Autophagy Is Critical for Pancreatic Tumor Growth and ...€¦ · to constrain tumor progression,...

AUGUST 2014�CANCER DISCOVERY | 905

RESEARCH BRIEF

Autophagy Is Critical for Pancreatic Tumor Growth and Progression in Tumors with p53 Alterations Annan Yang 1 , N.V. Rajeshkumar 3 , Xiaoxu Wang 1 , Shinichi Yabuuchi 3 , 7 , Brian M. Alexander 1 , Gerald C. Chu 2 , Daniel D. Von Hoff 4 , Anirban Maitra 3 , 5 , 6 , and Alec C. Kimmelman 1

ABSTRACT Pancreatic ductal adenocarcinoma is refractory to available therapies. We have

previously shown that these tumors have elevated autophagy and that inhibition

of autophagy leads to decreased tumor growth. Using an autochthonous model of pancreatic cancer

driven by oncogenic Kras and the stochastic LOH of Trp53 , we demonstrate that although genetic abla-

tion of autophagy in the pancreas leads to increased tumor initiation, these premalignant lesions are

impaired in their ability to progress to invasive cancer, leading to prolonged survival. In addition, mouse

pancreatic cancer cell lines with differing p53 status are all sensitive to pharmacologic and genetic inhi-

bition of autophagy. Finally, a mouse preclinical trial using cohorts of genetically characterized patient-

derived xenografts treated with hydroxychloroquine showed responses across the collection of tumors.

Together, our data support the critical role of autophagy in pancreatic cancer and show that inhibition of

autophagy may have clinical utility in the treatment of these cancers, independent of p53 status.

SIGNIFICANCE: Recently, a mouse model with embryonic homozygous Trp53 deletion showed paradoxi-

cal effects of autophagy inhibition. We used a mouse model with Trp53 LOH (similar to human tumors),

tumor cell lines, and patient-derived xenografts to show that p53 status does not affect response

to autophagy inhibition. These fi ndings have important implications on ongoing clinical trials. Cancer

Discov; 4(8); 905–13. ©2014 AACR.

See related commentary by Amaravadi and Debnath, p. 873.

Authors’ Affi liations: 1 Division of Genomic Stability and DNA Repair, Department of Radiation Oncology, Dana-Farber Cancer Institute; 2 Department of Pathology, Brigham and Women’s Hospital, Boston, Mas-sachusetts; 3 Department of Oncology and Pathology, Johns Hopkins Uni-versity School of Medicine, Baltimore, Maryland; 4 Translational Genomics Research Institute, Phoenix, Arizona; Departments of 5 Pathology and 6 Translational Molecular Pathology, The University of Texas MD Anderson Cancer Center, Houston, Texas; and 7 Department of Surgery, Tohoku Uni-versity Graduate School of Medicine, Sendai, Japan

Note: Supplementary data for this article are available at Cancer Discovery Online (http://cancerdiscovery.aacrjournals.org/).

A. Yang and N.V. Rajeshkumar contributed equally to this article.

Corresponding Author: Alec C. Kimmelman, Dana-Farber Cancer Institute, 450 Brookline Avenue, Boston, MA 02215. Phone: 617-632-5195; Fax: 617-249-0371; E-mail: [email protected]

doi: 10.1158/2159-8290.CD-14-0362

©2014 American Association for Cancer Research.

INTRODUCTION

Pancreatic ductal adenocarcinoma (PDAC) accounts for

95% of pancreatic cancers and is one of the most lethal

cancers, with a 5-year survival rate of approximately 6% ( 1 ).

More than 80% of patients are diagnosed at an advanced

stage, and treatment is very limited. Previously, we identi-

fi ed that human PDACs have elevated basal autophagy, and

either genetic or pharmacologic inhibition of autophagy

[the latter with chloroquine (CQ) treatment] showed robust

Research. on September 14, 2020. © 2014 American Association for Cancercancerdiscovery.aacrjournals.org Downloaded from

Published OnlineFirst May 29, 2014; DOI: 10.1158/2159-8290.CD-14-0362

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906 | CANCER DISCOVERY�AUGUST 2014 www.aacrjournals.org

Yang et al.RESEARCH BRIEF

responses in human and mouse PDAC cell lines and tumor

models ( 2 ). Because of its extensive use in human patients

for other indications, multiple clinical trials have opened

using the CQ derivative hydroxychloroquine (HCQ) to

inhibit autophagy in patients with pancreatic cancer ( www.

clinicaltrials.gov ).

Autophagy is a catabolic mechanism that recycles cellular

components, and the level is dynamically controlled to main-

tain cell function. When cells are under stress such as starva-

tion, autophagy is elevated to redistribute energy to sustain

cell survival; however, it could also lead to cell death when

the attempts to sustain viability have failed ( 3 ). The dual role

of autophagy also applies to tumorigenesis as it can both

serve as a barrier to cancer initiation and promote cancer

growth by providing energy for advanced malignancies ( 4 ).

This dynamic role of autophagy in cancer has been supported

using various mouse models. Mosaic deletion of Atg5 predis-

poses mice to benign liver adenomas that do not progress to

malignant tumors ( 5 ). Previous data have also shown that

autophagy may play a particularly important role in cancers

driven by oncogenic Kras ( 2 , 6 , 7 ). Indeed, mice engineered to

express oncogenic Kras in the lung with concurrent Atg5 or

Atg7 loss develop increased benign lesions (adenomas), but

fail to progress to malignancy ( 8, 9 ).

Sequence analysis of PDAC has shown that more than 90%

of tumors possess KRAS mutations ( 10 ), and mouse models

have validated the critical role of this oncogene in driving

PDAC formation ( 11, 12 ) and in tumor maintenance ( 13 ). In

addition, PDACs demonstrate a mixture of tumor-suppressor

gene mutations at varying frequencies that have been shown

to constrain tumor progression, including TP53 , CDKN2A ,

and SMAD4 ( 14 ). TP53 is one of the most commonly altered

genes in all of human cancer, and it is found mutated or lost

in the majority (∼75%) of PDAC ( 15 ). In most cases, these

genetic mutations, including in the tumor suppressor gene

TP53 , are acquired via LOH during tumor development ( 15 ).

As a result, a conditional Trp53 deletion allele or a Trp53

mutant allele is often crossed to an activated Kras allele

to accelerate tumorigenesis in PDAC genetically engineered

mouse models (GEMM). This is typically done as a single

copy, as mice with homozygous Trp53 deletion have rapidly

progressive disease that is nonmetastatic ( 16 ), and for these

reasons most treatment studies using PDAC GEMMs have

focused on heterozygous Trp53 models ( 17 ). The hetero-

zygous Trp53 mice develop tumors dependent on the stochas-

tic LOH at the Trp53 locus, analogous to human PDAC ( 18 ).

Recently, the role of autophagy in PDAC progression was

explored using a GEMM ( 19 ). Although loss of Atg5 or Atg7

in the pancreas with an activating Kras mutation completely

inhibited progression of premalignant lesions to invasive can-

cer, in the context of an embryonic homozygous Trp53 dele-

tion in the pancreas, autophagy loss seemed to accelerate the

progression to invasive cancer ( 19 ). However, because of the

nature of the Trp53 homozygous model used, the conclusion

generated from this model might not be fully representative

of human tumors where Tp53 alterations occur by LOH. In

this study, we used a Trp53 heterozygous PDAC GEMM to

study how loss of autophagy affects tumor development in

a situation where p53 is present before tumor initiation and

lost in fully developed tumors, mimicking human PDAC

development. We also explored how tumor cell lines with

TP53 deletions or mutations responded to autophagy inhi-

bition. Finally, we determined how human patient–derived

xenografts responded to autophagy inhibition using HCQ

treatment to assess the clinical signifi cance of TP53 genotypes

on human tumor response.

RESULTS To study the function of autophagy in pancreatic can-

cer development, we generated pancreatic conditional Atg5

knockout mice using a Pdx1 promoter–driven Cre recom-

binase ( Fig. 1A ; ref. 20 ). We fi rst assessed the impact of

homozygous deletion of Atg5 ( Atg5 L/L ) in the pancreas. Pan-

creatic deletion of Atg5 was not embryonic lethal and did not

cause signs of malignant transformation; however, it caused

a cellular disruption in endocrine tissues starting at 12 weeks,

leading to a reduction in insulin-producing β-cells, as had

been shown previously with Atg7 deletion in that compart-

ment (ref. 21 ; Supplementary Fig. S1A and S1B). Thus, Atg5 L/L

mice had a reduced long-term survival that was consistent

with previous reports ( Fig. 1B ; ref. 19 ). Given the relatively

restricted phenotype of the Atg5 L/L mice and the long sur-

vival of this cohort, we crossed Atg5 L/L mice into the PDAC

GEMM ( Pdx1Cre + ; lslKras G12D/+ ; Trp53 L/+ ) to assess the role of

autophagy in PDAC development. To this end, we generated

three cohorts with differing Atg5 allelic status: Atg5 +/+ , Atg5 L/+ ,

and Atg5 L/L . This GEMM recapitulates the human condition

beginning from premalignant pancreatic intraepithelial neo-

plasia (PanIN) to invasive and malignant PDAC ( 16 , 18 ). We

fi rst compared the overall survival between the groups and

found that the Atg5 L/L cohort had the longest median survival

( Fig. 1C ). Indeed, there were signifi cantly more long-term

survivors (>30 weeks of age) in this group compared with

either the Atg5 L/+ or Atg5 +/+ cohorts ( P < 0.0005). Interestingly,

we observed that approximately 20% of the Atg5 L/L mice died

before 6 weeks of age, which was not seen in the Atg5 +/+ cohort

(0%) and infrequently (4%) in the Atg5 L/+ group. Analysis of

this small population of Atg5 L/L mice revealed that they did

not die of PDAC but due to acinar destruction as shown by

massive acinar to ductal metaplasia, which led to the disrup-

tion of most of the exocrine pancreas and left on average

less than 10% normal pancreas (Supplementary Fig. S2A and

S2B). The majority of the Atg5 L/L cohort survived past this

early insult and when death due to PDAC was assessed, the

Atg5 L/L mice had a signifi cantly prolonged survival compared

with the Atg5 +/+ and Atg5 L/+ cohorts ( P = 0.0032; Fig. 1D ). To

investigate the mechanism behind the prolonged survival in

the Atg5 L/L mice, we performed a detailed histologic assess-

ment of pancreata at an early time point (6 weeks) and a

later time point (15 weeks) to determine how Atg5 loss affects

both tumor initiation and progression. At 6 weeks, there was

a marked difference in formation of noninvasive precursor

lesions, PanIN, between the cohorts, with the Atg5 L/L group

having on average more than 50 PanINs per mouse compared

with the Atg5 L/+ group, which showed only 2 to 5 PanINs per

mouse ( Fig. 1E ). The difference in PanIN formation was fur-

ther quantifi ed by comparing the fraction of normal pancreas

remaining with that containing PanIN and surrounding

infl ammation ( 22 ). The Atg5 L/L cohort showed 41.7% normal





Autophagy Is Critical for Pancreatic Tumor Growth RESEARCH BRIEF

Figure 1. Loss of autophagy extends PDAC survival. A, pancreatic sections stained by IHC for ATG5 expression, showing Pdx1 -driven Cre-mediated deletion of Atg5 . Scale bar, 50 μm. B, Kaplan–Meier analysis comparing overall survival of Pdx1Cre + Atg5 L/+ (blue) and Atg5 L/L (red) mice. Representative sections stained with hematoxylin and eosin (H&E), illustrating the β-islet disruption in the Atg5 L/L mice. Atg5 L/+ mice are shown as controls. Scale bar, 100 μm. C, Kaplan–Meier analysis comparing overall survival of Trp53 L/+ lslKras G12D/+ Pdx1Cre + mice with Atg5 +/+ (black), Atg5 L/+ (blue), and Atg5 L/L (red). There are signifi cantly more Atg5 L/L mice with long-term survival (>30 weeks) than in the Atg5 +/+ or Atg5 L/+ cohorts ( P < 0.0005, Fisher exact test). H&E staining of representative Trp53 L/+ lslKras G12D/+ Pdx1Cre + Atg5 L/L mice that died before 6 weeks of each due to pancreatic disruption . Scale bar, 100 μM. D, Kaplan–Meier analysis comparing PDAC-specifi c survival of Atg5 +/+ (black), Atg5 L/+ (blue), and Atg5 L/L (red) mice with Trp53 L/+ lslKras G12D/+ Pdx1Cre + . The survival of the Atg5 L/L cohort was signifi cantly longer than that of the Atg5 +/+ cohort (*, P = 0.003, log-rank test). E, quantifi cation of the normal pancreas area of Atg5 L/+ and Atg5 L/L mice at 6 weeks. H&E staining of representative Atg5 L/+ and Atg5 L/L pancreata at 6 and 15 weeks. Proportion of mice that developed PDAC (vs. only PanIN) at 15 weeks in Atg5 L/+ ( n = 10) and Atg5 L/L ( n = 8) mice. Scale bar, 100 μm. F, pancreatic tumors from indicated genotypes stained with cleaved caspase-3, γH2AX, and Ki67 antibody. The number of cleaved caspase-3 or γH2AX-positive cells per fi eld was counted from fi ve random fi elds per mouse. Scale bar, 50 μm. *, P < 0.05 by t test. G, growth curve comparing proliferation of Atg5 +/+ (black, n = 4) and Atg5 L/L (red, n = 4) tumor cell lines at both high- and low-serum conditions. Error bars, SD of the results from four cell lines of each genotype. *, P < 0.05 by t test.

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pancreatic parenchyma versus 98.6% in the Atg5 L/+ group

( P = 0.0015; Fig. 1E ). At 15 weeks, the normal pancreatic

parenchyma in the Atg5 L/L group further decreased to 31.7%,

but interestingly only 25% of the mice (2 of 8) had invasive

PDAC, whereas 60% of the Atg5 L/+ mice (6 of 10) developed

PDAC ( Fig. 1E ). Together, these data indicate that although

autophagy defi ciency increased PanIN development (tumor

initiation), it inhibits PanIN progression to PDAC. IHC for

ATG5 and LC3 confi rmed that all tumors developed in Atg5 L/L

mice lacked ATG5 expression and had absent autophagy






(Supplementary Fig. S3A and S3B). Interestingly, cell lines

derived from two of the deleted tumors reestablished Atg5

expression after a few passages, indicating that rare subclones

escaped Pdx1-cre–mediated excision and can grow out in a

small fraction of the tumors (Supplementary Fig. S4A). As

expected, tumors from all three genotypes showed LOH of the

remaining Trp53 allele and did not express p53 protein (Sup-

plementary Fig. S4B). We also assessed tumors from the three

groups for the expression of cleaved caspase-3, γH2AX, and

Ki67 ( Fig. 1F ) to measure apoptosis, DNA damage, and prolif-

eration, respectively, and found that there was elevation of both

apoptosis and DNA damage as well as a decrease in prolifera-

tion in the Atg5 L/L group compared with the Atg5 +/+ or Atg5 L/+

cohorts ( Fig. 1F ). Therefore, in the tumors that were able to

form in the setting of autophagy loss, there was increased DNA

damage and cell death. This is consistent with the fact that

cell lines derived from these autophagy-incompetent tumors

proliferate at a signifi cantly slower rate than Atg5 +/+ tumors

under both high-serum and low-serum conditions ( Fig. 1G ).

Together, our data suggest that autophagy is required for

proper progression of premalignant lesions to invasive PDAC,

and those tumors that do progress are less robust.

Previously, we reported that human PDAC cell lines and

tumors have elevated basal autophagy, and CQ treatment, as

well as suppression of autophagy using RNAi against critical

autophagy genes, could inhibit cell growth in vitro as well as in

xenografts. In addition, CQ treatment signifi cantly prolonged

survival in the lsl Kras G12D ; Trp53 L/+ PDAC GEMM ( 2 ). A recent

study using a Kras -driven PDAC GEMM with an embryonic

Trp53 homozygous deletion showed that either Atg5 or Atg7

deletion promoted tumor progression, thus reducing mouse

survival. In their model, CQ treatment seemed to have a mod-

est reduction in lifespan as well. Given the differences between

our prior and current data using the Trp53 L/+ PDAC GEMM,

and that reported by Rosenfeldt and colleagues ( 19 ) using

the Trp53 L/L GEMM, we examined the impact of autophagy

inhibition in a panel of murine PDAC cell lines with vary-

ing Trp53 genotypes ( Trp53 L/+ , Trp53 L/L , and p53 R172H/+ ). As

the majority of the human clinical trials are using CQ or its

derivative, HCQ, we focused on response to CQ given its clini-

cal relevance. As has been reported in the past ( 18 ), the wild-

type (WT) allele of Trp53 was lost in Trp53 L/+ , Trp53 L/L , and

the Trp53 R172H/+ lines, as confi rmed by PCR (Supplementary

Fig. S5A). Western blot analysis showed loss of p53 expression

in the Trp53 L/+ and Trp53 L/L lines, whereas Trp53 mutations

were detected in the Trp53 R172H/+ lines (DNA point mutation

confi rmed by sequencing; Supplementary Fig. S5B and S5C).

All PDAC lines, independent of the Trp53 genotype, showed a

signifi cant, dose-dependent reduction in clonogenic growth

when treated with CQ ( Fig. 2A ). We had previously shown that

one of the consequences of autophagy inhibition in human

PDAC cells was a decrease in oxidative phosphorylation

(measured by oxygen consumption; ref. 2 ). Consistent with

these fi ndings, all cell lines, independent of TP53 status, had a

signifi cantly reduced baseline oxygen consumption rate (OCR;

Fig. 2B ). To validate the CQ data, we repeated the clonogenic

assays using shRNAs to either ATG5 or ATG7 . Suppression of

expression of both ATG genes and autophagy inhibition was

confi rmed by Western blot analysis (Supplementary Fig. S6).

Similar to the CQ data, suppression of autophagy via RNAi

signifi cantly attenuated clonogenic growth independent of

the TP53 genotype ( Fig. 2C ).

Finally, to model the therapeutic situation that is occur-

ring in ongoing human clinical trials, we performed effi cacy

trials using 12 individual human patient–derived pancreatic

cancer xenografts (PDX) treated with HCQ or saline control.

Mice with established pancreatic tumors were treated with

HCQ, and tumor growth was compared with the vehicle-

treated mice. The overwhelming majority of the PDX lines

showed a reduction in tumor volume compared with controls

( P < 0.05), with a third of the PDX lines showing more than

20% inhibition of tumor growth compared with the tumors

in the vehicle-treated mice ( Fig. 3B ). All tumors had KRAS

mutations (except P410) and TP53 mutations (except JH024;

Supplementary Table S1). Interestingly, consistent with pre-

vious fi ndings of the role of autophagy in KRAS -mutant can-

cers ( 2 , 8 ), the KRAS WT tumor did not seem to have elevated

autophagy by transmission electron microscopy (TEM) and

did not respond to HCQ treatment ( Fig. 3A and B ). IHC for

LC3 in the treated tumors showed that HCQ increased the

LC3 punctate staining in the HCQ-treated samples, consist-

ent with effective autophagy inhibition. In line with the TEM

data, the KRAS WT tumor had the lowest amount of basal

puncta that did show an increase upon HCQ treatment ( Fig.

3C ). In addition, the fact that all the TP53 -mutant tumors

showed varying degrees of response further supports the fact

that disruption of the p53 axis does not affect response to

antiautophagy therapies. Ki67 and cleaved caspase-3 stain-

ing of the three best responders versus the KRAS WT non-

responder was consistent with its effect on tumor volume:

HCQ treatment signifi cantly inhibited tumor cell prolifera-

tion and increased apoptosis in the responders but had mini-

mal impact on the nonresponder ( Fig. 3D and E ).

DISCUSSION In this study, we have used multiple orthogonal approaches

(autochthonous models, cell lines, and human tumor

xenografts) to demonstrate that disruption of the p53 axis

(a fi nding observed in 75% of PDAC) has no impact on the

effi cacy of autophagy inhibition. We used a PDAC GEMM

( Pdx1Cre + ; lslKras G12D/+ ; Trp53 L/+ ) in which Trp53 is lost by

stochastic LOH as seen in human tumors and determined

the role of autophagy in PDAC progression using a condi-

tional Atg5 allele. We found that deletion of Atg5 predisposed

mice to premalignant pancreatic lesions as evidenced by the

increased occurrence of PanINs. On the other hand, mice

with Atg5 deletion were signifi cantly less likely to develop

PDAC and therefore had improved survival.

The role of autophagy in tumorigenesis is controversial

because there are studies supporting both its being a sup-

pressor and a promoter. Evidence to support autophagy as

a tumor suppressor comes from studies in which autophagy

genes were deleted in mice. With the exception of Becn1, in

which the heterozygote was used and autophagy was only

partially attenuated, these studies have shown that loss of

autophagy predisposes mice to benign tumors ( 4 , 23 , 24 ). In

contrast, evidence to support autophagy as a tumor promoter

comes from studies of advanced tumors, in which blocking

autophagy inhibits tumor growth and can synergize with






Figure 2. Autophagy inhibition reduces colony formation and reduces baseline OCR independent of p53 status. A, CQ reduces clonogenic growth (blank bar, 0 μmol/L CQ; 10% dotted bar, 7.5 μmol/L CQ; 25% dotted bar, 15 μmol/L CQ). Error bars, SD of a representative experiment performed in triplicate. *, P < 0.005 by t test. B, CQ reduces baseline OCR in tumor cell lines. A representative OCR plot is shown for one cell line of each genotype. Error bars, SD of triplicate wells. *, P < 0.05 by t test. Bottom, quantifi cation of the reduction in baseline OCRs from cell lines of each indicated Trp53 genotype. Error bars, SD from three experiments. C, clonogenic assay shows that knocking down autophagy reduces colony formation independent of Trp53 genotype. Blank bar, shGFP; 10% dotted bar, shAtg5; 25% dotted bar, shAtg7. Error bars, SD of a representative experiment performed in triplicate. *, P < 0.05 by t test.

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chemotherapy in mouse models ( 2 , 25 ). Our data suggest

a dual role for autophagy in PDAC development, whereby

autophagy loss increases the initiation of tumors, but abro-

gates the effi cient progression to invasive cancer.

Several prior studies have shown that autophagy deletion

attenuates malignant tumor formation in lslKras G12D/+ tumor

models ( 8, 9 ). However, the role of p53 in this process is com-

plex, with studies reporting results that differ in terms of how

tumorigenesis affects autophagy loss with concurrent Trp53

deletion ( 9 , 19 ). A recent study using a PDAC GEMM showed

that the impact of autophagy inhibition differed depending

on whether Trp53 was concurrently deleted or not ( 19 ). Some

of these differences could be due to the models used, or, alter-

natively, that particular ATG genes may have nonoverlapping






Figure 3. HCQ treatment selectively suppressed tumor growth and proliferation in PDXs with active baseline autophagy. A, representative TEM pho-tomicrographs of autophagy in PDXs. Arrows, autophagosomes. B, effi cacy of HCQ in a panel of 12 individual patient-derived PDXs ( n = 8–10 tumors per PDX). C, LC3 staining of PDX tumors treated with vehicle or HCQ. Small black squared area is enlarged in the left corner. Scale bar, 20 μm. Numbers of LC3 puncta per fi eld were quantifi ed (*, P < 0.05, t test). D, representative photomicrographs of Ki67-stained tumor sections in PDXs sensitive or resistant to HCQ treatment. The number of Ki67 + cells was quantifi ed for each line. Five fi elds were counted from two tumors for each PDX (*, P < 0.05; **, P < 0.01, t test). Scale bar, 50 μm. E, cleaved caspase-3 staining of PDX tumors and positive cells per fi eld were quantifi ed (*, P < 0.05). Scale bar, 50 μm.

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functions that are independent of autophagy. In addition,

there are likely differences regarding whether Trp53 is deleted

homozygously in the germline, or if one copy is lost by

somatic LOH (mimicking the cognate human phenomenon).

Our data are consistent with prior reports showing that loss

of autophagy can promote the initiation of tumorigenesis in

the pancreas, and prevent the progression to cancer in the

setting of oncogenic Kras mutations ( 19 ). However, unlike

the data from Rosenfeldt and colleagues ( 19 ), our work shows

that Atg5 deletion impairs the progression of premalignant

PanIN to invasive PDAC in the setting of Trp53 loss. We

believe that this difference stems from the fact that in their

study, Trp53 was homozygously deleted during embryogen-

esis. Therefore, in the physiologic setting of Trp53 loss during






tumor progression, autophagy seems to be required for opti-

mal PDAC development. The intricate and complex relation-

ship of autophagy and p53 is of great importance and awaits

further study ( 26 ).

Perhaps most relevant to cancer treatment, we showed that

acute inhibition of autophagy by CQ treatment or RNAi

inhibited growth of murine PDAC cell lines with various Trp53

alterations, and is consistent with our prior data using human

PDAC cell lines, which almost all harbor TP53 mutations

( 2 ). Finally, we used a large panel of patient-derived PDAC

xenografts and performed treatment studies using HCQ. HCQ

treatment attenuated the growth of the majority of primary

patient-derived PDAC xenografts that harbor TP53 mutations.

Together, our data continue to support the integration of

antiautophagy therapies into the treatment of PDAC. Ongo-

ing human clinical trials in PDAC will determine whether this

approach is feasible and effective in patients.

METHODS Genetically Engineered Mice

Atg5 L/L mice were kindly provided by Dr. Noboru Mizushima

( The University of Tokyo, Tokyo, Japan; ref. 27 ). Trp53 L/L mice were

obtained from Anton Berns ( Netherlands Cancer Institute, Amster-

dam, The Netherlands; ref. 28 ). Pdx1 -Cre was obtained from Doug

Melton ( Harvard University, Boston, MA; ref. 20 ). All animal experi-

ments were approved by the Institutional Animal Care and Use Com-

mittee under protocol 10-055 at the Dana-Farber Cancer Institute

(Boston, MA). Mice were maintained on a mixed background. Survival

was determined by humane endpoints as specifi ed by the protocol,

including showing signs of being moribund, signifi cant weight loss,

skin ulceration, or in rare cases being found dead. All mice with

PDAC-specifi c death were histologically confi rmed.

Histology All tissues were fi xed in 10% formalin overnight and embedded

in paraffi n. For IHC, tumors were deparaffi nized and rehydrated.

After antigen retrieval in citrate buffer (pH, 6.0), tumors were

labeled with primary antibody overnight and then detected using

the VECTASTAIN Elite ABC Kit (pk-6100; Vector Labs) and DAB

(sk-4100; Vector labs). Antibodies used for immunohistology are

ATG5 (1:200; NB110-53818; Novus Biologicals), cleaved caspase-3

(1:200; D175; Cell Signaling Technology), γH2AX (1:100; clone

JBW301; Millipore), and LC3 (1:100, NBP1-19167; Novus Bio-

logicals). Ki67 staining was performed using an anti–MIB-1 (Ki67)

antibody (Ventana Medical Systems; clone K2; 1:100 dilutions) as

previously described ( 29 ). Sections from two tumors per treatment

group were examined microscopically, and more than fi ve repre-

sentative fi elds from each slide were photographed under ×200

magnifi cations (except LC3, which was taken at ×1,000 magnifi ca-

tion; ref. 30 ).

Cell Culture All cell lines were derived from mouse primary tumors and grown

in DMEM (11965; Invitrogen) with 10% FBS and 1% Penstrep. The

Trp53 L/L and Trp53 R172H lines were obtained from Dr. H. Ying (The

University of Texas MD Anderson Cancer Center, Houston, TX)

and Dr. D. Tuveson (Cold Spring Harbor Laboratory, Cold Spring

Harbor, New York). Cells were regenotyped to verify p53 status, and

Western blot analyses were used to confi rm p53 protein expression.

Trp53 -mutant cell lines were sequenced to verify the presence of the

mutation. Atg5 L/L lines were harvested from an approximately 5-mm 2

chunk of tumor, minced, and digested in 4% collagenase/dispase

for 1 hour. Cell lines were routinely tested and all were negative for

mycoplasma infection.

Clonogenic Assay Cells were plated in 6-cm dishes at 500 cells per dish in growth

medium with 10% FBS and treated with CQ the day after seeding.

After 7 days, cells were fi xed in 80% methanol and stained with 0.2%

crystal violet, and colonies were counted. The surviving fraction was

calculated using the plating effi ciency.

Growth Curves Cells were plated in 24-well plates at 3,000 cells per well in 1 mL of

media. Media were not changed throughout the course of the experi-

ment. At the indicated time points, cells were fi xed in 10% formalin

and stained with 0.1% crystal violet. Dye was extracted with 10% ace-

tic acid, and the relative proliferation was determined by attenuance

(D) at 595 nm.

OCR Oxygen consumption measurements: 1.5 × 10 4 cells were seeded

in a 96-well Seahorse plate, and OCRs were measured using the Sea-

horse XF96 Instrument (Seahorse Biosciences). Basal mitochondrial

respiration (3 mmol/L glucose) and ATP production (2 mmol/L

oligomycin) were measured. Maximal respiration was obtained by

carbonyl cyanide 4-(trifl uoromethoxy)phenylhydrazone (FCCP;

0.5 mmol/L), and non-mitochondrial OCRs were obtained by adding

2 mmol/L antimycin A. Values were normalized by protein concen-

tration to account for cell number.

Western Blot Analysis Proteins were extracted by RIPA buffer and separated on 4% to 12%

stacking SDS–PAGE gel. Proteins were then transferred to polyvinyli-

dene difl uoride (PVDF) membrane (Bio-Rad). Membranes were blocked

with 5% nonfat dry milk and then incubated with the primary antibody

overnight at 4°C. Following Tris Buffered Saline with Tween 20 (TBST)

washing, membranes were incubated with peroxidase-conjugated sec-

ondary antibody for 1 hour and exposed on fi lm using the Enhanced

Chemiluminescence (ECL) Detection System (Thermo Scientifi c).

Antibodies used were as follows: ATG5 (1:500; NB110-53818; Novus

Biologicals), ATG7 (1:300; A2856; Sigma), LC3B (1:500; NB600-1384;

Novus Biologicals), p53 (1:1,000; FL-393; Santa Cruz Biotechnology),

and β-actin (1:3,000; A2066; Sigma).

Lentivirus-Mediated shRNAs All shRNA vectors were obtained from the RNA Interfer-

ence Screening Facility of Dana-Farber Cancer Institute. Atg5

(TRCN0000375819) and Atg7 (TRCN0000092163) shGFP: forward 5′, CCGGCGCAAGCTGACCCTGAGTTCATTCAAGAGATGAACTTCA

GGGTCAGCTTGCTTTTT; reverse 5′, AATTAAAAAGCAAGCTGAC

CCTGAAGTTCATCTCTTGAATGAACTCAGGGTCAGCTTGCG ( 31 ).

Lentivirus was produced using 293T cells, as previously described ( 2 ).

Electron Microscopy Freshly harvested subcutaneous tumors from mice were fi xed

immediately with 3% paraformaldehyde, 1.5% glutaraldehyde, and

2.5% sucrose in 0.1 mol/L sodium cacodylate and cut into 1-mm 3

squares. After postfi xation in 1% osmium tetroxide for 1 hour on ice

and dehydration, the samples were embedded in a mixture of Epon–

araldite. Thin sections from four blocks were collected on uncoated

grids, stained with uranil and lead citrate. Samples were sectioned

and examined using an FEI Tecnai 12 Transmission Electron Micro-

scope equipped with a 16-bit 2K × 2K FEI Eagle bottom-mount

camera and an SIS MegaView III wide-angle camera. Images were

captured at ×12,000 magnifi cation ( 32 ).






In Vivo Effi cacy of HCQ in Human PDXs Animal experiments were conducted following approval and in

accordance with the Institutional Animal Care and Use Committee

guidelines of the Johns Hopkins University under protocol

MO06M385. A total of 12 human PDXs established from the pri-

mary tumors, resected from patients with pancreatic cancer at

the Johns Hopkins Hospital, were used for the study ( 33 ). The

mutational status of these tumors was previously reported ( 14 )

and is shown as Supplementary Table S1. Fresh tumors resected

from mice were cut into cubes of 2 mm 3 , and were subcutaneously

implanted on both fl anks of 6-week-old female nu/nu athymic mice

(Harlan). When cohorts of tumors reached approximately 150 mm 3 ,

animals (5 mice per group, each group with 8–10 tumors) were ran-

domly assigned to receive vehicle or HCQ (60 mg/kg, i.p., once daily

for 4 weeks) treatments ( 2 ). Tumors were measured twice per week,

and tumor volumes were calculated using the following formula:

V = ( a × b 2) /2, where a is the largest dimension and b the smallest.

Tumor growth in HCQ-treated animals was compared with that in

vehicle-treated mice.

Statistical Analysis Overall survival events included death as defi ned by the protocol

with censoring for alive at last follow-up. Events for PDAC-specifi c

survival included deaths attributable to PDAC with uninformative

censoring for deaths related to other causes or at last follow-up. Sur-

vival plots were generated using the Kaplan–Meier method. The log-

rank test was used to compare survival distributions between groups.

The proportion of mice alive at 30 weeks or longer in the Atg5 L/L

group was compared with the other two groups using a Fisher exact

test. Statistical analyses were performed using R version 3.0.2 ( 34 ).

Disclosure of Potential Confl icts of Interest A.C. Kimmelman has received honoraria from the speakers’ bureau

of US Oncology and is a consultant/advisory board member for

Forma Therapeutics and Gilead. No potential confl icts of interest

were disclosed by the other authors.

Authors’ Contributions Conception and design: A. Yang, N.V. Rajeshkumar, B.M. Alexan-

der, A. Maitra, A.C. Kimmelman

Development of methodology: A. Yang, N.V. Rajeshkumar,

B.M. Alexander, A. Maitra

Acquisition of data (provided animals, acquired and managed

patients, provided facilities, etc.): A. Yang, S. Yabuuchi, A. Maitra

Analysis and interpretation of data (e.g., statistical analysis,

biostatistics, computational analysis): A. Yang, N.V. Rajeshkumar,

X. Wang, S. Yabuuchi, B.M. Alexander, G.C. Chu, A.C. Kimmelman

Writing, review, and/or revision of the manuscript: A. Yang,

N.V. Rajeshkumar, B.M. Alexander, D.D. Von Hoff, A. Maitra,

A.C. Kimmelman

Administrative, technical, or material support (i.e., reporting

or organizing data, constructing databases): A. Yang, X. Wang,

B.M. Alexander, D.D. Von Hoff

Study supervision: A. Maitra, A.C. Kimmelman

Acknowledgments The authors thank David Tuveson and Haoqiang Ying for provid-

ing mouse PDAC lines and Noboru Mizushima for the conditional

Atg5 -null mice.

Grant Support This work was supported by National Cancer Institute grant

R01CA157490, American Cancer Society (ACS) Research Scholar

grant RSG-13-298-01-TBG, and the Lustgarten Foundation

(to A.C. Kimmelman), and an SU2C–AACR Translational Research

Dream Team SU2C–AACR-DT0509 grant (to A. Maitra, N.V. Rajesh-

kumar, and D.D. Von Hoff).

The costs of publication of this article were defrayed in part by

the payment of page charges. This article must therefore be hereby

marked advertisement in accordance with 18 U.S.C. Section 1734

solely to indicate this fact.

Received April 4, 2014; revised May 19, 2014; accepted May 21,

2014; published OnlineFirst May 29, 2014.

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2014;4:905-913. Published OnlineFirst May 29, 2014.Cancer Discovery Annan Yang, N.V. Rajeshkumar, Xiaoxu Wang, et al. Progression in Tumors with p53 AlterationsAutophagy Is Critical for Pancreatic Tumor Growth and

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