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Can-14-3362 227.238
Published OnlineFirst December 4, 2015; DOI: 10.1158/0008-5472.CAN-14-3362
Microenvironment and Immunology
Control of PD-L1 Expression by OncogenicActivation of the AKT–mTOR Pathway inNon–Small Cell Lung CancerKristin J. Lastwika1,2, Willie Wilson III3, Qing Kay Li4, Jeffrey Norris1, Haiying Xu5,Sharon R. Ghazarian6, Hiroshi Kitagawa1, Shigeru Kawabata1, Janis M. Taube5,Sheng Yao7, Linda N. Liu7, Joell J. Gills1, and Phillip A. Dennis1
Alterations in EGFR, KRAS, and ALK are oncogenic drivers in
squamous cell carcinomas, membranous expression of PD-L1
lung cancer, but how oncogenic signaling influences immunity in
was significantly associated with mTOR activation. These data
the tumor microenvironment is just beginning to be understood.
suggest that oncogenic activation of the AKT–mTOR pathway
Immunosuppression likely contributes to lung cancer, because
promotes immune escape by driving expression of PD-L1, which
drugs that inhibit immune checkpoints like PD-1 and PD-L1 have
was confirmed in syngeneic and genetically engineered mouse
clinical benefit. Here, we show that activation of the AKT–mTOR
models of lung cancer where an mTOR inhibitor combined with a
pathway tightly regulates PD-L1 expression in vitro and in vivo.
PD-1 antibody decreased tumor growth, increased tumor-infil-
Both oncogenic and IFNg-mediated induction of PD-L1 was
trating T cells, and decreased regulatory T cells. Cancer Res; 76(2);
dependent on mTOR. In human lung adenocarcinomas and
227–38. 2015 AACR.
cells. Mice that lacked FoxP3 cells developed fewer lung tumorsthan mice with mutant KRAS alone.
Despite the development of targeted therapies, lung cancer
Although multiple mechanisms can contribute to immune
remains the leading cause of cancer-related death worldwide
suppression in the tumor microenvironment, programmed death
(1). Most of the oncogenic drivers in non–small cell lung cancer
ligand 1 (PD-L1 and B7-H1), an inhibitory member of the B7
(NSCLC), such as EGFR or KRAS, activate the PI3K–AKT–mTOR
family, plays a central role in many cancer types (6). This cell
pathway, which increases cell proliferation, metabolism, and
surface protein is normally found on immune cells and in
survival. Activation of this pathway is a critical event during lung
immune privileged tissues, but its expression is upregulated in
tumorigenesis. Previously, we showed that genetic deletion of
many epithelial tumors, including lung cancer (7). PD-L1 binds to
AKT1 or inhibitors of mTOR such as rapamycin or metformin
either PD-1 or CD80 receptors on activated immune cells to
prevent KRAS-driven lung tumorigenesis (2–4). We also demon-
inhibit their activation and effector responses (8). The interaction
strated a relationship between AKT/mTOR signaling and immune
of PD-L1 and PD-1 induces differentiation of na€ve CD4þ T cells
suppression, because inhibition of tumorigenesis by rapamycin
into Tregs and maintains Treg-suppressive functions. PD-L1 can
was associated with reduced influx of lung associated FoxP3þ
also act as a receptor by sending reverse signals to limit tumor cell
regulatory T cells (Tregs) into the tumors (5). This was confirmed
apoptosis. The importance of PD-L1 and PD-1 in lung cancer is
by creating mice that harbored mutant KRAS but lacked FoxP3
reflected by the antitumor activity observed using PD-1– or PD-L1–blocking antibodies as single agents in heavily pretreatedNSCLC patients (9, 10). Clinical responses were sometimes
sustained over many months, suggesting recovered ability of
Department of Oncology, Johns Hopkins University, Baltimore, Mary-
land. 2The George Washington University, Institute for Biomedical
immune effectors to control tumor growth (11). This clinical
Sciences, Washington, DC. 3Cancer Biology and Genetics Branch,
benefit supports efforts to study the mechanisms that regulate
Center for Cancer Research, National Institutes of Health, Bethesda,
tumor PD-L1 expression and therapeutic interventions to decrease
Maryland. 4Department of Pathology, Johns Hopkins University, Bal-
PD-L1 levels.
timore, Maryland. 5Department of Dermatology, Johns Hopkins Uni-versity, Baltimore, Maryland. 6Biostatistics, Epidemiology and Data
Tumors can express PD-L1 either constitutively or through
Management Core, Johns Hopkins University, Baltimore, Maryland.
flammatory cytokines, especially members of the
Amplimmune, Inc., Gaithersburg, Maryland.
interferon family. Cytokine-driven PD-L1 expression is indicative
Note: Supplementary data for this article are available at Cancer Research
of an ongoing immune response in the tumor microenvironment,
whereas intrinsic PD-L1 expression does not depend on the
Corresponding Author: Phillip A. Dennis, Johns Hopkins University School of
presence of tumor-infiltrating lymphocytes.
Medicine, 4940 Eastern Avenue, 301 Building/Suite 4500 Baltimore, MD 21224.
Multiple mechanisms can contribute to intrinsic tumor PD-L1
Phone: 410-550-9250; Fax: 410-550-5445; E-mail:
[email protected]
expression. Expressions of PD-L1 and PD-L2 (another ligand for
PD-1) are increased in Hodgkin's disease and mediastinal large B-
2015 American Association for Cancer Research.
cell lymphoma through chromosomal amplification (12). T-cell
Published OnlineFirst December 4, 2015; DOI: 10.1158/0008-5472.CAN-14-3362
Lastwika et al.
lymphomas carrying NPM–ALK fusions induce PD-L1 expression
In vivo treatments
through STAT3 activation (13). PTEN loss or PIK3CA mutations
For the transgenic KRASLA2 mouse model treatment began
in glioma, breast, and prostate cancers have been shown to
at weaning and lasted 4 weeks. One hundred and fifty micrograms
activate the AKT–mTOR pathway and subsequently increase
of anti–PD-1 blocking antibody (Amplimmune) was given on
PD-L1 expression (14, 15). A correlation between activating
the first treatment day in combination with rapamycin. The
mutations in EGFR and increased immunosuppression markers,
control vehicle was given on treatment day 1 in combination
including PD-L1 and PD-1, was established (16). Recently, a
with 150 mg IgG (Rockland) . A previously optimized rapamycin
mouse model of lung squamous carcinoma demonstrated high
dosing schedule was used to obtain trough levels that are readily
PD-L1 expression in tumor-promoting cells with loss of LKB1 and
tolerated in humans (25). The control and anti–PD-1 antibody
PTEN (17). In NSCLC patients, the relationship of oncogenic
were given by i.p. injection once a week for 3 wks and tumors were
drivers with PD-L1 expression is still unclear with one study
harvested 1 hour after the last injection. Mice were weighed QOD
associating PD-L1 expression with mutant EGFR but not KRAS
to monitor for toxicity. Tumor burden was calculated as the sum
or ALK (18), and another demonstrating no clear difference in PD-
of individual lung tumor volumes per mouse.
L1 staining between samples with mutations in EGFR, KRAS, orALK (19). Because the AKT–mTOR pathway serves as a conver-
gence point for activation of many of the oncogenes involved in
Cell lysates were prepared in 2xLSB. Antibodies were from Cell
NSCLC, we hypothesized that this pathway was likely responsible
Signaling Technology unless otherwise noted and included
for the control of PD-L1 expression. We used NSCLC cell lines,
anti–PD-L1 antibody (AbCam; ab58810), anti–phospho-
mouse models, and primary human lung cancers to show that PD-
AKTS473(9271), anti-AKT(9272), anti–phospho-S6(4858), anti-
L1 protein expression is dependent on active AKT–mTOR signal-
ing, regardless of specific oncogenic or cytokine stimuli. These
data identify a common mechanism of PD-L1 regulation in lung
cancer, and provided rationale for clinical trials of oncogenic
(2532), anti–phospho-ACCS79(3661), anti-ACC(3662), anti-
pathway inhibitors combined with inhibitors of immune
Materials and Methods
STAT3(8768), anti-p53(2524), anti-p21(2947), and anti-atubulin
CL30, IO33, CL13, and CL25 cell lines were derived from 4-
Quantitative RT-PCR for PD-L1
lung adenocarcinomas developed in A/J mice, and were a gener-
RNA was isolated from CL13 cells using the Qiagen RNeasy
ous gift from Dr. Steven Belinsky (Lovelace Respiratory Research
Mini Kit (Qiagen). cDNA was made using the SuperScript
Institute, Albuquerque, NM) in 1999 (20). Immortalized Beas2B
II RT Reaction Kit (Invitrogen) from 2 mg of isolated RNA.
and isogeneic Beas2B transformed with NNK have been described
previously (21). HCT-116 parent, PTEN/, PIK3CA mutant or
(Mm03928990_g1) primers were purchased from Applied
KrasD13/ isogeneic cells were obtained from the JHU Genetic
Biosystems. Samples were analyzed on a StepOnePlus RT-PCR
Resources Core Facility. Human lung cancer cell lines were early
System Instrument using TaqMan Universal PCR Master Mix, No
passages (<20) of the original cell lines from the National Cancer
AmpErase UNG (Applied Biosystems) according to the manufac-
Institute obtained in the years spanning 2000 to 2012. H1975 and
H157 cell lines had higher passages (>20) and were authenticatedby JHU Genetic Resources Core Facility. H1299 was authenticated
by DDC Medical in 2012. Immortalized Beas2B and isogeneic
A total of 1 106 human and mouse lung cancer cells were
Beas2B transformed with NNK were a gift in 1996. HCT-116
harvested and stained for 30 minutes at 4C with primary anti-
parent, PTEN/, PIK3CA mutant or KrasD13/ isogeneic cells
body to PE-anti–mouse-PD-L1 (BioLegend; #10F.9G2), PE-anti–
were obtained from the JHU Genetic Resources Core Facility in
mouse-B7-H4 (eBioscience; Clone 188), PE-anti–human-PD-L1
2012. All cell lines were passaged for fewer than 6 months after
(eBioscience; Clone M1H1), APC-anti–human-B7-H4 (BD Phar-
mingen; Clone M1H43) or isotype-matched controls. Sampleswere run on a FACS Caliber (BD Biosciences) and analyzed using
FlowJo software (TreeStar).
CL13 cells were transfected with DharmaFECT (Thermo Scien-
tific) and a pool of 4 mouse PTEN siRNA or scrambled siRNA (L-040700-00-0005; Thermo Scientific). The pLKO.1 plasmids con-
taining shRNA targeted to human RAPTOR or RICTOR have been
Formalin-fixed lung tissues were incubated in PD-L1 (CST#
described previously (22).
13684), pS6S235/235 (CST#4858), FoxP3 (eBio #14-5773-82),CD3 (A0452 Dako), Ki67 (Ab16667 AbCam), Cleaved caspase-
3 (CST#9664), pHP-1 gamma (ab45270 AbCam), and detection
All animal studies were conducted using a protocol approved
was completed using the VECTASTAIN Elite ABC Kit (Vector
by the Animal Care and Use Committee at the National Cancer
Laboratories) per the manufacturer's instructions. Tissues were
Institute. The genetically engineered KRASLA2 and CC10þ
also incubated in the presence of an isotype-matched control
EGFRL585R/T790M mice, as well as, the NNK-induced A/J mouse
antibody (sc-2027; Santa Cruz Biotechnology). All stains were
lung tumor model have been described previously (3, 23, 24).
quantified in 10 tumor-containing 40 magnification fields. For
Cancer Res; 76(2) January 15, 2016
Published OnlineFirst December 4, 2015; DOI: 10.1158/0008-5472.CAN-14-3362
Control of PD-L1 by Oncogenic Activation of AKT/mTOR in NSCLC
murine PD-L1, the percentage of positive tumor cell surface
immunoblotting was analyzed by unpaired the Student t test.
staining was scored as (<5%), 1þ(5–20%), 2þ(20%–50%)
Tumor volume and tumor-infiltrating lymphocytes were analyzed
or 3þ(50%). pS6 staining was quantified by assigning a score of
by two-way ANOVA followed by Tukey's post hoc test. Statistical
absent (0), minimal (1), moderate (2), or strong (3) to each
significance was reached with a P value less than or equal to 0.05.
tumor. The staining index was calculated for each tumor mymultiplying the staining intensity by its distribution. FoxP3, CD3,
Ki67, Cl. Caspase-3 and pHP-1g stains were quantified by count-
Expression of PD-L1 in mutant EGFR and mutant KRAS murine
ing the number of positive cells. The investigator was blinded to
sample identities during scoring.
PD-L1 expression was examined in mouse models of lung
TMA slides were stained with the 5H1 antibody for PD-L1
cancer driven by activating mutations in KRAS or EGFR that are
expression and a mouse IgG isotype antibody using a previously
used to model lung cancer in smokers and never smokers, respec-
described protocol by a board certified pathologist (Q.K. Li;
tively. In the KRASLA2 mouse model, lung adenocarcinomas
ref. 26). Approximately 10% of randomly chosen cores were
develop after spontaneous recombination events induce onco-
scored to confirm PD-L1 by a second board certified pathologist
genic KRASG12D expression. Immunoblotting of lung lysates from
(J.M. Taube). Both TMAs were also analyzed for pS6S235/236
KRASLA2 mice demonstrated increased activation of AKT/mTOR
(CST#4858) expression. Tumor with >10% phospho-S6 expres-
and PD-L1 expression compared with age-matched, wild-type
sion were considered positive.
littermates (Fig. 1A). EGFRL858R/T790M mice have doxycycline-inducible expression of human mutant EGFR. Lung lysates har-
Statistical analysis
vested from mice exposed to doxycycline for 3 weeks show
Data in bar graphs are presented as mean SE. c2 analyses
increased expression of EGFR, active AKT/mTOR signaling and
tested for differences between the distributions of clinical vari-
PD-L1 (Fig. 1A, middle). The tobacco-specific carcinogen NNK
ables across histologic samples. The Fisher exact test examined
induces KRAS mutations and causes primarily lung adenomas in
potential statistical associations of the association between PD-L1
susceptible mouse strains. We previously showed that activation
and phospho-S6 expression in both TMAs. Quantification of
of the AKT–mTOR pathway is critical for NNK-induced lung
C57Bl/6 lung lysates
cells + 55
Figure 1.
Expression of PD-L1 and oncogenic activation of the Akt–mTOR pathway. A, lung lysates were harvested from C57BL/6 KRAS LA2 or wt littermates (left),from FVB mEGFRþ/CC10þ littermates treated with or without doxycycline (middle), or from A/J mice exposed to i.p. saline or the tobacco carcinogen NNK (right),and processed for immunoblotting. Each lane represents one mouse. B, A/J mice treated as in A showing PD-L1 expression in lung lesions but not in normallung epithelium. Scale bar, 10 mm. C and D, human NSCLC cell lines (C) and NNK-derived murine lung adenocarcinoma cell lines (D) have activationof AKT–mTOR, as well as expression of PD-L1 as shown by flow cytometry and immunoblotting.
Cancer Res; 76(2) January 15, 2016
Published OnlineFirst December 4, 2015; DOI: 10.1158/0008-5472.CAN-14-3362
Lastwika et al.
tumorigenesis (27). In lung lysates from 1-year-old mice previ-
PD-L1 by immunoblotting and flow cytometry. The H1770
ously treated with NNK, PD-L1 expression was observed in NNK-
(NOTCH1) cell line did not have active AKT/mTOR signaling or
but not saline-exposed lungs. Lungs from NNK-treated mice also
express PD-L1. Murine cell lines established from NNK-induced
had higher activation of AKT and mTOR (Fig. 1A, right). IHC
lung adenocarcinomas also had AKT/mTOR activation and PD-L1
staining of lung tissues from mice demonstrates PD-L1 expression
expression (Fig. 1D). Expression of PD-L1 in these cell lines
in resident immune cells but not in normal lung epithelium (Fig.
appeared selective, because expression of another immunosup-
1B, top and middle). In contrast, PD-L1 was detected in early lung
pressive ligand, B7-H4, was only observed in 10% of cells for all
lesions after a single NNK exposure. Collectively, these data
but one cell line tested (H520; Supplementary Fig. S1A). These
demonstrate that PD-L1 is expressed in mouse models of NSCLC
studies show that activation of AKT and mTOR is associated with
driven by mutations in KRAS or EGFR.
PD-L1 expression in NSCLC lines that harbor a wide spectrum ofdriver mutations.
Expression of PD-L1 and AKT/mTOR activation in NSCLC celllines
Inhibition of PI3K, AKT, or mTOR decreases PD-L1 expression
NSCLC cell lines were examined for total PD-L1 expression by
in NSCLC cell lines
immunoblotting, and membranous PD-L1 expression by flow
To test whether PD-L1 expression was dependent on active
cytometry. The panel of human cell lines was chosen to include a
PI3K–AKT–mTOR signaling, murine and human NSCLC cell lines
variety of oncogenic drivers, in an effort to reflect the mutational
with mutations in KRAS or EGFR and high PD-L1 expression were
spectrum seen in patients. AKT/mTOR activation was detected in
treated with pharmacologic inhibitors of components in the
adenocarcinoma and squamous cell carcinoma cell lines with
pathway. Inhibitors of PI3K (LY294002), AKT (TCN-P), or mTOR
mutations in NRAS (H1299), KRAS (H157/A549), EGFR
(rapamycin) decreased PD-L1 expression in a time-dependent
(H1975/H1650), BRAF (H2087), PIK3CA (H1650), EML4-ALK
manner (Fig. 2A–C). Although some cell line specificity was
(H3122), RET (H1563), autocrine production of FGF2 (H226), or
observed, inhibition of PI3K, AKT, and mTOR activity appeared
FGFR1 amplification (H520; Fig. 1C). These cell lines expressed
to coincide with or precede decreased PD-L1 expression. After
Figure 2.
Inhibition of the PI3K–AKT–mTOR pathway decreases PD-L1 expression. A–C, NSCLC cell lines were treated with 10 mmol/L of a PI3K inhibitor (LY294002; A)1 mmol/L of an AKT inhibitor (TCN-P; B), or 100 nmol/L of an mTOR inhibitor (rapamycin; C). D, cells were treated with 2 mmol/L AICAR or vehicle.
E, cell lines were treated with 100 nmol/L of a dual mTORC1/2 inhibitor (AZD8055). F, stable shRNA knockdown of RAPTOR (mTORC1) but not RICTOR(mTORC2) decreases PD-L1 in H157 cells (A–F).
Cancer Res; 76(2) January 15, 2016
Published OnlineFirst December 4, 2015; DOI: 10.1158/0008-5472.CAN-14-3362
Control of PD-L1 by Oncogenic Activation of AKT/mTOR in NSCLC
48 hours of PI3K or AKT inhibition, recovery of pathway activa-
(Supplementary Fig. S1A). Although the majority of these cell
tion and expression of PD-L1 occurred with similar kinetics in cell
lines did not express B7-H4, the highest B7-H4–expressing cell
lines. In contrast, recovery of mTOR activity or PD-L1 expression
line, H520, was treated with rapamycin. Rapamycin did not alter
was not observed with rapamycin at the time points examined,
B7-H4 protein in H520 cells, suggesting that mTOR specifically
possibly due to its long half-life. To investigate whether PI3K and
regulates PD-L1 (Supplementary Fig. S1B). To determine whether
AKT inhibition were required for mTOR inhibition and decreased
other signaling pathways downstream of oncogenic drivers such
expression of PD-L1, we used AICAR, an activator of AMPK that
as the MEK–ERK pathway might play a role in regulating PD-L1,
can inhibit mTOR independently of PI3K and AKT. AICAR acti-
cells were treated with an MEK inhibitor, U0126. U0126 did not
vated AMPK, increased phosphorylation of the AMPK substrate
alter PD-L1 expression despite inhibiting ERK phosphorylation
ACC, inhibited mTORC1 activation, and decreased PD-L1 expres-
and proliferation (Supplementary Fig. S2), indicating that control
sion at 16 hours (Fig. 2D). Taken together, these results demon-
of PD-L1 expression was specific to the PI3K–AKT–mTOR path-
strate that inhibition of PI3K, Akt, or mTOR (through allosteric
way and was not due to stimulation of the MEK–ERK pathway or
inhibition with rapamycin or AMPK activation), decreases PD-L1
to indirect effects on cellular proliferation.
To confirm the results obtained with rapamycin and AICAR, we
Rapamycin decreases PD-L1 expression in murine lung tumors
also tested a dual mTORC1/2 inhibitor, AZD8055. AZD8055
To validate these in vitro studies, we examined the effects of
decreased PD-L1 expression coincident with decreased activation
rapamycin on PD-L1 expression in vivo. One year after exposure
of AKT and mTOR (Fig. 2E). Because inhibition of PD-L1 by
to NNK, 1 week of rapamycin treatment significantly reduced
rapamycin correlated more closely with inhibition of pS6 but not
mTOR signaling and decreased PD-L1 expression in A/J mouse
pAKT at early time points, this suggested that mTORC1 exerts
lung tumors compared with vehicle-treated lung tumors (Fig.
more control over PD-L1 expression than mTORC2. To discern
3A). Similarly, lung tumors from KRASLA2 mice treated for 10
whether PD-L1 expression is dependent on mTORC1 or
weeks with rapamycin also had lower PD-L1 expression and
mTORC2, shRNA-mediated knockdown of a key component in
mTOR activation compared with vehicle-treated littermates
mTORC1 (RAPTOR) or mTORC2 (RICTOR) was performed in
(Fig. 3B). Six weeks after doxycycline administration, mutant
H157 cells. Knockdown of RAPTOR but not RICTOR decreased
EGFRL858R/T790M mice were treated for 1 week with vehicle or
PD-L1 expression, even though RICTOR knockdown decreased
rapamycin. Lung tumors from mice treated with rapamycin had
phosphorylation of AKT at serine 473 (Fig. 2F).
reduced mTOR activation and PD-L1 expression compared to
To determine whether mTOR could regulate other immuno-
vehicle-treated mice (Fig. 3C). These studies indicate that
suppressive ligands expressed on tumors, we performed flow
mTOR activation is correlated with PD-L1 expression in murine
cytometry for B7-H4 using the same panel of NSCLC cells
lung tumors.
Figure 3.
Rapamycin decreases PD-L1 expression in lung tumors in vivo. Immunohistochemical staining for pS6 or PD-L1 in lung tumors treated with vehicle or rapamycinfrom A/J mice exposed to the tobacco-carcinogen NNK (A), C57BL/6 KRAS LA2 mice (B), or dox-exposed FVB mEGFRþ/CC10þ littermates (C). Bar graphsquantify decreased staining of pS6 and PD-L1. Scale bar, 10 mm. , P 0.05 by unpaired Student t test.
Cancer Res; 76(2) January 15, 2016
Published OnlineFirst December 4, 2015; DOI: 10.1158/0008-5472.CAN-14-3362
Lastwika et al.
Activation of the AKT–mTOR pathway increases PD-L1
genetic results linking active AKT/mTOR signaling to PD-L1
expression in NSCLC, pairs of isogenic HCT116 cells were used
On the basis of the observation that inhibition of the PI3K–
to determine whether single genetic alterations of the pathway
AKT–mTOR pathway decreases PD-L1 expression, we tested
could increase PD-L1 expression. Increased activation of Akt and
whether cell lines with low basal levels of PD-L1 could increase
mTOR, as well as increased expression of PD-L1 was observed in
PD-L1 expression after stimulation of the AKT–mTOR pathway.
HCT116 PTEN/ cells, suggesting that regulation of PD-L1 by
Administration of EGF to NSCLC cell lines activated the pathway
PTEN may occur in several tumor types. HCT116 cells that express
and increased PD-L1 expression (Fig. 4A). Likewise, mouse and
mutant KRAS or mutant PIK3CA alleles also had higher AKT
human cell lines rapidly activated AKT and mTOR and increased
activation and PD-L1 expression compared with isogeneic cells
PD-L1 expression (Fig. 4B). Comparison of BEAS-2B cells with
with corresponding wild-type alleles (Fig. 4E).
BEAS-2B cells fully transformed by NNK showed increased PD-L1expression and activation of AKT/mTOR in cells fully transformed
EGF and IFNg increase PD-L1 protein expression through
by NNK (Fig. 4C). Knockdown of PTEN, a negative regulator of
activation of mTOR
PI3K, increased the activation of AKT and PD-L1 expression in
PD-L1 expression can also be induced in tumors in response to
CL13 cells (Fig. 4D). To complement the pharmacologic and
proinflammatory cytokines like IFNg via JAK/STAT signaling and
Figure 4.
Activation of the AKT–mTOR pathway increases PD-L1 expression. A, NSCLC cell lines were treated with 5 ng/mL EGF or vehicle in 1% serum. B, NSCLC cell lines weretreated with 100 nmol/L NNK or vehicle in 1% serum. C, lysates from B2B and B2B-NNK isogeneic cell lines were evaluated by immunoblotting. D, cells weredirectly transfected with scrambled siRNA or PTEN-targeted siRNA for 24 hours in serum-free media. Cells with wt PTEN or that had lost PTEN are shown on theright. E, comparison of cells that have lost either the mutant KRAS or PIK3CA alleles or the corresponding wt alleles through homologous recombination (HR).
Cancer Res; 76(2) January 15, 2016
Published OnlineFirst December 4, 2015; DOI: 10.1158/0008-5472.CAN-14-3362
Control of PD-L1 by Oncogenic Activation of AKT/mTOR in NSCLC
interferon-stimulated response elements in the PD-L1 promoter
ylation events are rapidly controlled (Fig. 4C), we also included a
(7, 28). STAT1 and to a lesser extent STAT3 typically mediates
30-minute time point to observe EGF-induced AKT and mTOR
IFNg signaling. Although both STAT1 and STAT3 can bind to the
activation. At 16 hours, EGF and IFNg increased PD-L1 expression
PD-L1 promoter, STAT3 binds with higher affinity and stimulates
and activated mTOR signaling. Upregulation of PD-L1 was depen-
more PD-L1 transcript in dendritic cells (DC; ref. 29). Phosphor-
dent on mTOR activation, because rapamycin pretreatment pre-
ylation of JAK/STAT occurs minutes after exposure to IFNg, but in
vented EGF- and IFNg-mediated increases in PD-L1 expression,
multiple cancer cell lines maximum induction of PD-L1 occurs
but not IFNg-induced p-STAT3 (Fig. 5B).
much later (9–24 hours; ref. 28). For these reasons, we chose to
Although EGF and IFNg induce PD-L1 protein expression in an
use phospho-STAT3 as a readout for IFNg signaling and evaluated
mTOR-dependent manner, it is unclear whether mTOR exerts
PD-L1 expression at later time points. Cells were treated with IFNg
transcriptional control of PD-L1. Therefore, we measured PD-L1
for 16 and 24 hours (Fig. 5A). IFNg activated JAK2 and STAT3
transcription. IFNg increased transcription of PD-L1 but EGF did
signaling, as well as PD-L1 expression. Parallel cultures of cells
not. Rapamycin did not inhibit IFNg-induced transcription,
were also treated with EGF to compare PD-L1 regulation and
suggesting that mTOR provides translational control of PD-L1
signaling pathway activation. Because EGF-stimulated phosphor-
(Fig. 5C). To confirm translational regulation of PD-L1, NSCLC
Figure 5.
The AKT–mTOR pathway controls PD-L1 protein expression. A, CL13 and H1299 cell lines were treated with 10 ng/mL IFNg or 5 ng/mL EGF in 1% serum forthe indicated times. An early time point (30 m) was included after EGF addition to confirm pathway activation. B, cells were treated for 24 hours with100 nmol/L rapamycin alone, for 23 hours with 5 ng/mL EGF or 10 ng/mL IFNg alone, or the combination by treating with rapamycin for 1 hour, then addingEGF or IFNg to culture media and harvesting 23 hours later. C, CL13 cells treated as in B and RNA was harvested for RT-PCR. D, cells were treated with100 mg/mL cycloheximide for the indicated time points. E, cells were treated with 5 mg/mL actinomycin D for the indicated time points. Immunoblotsshown are representative of three independent experiments. F, cells were treated for 6 hours with 300 mmol/L chloroquine (CLQ) alone, for 4 hours with100 nmol/L rapamycin alone, or the combination by treating with CLQ for 2 hours, then adding rapamycin to culture media and harvesting 4 hours later.
G, cells were treated for 6 hours with 100 nmol/L PS-341 alone, for 5 hours with 100 nmol/L rapamycin alone, or the combination by treating with PS-341for 2 hours, then adding rapamycin to culture media and harvesting 5 hours later.
Cancer Res; 76(2) January 15, 2016
Published OnlineFirst December 4, 2015; DOI: 10.1158/0008-5472.CAN-14-3362
Lastwika et al.
Table 1. mTOR activation is required, but may not be sufficient to induce PD-L1
combined for further analyses. Approximately 90% of tumors
expression in primary lung adenocarcinoma and squamous cell carcinoma
with PD-L1 expression had activation of mTOR and 54% of
tumors with mTOR activation also expressed PD-L1, suggesting
that mTOR activation was necessary, but not sufficient, for PD-L1
expression (Table 1). Distribution of mTOR activation tended to
be similar to staining patterns for PD-L1, suggesting that the same
NOTE: Correlation between p-S6S235/236 and PD-L1 markers in the TMAs.
cells co-express both markers (Supplementary Fig. S4). The major-
Statistical analyses were performed using the Fisher exact test.
ity (83%) of tumors negative for pS6 were also negative for PD-L1.
A small subset of tumors expressed PD-L1 without mTOR acti-vation, indicating that there may be additional mechanisms
cell lines were exposed to the protein translation inhibitor cyclo-
inducing PD-L1 expression. Sixty-three of 158 (40%) of lung
heximide (Fig. 5D). Cycloheximide rapidly decreased PD-L1
tumors had both active mTOR signaling and PD-L1 expression,
protein expression, indicating that PD-L1 likely has rapid turnover
and a Fisher exact test revealed a statistically significant correlation
in lung cancer cells. In contrast with cycloheximide, inhibiting
between the two markers (P ¼ 0.0001; Table 1). These results
transcription with actinomycin D did not change PD-L1 expres-
underscore the clinical relevance of our preclinical associations.
sion, even at later time points (Fig. 5E). The accumulation of p53in IO33 cells and the accumulation of p21 in the mutant p53 cellline H1975 demonstrated that transcription was successfully
The combination of rapamycin and a PD-1 blocking antibody
inhibited. These results suggest that PD-L1 expression is predom-
decreases lung tumor growth
inantly controlled at the protein level and that mTOR exerts its
Monoclonal antibodies that block PD-L1 or PD-1 have shown
regulation at this level.
clinical benefit in NSCLC (9, 10). However, it is possible that
To examine how rapamycin was decreasing PD-L1 protein
simultaneous inhibition of both PD-L1 and PD-1 may increase
expression, we studied two main pathways of protein degradation
therapeutic benefit because each has additional immunosuppres-
via the lysosome or the proteasome. Pretreatment with a lysosome
sive binding partners. To test the efficacy of systemically blocking
acidification inhibitor (chloroquine) but not a proteasome inhib-
PD-1 while reducing the expression of PD-L1 in tumor tissue, a
itor (PS-341) prevented rapamycin-mediated decreases in PD-L1
murine anti–PD-1 antibody and rapamycin were administered in
protein (Fig. 5F and G). This suggests that rapamycin inhibits PD-
the KRASLA2 mouse model (Fig. 6A). Rapamycin alone decreased
L1 expression through a combination of decreased protein syn-
the tumor burden of KRAS-driven lung tumors by approximately
thesis and increased lysosomal protein degradation.
50% whereas PD-1 blockade had no effect as a single agent (Fig.
6B). The combination of rapamycin and anti–PD-1 significantlyreduced lung tumor burden by comparison with any other treat-
Expression of PD-L1 and activation of mTOR in human lung
ment group. The combination therapy increased CD3þ T cells and
adenocarcinomas and squamous cell carcinomas
reduced FoxP3þ Tregs (Fig. 6C). This led to a higher ratio of CD3þ
To determine whether these findings are clinically relevant, two
T cells to Tregs, indicating a shift towards an immune activated
human lung tissue microarrays (TMA) were stained and scored for
rather than immunosuppressive microenvironment. Lung tumors
membranous and/or cytoplasmic PD-L1 expression (Supplemen-
from mice treated with rapamycin had a reduction in PD-L1
tary Fig. S3). One TMA included 63 lung adenocarcinomas with
expression and mTOR activation (Fig. 6D and E). A marker of
matched normal lung and assorted normal tissues. The other TMA
apoptosis, cleaved caspase-3, was increased in tumors treated with
contained 96 lung squamous cell carcinomas with assorted nor-
the combination. Although rapamycin alone inhibited tumor
mal tissues. These normal tissues served as internal positive
proliferation, tumors treated with the combination also had more
(placenta) or negative (soft tissue) controls for PD-L1 expression.
pHP1gþ cells, suggesting that these tumors had undergone senes-
Each TMA was simultaneously stained with an IgG antibody to
cence. These findings demonstrate enhanced antitumor efficacy
control for background. Clinical and pathologic characteristics of
with the combination of rapamycin and a PD-1 blocking antibody
the patient population are summarized in Supplementary Table
through increased apoptosis and cellular senescence. We con-
S1. Sixty-two of 63 adenocarcinoma and 96 of 96 squamous cell
firmed the efficacy of rapamycin and PD-1 blockade in a second
carcinoma tumors were evaluable. Twenty of 62 (32.2%) lung
mouse model of KRAS-driven lung cancer (Supplementary Figs.
adenocarcinomas and 50 of 96 (52.1%) lung squamous cell
S5 and S6). These findings demonstrate enhanced antitumor
carcinomas expressed membranous PD-L1, which is consistent
efficacy against two mutant KRAS mouse models of lung cancer
with previous observations (Supplementary Fig. S3B and S3D;
when rapamycin and a PD-1 blocking antibody are combined.
refs. 30–34). No clinical or pathologic characteristics were asso-ciated with PD-L1 expression. PD-L1 membranous expression
was observed on lung tumor tissue and on resident alveolarmacrophages, but not on non-neoplastic lung tissue. These data
PD-L1 plays a prominent role in the balance of the immune
support a potential common role of this protein in mediating
system between the stimulatory signals needed for effective
immunosuppression in NSCLC.
immune responses and maintenance of self-tolerance or tissue
To explore the potential regulation of PD-L1 in human primary
integrity. PD-L1 can be expressed on hematopoietic and non-
lung tumors by mTOR activation, both TMAs were also stained
hematopoietic cells, as well as in lymphoid and peripheral tissues.
with an antibody specific for phosphorylation of S6 at S235/236
Consequently, the regulation of PD-L1 is complex and most likely
(Supplementary Fig. S3C and S3D). Because there were no sig-
depends on the status of underlying transcriptional and signaling
nificant differences between the TMA characteristics (Supplemen-
networks. Here, our studies reveal a strong association between
tary Table S1; stage significance is likely due to a sample size bias),
PD-L1 protein and activation of the AKT–mTOR pathway in lung
the adenocarcinomas and squamous cell carcinomas were
cancer. The dependence of PD-L1 expression on mTOR is
Cancer Res; 76(2) January 15, 2016
Published OnlineFirst December 4, 2015; DOI: 10.1158/0008-5472.CAN-14-3362
Control of PD-L1 by Oncogenic Activation of AKT/mTOR in NSCLC
Sacrifice 7 weeks
4.5 mg/kg LD; 1.5 mg/kg veh QOD and
N = 12
μg IgG weekly i.p.
N = 12
4.5 mg/kg LD; 1.5 mg/kg rapa QOD
N = 12
μg αPD-1 weekly i.p.
4.5 mg/kg LD; 1.5 mg/kg rapa QOD and
N = 12
150 μg αPD-1 weekly i.p.
pS6S235/236
P < 0.0001
P < 0.0001
cells/HPF 0
Cl. Caspase
Tumor burden
g+ cells/HPF 20
cells/HPF 30
/FoxP3 10
Figure 6.
The combination of rapamycin and aPD-1 blockade significantly reduces lung tumor burden in the KRAS LA2 mouse model. A, KRAS LA2 mice were treatedwith either IP vehicle and IgG, rapamycin, aPD-1 antibody, or rapamycin and aPD-1 for 4 weeks beginning at the time of weaning. B, tumor burden after4 weeks of treatment; , P 0.05 by Mann–Whitney. C, quantification of IHC staining for CD3þ or FoxP3þ cells. The ratio of CD3þ over FoxP3þ cells is also shown;
, P 0.05 by two-way ANOVA. D, images represent IHC staining for PD-L1, pS6, cleaved caspase-3, Ki67, and pHP1g. Scale bar, 10 mm. E, quantification of IHCstains in D. , P 0.05 by two-way ANOVA.
consistent with studies in glioma, breast, prostate, ovarian, and
regulatory steps on PD-L1 expression will probably depend on cell
pancreatic cancer. Interestingly, this relationship does not extend
type, context, and may vary over the course of response to a
to melanoma, emphasizing multiple mechanisms for PD-L1
regulation in solid tumors (35).
Transcription of PD-L1 can be induced by many cytokines, of
Our TMA study suggested that mTOR activation is necessary but
which IFNg is the most potent (7). Activation of the AKT–mTOR
not sufficient for PD-L1 expression. It is possible that tumors with
pathway plays a central role in the initiation of IFN-stimulated
mTOR activation but no PD-L1 protein lack PD-L1 transcripts,
gene translation, in a mechanism parallel to but independent of
which would preclude mTOR-dependent translation. Approxi-
activation of the JAK–STAT pathway (39). Therefore, although
mately 53% (810/1,537) of lung cancer specimens in The Cancer
PD-L1 transcription does not depend on mTOR activation, trans-
Genome Atlas set do not have detectable PD-L1 mRNA (36).
lation of IFNg-induced transcripts, including PD-L1, may be
Other studies have identified that PD-L1 mRNA levels were only
dependent on activation of PI3K, AKT, and mTOR kinase activity.
higher than normal lung tissue in stage IV lung tumors (37). Thus,
The dependence of PD-L1 translation on PI3K–AKT–mTOR activ-
there may be additional levels of PD-L1 regulation between
ity is also observed during viral infections. In HIV-1–infected
transcription and translation. A recent study directly compared
macrophages and dendritic cells, the viral protein Nef induces PD-
samples for PD-L1 mRNA and protein expression and observed
L1 transcription by binding to the promoter but PD-L1 protein
that PD-L1 mRNA had a complex, nonlinear positive association
expression depends on active PI3K/AKT signaling (40). Our data
with PD-L1 protein expression. This finding was consistent in two
indicate that multiple types of stimuli, including growth factors
separate TMA cohorts and suggests that PD-L1 is regulated at both
cytokines and oncogenes, converge at mTOR to increase PD-L1
transcription and translational levels (34). In DCs, LPS and IFNg-
mediated induction of PD-L1 protein depends on both active
Inhibiting ligation of tumor-derived PD-L1 with PD-1 on T cells
transcription and translation (38). The relative contribution of
is proposed as a major therapeutic target to revert tumor-mediated
Cancer Res; 76(2) January 15, 2016
Published OnlineFirst December 4, 2015; DOI: 10.1158/0008-5472.CAN-14-3362
Lastwika et al.
immunosuppression. However, because PD-L1 and PD-1 have
PD-L1 expression, it is possible that response to PD-1 or PD-L1
additional binding partners perhaps blocking multiple interac-
blockade depends on a critical threshold of TILs at the start of
tions is needed to fully rescue antitumor immunity. Combining
therapy (50). We have observed that the number of tumor-
PD-1 blockade with rapamycin, which inhibited oncogenic KRAS
infiltrating CD3þ T cells per high powered field is doubled in
signaling and PD-L1 expression, resulted in a significant reduction
the mutant EGFRL858R/T790M compared with the KRASLA2 mouse
in tumor burden compared with either drug alone. Only the
model (88.9 vs. 37.5 CD3þ T cells; unpublished data). Further-
combination therapy significantly increased the ratio of CD3þ to
more, a recent study demonstrated higher nonsynonymous muta-
FoxP3þ cells, supporting this change in T-cell populations as a
tional burden is associated with response to PD-1 blockade as a
readout for antitumor activity. In addition, only the combination
single agent, in part by enhancing neoantigen-specific CD8þ T-cell
was associated with decreased proliferation and increased apo-
responses (51). Identifying the mechanisms responsible for the
ptotic and senescent markers. Drug-induced senescence with
differences in lung tumors and TILs between responders and non-
DNA-damaging agents is well established, but a role for adaptive
responders of PD-1 blockade would have important insight into
immunity in driving cancer cell senescence was recently identified.
In multiple murine models and in human cancers, T helper 1 cell
Activation of PI3K–AKT–mTOR signaling is driven by mul-
production of IFNg and TNFa induce immune-dependent tumor
tiple mechanisms in NSCLC and is vital to tumor develop-
cell senescence (41). Although this is the first demonstration of
ment, progression, and prognosis. We show that activation of
immune-induced senescence in tumors, immune cells promote
AKT–mTOR, regardless of the driving oncogene or exogenous
senescence to regulate other leukocytes. For example, Tregs can
stimulus, increases PD-L1 protein expression in NSCLC. Our
induce senescence in na€ve and memory T cells through a mech-
data extend a growing body of evidence that oncogenes have
anism dependent on toll like receptor 8, p38, and ERK1/2 (42). A
tumor cell autonomous effects by altering the immune system
remarkable aspect of checkpoint blockade with PD-1 or PD-L1 is
in the tumor microenvironment. Clinical trials combining
the generation of long-term stable disease in the absence of
anti–PD-1 antibodies and current standard-of-care treatments
complete tumor regression, raising the possibility that these
are already underway and include combining targeted thera-
tumors have undergone senescence.
pies with immunotherapy (73–75). Our studies provide ratio-
Implementing rapamycin as a cancer therapy raises issues about
nale to combine and optimize PI3K–AKT–mTOR inhibitors
its own role in immunosuppression. Rapamycin has a black box
with anti–PD-1 antibodies.
warning from the FDA stemming from a study of renal transplantpatients who were also taking cyclosporine and corticosteroids
Disclosure of Potential Conflicts of Interest
(43), but multiple trials of single-agent rapamycin or rapamycin
J.M. Taube reports receiving a commercial research grant and is a consultant/
analogues in cancer patients have shown no evidence of increased
advisory board member for Bristol Myers Squibb. No potential conflicts of
incidence of immunosuppression (25, 44). In fact, many basic
interest were disclosed by the other authors.
and clinical studies have associated rapamycin with activeimmune responses (45, 46). Our studies in the NNK-induced
Authors' Contributions
lung cancer model have shown only modest decreases in CD4þ
Conception and design: K.J. Lastwika, W. Wilson III, S. Yao, L.N. Liu, P.A.
levels with short-term or continuous rapamycin treatment. Fac-
tors that are likely to play important roles in the cumulative effects
Development of methodology: W. Wilson III, J. Norris, H. Xu, P.A. Dennis
of rapamycin on the immune system include the timing and
Acquisition of data (provided animals, acquired and managed patients,
degree of mTOR inhibition, as well as cell type and modulation of
provided facilities, etc.): K.J. Lastwika, J. Norris, H. Kitagawa, S. Kawabata,
mTORC2 signaling. Although precise mechanisms remain
unclear, we demonstrate the potential to use rapamycin in com-
Analysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): K.J. Lastwika, S.R. Ghazarian, S. Kawabata, J.M.
bination with a PD-1 blocking antibody to increase antitumor
Taube, P.A. Dennis
immunity. Rapamycin administration has been shown to sensi-
Writing, review, and/or revision of the manuscript: K.J. Lastwika, W. Wilson
tize tumors to immunotherapy in other mouse model systems.
III, Q.K. Li, S.R. Ghazarian, H. Kitagawa, J.M. Taube, S. Yao, J.J. Gills, P.A. Dennis
For example, treatment of fibrosarcoma or colorectal cancers with
Administrative, technical, or material support (i.e., reporting or organizing
rapamycin increased tumor sensitivity to adoptive cellular immu-
data, constructing databases): Q.K. Li, S. Kawabata, S. Yao, P.A. Dennis
notherapy (47). Although PD-L1 expression was not examined in
Study supervision: P.A. Dennis
this study, it is tempting to speculate PD-L1 as a contributingfactor in immunosuppression.
Responses to PD-1 and PD-L1 blockade have been proposed to
The authors thank Dr. Leiping Chen for providing the anti–PD-L1 mono-
be associated with the presence of PD-L1 and many ongoing
clonal antibody 5H1.
clinical trials require PD-L1þ pretreatment biopsies. Despitestrong expression of PD-L1 in lung tumors, PD-1 blockade had
no effect on tumorigenesis in the KRASLA2 mouse model. This is in
This work was supported by intramural funding from the National
agreement with a report demonstrating PD-1 blockade reduced
Cancer Institute, the George Washington University, and NIH grant P30
tumor burden in mouse models of mutant EGFR- but not KRAS-
driven lung cancer (16). Because both mutant EGFR and KRAS
The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
tumor models express PD-L1, this may indicate specific genomic
advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate
subsets of lung tumors predict response to single-agent anti–PD-1
outside of PD-L1 expression. However, multiple clinical studieshave not identified the presence of mutant KRAS or EGFR as
Received November 19, 2014; revised August 30, 2015; accepted September
predictors for successful PD-1 blockade (48, 49). In addition to
20, 2015; published OnlineFirst December 4, 2015.
Cancer Res; 76(2) January 15, 2016
Published OnlineFirst December 4, 2015; DOI: 10.1158/0008-5472.CAN-14-3362
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