Targeting autophagy inhibits melanoma growth by enhancing NK cells infiltration in a CCL5-dependent manner

Article abstract of DOI:10.1073/pnas.1703921114

While blocking tumor growth by targeting autophagy is well established, its role on the infiltration of natural killer (NK) cells into tumors remains unknown. Here, we investigate the impact of targeting autophagy gene Beclin1 (BECN1) on the infiltration of NK cells into melanomas. We show that, in addition to inhibiting tumor growth, targeting BECN1 increased the infiltration of functional NK cells into melanoma tumors. We provide evidence that driving NK cells to the tumor bed relied on the ability of autophagy-defective tumors to transcriptionally overexpress the chemokine gene CCL5. Such infiltration and tumor regression were abrogated by silencing CCL5 in BECN1-defective tumors. Mechanistically, we show that the up-regulated expression of CCL5 occurred through the activation of its transcription factor c-Jun by a mechanism involving the impairment of phosphatase PP2A catalytic activity and the subsequent activation of JNK. Similar to BECN1, targeting other autophagy genes, such as ATG5, p62/SQSTM1, or inhibiting autophagy pharma-cologically by chloroquine, also induced the expression of CCL5 in melanoma cells. Clinically, a positive correlation between CCL5 and NK cell marker NKp46 expression was found in melanoma patients, and a high expression level of CCL5 was correlated with a significant improvement of melanoma patients’ survival. We believe that this study highlights the impact of targeting autophagy on the tumor infiltration by NK cells and its benefit as a novel therapeutic approach to improve NK-based immunotherapy.

Full text of DOI:10.1073/pnas.1703921114

Targeting autophagy inhibits melanoma growth
by enhancing NK cells infiltration in a
CCL5-dependent manner
Takouhie Mgrditchiana, Tsolere Arakeliana, Jérôme Paggettia, Muhammad Zaeem Nomana, Elodie Virya,
Etienne Moussaya, Kris Van Moera, Stephanie Kreisb, Coralie Guerinc, Stephanie Buartd, Caroline Roberte,
Christophe Borgf, Philippe Vielhg, Salem Chouaibd, Guy Berchema,h, and Bassam Janjia,1
aLaboratory of Experimental Cancer Research, Department of Oncology, Luxembourg Institute of Health, L-1526 Luxembourg City, Luxembourg; bLife
Sciences Research Unit, University of Luxembourg, L-4367 Belvaux, Luxembourg; cNational Cytometry Platform, Department of Infection and Immunity,
Luxembourg Institute of Health, L-4354 Esch-sur-Alzette, Luxembourg; dINSERM UMR 1186, Gustave Roussy Cancer Center, 94805-Villejuif, France; eINSERM
UMR 981, Gustave Roussy Cancer Center, 94805-Villejuif, France; fDepartment of Medical Oncology, University Hospital of Besançon, 25000 Besançon,
France; gLaboratoire National de Santé, L-3555 Dudelange, Luxembourg; and hDepartment of Hemato-oncology, Centre Hospitalier du Luxembourg, 1210
Luxembourg City, Luxembourg
Edited by Charles G. Drake, Johns Hopkins Medical Institutions, Baltimore, MD, and accepted by Editorial Board Member Philippa Marrack September 20, 2017
(received for review March 8, 2017)
While blocking tumor growth by targeting autophagy is well
established, its role on the infiltration of natural killer (NK) cells
into tumors remains unknown. Here, we investigate the impact of
targeting autophagy gene Beclin1 (BECN1) on the infiltration of NK
cells into melanomas. We show that, in addition to inhibiting tumor
growth, targeting BECN1 increased the infiltration of functional NK
cells into melanoma tumors. We provide evidence that driving NK
cells to the tumor bed relied on the ability of autophagy-defective
tumors to transcriptionally overexpress the chemokine gene CCL5.
Such infiltration and tumor regression were abrogated by silencing
CCL5 in BECN1-defective tumors. Mechanistically, we show that the
up-regulated expression of CCL5 occurred through the activation of
its transcription factor c-Jun by a mechanism involving the impair-
ment of phosphatase PP2A catalytic activity and the subsequent
activation of JNK. Similar to BECN1, targeting other autophagy
genes, such as ATG5, p62/SQSTM1, or inhibiting autophagy pharma-
cologically by chloroquine, also induced the expression of CCL5 in
melanoma cells. Clinically, a positive correlation between CCL5 and
NK cell marker NKp46 expression was found in melanoma patients,
and a high expression level of CCL5 was correlated with a significant
improvement of melanoma patients’ survival. We believe that this
study highlights the impact of targeting autophagy on the tumor
infiltration by NK cells and its benefit as a novel therapeutic ap-
proach to improve NK-based immunotherapy.
In this regard, it should be emphasized that the infiltration of
functional cytotoxic immune cells, including NK and cytotoxic T
lymphocytes (CTLs), will likely become a major factor in achieving
successful immunotherapies, notably those based on the use of im-
mune checkpoint inhibitors. Accumulating new evidence highlights
that, similar to CTLs, activated NK cells can express, under some
circumstances, the immune checkpoint programmed cell death
protein 1- (PD-1) (9–11) and CTL-associated antigen 4 (CTLA4)
(12). Thus, it stands to reason that improving the infiltration of cy-
totoxic immune cells, including NKs, into the tumor bed could en-
hance the therapeutic benefit of NK cell-based immunotherapy and
provide novel therapeutic targets that could complement the
expanding armamentarium of cancer immunotherapies.
Chemokines are chemotactic cytokines playing a major role in
the infiltration of immune cells into the tumor bed, and are
therefore supposed to play a tumor-suppressive role (13). How-
ever, depending on the balance between several tumor-promoting
and tumor-inhibiting factors, some cytokines may play a dual role
in tumor promotion or tumor suppression. For example, several
cytokines expressed by melanomas are involved in tumor growth
Significance
The failure in achieving a durable clinical immune response
against cancer cells depends on the ability of cancer cells to
establish a microenvironment that prevent cytotoxic immune
cells to infiltrate tumors and kill cancer cells. Therefore, the key
approach to achieving successful antitumor immune response
is to harness strategies allowing the reorientation of immune
cells to the tumor. Herein we reveal that inhibiting autophagy
induces a massive infiltration of natural killer immune cells into
the tumor bed, and a subsequent dramatic decrease in the
tumor volume of melanomas. These results highlight the role
of targeting autophagy in breaking the immunosuppressive
tumor microenvironment barrier, thus allowing the infiltration
of natural killer cells into the tumor to kill cancer cells.
autophagy melanoma natural killer CCL5 immunotherapy
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atural killer (NK) cells are known to be a critical part of the
immune system involved in tumor control. In human and
N
animal models, NK cell deficiency leads to increased incidence
of different types of tumors (1). While the role of NK cells in
tumor immune surveillance is well established and experimen-
tally supported (2), the use of NK cells is far from being suc-
cessfully and fully used in the clinic, although efforts are now
being undertaken to exploit their antitumor properties (3). This
might be in part related to the lack of crucial knowledge about
NK cell-homing capacities (4, 5) and their poor infiltration into
solid tumors (6). Indeed, the long-lasting observations showing
that NK cells are infrequently detected in tumor biopsies suggest
that intratumoral NK cells can be associated with increased
survival of cancer patients (7). Therefore, strategies aiming at
increasing the infiltration of NK cells into tumors would be of
great interest to improve NK-based tumor immunotherapies (8).
Consequently, a deeper understanding of the mechanisms reg-
ulating NK cell infiltration will allow us to take full advantage of
the tremendous antitumor capacities of NK cells and rapidly
bring them to the clinical use.
Author contributions: M.Z.N., G.B., and B.J. designed research; T.M., T.A., J.P., M.Z.N., E.V.,
E.M., and K.V.M. performed research; S.K., S.B., and C.R. contributed new reagents/analytic
tools; T.M., T.A., J.P., M.Z.N., E.V., E.M., C.G., C.B., P.V., S.C., and G.B. analyzed data; and B.J.
wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission. C.G.D. is a guest editor invited by the
Editorial Board.
This is an open access article distributed under the PNAS license.
1To whom correspondence should be addressed. Email: bassam.janji@lih.lu.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1703921114/-/DCSupplemental.
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and progression, including CXCL1, CXCL2, CXCL3, CXCL8, revealed a massive infiltration of NK cells into BECN1− com-
CCL2, and CCL5 (14). In contrast, it has been shown that che-
motherapy can also induce the expression of cytokines, including
CCL5, involved in the trafficking of T cells into the tumor bed
(15). Therefore, a positive correlation between the expression of
some cytokines and the clinical outcome has been proposed (16).
It is now well defined that the dual role played by some cytokines
as tumor-promoting or tumor-suppressing depends on the balance
between tumor-promoting and tumor-inhibiting factors. There-
fore, understanding the complex role of chemokines in tumor
biology and the context by which they play such a dual role will
contribute to the improvement of the efficacy of cancer immu-
notherapeutic strategies and the induction of long-lasting host
antitumor immunity.
Using syngeneic melanoma and breast mouse models, we have
previously reported that targeting the autophagy gene Beclin1
(BECN1) significantly inhibited tumor growth by improving NK-
mediated antitumor immune response (17). Briefly, autophagy is
a lysosomal degradation pathway executed at the basal level that
allows cells to self-digest their own components and rid them-
selves of damaged organelles and misfolded proteins in well-
defined structures, named autophagosomes. Such a degrada-
tion process provides nutrients to maintain cellular functions
under environmental stresses, thus allowing survival of cancer
cells under stress conditions (18, 19). Autophagy involves a
BECN1/class III phosphoinositide-3-kinase (PI3K) complex to
initiate the formation of an isolation membrane, called the
phagophore (20). ATG5, another autophagy-related protein,
plays a role in the elongation of the phagophore and in its sub-
sequent maturation into the complete autophagosome (21).
Consistent with the degradation property of autophagy, we have
demonstrated that targeting BECN1 prevented the degradation
of NK-derived GranzymeB (GzmB) in hypoxic melanoma cells,
and therefore restored their susceptibility to NK cell-mediated
killing (17). A growing body of evidence suggests that autophagy
could operate in tumor cells as an immunosuppressive and cell-
resistance mechanism, because autophagy blockade not only
sensitizes tumor to chemotherapy (22) but also improves the
antitumor immune response (23, 24).
pared with control (Ctrl) tumors (Fig. 1A).
Since NK cell-infiltrating tumors could be functionally impaired/
exhausted (25), we then assessed the functional properties of the
NK cells that infiltrated BECN1− tumors. Flow cytometry analysis,
performed based on the gating strategy described in Fig. S1, on Ctrl
and BECN1− B16-F10 tumors confirmed our results obtained by
immunohistochemistry result and revealed an almost twofold in-
crease in the number of NK cell-infiltrating BECN1− tumors
compared with Ctrl (Fig. 1 B and C). Furthermore, our data show
that infiltrated NK cells were CD69+ and GzmB+, indicating that
they were fully functional in the BECN1− tumor bed (Fig. 1 B and
C). Interestingly, when reported to the total number of NK cells,
no difference in the activation status of NK cells infiltrating both
Ctrl and BECN1− tumors was found (Fig. 1D). This result shows
that targeting BECN1 increases the number of infiltrated NK cells
without affecting their activation properties. Together, our data
highlight that targeting BECN1 presumably inhibits the growth of
B16-F10 tumors at least in part by improving the homing of
functional NK cells into the tumor bed.
In this study, we investigated the impact of targeting auto-
phagy on the infiltration of NK cells into the tumor bed. We
show that targeting BECN1 in melanoma tumor induced a
massive infiltration of functional NK cells into the tumor by a
mechanism involving the secretion of high levels of CCL5, since
the infiltration was no longer observed after silencing CCL5. In
autophagy-defective tumor cells, we provide evidence that the
induction of CCL5 is transcriptionally related to the increase in
the phosphorylation of c-Jun at Ser-63 and -73. We also provide
clinical evidence supporting our data by showing a positive cor-
relation between the expression of CCL5 and the NK cell marker
NKp46 in melanoma patients. In addition, high expression of
CCL5 was correlated with an overall improved survival of
melanoma patients.
Results
Fig. 1. BECN1− B16-F10 tumors contain higher numbers of active NK cells
compared with Ctrl tumors. (A) Immunohistochemical staining of NK cells
performed on control (Ctrl) or BECN1− B16-F10 tumor sections using anti–
Asialo-GM1. (Scale bar, 200 μm.) (B) Tumor-infiltrating NK cells were stained
with NK1.1, CD69-, and GzmB-specific antibodies. Total NK cells (CD45+NK1.1+)
were gated and the percentage of active NK cells (NK1.1+CD69+ and
NK1.1+GzmB+) was determined by multicolor flow cytometry. Images repre-
sent flow cytometric plots of CD45+NK1.1+, NK1.1+CD69+, and NK1.1+GzmB+
cells infiltrating Ctrl and BECN1− B16-F10 tumors. (C) Quantification of total
NK1.1+ and active (NK1.1+CD69+ and NK1.1+GzmB+) NK cells infiltrating Ctrl
and BECN1- B16-F10 tumors, reported as a percentage (%). (D) Quantification
of CD69+/NK1.1+ (Left) and GzmB+/NK1.1+ (Right) cells infiltrating control Ctrl
and BECN1− B16-F10 tumors, reported as a percentage (%). Results are the
average SEM of three mice from each group. *P < 0.05; **P < 0.01; ns, not
significant (two-tailed Student’s t test).
Targeting Autophagy Related Gene BECN1 Improves the Recruitment
of Functional NK Cells into the Tumor Bed. We have previously
reported that targeting BECN1 significantly reduces the growth
of B16-F10 mouse tumors. This effect is primarily related to the
improvement of NK-mediated tumor cell killing, as such re-
duction was no longer observed in NK cell-depleted mice (17).
Although NK cells seem to play a major role in the control of
B16-F10 tumor growth, it should be emphasized that the reduced
BECN1-defective (BECN1−) B16-F10 tumor volume was also
rescued, but to a lesser extent, by the depletion of CD8 T cells. In
this study, we investigated whether targeting BECN1 inhibits the
tumor growth by improving the infiltration of NK cells into the
tumor bed. Immunohistochemistry staining of tumor sections
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Targeting Autophagy Induces the Expression of CCL5, Which Is
Involved in the Migration of NK Cells. It is well established that
tumors displaying high number of cytotoxic immune cells secrete
high amounts of attracting chemokines (26, 27). Consequently,
we evaluated whether the improvement of the infiltration of NK
cells into BECN1− B16-F10 tumors is related to the regulation
of some chemokines involved in the migration of NK cells. A
cytokine profiling assay, performed using the conditioned me-
dium of tumor cells, revealed that targeting BECN1 increased
the secretion of several chemokines, including CCL5, CXCL10,
and TIMP1 (Fig. 2A, Left). The quantification of the results
obtained by cytokine arrays revealed that CCL5 was the pre-
dominant up-regulated cytokine in BECN1− tumor cells com-
pared with CXCL10 or TIMP1 (Fig. 2A, Right). Based on these
data, we focused our interest on CCL5 and quantified its level in
the conditioned media of Ctrl and BECN1− B16-F10 tumor cells
by ELISA. Fig. 2B shows that BECN1− cells secreted 4.5-fold
more CCL5 compared with Ctrl cells (525 and 112 pg/mL for
BECN1− and Ctrl, respectively).
The impact of targeting BECN1 on the expression of CCL5
was further assessed in human melanoma cell lines displaying
low levels of CCL5. Indeed, according the expression value (log2)
of CCL5 in 62 human melanoma cell lines reported by the
Cancer Cell Line Encyclopedia (https://software.broadinstitute.
org/morpheus/), we selected three human melanoma cell lines—
A375, IPC298, and MelJuso—displaying a relatively low ex-
pression level of CCL5 mRNA (Fig. S2). Fig. 2C shows that,
similarly to B16-F10 cells, targeting BECN1 in A375, IPC298,
and MelJuso human melanoma cell lines induced a significant
increase in the secretion of CCL5, indicating that the up-
regulation of CCL5 resulting from targeting BECN1 is not re-
stricted to B16-F10 but can also be observed in other human
melanoma cell lines.
To further determine the involvement of CCL5 in the migration
of NK cells, we tracked the migration of human NK cells toward a
CCL5 gradient, using ibidi slides and a cell IQ platform. Fig. 2D
shows that, in the absence of CCL5, NK cells migrate randomly
(50% of cells to the upper and 50% to the lower compartments) in
short-distance migration trajectories. Interestingly, the presence of
a CCL5 gradient in the upper compartment induced directional
long-distance migration of 80% of the NK cells toward that
compartment. These results strongly suggest that CCL5 is a key
determinant for the migration of NK cells.
A growing body of evidence has revealed that, in addition to
its role in autophagy, BECN1 has several nonautophagic func-
tions (28). We therefore investigated whether targeting other
autophagy genes could also induce CCL5. We first determined
whether CCL5 is regulated at mRNA level. Our results (Fig. 2E)
show that, in BECN1− tumor cells, CCL5 is up-regulated at the
transcription level as a significant increase in the mRNA of
CCL5 was observed in BECN1− B16-F10 cells. Next, to de-
termine to what extent the autophagy process is involved in the
regulation of CCL5, we evaluated the impact of targeting other
autophagy genes on the expression of CCL5. We show that,
similarly to BECN1, targeting ATG5 or p62/SQSTM1 or treating
cells with chloroquine, an autophagy inhibitor (Fig. 2F), resulted
in a significant increase in the expression of CCL5 mRNA in
B16-F10 melanoma cells. Taken together, these data provide
clear evidence that—not only BECN1 but also the autophagy
process—are involved in the regulation of CCL5.
Fig. 2. BECN1− melanoma cells secrete high level of CCL5. (A, Left) Identifi-
cation of cytokines in the supernatants of Ctrl (up) and BECN1− (down) B16-
F10 cells using a Mouse Cytokine Array. Highly expressed cytokines (CCL5,
TIMP1, and CXCL10) regulated in BECN1− cells are highlighted in red. (Right)
Quantification of CCL5, TIMP1, and CXCL10 spots of the Left panel reported as
fold-change compared with the Ctrl. (B) Quantification of CCL5 in the super-
natant of Ctrl and BECN1− B16-F10 cells by ELISA. Results are the average
SEM of three independent experiments. *P < 0.05 (two-tailed Student’s t test).
(C) Expression of CCL5 in A375, IPC298, and MelJuso human melanoma cell
lines. (Upper) Quantification of CCL5 secretion by ELISA in the supernatants of
the indicated human melanoma cells transfected with either control (Ctrl) or
BECN1 siRNA (BECN1−). Data represent the average SEM of four (Left and
Right) or three (Center) independent experiments. *P < 0.05; ***P < 0.001
(two-tailed Student’s t test). (Lower) Expression of Beclin1 protein was assessed
by Western blot. Actin was used as loading control. (D) Migration of NK92MI
cell line toward recombinant CCL5 gradient. Migration of NK cells toward
serum-free medium in the absence (Left) or presence of 20 ng/mL of
recombinant CCL5 (Right). Red and black points represent NK cells migrating
to the top and bottom compartments, respectively. The percentage of mi-
grating NK cells are reported in the bottom of each panel. (E) Expression of
CCL5 mRNA in BECN1− B16-F10 cells (Upper). The expression of Beclin1 protein
was assessed by Western blot. Actin was used as loading control. **P < 0.01
(two-tailed Student’s t test). (F) Expression of CCL5 in B16-F10 cells transfected
with ATG5 siRNA (ATG5-) and in cells treated with chloroquine (CQ) or
transfected with p62/SQSTM1 siRNA (p62-) (Upper). The expression of ATG5,
LC3-I and -II, as well as p62/SQSTM1 proteins was assessed by Western blot.
Actin and GAPDH were used as loading controls. Results are reported as a fold-
change (FC) compared with Ctrl. Data represent the average SEM of three
independent experiments. **P < 0.01 (two-tailed Student’s t test).
Targeting BECN1 Induces the Expression of CCL5 Transcription Factor
c-Jun. To gain more insight into the molecular mechanisms in-
volved in the up-regulation of CCL5 mRNA in BECN1− B16-
F10 cells, we focused our interest on the major transcription
factor involved in the regulation of the CCL5 transcript. Indeed,
the promoter region of mouse and human CCL5 contains several
binding sites for well-defined transcription factors (29), including
AP-1. We first assessed whether c-Jun binds to the CCL5 pro-
moter in B16-F10 cells. We performed in silico identification of
consensus (5′-TGA[C/G]TCA-3′) (30) and nonconsensus (5′-
TGACTgA-3′ or 5′-TGACTCc-3′) (31) motifs for the Jun/Fos
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AP-1 binding site on the mouse CCL5 promoter. The promoter
containing fragment has been defined using the Eukaryotic Pro-
moter Database (Swiss Institute of Bioinformatics) from −8,000
to +100 bp relative to the transcription start site (TSS) of the
mouse CCL5 gene (RefSeq NM_013653.3). Fuzznuc EMBOSS
searches for patterns in nucleotide sequences software identified
two consensus and two nonconsensus AP-1 binding sites, high-
lighted in red and green, respectively, on the sequence (shown in
Fig. S3A). The positions of the consensus motifs were −7,668 and
−5,489 and those of the nonconsensus motifs were −7,636 and
−5,129 (Fig. S3B). ChIP was performed on CCL5-overexpressing
BECN1− B16-F10 cells using anti–c-Jun ChIP grade antibody.
Our results, depicted in Fig. S3C, show that c-Jun strongly binds
to the nonconsensus 5′-TGACTgA-3′ motif containing region C
of the CCL5 promoter, indicating that CCL5 is a direct c-Jun
target gene in BECN1− B16-F10 cells.
We next investigated the expression and the activation of c-Jun,
the AP-1 founding member (32). It is known that c-Jun is trans-
activated by phosphorylation at the N terminal Ser-63 and Ser-
73 residues (33, 34). Our results showed that the total and the
phosphorylated level of c-Jun at both Ser-63 and Ser-73 residues
were higher in BECN1− compared with control B16-F10 cells
(Fig. 3A, Left). An increase in the phosphorylation of c-Jun was
also observed in BECN1− tumor extracts (Fig. 3A, Right).
c-Jun phosphorylation is mediated by the upstream kinase JNK,
which in turn is exclusively phosphorylated by MKK4 kinase (35).
While no difference was observed in the total expression and the
phosphorylation levels of MKK4, a significant increase in the
phosphorylation of JNK at Thr-183 and Tyr-185 was detected in
BECN1− compared with Ctrl cells (Fig. 3B), suggesting that the
increased phosphorylation level of c-Jun in BECN1− cells is most
likely related to an increased JNK activity. This assumption was
further supported by our data showing that pharmacological (Fig.
3C, Upper) and genetic (Fig. 3D, Upper) inhibition of JNK com-
pletely suppressed, as expected, the phosphorylation of c-Jun at
Ser-63 and Ser-73 residues and, more interestingly, led to a sig-
nificant down-regulation of CCL5 mRNA in BECN1− tumor cells
(Fig. 3 C and D, Lower).
It has long been postulated that the regulation of protein
phosphorylation is partially balanced by the activity of kinases
and phosphatases. The serine/threonine phosphatase, PP2A, has
been described to be involved in the regulation of JNK phos-
phorylation and subsequently in that of its downstream protein
c-Jun (36). In accordance with this, we assessed PP2A activity in
Ctrl and BECN1− B16-F10 cells to determine whether the in-
creased levels of phosphorylated JNK and phosphorylated c-Jun
are related to a defect in PP2A activity. The results (Fig. 3E)
revealed that PP2A activity was significantly lower in BECN1−
compared with Ctrl B16-F10 cells, suggesting that the increased
levels of phosphorylated JNK and phosphorylated c-Jun resulted
from a decrease in the phosphatase activity of PP2A. This was
further supported by our data showing that targeting PP2A
subunit A genetically by siRNA in control cells increased the
phosphorylation of both JNK and c-Jun (Fig. 3F, Left), and
subsequently increased the expression of CCL5 (Fig. 3F, Right).
Similarly, treatment of control cells with PP2A inhibitor okadaic
acid inhibited its catalytic activity by increasing the phosphory-
lation at Tyr-307 (Fig. 3G, Upper). Such inhibition enhanced the
phosphorylation of c-Jun at Ser-63 and increased the expression
of CCL5 (Fig. 3G, Lower). Overall, these results provide a
mechanistic clue on how targeting autophagy increases the ex-
pression of CCL5 by enhancing the phosphorylation of c-Jun.
Fig. 3. Targeting autophagy induces the up-regulation of CCL5 mRNA by
increasing the phosphorylation of c-Jun. (A, Left) Expression of Beclin1, total
c-Jun, phosphorylated c-Jun on serine 63 (pS63), and serine 73 (pS73) proteins
in cell extracts from Ctrl and BECN1− B16-F10 cells. Actin was used as loading
control. (Right) Expression of Beclin1, phosphorylated c-Jun on serine 63
(pS63), and serine 73 (pS73) proteins in three regions (#1, #2, #3) of Ctrl and
BECN1− B16-F10 tumor extracts. Actin was used as loading control. (B) Ex-
pression of Beclin1, total and phosphorylated SEK/MKK4 on Ser-80, Thr-261,
and Ser-257, and phosphorylated JNK on Thr-185/Tyr-183 in Ctrl and BECN1−
B16-F10 melanoma cells. (C, Upper) Expression of Beclin1, phosphorylated JNK
on Thr-185/Tyr-183, and phosphorylated c-Jun on serine 63 (pS63) and serine
73 (pS73) proteins in Ctrl and BECN1− cells untreated (−) or treated (+) with
JNK inhibitor SP600125. Actin was used as loading control. (Lower) The expres-
sion of CCL5 mRNA in cells described in the Upper panel. Data are reported as
fold-change (FC) compared with the Ctrl. *P < 0.05 (two-tailed Student’s t test).
(D, Upper) BECN1− B16-F10 melanoma cells were transfected with control (Ctrl)
or JNK1/2 (JNK) siRNA. The expressions of JNK, Ser-63 (pS63), and Ser-73 (pS73)
phosphorylated c-Jun were assessed 72 h after transfection. Vinculin was used as
loading control. (Lower) The expression of CCL5 mRNA by real-time RT-PCR in
cells described in A. Data represent the average
SD of three independent
experiments. ***P < 0.005 (two tailed Student’s t test). (E) PP2A phosphatase
activity assay in Ctrl and BECN1− B16-F10 tumor cells. Data reported as picomoles
of free phosphate released following incubation of PP2Ac immunoprecipitated
from each cell with Threonine phosphopeptide. The result shown is the
average SEM of five independent experiments performed in duplicate. *P <
0.05 (two-tailed Student’s t test). (F, Left) Expression of PP2A subunit A, total and
phosphorylated c-Jun on serine 63 (pS63) and serine 73 (pS73) and total and
phosphorylated JNK on Thr-185/Tyr-183 proteins in Ctrl B16-F10 cells untrans-
fected (−) or transfected with PP2A siRNA targeting A subunit. Actin was used as
loading control. (Right) Expression of CCL5 mRNA in the cells described in the
Left panel. Data are reported as fold-change (FC) compared with the Ctrl. Results
represent the average SEM of three independent experiments. **P < 0.01
(two-tailed Student’s t test). (G, Upper) Expression of phosphorylated PP2A on
Tyr-307 PP2A and phosphorylated c-Jun on serine 63 (pS63) in Ctrl B16-F10 cells
untreated (−) or treated (+) with okadaic acid (OA). (Lower) Quantification of
CCL5 secreted by cells described in the Upper panel as determined by ELISA.
Targeting CCL5 in BECN1− Tumors Significantly Reduces the Infiltration
of NK Cells into the Tumor Bed and Prevents Tumor Regression. We
next investigated the impact of silencing CCL5 on the tumor
growth and on the infiltration of NK cells in BECN1− B16-
F10 tumors. We generated B16-F10 cells defective for both
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BECN1 and CCL5 (BECN1−/CCL5−) and characterized them in
terms of BECN1 expression and CCL5 secretion. Our results,
shown in Fig. 4A, demonstrate a significant inhibition of the se-
cretion of CCL5 in BECN1−/CCL5− cells. Fig. 4B clearly shows
that the reduction of tumor growth, seen in BECN1− tumors (Fig.
4B, red curve) compared with controls (Fig. 4B, black curve), was
no longer observed when CCL5 was silenced (Fig. 4B, gray curve).
Interestingly, immunohistochemistry staining demonstrated that
targeting CCL5 in BECN1− tumors dramatically prevented the
infiltration of NK cells into the tumor bed (Fig. 4C). The quan-
tification of the percentage of NK cells infiltrating the tumors is
shown in Fig. 4D. These data provide additional support that the
recruitment of NK cells into BECN1− B16-F10 tumors is driven by
CCL5. Because the JNK/c-Jun pathway has been identified in
vitro as the major transcription factor involved in the up-
regulation of CCL5 in BECN1− tumor cells, our results showed
that genetic targeting of JNK/c-Jun pathway (Fig. 4E) resulted in a
dramatic decrease in CCL5 expression (Fig. 4F) and an increase in
BECN1− tumor volume (Fig. 4G) and weight (Fig. 4H). In-
terestingly, there was a significant decrease in both the expression
of tumoral CCL5 (Fig. 4I) and the infiltration of NK cells into the
tumor bed (Fig. 4J and Fig. S4). Overall, our data provide evi-
dence that JNK/c-Jun/CCL5 pathway regulates the recruitment of
NK cells into BECN1− tumors.
The Expression of CCL5 Positively Correlates with Expression of the NK
Cell Marker NKp46 and Predicts Improvement of Melanoma Patients’
Survival. We next investigated whether a positive correlation could
be found between the expression of CCL5 and the infiltration of
NK cells in melanoma patients. Sections of melanoma tumor bi-
opsies were first stained with CCL5 and assigned, according to the
expression level of CCL5, as no, low, moderate, high, and very-
high CCL5 staining. These tumors were further stained with the
NK cell marker NKp46. Our results reveal a striking positive
correlation between the expression levels of CCL5 and NKp46
(Fig. 5A). Our data were further confirmed by quantitative real-
time PCR performed using mRNA isolated from a panel of 22
melanoma biopsies (Fig. 5B). To extend our study to an even
larger cohort of melanoma patients, we used data from 471 skin
cutaneous melanomas described in the TCGA database. As
expected, a strong positive correlation was also observed between
the expression of CCL5 and the expression of the NKp46
encoding gene NCR1 (Fig. 5C). Taken together, these results
highly suggest that melanoma tumors expressing high levels of
CCL5 are strongly infiltrated by NK cells.
We next assessed the impact of CCL5 level on the survival of
458 melanoma patients using the recently described OncoLnc
resource (www.oncolnc.org). This resource links RNAseq data of
particular genes with patients’ survival using clinical data from
The Cancer Genome Atlas (TCGA) (37). Based on RNAseq
data, the median log expression level of CCL5 in melanoma
patients was 588.44 (reported as a “log”). According to the ex-
pression level of CCL5, the 458 melanoma samples were
assigned into low and high CCL5-expressing groups, each of
them containing 229 samples. The expression of CCL5 in the low
and high groups ranged from 7.92 to 588 and from 588 to 32,675,
respectively. The Kaplan–Meier survival plot reported in Fig. 5D
depicts that melanoma patients with an elevated expression level
of CCL5 had a significant longer survival. Taken together, these
data strongly suggest that the improvement of the survival of
melanoma patients displaying a high level of CCL5 is most likely
due to an increase in the infiltration of NK cells into the tumors.
Fig. 4. CCL5 is the major chemokine involved in the infiltration of NK cells
into BECN1− B16-F10 tumor. (A, Upper) Expression of Beclin1 protein in Ctrl,
BECN1−, and BECN1−/CCL5− cells. Actin was used as loading control. (Lower)
Quantification of CCL5 secreted into the supernatants of the cells described
in the Upper panel. Data represent the average SEM of three independent
experiments. ***P < 0.001 (two-tailed t test). (B) Tumor cells described above
were engrafted subcutaneously in C57BL/6 mice (n = 8 per group). Tumor
volume was measured at the indicated days and the average
SEM was
reported. *P < 0.05; **P < 0.01 (two-tailed Student’s t test). (C) Immuno-
histochemical staining of NK cells (Upper) or CCL5 (Lower) performed on
indicated tumor sections using anti–Asialo-GM1 and CCL5 antibodies, re-
spectively. (Scale bars, 200 μm.) (D) Quantification of NK cells infiltrating Ctrl,
BECN1− and BECN1−/CCL5− tumors reported as a percentage (%) of the total
number of cells for each tumor. Data represent the average SEM of two
sections from two independent tumors. *P < 0.05; **P < 0.01; ns, not-
significant (two-tailed Student’s t test). (E) BECN1− B16-F10 melanoma cells
were infected with control (Ctrl) or JNK1/2 (JNK) shRNA. The expressions of
JNK, c-Jun, and Beclin1 were assessed. Vinculin was used as loading control.
The panel corresponding to the expression of Beclin1 was generated from
the same blot but consolidated from noncontiguous loading. (F) The ex-
pression of CCL5 mRNA by real-time RT-PCR in cells described in E. Data
represent the average
SEM of three independent experiments. ***P <
0.005 (two tailed Student’s t test). (G) BECN1− and BECN1−/JNK− tumor cells
described in E were engrafted subcutaneously in C57BL/6 mice (n = 5 or 6 per
group). Tumor volume was measured at the indicated days and the
average
SEM was reported. *P < 0.05; **P < 0.01 (two-tailed Student’s
t test). (H) The weight of BECN1− and BECN1−/JNK− tumors described in G at
day 18. *P < 0.05 (two-tailed Student’s t test). (I) Immunohistochemical
staining of CCL5 performed on BECN1− and BECN1−/JNK− tumor sections
using anti-CCL5 antibody. (Scale bars, 100 μm.) (J) Quantification of NK cells
infiltrating BECN1− and BECN1−/JNK− tumors reported as a percentage (%)
of the total number of cells for each tumor. Data represent the average
SEM of two sections from one independent tumor. *P < 0.05 (two-tailed
Student’s t test).
Discussion
The role of NK cells in the improvement of immune responses to
cancer has been extensively investigated during the past few
decades, and several NK cell-based immunotherapies have been
proposed and are used in the clinic (38).
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are available so far about its role in the recruitment of NK cells
into the tumor microenvironment. In line with our previous work,
which showed that targeting autophagy improves the NK-
mediated antitumor immune response (17), we show here that
inhibiting autophagy increases the expression and the release of
CCL5 by murine B16-F10 tumor cells both in vitro and in vivo,
which subsequently led to a massive infiltration of NK cells into
the tumor bed. Because the expression of CCL5 was also observed
in ATG5-defective cells and after chloroquine treatment, we
strongly believe that targeting the autophagy mechanism as a
whole, rather than specifically targeting BECN1, results in the
improvement of the NK-mediated antitumor immune responses
by inducing NK cell-homing into tumors.
It has been previously reported that CCL5 can increase the cy-
totoxicity of CD56+ human NK cells (44). Other studies also showed
that CCL5 promotes the release of cytotoxic granules by human NK
cells (45–47). We therefore asked whether an elevated level of
CCL5 could enhance the cytolytic activity of NK cell-infiltrating
tumors. We strongly believe that CCL5 released by BECN1− tu-
mor cells has no impact on the activation status of NK cells as, when
reported to the total number of NK cells, no differences in the
expression of NK cell-activation markers CD69 and GzmB were
observed in NK cell-infiltrating control or BECN1− tumors.
Another important question is to determine whether CCL5 is
the only factor involved in the recruitment of NK cells. Indeed,
in addition to CCL5, our cytokine array revealed that CXCL10
was found to be up-regulated in BECN1− tumor cells, although
at a lower extent. Based on the fact that CXCL10 has also been
described to induce the migration of NK cells (27), the in-
volvement of CXCL10 in the homing of NK cells cannot be ruled
out, and additional experiments need to be conducted to de-
termine the relative contribution of CXCL10. Nevertheless, our
data showed that targeting CCL5 was sufficient to significantly
suppress the infiltration of NK cells and subsequently enhance
the tumor growth in BECN1− tumors, strongly suggesting that
CCL5 is the major factor involved in the recruitment of NK cells
to B16-F10 melanoma tumors.
Fig. 5. CCL5 overexpression is correlated with the increase of NKp46 and the
improvement of melanoma patients’ survival. (A) Twelve FFPE melanoma bi-
opsy sections were stained with CCL5 (Left) or NKp46 (Right) antibodies and
immunohistochemical analysis showed that tumor sections strongly positive
for CCL5, also had an abundant number of NK cells. Five representative sections
(P1 to P5) are reported. (Scale bars, 100 μm.) (B) RNA extracted from 22 mela-
noma biopsies were assessed for the expression of CCL5 and NKp46 encoding
NCR1 gene. The Spearman test showed a significant and positive correlation
between the expression of CCL5 and NCR1. (C) A similar Spearman correlation
test was set up using RNA-Seq data of 471 skin cutaneous melanomas described
in the TCGA database. A positive correlation was found between the expression
of CCL5 and NCR1. The expression of each gene was reported as RNA-Seq
V2RSEM (RNA-Seq expression estimation by expectation-maximization). (D)
Kaplan–Meier survival plot of skin cutaneous melanoma (SKCM) patients gen-
erated using OncoLnc resource. TCGA data of 458 melanoma patients were
assigned into low or high groups according to the expression level of CCL5
reported as RNASeq values. Patients having a CCL5 expression level ranging
from 7.92 to 588 were considered as low (blue curve) and those having a CCL5
expression level ranging from 588 to 32,675 were considered as high (red
curve). Statistically significant difference is indicated by P value.
CCL5 elicits its chemo-attracting activity by acting via CCR1,
CCR3, and CCR5 (48). As such, the increased migration of NK
cells toward a CCL5 gradient could be related to the increased
expression of CCL5 receptors on the surface of NK cells in-
filtrating BECN1− tumors. This possibility can be ruled out since
no difference in the expression of CCR1, CCR3, and CCR5 was
found on the surface of NK cells infiltrating both control and
BECN1− tumors (Fig. S5). Interestingly, we even observed that
BECN1− B16-F10 cells isolated from tumors exhibited signifi-
cantly lower levels of CCR1, CCR3, and CCR5 compared with
controls (Fig. S6). This implies that the autocrine CCL5 signal-
ing, previously described to promote the invasion and migration
of tumor cells (49), is attenuated in BECN1− tumor cells, and
that CCL5 released by tumor cells primarily acts via paracrine
signaling to attract NKs to the tumor bed. However, how the
CCL5 receptors are regulated in BECN1− tumors remains, so
far, an unresolved issue that needs to be further investigated.
Mechanistically, we provide strong evidence that targeting
BECN1 induces a dramatic phosphorylation of c-Jun at both Ser-
63 and Ser-73 residues through PP2A-dependent increase of
JNK phosphorylation. Therefore, our results predict that c-Jun is
constitutively present in control cells in an inactive form and that
targeting autophagy induces the phosphorylation of c-Jun at Ser-
63 and Ser-73 residues located within its transactivation domain.
Such phosphorylation serves to increase both its stability and
transcriptional activity, as previously reported (50, 51).
CCL5, also known as RANTES, is a small protein that belongs
to a large family of cytokines and displays chemotaxis activity, as it
is involved in inducing the migration of several leukocytes into
inflammation sites (39). CCL5 is secreted by a wide variety of
cells, including T cells (40), NKs (27), and some tumor cells (41).
CCL5 interacts with different receptors including C-C chemo-
kine receptor types 1, 3, and 5 (CCR1, CCR3, and CCR5) (42).
Controversial data exist about the role of CCL5 in the tumor
microenvironment. While some reports highlight the role of
CCL5 in promoting breast cancer metastasis (43), others refer the
importance of CCL5 in T cell recruitment into melanomas (26). It
seems that the pro- or the antioncogenic roles of CCL5 depends
on the tumor type, the nature of the cells secreting CCL5, and the
different interactions within the tumor microenvironment. Al-
though the role of CCL5 in the recruitment of NK cells in in-
The increase in the phosphorylation of JNK/c-Jun in BECN1−
cells seems to be tightly linked to the decreased activity of PP2A.
Although the causal mechanisms underlying the decrease in PP2A
activity in BECN1− cells remains largely unknown, our data provide
flammation and allergic diseases has been widely studied, no data an important mechanistic clue on how CCL5 is transcriptionally
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Mgrditchian et al.

compromise the antitumor immunity by inducing an immuno-
suppressive microenvironment (59). Our data reported here re-
veal the impact of targeting autophagy in tumor cells on the
release of chemokine involved in the infiltration of NK cells.
Although much remains to be learned mechanistically, this study
provides a cutting-edge approach on how to switch the immu-
nosuppressive tumor microenvironment to an immunosupportive
one, allowing for the redirection of NK cells to the tumor bed.
One issue of great interest that remains to be addressed is to
investigate the impact of targeting autophagy on the immune
landscape of tumors and on the infiltration of other populations
of immune cells into the tumor bed. Nevertheless, it is our belief
that the knowledge generated from this study will pave the way
to fully exploit NK cells’ antitumor properties in clinical settings.
Autophagy targeting
in tumor cells
Decreased PP2A
Catalytic activity
JNK
p-JNK
c-Jun p-c-Jun
Ser-63/-73
CCL5 expression
and secretion
Materials and Methods
Attracting NK cells
Into the tumor bed
Immunoblot Assays. Cell lysates were prepared using RIPA lysis buffer (20-188;
Millipore) supplemented with a protease inhibitor mixture (11873580001;
Prophac) and phosphatase inhibitor mixture 2 and 3 (P5726-5ML and P0044-
5ML; Sigma). Protein concentration was determined using Bio-Rad protein
assay dye reagent (500-0006; Bio-Rad). Equal amounts of proteins were loaded
on SDS/PAGE gel, transferred to nitrocellulose membranes, and blocked with
either 5% of milk or 5% of BSA according to the manufacturer’s instructions for
each antibody. The following primary antibodies were used: Beclin1 (3738; Cell
Signaling), c-Jun (9165S; Cell Signaling), phospho c-Jun S63 (9261S; Cell Sig-
naling), phospho c-Jun S73 (3270S; Cell Signaling), LC3II (2775S; Cell Signaling),
ATG5 (MBL 153-3; MBL), GAPDH (5174S; Cell Signaling), SAPK/JNK (9552S; Cell
Signaling), phospho SAPK/JNK (Thr183/Tyr185) (9251S; Sell Signaling), PP2A
subunit A (2041; Cell Signaling), phospho PP2A tyr307 (sc-271903; Santa Cruz),
SEK1/MKK4 (9152S; Cell Signaling), phospho SEK1/MKK4 S257 (4514P; Cell
Signaling), phospho SEK1/MKK4 S80 (9155S; Cell Signaling), phospho SEK1/
MKK4 Thr261 (9151S; Cell Signaling), and actin (A5441; Sigma Aldrich). Blots
were then incubated with horseradish peroxidase-conjugated secondary an-
tibodies (Jackson ImmunoResearch Laboratories). Proteins were detected using
enhanced chemiluminescence ECL (GE Healthcare).
Tumor shrinking
Fig. 6. Schematic representation of the mechanism involved in the over-
expression of CCL5 in BECN1− tumor cells. Inhibition of autophagy by targeting
BECN1 decreases the catalytic phosphatase activity of PP2A by a mechanism
that is not fully understood. The decreased PP2A activity leads to an increased
phosphorylation of both JNK (p-JNK) and its downstream c-Jun at Ser-63 and
Ser-73 (p-c-Jun Ser-63/-73). The activation of c-Jun by phosphorylation en-
hances the expression of CCL5 gene. CCL5 released by tumor cells attracts NK
cells into the tumor bed leading to the decrease in tumor growth.
regulated in BECN1− tumor cells. A schematic representation of
the mechanism underlying the expression of CCL5 in BECN1−
cells is reported in Fig. 6. In addition, we strongly believe that the
increased-phosphorylation of c-Jun at Ser-63 residue is a conse-
quence of targeting Beclin1 rather than the up-regulation of CCL5,
as the level of phosphorylated c-Jun was not affected following
CCL5 silencing in BECN1− tumor cells (Fig. S7).
RNA Extraction and Reverse-Transcriptase PCR. Total RNAs were extracted using
the miRCURY RNA isolation kit (300110; Exiqon). RNA (1 μg) from each sample
was reverse-transcribed using
a reverse-transcription reaction mix (Euro-
It is now well established that tumor infiltration by NK cells
represents a positive prognostic marker in different cancers, in-
cluding colorectal (7), gastric (52), and lung carcinomas (53). It
has been suggested that low NK cell numbers in tumors are likely
due to their inefficient homing into the tumor bed, which could
be overcome by cytokine-mediated activation (2). Furthermore,
convincing evidence for a beneficial role of NK cells in the
control of tumor growth was provided from clinical studies of
leukemia patients who received alloreactive NK cells in the
course of allogenic hematopoietic stem cell transplantation (2).
Using a small cohort of melanoma tumor biopsies, we show here
a striking correlation between the expression of CCL5 and NK
cell marker NKp46 at the protein and mRNA levels. As tumoral
CCL5 has been described to recruit T cells (54) and macro-
phages (55) into the tumor bed as well, we cannot exclude that
melanoma tumors expressing a high level of CCL5 are not only
highly infiltrated by NK cells but also by T cells and macro-
phages. Furthermore, it should be noted that the CCL5 staining
detected in melanoma biopsies may not be only produced by
tumor cells, since other cells in the tumor microenvironment—
such as T cells—can also secrete CCL5, as previously reported
(56, 57). Nevertheless, the positive correlation between
CCL5 and NK cells was further confirmed using more than
400 melanoma patients from the TCGA database. Consistent
with the fact that CCL5 induces the homing of NK cells into the
tumor bed, it stands to reason that CCL5 overexpression impacts
on the survival of melanoma patients. Accordingly, we show that
the long-term survival of melanoma patients overexpressing
CCL5 was significantly improved.
gentec). The reverse transcription was performed at 48 °C for 30 min. The
resulting cDNA was subjected to amplification by qPCR using power SYBR
green PCR master mix (Life Technologies). The RPL13 gene encoding Ribosomal
Protein L13 was used as internal control. For mouse RPL13, the following
primers were used: forward 5′-GGAGGGGCAGGTTCTGGTAT-3′ and reverse
5′-TGTTGATGCCTTCACAGCGT-3′. For mouse CCL5, the following primers
were used: forward 5′-CTGCTGCTTTGCCTACCTCT-3′ and reverse 5′-CGAGT-
GACAAACACGACTGC-3′. RNAs from melanoma patients were provided by the
Gustave Roussy Cancer Center, Villejuif, France. Briefly, RNAs from frozen
melanoma were extracted using the Qiagen miRNEasy kit (217004; Courta-
boeuf) and those from formalin-fixed paraffin-embedded (FFPE) were
extracted using the Qiagen RNeasy FFPE kit (73504; Qiagen). RNAs were
quantified by Nanodrop and their quality was verified by Bioanalyzer. The
reverse-transcription qPCR was performed by AnyGenes Company (Tenon
Hospital, Paris). Information related to the primers used amplification is the
following. PPIA and ACTB genes encoding Peptidylprolyl isomerase A and actin
β, respectively, were used as internal loading controls; for NKp46 encoding
NCR1 gene, the forward primer is localized in exon 4 and the reverse primer is
localized at the junction of exons 4 and 5; for CCL5, the forward and reverse
primers are localized in exon 1 and 2, respectively; for PPIA, the forward and
reverse primers are localized in exon 1 and 2, respectively; and for ACTB, the
forward and reverse primers are localized in exon 6.
Cytokine Array and ELISA Measurement of Mouse and Human CCL5. The profiling
of different cytokines and chemokines was performed by Mouse cytokine array,
and panel A (R&D systems) according to the manufacturer’s instructions using
serum-free supernatant harvested from equal number of cells. The signal in-
tensities of the spots were quantified by ImageJ. CCL5 secretion by cells was
quantified using the CCL5/RANTES Quantikine Elisa kit (R&D Systems)
according to the manufacturer’s instructions. The sensitivity of ELISA kits was
2.0 pg/mL for mouse CCL5 and 5.0 pg/mL for human CCL5.
It is now well accepted that NK cells are capable of driving
potent antitumor responses (58). However, tumor cells often
In Vivo Tumor Growth and Flow Cytometry Analysis. Nine-week-old C57BL/6
mice were engrafted subcutaneously with B16F10 melanoma cells (2.3 × 105
Mgrditchian et al.
PNAS Early Edition
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cells per mouse). Tumor growth was measured using a caliper every other
day starting from day 11. Tumor volume was calculated as follows: volume
(cm3) = 3.14159/6 × (width × length × height). B16-F10 tumor-bearing
C57BL/6 mice were killed at day 21. The mouse experiments were per-
formed according to the Luxembourg Institute of Health’s instructions and
guidelines and approved by the Luxembourg Institute of Health Ethical
Committee. For flow cytometry analysis, cells were dissociated from tu-
mors in DMEM complete medium, and centrifuged for 10 min at 4 °C. Red
blood cells were lysed using ACK lysing buffer (10-548E; Lonza). Cells were
counted and Fc-receptors were blocked with CD16/CD32 for 30 min before
staining with appropriate antibodies. The following antibodies were used:
BV421 anti-mouse NK1.1 (108731; Biolegend), Al700 anti-mouse CD19
(115528; Biolegend), APC-R700 rat anti-mouse CD11b (564985; BD Hori-
zon), BUV395 rat anti-mouse CD45 (564279; BD Horizon), BV605 Hamster
anti-mouse CD69 (563290; BD Horizon), Pacific blue anti-human/mouse
GzmB (515407; Biolegend) and live/dead near IR (L10119; Life Technolo-
gies). CD45+/CD11b−/CD19− cells were gated to determine the percentage
of NK1.1+; NK1.1+GzmB+, and NK1.1+CD69+.
Generation of BECN1− and CCL5− B16-F10 Cell Line. Scrambled and mouse
BECN1 short hairpin (shRNA) lentiviral particles were purchased from Santa
Cruz Biotechnology. Cells were transduced in the presence of puromycin.
Control and mouse CCL5 SureSilencing shRNA encoding plasmids were
purchased from Qiagen. Transfected cells were maintained in the presence
of hygromycin. Mouse JNK1 and JNK2 shRNA Lentiviral Particles were pur-
chased from Santa Cruz Biotechnology.
In Vitro Chemotaxis Experiment. The migration of NK92MI cells was evaluated
in μ-Slide Chemotaxis3D from ibidi according to the manufacturer’s instruc-
tions. NK92MI cells (9 × 106 cells/mL) cultured in serum-free medium in the
absence or presence of 20 ng/mL of human recombinant CCL5/RANTES were
transferred to the ibidi slide reservoirs. The time-lapse experiment was
performed using Cell IQ platform during 48 h. One image was recorded
every 15 min and the migration of NK92MI cells was analyzed by Tracking
Tool PRO v2.0 software.
siRNA Transcfections. Small interference RNAs (siRNA) were purchased from
Qiagen. Cells were transfected using Lipofectamine RNAiMax reagent (13778-
075; Life Technologies) in Opti-MEM reduced serum medium (31985070;
Thermo Fisher Scientific). The following siRNAs against human Beclin1 were
used: SI0005594 for IPC298 and A375, SI00055573 for MelJuso. siRNA against
mouse ATG5 was SI02696806. siRNA against mouse p62 was SI02713445
(Qiagen). siRNA against mouse JNK1 was SI01300691. siRNA against JNK2 was
SI01300803. Transfected cells were harvested for RNA and protein extractions.
Immunohistochemistry Staining. FFPE tumor sections (5 μm) from B16-
F10 tumors were used to determine the expression of NK and CCL5/RANTES by
immunohistochemistry using rabbit anti-Asialo GM1 polyclonal antibody (986-
10001; Wako Chemicals) and CCL5/RANTES antibody (NBP1-19769; Novus Bi-
ologicals) respectively. The staining was performed at the Laboratory of Ex-
perimental Pathology, Gustave Roussy Cancer Center, Villejuif, France. Stained
sections were scanned with the Leica Aperio AT2 scanner and the number of
NK+ and CCL5+ cells were quantified by HistoWiz Company using HALO
software from Indica laboratories. FFPE sections from melanoma patients were
assessed for the expression of CCL5 and NKp46 using NBP1-19769 and NBP2-
11820 antibodies (Novus Biological), respectively. Stained sections were scan-
ned with Leica Aperio AT2 by HistoWiz.
In Vivo Experiments and Clinical Samples. In vivo protocols were approved by
the “Comité National d’Ethique de Recherche” Luxembourg, LHCE-2014-02.
Clinical samples were collected from patients after having given their writ-
ten informed consent in accordance with the declaration of Helsinki.
Statistical Analysis. Statistically significant differences were evaluated using the
unpaired two-tailed t test (SigmaPlot 12.5). A P value of less than 0.05 was
considered statistically significant. Data were expressed as average SEM. The
Spearman test was used to determine the correlation between the expression
of CCL5 and NKp46-encoding NCR1 genes in melanoma patients. Melanoma
patients’ survival curves were generated by the OncoLnc tool (open access),
which uses the Kaplan–Meier method for generating the curves and a log-rank
test for calculating P values.
Cell Culture and Treatments. B16-F10 cells from ATCC were maintained in
DMEM supplied with 10% of FBS and 1% of penicillin/streptomycin. IPC298,
MelJuso and A375 cells were provided by S.K. and were maintained in RPMI
medium supplied with 10% of FBS and 1% of penicillin/streptomycin. B16-
F10 cells were treated with 60 μM of chloroquine during 4 h or with 20 μM of
SP600125 overnight and washed before harvesting RNAs and proteins.
ACKNOWLEDGMENTS. We thank Lea Guyonnet from the National Cytome-
try Platform at Luxembourg Institute of Health for her assistance in flow
cytometry analysis; Olivia Bawa from the Gustave Roussy Cancer Center for
her assistance in immunohistochemistry staining; and Cristina Maximo from
Luxembourg Institute of Health for proofreading the manuscript. This work
was supported by grants from Fond National de la Recherche, Luxembourg
(Grant FNR-AFR 7842786 to T.M.), Luxembourg Institute of Health (Grant
LHCE 2013 11 05 to B.J.), Fonds de la Recherche Scientifique FNRS Televie
(Grants 7.6503.16 to T.A. and B.J., 7.4664.15 to M.Z.N. and B.J., and 7.4571.15
to E.V. and B.J.), Fondation Cancer, Luxembourg (Grants FC/2012/02 and F/2016/
01 to B.J. and G.B.), Calouste Gulbenkian Foundation (Grant P.1333237 to T.M.
and B.J.), Ligue Contre le Cancer (Équipes Labellisées), and Institut National
du Cancer (to S.C.).
PP2A in Vitro Protein Phosphatase Assay.
A PP2A immunoprecipitation
phosphate assay kit (Millipore) was used to detect PP2A activity according to
the manufacturer’s instructions. Briefly, 200 μg of Ctrl and BECN1− B16-
F10 cells lysates were used to immunoprecipitate PP2A using anti-PP2Ac
antibody. PP2A bound beads were washed with phosphatase assay buffer
and phosphopeptide (KRpTIRR) in serine/threonine assay buffer (750 μM)
was added and incubated 10 min at 30 °C. After centrifugation, 25 μL of the
supernatant was transferred to a microplate and 100 μL of Green phosphate
assay was added for 15 min. The relative absorbance was measured at
630 nm. The absorbance values were compared with negative controls
containing no enzyme.
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