 and 5 hr of starvation

(Figure 1A). Ras expression levels in these iBMK cells are com-

(D and E) Starvation robustly induces autophagy in iBMK cells and TDCLs. iBMK c

lysed, and immunoblotted for the indicated antibodies. Quantitation of the LC3-II

(n = 2).

Data are representative of at least two independent experiments.

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parable to those of human cancer cell lines with activating Ras

mutations and render them tumorigenic, although Atg5 defi-

ciency compromises tumor growth (Guo et al., 2011).

Western blot analysis revealed induction of autophagy in the

WT cells in starvation, and was completely blocked by Atg5

and Atg7 deletion as indicated by the absence of LC3-I-to-

LC3-II processing in the autophagy-deficient cell lines, which

instead accumulated the autophagy substrate p62 (Figure 1B).

Autophagy defects also caused accumulation of ER chaperones

GRp170 and GRp78 and protein disulphide isomerase (PDI) in

both iBMK cell lines (Figure 1C; top panel), consistent with

previous findings (Mathew et al., 2009). We then examined the

autophagy flux with the lysosomal inhibitor Bafilomycin A1,

which resulted in the accumulation of LC3-II in the WT cells,

but not in the autophagy-deficient cells (Figure 1D). In tumor-

derived cell lines (TDCLs) from the KRasG12D;p53�/� genetically

engineeredmousemodel (GEMM) for non-small-cell lung cancer

(NSCLC) (Guo et al., 2013a), starvation robustly induced auto-

phagy in the WT cells, while autophagy defects resulted in p62

accumulation and elevated expression of ER stress markers

(Figures 1B, 1C, and 1E). Thus, the Ras-transformed Atg5

iBMK cells used for the SILAC are representative of autophagy

functionality independent of tissue type and Ras subfamily.

SILAC-based mass spectrometry coupled with strong cation

exchange (SCX) and off-gel fractionations (OG) and protein iden-

tification by MaxQuant (MQ) and Proteome Discoverer (PD)

(Supplemental Experimental Procedures) enabled identification

of 7,184 proteins (�25% of the total estimated mouse proteome)

present during at least one of the conditions tested (0, 3, and 5 hr

of starvation) (Figure 2A; red) comparable to the most compre-

hensive description of the mouse kidney proteome to date

(dotted circle) (Huttlin et al., 2010) (Figure 2A). Of these, 5,300

proteins were identified by both MQ and PD algorithms, with

845 unique to MQ and 1,039 unique to PD (Figure 2B). Similarly,

5,441 proteins were identified by both fractionation techniques,

with 931 unique to SCX and 812 unique to OG separations

(Figure 2B), consistent with other observations of partial comple-

mentarity between the two fractionation techniques and algo-

rithms (Barbhuiya et al., 2011; Chang et al., 2014). A substantial

overlap between the proteins was identified in each starvation

condition, indicating the relatively minor qualitative alterations

to the proteomes (Figure 2B).

We normalized the data in two ways. First, we took into

account difference in viability during starvation, to which the

autophagy-deficient cells are more sensitive, by normalizing

the proteomes on a per-cell basis. The first observation was

that a strikingly large percentage of the observed proteome

was impacted by the functional status of autophagy, as evident

by differential relative protein abundances between WT and

autophagy-deficient cell lines consistent across the duration of

starvation. We observed that autophagy was involved in the

predicted degradation of nearly half of the overall proteome on

ells or TDCLswere starvedwith andwithout lysosomal inhibitor Bafilomycin A1,

/LC3-I ratio is shown as bar graphs. Quantitation is represented as mean ± SD


Figure 2. Effect of Autophagy Deficiency on the Cellular Proteome

(A) Venn diagrams showing statistically validated proteins by SILAC-MS, compared to the total proteins identified in our study (red), a previously publishedmouse

proteome (gray), and the total estimated mouse proteome (turquoise).


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a per-cell basis within 3 hr of starvation, which increased to 70%

at 5 hr (Figure 2C). This suggests that autophagy is a significant

mechanism for turnover and remodeling of the cellular proteome.

Second, to examine the specificity by which autophagy im-

pacts the global proteome, proteins in each SILAC channel

were normalized to total protein for each cell type and examined

for relative protein abundance ratios (ratios of levels in auto-

phagy-deficient compared to WT; Atg5�/�/Atg5+/+: Heavy/Light[H/L]) in normal growth and starvation conditions. Each condition

contained groups of proteins with disproportionately higher or

lower levels in autophagy-deficient cells compared to the WT,

representing proteins that appear to be selectively eliminated

or spared by autophagy, respectively (Figure 2D). Out of the

7,184 total proteins identified, under normal conditions, 6.4%

(462 proteins) were elevated (>1.5-fold) in the autophagy-defi-

cient cells, while 6.7% (484 proteins) showed decreased levels

compared to the WT cells. Following 3 hr of starvation, 5.8%

(419 proteins) showed elevation and 7.2% (517 proteins) showed

a decrease in relative protein levels. Similarly, after 5 hr of starva-

tion, 4.9% (352 proteins) were elevated, while 7.2% (520 pro-

teins) showed reduced relative proteins levels (Figures 2B and

2D). Thus, autophagy has a dramatic impact on the overall pro-

teome, with its loss surprisingly producing both up- and downre-

gulation of specific proteins relative to the WT.

To examine if the changes in the proteome caused by auto-

phagy deficiency were specific to cellular activities, we looked

at the molecular functions enriched among proteins with

extreme changes in their relative abundance in each condition

using Gene Set Enrichment Analysis (GSEA) as described previ-

ously (Subramanian et al., 2005). Interestingly, the Rig-I like

receptor (RLR) signaling pathway, DNA replication, and one car-

bon pool by folate metabolism were highly enriched among pro-

teins elevated in autophagy-deficient cells at all the conditions

tested (Figure 2E). Alternatively, proteins involved in cellular pro-

cesses that include proteasomes and ribosomes were selec-

tively preserved with functional autophagy as indicated by low

H/L ratios.

We then examined how relative protein abundances changed

across time during starvation by subjecting the log geometric

means of the relative abundance values normalized to their basal

ratio (0 hr) to hierarchical clustering (Figure 2F). While the major-

ity of proteins had relative protein abundances (H/L ratio) consis-

tent across all conditions, there were clusters of proteins whose

relative levels decreased (green) or increased (red) during starva-

tion. Interestingly, pathways related to glycerophospholipid

metabolism, angiogenesis (VEGF signaling), SNARE vesicle

trafficking, etc. were enriched among proteins that showed a

(B) Venn diagrams showing proteins identified using MaxQuant (MQ), Proteome D

the distribution of protein identifications at three different time points (0, 3, and 5

(C) Bar graph showing the increase in the total number of proteins that showed r

compared to the WT cells at 0, 3, and 5 hr of starvation on a per-cell basis (norm

(D) S curve displaying relative protein abundances of all the proteins between auto

protein.

(E) Bar graphs showing NES for pathways enriched among extremes of chang

deficiency (only normalized p values < 0.05 are shown). Positive NES indicates

negative NES indicates enrichment for H/L ratios < 1 (relative increase in autoph

(F) Hierarchical clustered heatmap with the 0 hr normalized log2 H/L values sho

starvation using the top and bottom 10% most-pronounced changes.

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progressive decrease in protein abundance in starvation (Fig-

ure 2F). In contrast, pathways related to lysosome, protein

degradation (ubiquitin mediated proteolysis), etc. were enriched

among proteins that showed an increase (Figure 2F), consistent

with the need to preserve autophagy machinery and protein ho-

meostasis during starvation.

To examine any inherent basal difference due to the auto-

phagy defect, and to identify a subset of proteins that showed

statistically significant changes in normal and starvation condi-

tions, we compared the overall data in Figure 2D by t statistics

calculated for each methodology and performed hierarchical

clustering of relative abundance measurements across the

starvation time course. We then computed p values based on

t statistics to determine whether the time zero intercept of

proteins log2 (H/L) relative abundances differed significantly

over time from time zero (Figures 3A and 3B). We identified a

subset of 3,181 proteins that were either elevated (yellow) or

depressed (blue) due to autophagy deficiency (Figure 3A),

with over 50% of the proteins showing autophagy-dependent

alteration.

Autophagy-Mediated Protein Remodeling Is SelectiveIn the subset of 3,181 proteins that had statistically significant

changes in normal and starvation conditions that were attribut-

able to autophagy defect, 1,638 behaved as major autophagy

substrates (high H/L ratio) and thus are potential markers for

autophagy-mediated protein degradation (Figure 3C). Surpris-

ingly, 1,543 proteins behaved as if they were resistant to de-

gradation (low H/L ratio); that is, their levels were retained in

autophagy-competent cells in starvation (Figure 3C).

Among the proteins that were enriched on both extremes, a

common theme of molecular functions suggested that auto-

phagy selectively targets protein substrates to enable cell sur-

vival. For example, DNA replication proteins, including PCNA,

POLA2, MCM3, MCM, and LIG1, were among the proteins

elevated in autophagy-deficient cells (Figure 3C and Table S1).

Importantly, levels of poly(ADP-ribose) polymerase (PARP), a

critical component of the single-stranded DNA repair, energy

sensing machinery, and apoptotic cell death, and STAT1, an

important mediator of interferon-mediated cell death, were

significantly elevated in the autophagy-deficient cells (Figure 3C

and Table S1), suggesting a prosurvival role for autophagy

through selective targeting of protein substrates.

Proteins Preserved during AutophagyConsistent with a stress survival role for autophagy, specific pro-

teins with purported roles in cellular functions essential for stress

iscoverer (PD), and different fractionation techniques (SCX and OG), as well as

hr).

elative (>2-fold) increase (red) or decrease (green) in the autophagy-defective

alized to cell number).

phagy-deficient versusWT cells (log2 H/L ratios) shown in (A) normalized to total

es in relative protein abundances (H/L ratios) in starvation due to autophagy

enrichment for H/L ratios > 1 (relative increase in autophagy-defective), and

agy-competent).

wing time-dependent changes in relative protein levels across time points in


Figure 3. Pathway Enrichment in Autophagy-Deficient Cells Due to Defects in Protein Degradation(A) Volcano plot showing statistical significance of pre-starvation differences in protein abundances due to autophagy function. Proteins whose abundance is

significantly altered due to autophagy are shown: a total of 1,638 proteins (51.5%) were preferentially degraded in autophagy-competent cells (yellow), while

1,543 (46.7%) were selectively preserved (blue). Significance of PD-based quantification is shown.

(B) Scatter plot comparing statistical significance as determined by either MQ or PD. Proteins found to be elevated or depleted due to autophagy were those that

exceeded a false discovery rate of 0.05 when analyzed with both MQ and PD platforms.

(C) S plot showing relative protein abundance (H/L ratio) in 3,181 proteins found to be significantly altered due to autophagy deficiency. Some select examples of

proteins that showed extreme changes are highlighted.

(D) NES from GSEA analysis of the 3,181 proteins found to be significantly altered due to autophagy deficiency with normalized p value < 0.05 are shown.

(E) Enrichment plots showing relative enrichment for significantly enriched pathways in GSEA analysis in (D).

See also Table S1.

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survival, such as poly(ADP-ribose) glycohydrolase (PARG, poly

[ADP-ribose] catabolism), adenylate kinase 4 (AK4, ATP/ADP

homeostasis), CTP synthase 2 (CTPS, glutaminolysis), and cit-

rate synthase (CS), were among those that showed relative

accumulation in the WT cells (low H/L ratio) (Figure 3C and Table

S1). Similarly, several of the proteasome components (PSMC2,

PSMC4, PSMC5, PSMD6, etc.) were among the proteins highly

retained by autophagy in starvation (Figure 3C and Table S1).

Importantly, critical components of vesicle trafficking and endo-

cytosis, such as synaptosomal-associated protein 29 (SNAP29),

were also selectively preserved, indicating that maintaining func-

tional autophagy is critical in remodeling the proteome. This sug-

gests that autophagy selectively retains protein components of

distinct pathways that are essential for cell survival.

Defects in Proteome Remodeling Alter CellularSignaling PathwaysAlterations in protein homeostasis of signaling pathway compo-

nents may impact the function of these pathways. Therefore, in

order to test the functional consequences of this preferential pro-

teome remodeling by autophagy on cellular stress signaling,

3,181 proteins (UniProt IDs) ranked by change in relative abun-

dance were subjected to GSEA that allowed statistical enrich-

ment of pathways and cellular functions on both extremes of

changes. Interestingly, proteins related to DNA replication and

innate immunity-related pathways, such as RLR signaling, Toll-

like receptor (TLR) signaling, and RNA-degradation, were among

the most significantly enriched in the autophagy-defective cells

(Figures 3D and 3E). In contrast, pathways such as the protea-

some, ribosome, and lysosome pathways were enriched in the

autophagy-competent cells (Figures 3D and 3E), supporting

the importance of these processes in cell survival. This consis-

tently indicated a high degree of selectivity during starvation.

To verify the major implicated pathways by means orthogonal

to SILAC-based proteomics, we explored the constituents of

these pathways by western blotting. Consistent with the SILAC

data as indicated by H/L ratios for major members of the

pathway (Figure 4A; red indicates high, green indicates low or

unchanging), proteins in the RIG-I pathway were upregulated

in the autophagy-deficient compared to the WT cells. Members

of the RIG-I pathway, including the double-stranded RNA virus

(dsRNA) sensing RNA helicases, melanoma differentiation-

associated gene 5 (MDA-5), and RIG-I, and the downstream ki-

nases, the phosphorylated form of TANK-binding kinase 1 (p-

TBK1) and the inhibitor of NF-kB kinase subunit epsilon

(IKKε), accumulated in autophagy-deficient cells, consistent

with SILAC observations (Figures 4A and 4B). Autophagy-defi-

cient TDCLs showed similar accumulation of these proteins,

although MDA-5 or RIG-I were undetectable, consistent with

the requirement for p53 for the expression of these proteins

(Munoz-Fontela et al., 2008). Note that the TDCLs are p53 defi-

cient, whereas the iBMKs express dominant-negative p53 (p53-

DD) and perhaps do not have a complete p53 block.

In order to validate that autophagy selectively retains specific

proteins essential to preserve functional autophagy and protein

homeostasis, we examined proteins in different subdivisions of

the endosome-lysosome pathway. Proteins involved in vesicle

formation at the cell surface (caveolin and clathrin) and early

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endosome/vesicle fusion proteins, including DCC-interacting

protein 13-alpha (APPL1), early endosome antigen 1 (EEA1),

RAB4, RAB5, and SYNTAXIN 6, were generally retained in the

autophagy-WT cells, suggesting selective resistance to degra-

dation (Figure 4C). Interestingly, there were differences in the

mechanisms of vesicle internalization that were cell-type depen-

dent; higher levels of clathrin, but not caveolin, were detected in

WT iBMK cells, whereas the opposite trend was observed in the

TDCLs. Thus, although mechanisms may differ depending on

cell type, in general autophagy preserves functional vesicle

trafficking.

Autophagy Limits the Interferon Response bySuppressing IRF3 ActivationSelective accumulation of members of the RIG-I pathway in the

autophagy-defective cells suggested that autophagy may be

involved in suppressing innate immune and type I interferon

(IFN) responses. Therefore, we examined the activation of the

transcription factor interferon regulatory factor 3 (IRF3), which

is the target of p-TBK1 and IKKε. The dimerization of IRF3 is a

hallmark of its activation (Takahasi et al., 2003), andwe assessed

this by starving or treating cells with the synthetic dsRNA virus

analog poly I:C in complexed aswell as uncomplexed forms (Fig-

ure 5A). Uncomplexed poly I:C stimulates TLR3, and complexed

poly I:C is detected by MDA-5. Consistent with our hypothesis,

dimerized IRF3 was increased in autophagy-deficient cells,

especially in response to complexed poly I:C (Figure 5A). IRF3

activation and subsequent nuclear translocation results in the

transcription of genes involved in IFN response. Therefore, we

investigated if the activation of the innate immune response re-

sulted in the transcriptional activation of the IFN response by

gene expression profiling of these cells under similar conditions

of cellular stress (Figure 5B and Table S2). GSEA analysis iden-

tified the cytokine-cytokine receptor interactions, TLR, and JAK-

STAT signaling pathways as the most significantly enriched

pathways, consistent with our hypothesis (Figure 5C). Impor-

tantly, autophagy defects caused significant alterations in the

expression of several IRF3 and NF-kB target genes even under

basal conditions (Figure 5D). Interestingly, JAK-STAT mediates

the interferon response upon interferon alpha or beta (IFN-a or

IFN-b) stimulation, and autophagy-defective cells showed levels

of STAT1 by SILAC >10-fold higher (Table S1). This further sup-

ported our recent finding of an elevated inflammatory response

with tumor-specific Atg7 deletion in KRas-driven spontaneous

lung cancer (Guo et al., 2013a). Thus, autophagy functions to

suppress IRF3 activation and the interferon response.

Defects in protein homeostasis induced by autophagy defi-

ciency deregulate protein components of the innate immunity

pathway sufficient to trigger and elicit activation of IFN response.

To further confirm this hypothesis, we performed interleukin-6

(IL-6) and IFN-b luciferase reporter assays. Despite similar basal

levels, autophagy-deficient iBMKs showed increased IL-6 pro-

moter activity in response to different stimuli, including tumor ne-

crosis factor alpha (TNF-a), a known inducer of NF-kB signaling,

poly I:C, and starvation (Figure 5E). In contrast to IL-6, IFN-b

expression was increased in the autophagy-deficient cells by

poly I:C, but not by TNF-a or starvation. Uncomplexed poly I:C

significantly increased IL-6 and IFN-b expression, pointing to


Figure 4. Validation of Proteins Designated as Autophagy Substrates and Proteins Designated as Selectively Preserved

(A) Pathway diagram indicating t-intercept estimates fromMaxQuant log2 H/L ratios for RIG-I pathway members, showing high (red) and low or unchanging ratio

(green). Blank circles indicate proteins not designated by SILAC.

(B) Autophagy-deficient cells accumulate RIG-I pathway proteins. iBMK cells and TDCLs were starved, lysed, and immunoblotted for the indicated antibodies.

b-actin was used as a loading control.

(C) Autophagy-competent cells retain vesicle trafficking proteins in starvation. iBMK cells and TDCLs were starved, lysed, and immunoblotted for the indicated

antibodies. b-actin was used as a loading control. Quantitation of the ratio of protein between autophagy-deficient and autophagy-competent cells at the

indicated time point normalized to b-actin is shown as well. Quantitation is represented as mean ± SD (n = 2).

Data are representative of at least two independent experiments.

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simultaneous IFN and NF-kB activation. Recently, it was re-

ported that activation of RIG-I signaling activates the mitochon-

drial antiviral-signaling protein (MAVS) by aggregation, which in

turn activates IRF3 and NF-kB upon viral infection (Hou et al.,

2011). In order to test if the IRF3 activation due to autophagy

deficiency was mediated through MAVS, we expressed in-

creasing amounts of MAVS, which showed a dose-dependent

decrease in cell viability (Figures S1A and S1B). We then per-

formed luciferase assays with increasing levels of MAVS expres-


sion to see if we could further induce IRF3 signaling (Figure S1C).

In general, the reporter activity for IFN-b increased with ectopic

MAVS expression in a dose-dependent manner under basal and

poly I:C treatment, but not starvation. This suggests that the level

of MAVS protein, or potentially other RIG-I components, corre-

lates with levels of signaling through the pathway even under

basal conditions. To further confirm increased expression

of these cytokines, we performed ELISA for IL-6, IFN-a, and

IFN-b (Figures 5F and S1D), which showed dramatically

Figure 5. Autophagy Suppresses IRF3 Activation and Subsequent Transcription of IRF3-Target Genes

(A) Autophagy-deficient cells have increased IRF3 activation. iBMK cells were either starved or treated with poly I:C, lysed, and probed by native PAGE for

dimerized IRF3.

(B) Gene expression forHRasG12V-expressing Atg5+/+ (top) and Atg5�/� (bottom) iBMK cells in starvation. Functionally enriched genes in Atg5�/� iBMK cells (red)

included those involved in the inflammatory response as shown in (C) and (D) below.

(C) GSEA analysis of data in (B), showing enrichment of cytokine-cytokine receptor interaction and Toll-like receptor signaling pathways in Atg5�/� iBMK cells

under basal condition (0 hr).

(D) Table of inflammatory genes identified in (B) by gene expression as enriched in Atg5�/� iBMK cells (0 hr).

(E) Luciferase reporter assays in iBMK cells showing increased IL-6 and IFN-b activity in autophagy-deficient iBMK cells. Relative fold changes of Atg5�/� as

compared to Atg5+/+ iBMK cells are also shown.


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increased levels in media from poly I:C-treated autophagy-defi-

cient cells compared to those from the WT.

Autophagy Defects Disrupt Proteome Composition andPrime Nonimmune Cells for Innate Immunity and theInterferon ResponseWe hypothesized that accumulation of RIG-I-related proteins

produced by autophagy defects allows nonimmune cells to be

primed to mount an innate immune response, leading to cell

death. We assessed the viability and clonogenicity of these cells

when exposed to starvation or poly I:C and found that auto-

phagy-deficient iBMK cells were more sensitive to all stressors,

most prominently complexed poly I:C (Figures 6A, 6B, and S2A).

To confirm that cytokines in the media caused the reduced

viability, we treated cells with poly I:C in the presence of neutral-

izing antibodies against IFN-a and IFN-b, which efficiently

rescued the cell death (Figure 6C). Next, we performed condi-

tioned media experiments to determine if interferon acts through

a paracrine mechanism. Autophagy-deficient iBMK cells had

reduced viability and clonogenicity when givenmedia from either

autophagy-competent or -deficient cells that had been treated

with poly I:C (Figures 6D and S2B), which was rescued by the

addition of neutralizing antibodies to the media (Figure 6E, red

boxes). IFN-a/b activate JAK-STAT signaling, which ultimately

increases transcription of interferon-stimulated genes, possibly

leading to apoptosis (Stawowczyk et al., 2011). To determine

the mechanism of cell death, we blocked apoptotic cell death

by treating cells with a pan-caspase inhibitor Z-VAD-FMK. Unex-

pectedly, cell deathwasmuchmore pronounced upon treatment

with Z-VAD-FMK (Figures 7A and S3A). It was previously shown

inmacrophages that treatment with poly I:C and Z-VAD-FMK ac-

tivates programmed necrosis (He et al., 2011). Consistently, we

found that treatment with poly I:C in the presence of Z-VAD-FMK

amplifies cell death. Addition of the receptor-interacting serine/

threonine-protein kinase 1 (RIP1) inhibitor necrostatin-1 (Nec-1)

effectively reduced this increased cell death, while Nec-1 alone

did not have any effect (Figures 7A and S3A). Levels of RIP1

confirmed that Nec-1 had available substrate (Figure 7B). We

then sought to determine the mechanism of this cell death in

response to poly I:C by assessing caspase-3 cleavage. Indeed,

we observed higher levels of cleaved caspase-3 in autophagy-

deficient cells when treated with complexed poly I:C, consistent

with diminished viability and clonogenic survival. Together, these

results suggest that autophagy-deficient cells are primed to

mount a robust interferon response upon stress stimulus, such

as RNA-helicase-mediated detection of cytoplasmic dsRNAs.

Activation of interferon signaling occurs through a paracrine

mechanism, which leads to cell death by apoptosis.

DISCUSSION

Autophagy promotes the stress survival, growth, and aggres-

siveness of Ras-driven lung cancers (Guo et al., 2013a) by recy-

(F) Autophagy-deficient cells have elevated secretion of inflammatory cytokines.

collected, and ELISA was performed for IL-6 and IFN-b.

Data in (E) and (F) are represented asmean ± SD (n = 2). n.s., not significant; *p < 0

and Table S2.


cling macromolecules to fuel mitochondrial respiration and

metabolism (Mathew and White, 2011; Rabinowitz and White,

2010; White, 2013), but the mechanism is still under inves-

tigation. In the absence of external nutrients, a selective auto-

phagy-mediated degradation of the cellular proteome may

facilitate this process, promoting survival. Recently, a model

for selective autophagosome formation was proposed wherein

autophagy receptors p62 and NBR1 directly engage ubiquitin-

like modifiers (UBLs) for selective autophagosome formation

around the cargo in a context-dependent manner (Rogov et al.,

2014). In addition, it was shown that autophagosomes are en-

riched in specific cargo receptors that mediate iron metabolism,

indicating the selectivity of autophagy substrates (Mancias et al.,

2014). However, a comprehensive understanding of the selec-

tivity for autophagy substrates, and if or how this impacts cellular

function to promote tumor growth, has been lacking.

The finding that autophagy deficiency altered amino acid and

mitochondrial metabolism, rendering lung cancer cells suscepti-

ble to stress (Guo et al., 2013a), raised several important ques-

tions. First, does autophagy-mediated protein degradation alter

cellular proteome composition? Second, if true, do these auto-

phagy-mediated proteome alterations modify cellular function?

While defects in autophagy have been shown to have indirect

effects on cells, such as compromising the UPS, the extent to

which autophagy deficiency affects proteins with long half-lives,

and its impact on cell survival signaling, is unknown (Korolchuk

et al., 2009). Herein, we report that in starvation, Atg5 deficiency

in HRasG12V-transformed cells significantly altered relative pro-

teins levels of the majority of cellular proteins compared to those

in WT cells, revealing the magnitude of the impact of autophagy

on protein homeostasis. The major downstream consequence of

disruption in protein homeostasis was 2-fold. First, when auto-

phagy was functional, there was selective elimination of proteins

detrimental for cell survival to stress, while those that support cell

survival were preserved. Second, defects in autophagy caused

accumulation of putative autophagy substrates, many of which

were members of signaling pathways detrimental to cell survival.

Moreover, their accumulation was sufficient to mediate cell

death, providing an explanation, at least in part, for the require-

ment of autophagy for tumor maintenance. Importantly, auto-

phagy-deficient cells accumulate levels of PARP familymembers

while depleting their catabolic counterpart, PARG (Figure 3C).

This alteration could occur in response to alterations in NAD+

regulation and changes in cellular energy homeostasis and likely

leads to increased ADP-ribosylation of PARP targets. Alteration

in PARP levels is implicated in NAD+ depletion, mitochondrial

dysfunction, inflammatory-response gene expression, senes-

cence, and susceptibility to cell death, all of which are pheno-

typeswe and others have observed in autophagy-defective cells.

Most striking was the observation that autophagy defects

result in accumulation of proteins, such as RIG-I (6.75-fold)

and MDA-5 (12.36-fold), that in turn primed cells for subsequent

activation of the innate immune response. Ligand activation of

Supernatants from iBMK cells in normal media or poly I:C-treated cells were

.05, **p < 0.01, and ***p < 0.001 by unpaired Student’s t test. See also Figure S1

Figure 6. Autophagy Defects Prime Cells for Innate Immune Response and Subsequent Cell Death

(A) Autophagy-deficient cells are more susceptible to metabolic stress or poly I:C treatment. iBMK cells were treated with HBSS, uncomplexed poly I:C, or

complexed poly I:C, and cell viability was examined.

(B) Cells treated as in (A) were allowed to recover in normal medium and assessed for clonogenic survival. Triplicate wells are shown in Figure S2A.

(C) Neutralizing antibodies rescue poly I:C-induced cell death. iBMK cells were stimulated with complexed or uncomplexed poly I:C in the presence or absence of

neutralizing antibodies to IFN-a and IFN-b, and cell viability was assessed.

(D) Autophagy-deficient cells have reduced viability with conditioned media. iBMK cells were stimulated with complexed or uncomplexed poly I:C and then given

fresh media to allow for interferon production. This media was then collected and overlayed onto untreated cells overnight, after which cell viability or clonogenic

survival was assessed.

(E) Neutralizing antibodies rescue cell death in conditioned media. iBMK cells were treated as in (D), and collected media was mixed with either PBS or

neutralizing antibodies to IFN-a and IFN-b and overlayed onto untreated cells overnight, upon which cells were assessed for clonogenic survival.

Data are represented as mean ± SD (n = 3). n.s., not significant; *p < 0.05, **p < 0.01, and ***p < 0.001 by unpaired Student’s t test. Data are representative of at

least two independent experiments. See also Figure S2.

Molecular Cell


Molecular Cell 55, 1–15, September 18, 2014 ª2014 Elsevier Inc. 11


(legend on next page)

Molecular Cell




Molecular Cell



RIG-I induces apoptosis inmelanoma cells (Besch et al., 2009). A

recent report showed that the autophagy-activating kinaseULK1

inhibits IRF3 and the interferon response through phosphoryla-

tion of stimulator of interferon genes (STING) (Konno et al.,

2013). In addition, conditional knockout of the essential auto-

phagy gene FIP200 elevates interferon response inmammary tu-

mors (Wei et al., 2011). Hence, derepression of innate immunity

with autophagy deficiency was not surprising, although whether

accumulation of pathway proteins in tumor cells was sufficient to

activate interferon response was unclear.

The highlight of our study is the finding that selective protein

accumulation due to autophagy defects alone is sufficient to

prime cells for the interferon response, which upon activation

by ligands leads to cell death by apoptosis via a paracrine mech-

anism. It was recently shown that in the absence of viral infec-

tion, RIG-I induction alone could trigger apoptosis-independent

cell death via AKT-mTOR inhibition in an AMLmodel, supporting

our findings (Li et al., 2014). This inhibition also activated auto-

phagy, possibly as a stress response, indicating the importance

of autophagy in this setting. Inherent metabolic stress and cell

death in autophagy-deficient cancer cells can lead to incom-

pletely digested nucleic acids that may mimic an antiviral-like in-

flammatory response, leading to reduced tumor burden. Indeed,

failure to degrademitochondrial DNA throughmitophagy causes

inflammation and heart failure inmice (Oka et al., 2012), and DNA

and toxic radicals from depolarized mitochondria, a conse-

quence of defective autophagy, activate inflammasomes (Zhou

et al., 2011), producing proinflammatory cytokines such as

IL-1b and IL-18. Alternatively, autophagy may be required to

degrade the protein components of inflammatory signaling as

a negative feedback mechanism that is absent in autophagy-

deficient cells, explaining the activated state of these pathways.

Regardless, our data suggest that accumulation of autophagy

substrates in autophagy-deficient cells may contribute to activa-

tion of the interferon response, leading to apoptosis, andwarrant

further investigation.

In stark contrast to autophagy substrates, proteins whose

levels were refractory to autophagy-mediated downregulation

were found to be essential for maintaining autophagy-related

processes, such as vesicle-mediated trafficking, endocytosis,

and lysosomal protein degradation, among others. While auto-

phagy machinery has been known to interact with vesicle

trafficking components (Behrends et al., 2010), their selective

retention in starvation-induced autophagy was not known. This

underscores the importance of selective autophagy as a mech-

anism to preserve the autophagy process itself and cell survival

in starvation. Strategies to inhibit these processes may provide

Figure 7. Activation of Interferon Response Triggers Cell Death by Ap

(A) Z-VAD-FMK accelerates cell death under poly I:C treatment by activating necr

Nec-1, followed by HBSS, uncomplexed poly I:C, or complexed poly I:C. Cells we

(B) Poly I:C induces cell death by apoptosis. iBMK cells were treated as in (A), l

loading control.

(C) A model for autophagy-mediated immune suppression. In normal cells, autop

limiting IRF3 activation and the interferon response. Autophagy deficiency cau

members, which is sufficient to prime the host cells for interferon response. Up

signaling, leading to cell death by apoptosis, which when blocked reverts to nec

Data are represented as mean ± SD (n = 3). n.s., not significant; *p < 0.05, **p < 0

least two independent experiments. See also Figure S3.

M

additional opportunities to augment cell death by autophagy in-

hibition for enhanced therapeutic efficacy.

In summary, our results indicate that autophagy plays a major

role in modulating the cellular proteome and that deregulation of

this process alters cell function. Autophagy-mediated suppres-

sion of the antitumor immune response may be a mechanism

by which autophagy supports tumor growth (Figure 7C). We

report that autophagy specifically targets mediators of inflam-

mation to suppress inflammatory pathways, unraveling an

important consequence of defective autophagy in activating

the innate immune and interferon responses. This selective auto-

phagic degradationmay potentially explain the increased inflam-

mation in mice with tumor-specific autophagy defects (Guo

et al., 2011, 2013a). Together, these observations suggest that

autophagy selectively targets substrates to maintain cell survival

and protein homeostasis, the deregulation of which has the

potential to alter signaling pathways critical for survival in Ras-

driven cancers. Autophagy substrates and autophagy-resistant

proteins identified here will serve as biomarkers for monitoring

autophagy function in clinical settings.

EXPERIMENTAL PROCEDURES

Tissue Culture, SILAC, Viability, and Clonogenic Assays

All cell lines were cultured as previously described (Guo et al., 2011, 2013a).

SILAC labeling was performed using heavy arginine (613C, 415N) and lysine

(613C) (Pierce, cat. # 1860972, 89990). Cell viability was assessed by Vi-

CELL cell viability analyzer (Beckman Coulter). For clonogenic assays, cells

were treated, allowed to recover for 2 days in normal medium, fixed, and

stained with Giemsa.

Luciferase Assays

Luciferase assays were performed as described previously (Mathew et al.,

2009). A detailed description can be found in the Supplemental Experimental

Procedures.

Mass Spectrometry

SILAC-labeled cells were subjected to a starvation time course and lysed.

Extracts were trypsin digested and separated by strong cation exchange

(SCX) or off-gel fractionation (OG). Peptide fractions were subjected to

reverse-phase nano-LC-MS and MS/MS performed on Nano Ultra 2D plus

UPLC system (Eksigent) coupled to an LTQ-Orbitrap hybrid MS (Thermo

Fisher Scientific). The raw MS data set acquired for this study can be down-

loaded at the following URL: https://chorusproject.org/anonymous/download/

experiment/28d353ecd5924727900e231d0a11ecc5.

SUPPLEMENTAL INFORMATION

Supplemental Information includes Supplemental Experimental Procedures,

three figures, and two tables and can be found with this article online at

http://dx.doi.org/10.1016/j.molcel.2014.07.019.

optosis

optosis. iBMK cells were pretreated with Z-VAD-FMK, Nec-1, or Z-VAD-FMK +

re allowed to recover in normal medium and assessed for clonogenic survival.

ysed, and immunoblotted for the indicated antibodies. b-actin was used as a

hagy functions to suppress accumulation of RIG-I pathway proteins, thereby

ses deregulation of protein homeostasis and accumulation of RIG-I pathway

on activation by appropriate stress stimulus, host cells activate paracrine IFN

roptosis.

.01, and ***p < 0.001 by unpaired Student’s t test. Data are representative of at


https://chorusproject.org/anonymous/download/experiment/28d353ecd5924727900e231d0a11ecc5

https://chorusproject.org/anonymous/download/experiment/28d353ecd5924727900e231d0a11ecc5


Molecular Cell



AUTHOR CONTRIBUTIONS

R.M., S.K., D.H.P., and E.W. designed the experiments. R.M. and S.K. per-

formed the experiments and analyzed the data. S.R.H. performed the statisti-

cal analysis. R.M., S.K., D.H.P., and E.W. interpreted the data and prepared

the manuscript. J.D.R. provided advice.

ACKNOWLEDGMENTS

We thankmembers of theWhite and Rabinowitz laboratories for insightful sug-

gestions. We thank Drs. Z.J. Chen (UT Southwestern Medical Center, TX) for

IFN-b-luciferase and FLAG-MAVS plasmids, C. Gelinas (Rutgers University,

NJ) for pIL6kB-Luc plasmid, J.Y. Guo for generating TDCLs, and J.Y. Guo

and H.Y. Chen for generating iBMK cell lines. We also thank C. Hulderman

and J. Park for assistance with western blotting and cell culture. This work

was supported by grants from NIH (R37 CA53370 and R01 CA130893 to

E.W., RC1 CA147961 and R01 CA163591 to E.W. and J.D.R) and DOE (DE-

AC05-06OR23100 to S.R.H.).

Received: May 8, 2014

Revised: June 27, 2014

Accepted: July 24, 2014

Published: August 28, 2014

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