Chemerin reactivates PTEN and suppresses PD-L1 in tumor cells via modulation of a novel CMKLR1-mediated signaling cascade
Authors and affiliations
Keith Rennier1, Woo Jae Shin1, Ethan Krug1, Gurpal Virdi1, Russell K Pachynski*1,2,3
1Division of Oncology, John T. Milliken Department of Medicine, 2Alvin J. Siteman Cancer Center, 3The Bursky Center for Human Immunology & Immunotherapy Programs (CHiiPs); Washington University School of Medicine, St. Louis, MO, USA
*Corresponding Author
Russell K Pachynski, MD; 660 S Euclid Ave; Box 8056, St Louis, MO 63110; 314-286-
2341; [email protected]
Running Title: Favorable PTEN/PD-L1 modulation by chemerin in tumors
Key Words: Chemerin, RARRES2, CMKLR1, PTEN, PD-L1, anti-tumor immunity
Conflict of Interests: The authors declare that there are no relevant conflicts of interest
regarding this manuscript.
Acknowledgements: We would like to thank Dr. Brian Van Tine for the generous
donation of the sarcoma cell lines used in this publication.
Funding: This work was supported in part by American Cancer Society MSRG 125078-
MRSG-13-244-01-LIB, the Prostate Cancer Foundation, The Kimmel Foundation, and a
generous gift from Kerry Preete (RKP). KR was supported in part by a fellowship
provided by Ferring Pharmaceuticals.
Word Counts:
Statement of translational relevance 140, Abstract 250, Methods 1311, Text 5620
Main Figures 6, Supplemental Figures 8, Supplementary Tables 2
References 61
Research. on September 20, 2020. © 2020 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 30, 2020; DOI: 10.1158/1078-0432.CCR-19-4245
Favorable PTEN/PD-L1 modulation by chemerin in tumors Page 2 of 33
Statement of Translational Relevance
Loss of the tumor suppressor PTEN in human cancers has recently been shown to
contribute to resistance to immunotherapy; unfortunately, therapeutic reactivation of
PTEN has remained elusive. Chemerin (RARRES2) is a leukocyte chemoattractant
known to recruit effector immune cells, and is often downregulated in tumors. Recent
data links chemerin to PTEN expression, and thus we hypothesized that chemerin may
act to augment PTEN and result in improved responses to immunotherapy. Herein, we
describe a novel pathway in human tumors whereby chemerin, through its GPCR
receptor CMKRL1, induces PTEN expression and activity while concurrently
suppresses PD-L1 expression. We show that chemerin treatment significantly inhibits
tumor migration/invasion, increases T cell-mediated cytotoxicity, and suppresses in vivo
tumor growth. Taken together, these results identify chemerin as a promising clinical
therapeutic able to reactivate PTEN and suppress PD-L1 expression, thus potentially
improving responses to immunotherapy.
Research. on September 20, 2020. © 2020 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 30, 2020; DOI: 10.1158/1078-0432.CCR-19-4245
Favorable PTEN/PD-L1 modulation by chemerin in tumors Page 3 of 33
Abstract
Purpose: Chemerin (RARRES2) is an endogenous leukocyte chemoattractant that
recruits innate immune cells through its receptor, CMKLR1. RARRES2 is widely
expressed in non-hematopoietic tissues and often downregulated across multiple tumor
types compared to normal tissue. Recent studies show that augmenting chemerin in the
tumor microenvironment significantly suppresses tumor growth, in part by immune
effector cells recruitment. However, as tumor cells express functional
chemokine/chemoattractant receptors that impact their phenotype, we hypothesized
that chemerin may have additional, tumor-intrinsic effects.
Experimental Design: We investigated the effect of exogenous chemerin on human
prostate and sarcoma tumor lines. Key signaling pathway components were elucidated
using qPCR, Western blotting, siRNA knockdown, and specific inhibitors. Functional
consequences of chemerin treatment were evaluated using in vitro and in vivo studies.
Results: We show for the first time that human tumors exposed to exogenous chemerin
significantly upregulate PTEN expression/activity, and concomitantly suppress PD-L1
expression. CMKLR1 knockdown abrogated chemerin-induced PTEN and PD-L1
modulation, exposing a novel CMKLR1/PTEN/PD-L1 signaling cascade. Targeted
inhibitors suggest signaling is occurring through the PI3K/AKT/mTOR pathway.
Chemerin treatment significantly reduced tumor migration, while significantly increasing
T cell-mediated cytotoxicity. Chemerin treatment was as effective as both PD-L1
knockdown and the anti-PD-L1 antibody atezolizumab in augmenting T cell-mediated
tumor lysis. Forced expression of chemerin in human DU145 tumors significantly
suppressed in vivo tumor growth, and significantly increased PTEN and decreased PD-
L1 expression.
Research. on September 20, 2020. © 2020 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 30, 2020; DOI: 10.1158/1078-0432.CCR-19-4245
Favorable PTEN/PD-L1 modulation by chemerin in tumors Page 4 of 33
Conclusions: Collectively, our data show a novel link between chemerin, PTEN and
PD-L1 in human tumor lines, that may have a role in improving T cell-mediated
immunotherapies.
Introduction
Chemerin, or RARRES2 (retinoic acid receptor responder 2), is an endogenous
leukocyte chemoattractant, but has myriad roles in adipogenesis, metabolism,
angiogenesis, microbial defense, and cancer. Chemerin is widely expressed in non-
hematopoietic tissues, with low/no expression noted in leukocytes 1. Chemerin recruits
innate immune cells along its concentration gradient to sites of inflammation via its G-
protein coupled receptor (GPCR) chemokine-like receptor-1 (CMKLR1, aka ChemR23)
2,3. In humans, CMKLR1 expression on leukocytes has been shown in macrophages,
dendritic cells (DCs), and NK cells with comparable expression in the mouse3-6. While
data is limited, CMKLR1 expression has been detected on human tumor cells 7,8,
suggesting that interaction with its endogenous ligand chemerin may modulate tumor
cell phenotype, as seen with other chemokine/receptor pairs 9.
Chemerin/RARRES2 is commonly downregulated across several tumor types, including
melanoma, breast, prostate, and sarcoma, compared to their normal tissue counterparts
1. Our group was the first to show that forcible re-expression of chemerin in the tumor
microenvironment (TME) resulted in recruitment and increased tumor-infiltrating effector
leukocytes, leading to a significant reduction in the growth of aggressive B16 melanoma
in a mouse model 10. While recruitment of immune effector cells is important, tumor cell-
intrinsic oncogenic signaling pathways can also impact directed immune responses, and
thus play a key role in determining therapeutic efficacy.
Research. on September 20, 2020. © 2020 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 30, 2020; DOI: 10.1158/1078-0432.CCR-19-4245
Favorable PTEN/PD-L1 modulation by chemerin in tumors Page 5 of 33
PTEN (phosphatase and tensin homolog deleted on chromosome 10) is a critical tumor
suppressor whose expression is downregulated and/or lost in many tumor types 11.
PTEN loss has correlated with activation of the PI3K-AKT pathway, which is implicated
in the pathogenesis of these cancers, and is particularly relevant in prostate cancer 12.
Deleterious PTEN alterations are found in up to ~20-30% of primary prostate cancer
tissues and in ~40-60% of metastatic tissues, and are among the most common
genomic events in prostate cancer 13. While less commonly mutated in sarcoma, PTEN
downregulation has also been shown to play an important role in a subset of soft tissue
sarcomas (STSs), with one study showing 57% of STSs with decreased PTEN
expression 14. Futhermore, aberrations in the downstream PI3K/Akt pathway are almost
always implicated in the pathogenesis of sarcomas, with essentially 100% of advanced
stage osteosarcomas showing dysregulation in this pathway 15.
Here, we examine the effects of chemerin on tumor cell-intrinsic phenotype and
describe – for the first time – the ability of chemerin to upregulate the expression and
function of PTEN in human prostate and sarcoma tumor cell lines. Importantly, we show
– also for the first time – that chemerin treatment of tumor cells results in a concomitant
downregulation of PD-L1 expression, which directly translates into significantly
increased T cell-mediated cytotoxicity. These effects were dependent on CMKLR1, as
siRNA knockdown and specific inhibition with the CMKLR1 antagonist -NETA
completely abrogated these effects. In vivo studies using the human DU145 prostate
tumor line show that expression of chemerin in the TME significantly suppresses tumor
growth, increasing tumor PTEN and decreasing tumor PD-L1 expression compared to
controls. Collectively, these studies show that in addition to recruitment of effector
leukocytes into the TME, chemerin can also upregulate PTEN expression/function and
Research. on September 20, 2020. © 2020 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 30, 2020; DOI: 10.1158/1078-0432.CCR-19-4245
Favorable PTEN/PD-L1 modulation by chemerin in tumors Page 6 of 33
suppress PD-L1 expression, suppressing in vivo tumor growth and potentially rendering
tumor cells more susceptible to T cell mediated immunotherapies.
Materials and Methods
Cell Culture and Reagents: Cell lines were obtained from ATCC between the years of
2015 to 2018. For experiments, each cell line was used between passage 4-12. Cell line
authentication was verified by ATCC through PCR, karyotyping, and morphology based
techniques to confirm the tumor line status prior to use. DU145 (Human Prostate
Cancer, HTB-81), PC3 (Human Prostate Cancer, CRL-1435) cells were cultured using
RPMI 1640 complete media. SKES-1 (Human Ewing Sarcoma, HTB-86) and U2-OS
(Human Osteosarcoma, HTB-96) cells were cultured with McCoy’s 5A media. Cell lines
were tested for mycoplasma every 2-4 weeks, depending on the rate of usage, using
the Mycoprobe mycoplasma detection kit (CUL001B, R&D Systems). Recombinant
human chemerin (2325-CM, R&D Systems) was added at specified concentrations for
48h. Vehicle control (captisol) vs α-NETA (10uM) incubation for up to 24h with either
PBS or 6nM Chemerin, each reagent is replaced in fresh media every 24h. α-NETA
(10uM, Selleck Chemical) or CMKLR1 blocking peptide (5uM, sc-374570 P, Santa Cruz
Biotechnology) were used to block CMKLR1. Everolimus (RAD001, Sigma, mTOR) and
CCG-1423 (Cayman Chemical, RhoA/SRF) were used as inhibitors. mTOR inhibitor
(Everolimus, Selleck Chemical) – 200nM for complete inhibition for 24h. PI3k inhibitor
(BEZ235, Selleck Chemical) – 100nM for 24h pretreatment.
siRNA Transfection: X-tremegene siRNA transfection reagent (#4476093001, Roche)
and each siRNA were added drop-wise to the cell media. siRNAs (CMKLR1, PTEN, PD-
Research. on September 20, 2020. © 2020 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 30, 2020; DOI: 10.1158/1078-0432.CCR-19-4245
Favorable PTEN/PD-L1 modulation by chemerin in tumors Page 7 of 33
L1) were all 10uM stock concentration. The optimal ratio of transfection reagent to
siRNA (4:10), gave a final concentration of 40pM, and signal knockdown was evaluated
via Western blot (supplemental figures 1,8). ChemR23/CMKLR1 (sc-44633, Santa
Cruz), PD-L1 (sc-39699, Santa Cruz), PTEN (6251S, Cell Signaling) and Control (sc-
37007, Santa Cruz) siRNA were used for signal knockdown. The control siRNA-A is a
non-specific scrambled sequence used as a negative control in the siRNA-targeted
knockdown experiments.
Flow Cytometry: Cells were stained with the target-specific antibody (supplemental table
1) as labeled in each figure at 1uL/100k cells for 30 minutes at 4˚C. Cells were analyzed
using a FACScalibur (BD Biosciences).
Real-time RT-PCR: Sample RNA was isolated using Trizol (Invitrogen) and RNeasy
Mini RNA Isolation kit (Qiagen). RNA concentrations were verified using NanoDrop
2000 (Thermo). Bio-Rad iScript Advanced cDNA Synthesis kit converted RNA to cDNA
via manufacturer’s protocol. cDNA was amplified with iTaq Universal SYBR Green
Supermix (Bio-Rad) via the manufacturer’s protocol. A CFX96 Real-Time PCR system
(Bio-Rad) was used to quantify gene expression via the 2Ct analysis method. Primer
sequences were developed using Primer-Blast software
(https://www.ncbi.nlm.nih.gov/tools/primer-blast/). Each sample result was normalized to
its respective GAPDH loading control. See supplemental table 2 for primer sequences.
Immunoblot Analysis: RIPA lysis buffer (protease/phosphatase inhibitor cocktail
(Thermo)) was used to lyse cells post-experiment. Protein concentration was calculated
using Pierce BCA Protein Assay (Thermo) via manufacturer’s protocol. Bolt 4-12% Bis-
Tris SDS-PAGE gels (Invitrogen) were loaded with equal sample protein amounts
(50ug/sample). Gels were transferred to NitroBind Nitrocellulose membrane (Thermo).
Research. on September 20, 2020. © 2020 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 30, 2020; DOI: 10.1158/1078-0432.CCR-19-4245
Favorable PTEN/PD-L1 modulation by chemerin in tumors Page 8 of 33
Blots were illuminated using Thermo SuperSignal West Dura per manufacturer’s
protocol. Imaging was done using BioRad Molecular Imager ChemiDoc XRS+ System
and quantified using the BioRad Image Analysis Software.
PTEN Phosphatase Activity: Protein was processed as described above using
immunoprecipitation (IP) lysis buffer (Thermo). Samples were normalized
(200μg/sample) and Anti-human PTEN (138G6, Cell Signaling, 1:250) was added for
PTEN IP. To initiate activity, 3pM PIP3 (Echelon Biosciences, DiC8) was added to each
PTEN-IP protein sample (200μg/sample) for 2h at 37˚C. To measure free phosphate,
the Malachite Green Phosphate Detection kit (12776, Cell Signaling) was followed via
manufacturer’s protocol.
Tumor migration/invasion assay: A 24-well plate transwell inserts (6.5 mm, Costar, 8μm
pores) were pre-coated with 35μl of 1 mg/mL matrigel (BD Biosciences) at 37°C for 2h.
0.5 × 105 cells of each sample in serum-free medium were plated in the upper chamber
and media (10% FBS) was added to the bottom well. After 24h, the inserts were fixed
and stained with 0.1% crystal violet for imaging before being lysed with 10% acetic acid.
Absorbances were measured correlating to the number of migrated cells per insert
(BioTek Instruments).
T cell-mediated cytotoxicity: Human T cells were isolated from donor PBMCs using
Mojosort Human CD3 Isolation kit (Cat. #480022, Biolegend) via manufacturer’s
protocol. T cells were left untreated (naïve T cells) or treated with IL-2 + ImmunoCult
CD3/CD28/CD2 T cell tetramers (activated T cells, 25uL/mL, #10970, StemCell).
Trypsinized tumor cells were counted and stained with CFSE (1L/mL, #423801,
Biolegend) via manufacturer’s recommendation. CFSE+ target tumor cells were
Research. on September 20, 2020. © 2020 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 30, 2020; DOI: 10.1158/1078-0432.CCR-19-4245
Favorable PTEN/PD-L1 modulation by chemerin in tumors Page 9 of 33
incubated with naïve (untouched) or activated T cells overnight (approx. 18 hours) at
indicated E:T ratios (typically 3:1). Samples were stained with 7-AAD (5uL/1e6 cells,
#420404, Biolegend) to identify dead cells. Percent lysed was the fraction of cells that
stained positive for both CFSE and 7-AAD. As donor T cells and target tumor cells were
not HLA-matched (and thus measured alloreactivity), anti-MHCI, anti-human HLA-ABC
(311402, clone W6/32, Biolegend) was used for additional control experiments.
RNA In Situ Hybridization and Image Analysis: Manual chromogenic RNAScope was
performed with RNAScope 2.5 HD Reagent kit–brown (ACD, #322310), using
optimized company protocols. Single ISH detection for PTEN (ACD Probe: 408511),
PD-L1 (CD274 – ACD Probe: 600861), Positive Control Probe (PPIB - ACD Probe:
313901) and Negative Control Probe (Dapb - ACD Probe: 310043) was performed via
manufacturer’s protocols. Three comparable ROIs for each respective sample set were
analyzed using HALO Software (3 ROIs per sample, repeated for n = 3 independent
experiments).
In vivo studies: All mice were used in experiments were purchased from The Jackson
Laboratory. NOD/SCID/IL2R gamma (null) (NSG; #005557, NOD.Cg-
PrkdcscidIl2rgtm1Wjl/SzJ) male mice were used at approximately 9–10 weeks of age,
as indicated. Mice were maintained in the Washington University facilities under the
direction and guidelines of the Division of Comparative Medicine. All animal
experiments were conducted in accordance with approved Washington University and
National Institutes of Health Institutional Animal Care and Use Committee guidelines
under an approved protocol (#20170174). To evaluate the effect of constitutive
chemerin secretion on in vivo tumor growth, vector control or chemerin-expressing
DU145 tumor cells (2.5 × 106) were inoculated subcutaneously into 9–10 weeks old
Research. on September 20, 2020. © 2020 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 30, 2020; DOI: 10.1158/1078-0432.CCR-19-4245
Favorable PTEN/PD-L1 modulation by chemerin in tumors Page 10 of 33
male mice. The pcDNA3.1+ (Thermo Scientific) vector was used to produce either
vector control or human RARRES2 transfected DU145 cells. Transfected cells were
selected using Geneticin (G418). Prior to inoculation, DU145 lines were grown to ~70–
80% confluence to ensure log-growth kinetics, and cell viability was assessed using
trypan blue and cells used only if ~95% viable. Tumor growth was measured every 3–4
days by calipers, and size was expressed as the volume product of perpendicular length
by width in square millimeters. Mice were euthanized when tumor size reached
~400mm2 or at indicated time points for downstream analyses.
Primary Prostate Tumor Processing: Within one hour of resection, primary tissue was
processed into single cell suspensions. To digest the tissue, prostate tissue or
metastatic biopsy cores were cut into 1×1mm pieces and incubated with 100μl of
Liberase TL solution (28U/ml, Roche Applied Science) and DNase I (20U/mL, Thermo
Scientific) were added and samples were continuously rotated and incubated at 37 °C
for 1 hour. Digested cell suspensions were then homogenized by using a 1000μL wide
bore pipette tip and samples were passed through a 100μM strainer. Following
processing, cells were ready for use in investigative studies and downstream analysis.
All human subjects were consented under the approved IRB Protocol (# 201411135)
titled Tissue, Blood, and Urine Acquisition for Genomic Analysis and Collection of
Health Information for Patients with Malignancies of the Genitourinary Tract.
Statistics: All experiments were done independently (n = 3 or more). Each time sample
replicates were prepared and analyzed independently. Means and standard errors of
the mean (SEM) were calculated. Paired Student t-tests were used for comparison
between two groups in each experiment. One-way ANOVA was used to compare more
than two groups, including a post-hoc Tukey test to confirm differences between groups.
Research. on September 20, 2020. © 2020 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 30, 2020; DOI: 10.1158/1078-0432.CCR-19-4245
Favorable PTEN/PD-L1 modulation by chemerin in tumors Page 11 of 33
A p-value of less than 0.05 was considered statistically significant via Microsoft Excel
and GraphPad Prism v.8 software.
Results
Chemerin exposure can induce PTEN expression in tumor cell lines
We initially questioned whether chemerin, given its myriad roles, would have an impact
on tumor-intrinsic cell functions. Previous studies in mouse models have not shown
detectable levels of CMKLR1 on mouse tumor cell lines, nor direct effect of recombinant
chemerin exposure on tumor cell phenotype measured 10. Given the prominent role of
PTEN dysregulation in prostate and sarcoma tumors, we decided to study these tumor
types using human cell lines. Analysis of prostate and sarcoma TCGA data shows that
patients with higher levels of RARRES2 in their tumors have improved overall survival
compared to those with lower expression (figure 1A, 1B), in line with our and others’
analyses in other tumor types 16. We looked at human tumor lines that had detectable
CMKLR1 protein expression and genetically intact PTEN (DU145, U2OS, SKES) and
used a CMKLR1+, PTEN null (-/-) cell line (PC3) as a control. Both prostate and
sarcoma cell lines were analyzed for expression of CMKLR1, and showed detectable
levels of CMKLR1 protein at both the intracellular and cell surface levels
(supplementary figure 1). Cell lines had no detectable chemerin expression using anti-
human chemerin ELISA assays (supplemental figure 2F and data not shown). We then
investigated the effect of exogenous, recombinant chemerin on these cell lines.
Chemerin is found systemically in plasma and most non-hematopoietic tissues, and
engages CMKLR1 at low nanomolar concentrations 2, thus we chose to initially focus in
this range of concentrations. Cell lines were plated as indicated with complete media
Research. on September 20, 2020. © 2020 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 30, 2020; DOI: 10.1158/1078-0432.CCR-19-4245
Favorable PTEN/PD-L1 modulation by chemerin in tumors Page 12 of 33
containing 6nM recombinant chemerin protein or PBS (the diluent control) for 48h.
Following treatment, we found PTEN mRNA expression was significantly upregulated
over control-treated cells in PTEN wild type (WT) cell lines tested (figure 1C, 1D). As
expected, there was no detection of PTEN in the PC3 cells, while we saw an
approximately 2-fold increase in PTEN expression in the DU145 cells, and a ~2.5-fold
increase in the sarcoma SKES and U2OS cells after incubation with chemerin.
As mRNA expression does not perfectly correlate with protein production 17, we then
investigated protein expression after chemerin treatment. Western blot analyses
showed a noticeable upregulation of PTEN protein expression in all three cell lines after
a 48h incubation (figure 1E, 1F). Quantification showed that PTEN expression
increased with an increasing concentration of chemerin, suggesting a dose-response.
PTEN protein was increased ~1.5-fold in DU145 cells, and ~1.6-1.7-fold in U2OS and
SKES cells compared to the controls (figure 1G - 1I). Neither in vitro cell proliferation
nor apoptosis (supplemental figures 2 and 3) was significantly altered after chemerin
exposure over a 72h period, compared to controls. Collectively, these results show that
exogenous chemerin significantly induces PTEN mRNA and protein expression in a
dose-dependent manner, without significant impact on their in vitro proliferation or
apoptosis.
Chemerin treatment, mediated by CMKLR1, significantly reduces tumor migration and
invasion
PTEN has multiple roles in tumor suppression, including inhibition of tumor cell
proliferation, invasion and migration 18,19. While chemerin did not detectably impact cell
proliferation nor apoptosis, we hypothesized it might affect other aspects of tumor cell
Research. on September 20, 2020. © 2020 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 30, 2020; DOI: 10.1158/1078-0432.CCR-19-4245
Favorable PTEN/PD-L1 modulation by chemerin in tumors Page 13 of 33
phenotype. Using a tumor migration model, we examined the effects of chemerin
treatment for 48h, in line with our previous experimental conditions. Control or
chemerin-treated cells were allowed to migrate on matrigel for 24h. Imaging showed a
noticeable decrease in tumor migration/invasion in all three of the cell lines treated with
chemerin compared to control cells (figure 1J). Quantification of migrated tumor cells
shows that chemerin treatment significantly reduced tumor cell invasion by 29% in
DU145 cells, 31% in U2OS cells, and 22% in SKES cells compared to control-treated
samples, respectively (figure 1K).
Subsequently, we assessed whether the effect of chemerin treatment on reducing tumor
migration/invasion was mediated through its binding to CMKLR1, and not “off-target”
effects. CMKLR1 siRNA knockdown experiments were performed as described above,
using the matrigel invasion assay. Knockdown of CMKLR1 protein using siRNA was
confirmed at both the intracellular and cell surface levels (figure 2A, supplemental figure
1). Mock transfection and control siRNA cells continued to display significantly reduced
tumor cell invasion after chemerin treatment, compared to control-treated tumor cells. In
general, CMKLR1 siRNA knockdown decreased overall cell invasion in both control and
chemerin-treated groups compared to mock transfection or control siRNA groups.
However, CMKLR1 knockdown completely abolished the ability of chemerin to inhibit
tumor cell migration compared to control-treated cells in all three lines (supplemental
figure 4). Collectively, these studies show a significant functional impact of chemerin
treatment on tumor cell lines, and may represent a way to reduce tumor metastatic
potential in vivo.
CMKLR1 mediates chemerin-induced PTEN expression and function
Research. on September 20, 2020. © 2020 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 30, 2020; DOI: 10.1158/1078-0432.CCR-19-4245
Favorable PTEN/PD-L1 modulation by chemerin in tumors Page 14 of 33
Given the two other known receptors for chemerin (CCRL2 and GPR1) are either non-
signaling or have limited tissue expression20, we focused on the role of CMKLR1 –
chemerin’s main chemotactic receptor - in mediating chemerin-induced PTEN
upregulation in these cell lines. Compared to controls, significant increases in PTEN
expression, by both qPCR and Western blot, were seen with exposure to 6nM chemerin
during mock and control siRNA transfections in all cell lines. However, only with
CMKLR1 siRNA was there a complete abrogation of chemerin-induced PTEN
expression (figure 2B). This establishes the role of CMKLR1 in mediating the chemerin-
induced increase of PTEN expression, at both the mRNA and protein levels.
While PTEN increased due to chemerin in all cell lines, we tested if the augmented
PTEN was indeed functional. PTEN phosphatase activity modulates PI3K-induced
phosphatidylinositol-3,4,5-triphosphate (PIP3), which is a critical factor in mediating
subsequent signaling pathways involved in cell survival, proliferation, and migration 21.
We studied the ability of PTEN protein to dephosphorylate PIP3 phosphate, following
48h PBS or chemerin incubation. Protein lysates were collected following each 48h
experiment for PTEN immunoprecipitation. PTEN phosphatase activity was significantly
increased after 48hr chemerin exposure compared to control-treated cells (figure 2C),
suggesting the chemerin-induced PTEN retained its ability to function as a phosphatase.
Likewise, specific CMKLR1 knockdown – but not mock transfection nor control siRNA –
completely abrogated the chemerin-mediated increase in PTEN phosphatase activity
(figure 2C). Together, these findings support a role for chemerin to induce significant
functional PTEN expression via CMKLR1 in human tumor lines.
Research. on September 20, 2020. © 2020 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 30, 2020; DOI: 10.1158/1078-0432.CCR-19-4245
Favorable PTEN/PD-L1 modulation by chemerin in tumors Page 15 of 33
Chemerin treatment results in an increase in the transcription factors SRF and EGR-1,
and correlates with PTEN upregulation
To further elucidate this novel pathway, we investigated the underlying mechanisms of
the chemerin-PTEN interaction. A recent study showed that chemerin binding to
CMKLR1 leads to transcriptional activation of the serum response factor (SRF) 22. SRF
expression has been shown to modulate PTEN expression, as well as induce activation
of its target gene EGR-1 (early growth response 1), which directly regulates PTEN
expression23
24. Aberrant PI3K pathway activation leads to a decrease in SRF levels and
results in reduced binding to the EGR-1 promoter necessary for EGR-1 transcription 25.
Thus, we hypothesized these components may mediate signaling between chemerin
and PTEN, via CMKLR1, in DU145 cells. Therefore, we examined both SRF and EGR-1
expression in chemerin-treated DU145 cells as previously described above.
Concomitant with upregulated PTEN expression, our RT-qPCR results showed
significant increases for both SRF (1.75-fold) and EGR-1 (1.91-fold) mRNA expression
in the chemerin-treated DU145 cells compared to PBS alone (figure 3A). Similarly,
Western blot analysis showed a significant increase in SRF and EGR-1 protein
expression, 1.68-fold and 1.57-fold, respectively, directly correlating with increased
PTEN protein expression (1.67-fold) (figure 3A). Taken together, these results suggest
chemerin binding CMKLR1 induces increased SRF and EGR-1 expression upstream of
augmented PTEN expression and activity in DU145 cells.
Chemerin suppresses pAKT and pS6 expression, correlating with decreased PD-L1
expression
Research. on September 20, 2020. © 2020 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 30, 2020; DOI: 10.1158/1078-0432.CCR-19-4245
Favorable PTEN/PD-L1 modulation by chemerin in tumors Page 16 of 33
Next, we set out to further characterize the relationship between chemerin, PTEN and
PD-L1. We examined the protein levels of p-Akt (ser473) and pS6 (ser235/236) by
Western blot. PTEN negatively regulates the PI3K/Akt pathway and overall PTEN
activation inversely correlates with p-Akt expression 12,15,26,27. Furthermore, PTEN loss
or PI3K genetic alterations in prostate, breast, or glioma tumors result in significantly
augmented PD-L1 expression 28. Importantly, Lastwika et al. showed that PD-L1
expression is tightly regulated by the Akt-mTOR pathway, where activation can lead to
immune escape for some tumor types 29. Inhibition studies targeting mTORC1 (pAKT)
and mTORC2 (pS6) within the PI3k/Akt/mTOR pathway confirmed their role in control of
PD-L1 expression 29, thus we studied these key signaling constituents to further
investigate chemerin’s downstream impact on PD-L1. Figure 3B shows a significant
decrease in p-Akt (ser473) protein expression following chemerin incubation, compared
to the PBS treatment. Western blot data from 4 independent sample sets show a 29%
decrease in pAkt (ser473) protein expression in chemerin-treated cells compared to the
control PBS group. We also show that chemerin treatment leads to a significant
decrease in both phospho-S6 (pS6 ser235/236; 43% decrease) and PD-L1 protein
expression (32% decrease) compared to the control PBS group (figure 3B). Thus, our
experimental results show that chemerin exposure increases PTEN expression leading
to a subsequent negative regulation of the Akt–mTOR–PD-L1 signaling cascade. These
results are consistent with previous studies looking at the effects of augmented PTEN
expression on the PI3k/Akt/mTOR pathway and its signaling constituents 12,15,26,27,30,31.
To evaluate the effects of chemerin treatment on tumor PI3K/AKT/mTOR pathway
components, we used two well-studied inhibitors of PI3K/mTOR (BEZ235) and mTOR
(RAD001). PI3K inhibition by BEZ235 had no effect on the increase in tumor PTEN
expression seen with chemerin treatment (figure 3C) but did significantly reduce the
Research. on September 20, 2020. © 2020 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 30, 2020; DOI: 10.1158/1078-0432.CCR-19-4245
Favorable PTEN/PD-L1 modulation by chemerin in tumors Page 17 of 33
expression of downstream pAKT/AKT, pS6/S6, and PD-L1; these reductions seen with
this PI3K/mTOR inhibitor (aka Dactolisib) were not significantly different than those
seen with chemerin treatment in the DU145 tumor cells (figure 3C). Treatment with the
mTOR (mTORC1/2) inhibitor RAD001 suppressed PD-L1 and pS6 expression, as
expected 29,32 (figure 3D and supplemental figure 5) in both control and chemerin
treated tumor cells. Suppression of tumor PD-L1 was comparable and not statistically
different between chemerin and RAD001 treatment. Following treatment, pS6 protein
expression was completely knocked out, as expected (supplemental figure 5). RAD001
treatment completely abrogated the decrease in PD-L1 seen in the DU145 cells after
chemerin exposure (figure 3D), implicating mTOR as a critical factor in this signaling
pathway.
We next used the RhoA/SRF pathway inhibitor CCG-1423, given CMKLR1 has been
shown to signal through RhoA/SRF 22, and again found that both chemerin-induced
increases in PTEN (upper panel) and decreases in PD-L1 (lower panel) expression
were completely abrogated with use of the CCG-1423 inhibitor (Figure 3D). As siRNA
can have off target effects, we used a specific CMKLR1 small molecule antagonist, -
NETA, that recapitulates a CMKLR1 knockout phenotype 33. We again looked at
PTEN/PI3K/mTOR pathway components and found that treatment with -NETA
completely abrogated effects seen with chemerin treatment (figure 3E), suggesting
chemerin is signaling through CMKLR1 and mediating these effects. A CMKLR1
blocking peptide also showed similar abrogation of PTEN and PD-L1 changes induced
by chemerin (supplemental figure 5), suggesting our results seen with CMKLR1 siRNA
were unlikely due to off-target effects. Taken together, these data suggest that chemerin
treatment results in an increase in tumor PTEN expression, with associated changes in
Research. on September 20, 2020. © 2020 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 30, 2020; DOI: 10.1158/1078-0432.CCR-19-4245
Favorable PTEN/PD-L1 modulation by chemerin in tumors Page 18 of 33
the canonical PTEN–PI3K–AKT–mTOR signaling pathway as well as decreases in
pS6/S6 and PD-L1 expression, comparable to PI3K and mTOR inhibitors that have
been used in clinical trials.
Chemerin upregulates PTEN and concomitantly decreases PD-L1 expression on tumor
cells, via CMKLR1
Recent evidence correlates PTEN expression and function to programmed death
ligand-1 (PD-L1) expression in cancer, and this has been shown to be dependent on the
PI3K pathway and S6 kinase (S6K) activation 28,34-36. Thus, we decided to further study
PD-L1 expression in the context of chemerin exposure and PTEN expression. We
tested a wide range (3-62nM) of recombinant chemerin concentrations on DU145 tumor
cells, and then assessed for both PTEN and PD-L1 expression. Again, 6nM chemerin
treatment produced the most robust increase in PTEN expression, with an obvious
dose-response relationship seen. Importantly, we also saw a concomitant, significant
decrease in PD-L1 mRNA expression via qPCR (figure 4A).
To further elucidate, we looked at RNA in situ hybridization (ISH) staining for PTEN and
PD-L1 using RNA specific probes (ACDBio). Image analysis showed chemerin
significantly upregulated PTEN and simultaneously decreased PD-L1 RNA expression
(figure 4B). Further, we investigated the role of CMKLR1 in the chemerin-mediated
suppression of PD-L1 expression. We found that only knockdown of CMKLR1 – and
neither mock transfection nor control siRNA – completed abrogated both the significant
increase in PTEN and decrease in PD-L1 expression seen following chemerin treatment
(figure 2B, 4C). Additionally, we evaluated the impact of chemerin on tumor cell surface
Research. on September 20, 2020. © 2020 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 30, 2020; DOI: 10.1158/1078-0432.CCR-19-4245
Favorable PTEN/PD-L1 modulation by chemerin in tumors Page 19 of 33
expressed PD-L1 protein, as this ultimately mediates its immunosuppressive effects.
FACS staining analyses for PD-L1 revealed a significant decrease in surface expression
in the chemerin-treated tumor cells compared to controls by both percent positive
(based on isotype control) as well as mean fluorescence intensity (MFI) (figure 4D).
Further, chemerin treatment significantly reduced IFN--induced PD-L1 expression
compared to PBS treated DU145 cells (figure 4E), suggesting chemerin may blunt the
induction of PD-L1 expression in the setting of increased IFN- that can occur with some
immunotherapies.
Collectively, these data confirm a key inverse relationship between the tumor
suppressor, PTEN, and a key immune checkpoint inhibitor, PD-L1 28,34-36. More
importantly, our findings show - for the first time - that chemerin can directly modulate
this established PTEN/PD-L1 axis via CMKLR1 in human tumor cells.
Chemerin treatment, mediated through CMKLR1, significantly improves T cell-mediated
cytotoxicity of tumor cells
Our findings that chemerin treatment of tumor cells suppresses PD-L1 expression
suggest that it could play a role in T cell-mediated cytotoxicity. PD-L1 is known to inhibit
T cell function via its interaction with programmed cell death-1 (PD-1) on T cells 37. To
investigate, we isolated human T cells from donor PBMCs to target DU145 and U2OS
cells. We found that unstimulated, naïve T cells were only able to mediate low levels of
DU145 cytotoxicity, as previously published 38. This is not surprising, as our effector T
cells were donor-derived and not HLA-matched, thus measuring T cell alloreactivity to
tumors. Both activation (CD2/CD3/CD28 tetramers) and increasing effector to target
Research. on September 20, 2020. © 2020 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 30, 2020; DOI: 10.1158/1078-0432.CCR-19-4245
Favorable PTEN/PD-L1 modulation by chemerin in tumors Page 20 of 33
(E:T) ratio improved tumor cell killing (supplemental figure 6), generally in line with other
studies 39-41. Importantly, we found that chemerin treatment of DU145 tumor cells
resulted in a significant increase in activated T cell-mediated cytotoxicity, compared to
controls (figure 5A). Treatment of tumor cells with an anti-HLA antibody abrogated the
effect of chemerin on augmenting T cell mediated lysis, suggesting an MHC-dependent
mechanism. Treatment of tumor cells did not change MHCI surface expression in the
presence or absence of chemerin (supplemental figure 6).
The increase in cytotoxicity seen with chemerin treatment, however, was only seen at
lower E:T ratios (i.e. 0.5:1 to 3:1), while the effect seemed to lessen at higher E:T ratios
(supplemental figure 6). This suggests that higher numbers of activated T cells per
tumor target cell may act to obscure the effect mediated by chemerin treatment. It is
important to note that prostate cancers typically are less infiltrated with immune cells
(especially T cells) compared to most other tumor types 42, suggesting the lower E:T
ratios used in our assays may, in fact, be more physiologically relevant to the human
TME. Furthermore, prostate tumor infiltrating T cells show an exhausted phenotype, as
evidenced by high PD-1 expression and decreased IFN- 43, similar to T cells used in
our assays (supplemental figure 7). Thus, PD-L1 expression on prostate tumor cells is
likely to modulate prostate-infiltrating T cell function; indeed, recent clinical data shows
that blocking the PD-1/PD-L1 pathway in mCRPC patients led to disease control rates
of up to 22% 44.
We next investigated the mechanisms underlying chemerin’s ability to augment T cell-
mediated cytotoxicity. Using siRNA knockdown, we again examined the role of
CMKLR1 in mediating chemerin’s effects. siRNA significantly reduced both mRNA and
surface protein levels of CMKLR1 in tumor cells (supplemental figure 1). Neither control
Research. on September 20, 2020. © 2020 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 30, 2020; DOI: 10.1158/1078-0432.CCR-19-4245
Favorable PTEN/PD-L1 modulation by chemerin in tumors Page 21 of 33
nor CMKLR1 siRNA affected the low level of cytotoxicity seen using naïve T cells in
either PBS or chemerin-treated tumor cells. However, CMKLR1 knockdown completely
abrogated chemerin’s effect on tumor cell cytotoxicity by activated T cells, whereas
control siRNA had no effect (figure 5B).
Chemerin augmentation of cytotoxicity is mediated, in part, by PTEN and PD-L1
Given chemerin’s impact on both PTEN and PD-L1 expression, we then explored their
roles in the cytotoxicity assay. Control or PD-L1 siRNA were then used to look at the
role of PD-L1 in this setting (supplemental figure 8). Again, no effect of siRNA
transfections was seen with naïve T cells. Using activated T cells, control siRNA again
had no impact on the chemerin-mediated increase in tumor killing, while PD-L1
knockdown significantly increased cytotoxicity in both control and chemerin-treated
tumor cells (figure 5C). This is not surprising given the high levels of PD-1 found on the
activated T cells (supplemental figure 7), and known effects of blocking PD-L1 in this
setting 37. Interestingly, levels of cytotoxicity in the PD-L1 knockdown groups were
comparable to – and not statistically different from – the chemerin treated/control siRNA
groups: control siRNA + chemerin-treated cells displayed 29% lysis compared to 31%
and 33% lysis in the control PBS and chemerin-treated PD-L1 knockdown groups,
respectively. While there was a small difference in activated T cell lysis between
chemerin-treated control siRNA cells compared to chemerin-treated PD-L1 siRNA
DU145 cells, this was not statistically significant. Similarly, there was no significant
difference in activated T cell lysis between the PBS vs chemerin-treated PD-L1 siRNA
DU145 subsets, showing that PD-L1 was necessary for the effect of chemerin on T cell
mediated cytotoxicity (figure 5C). We then examined the effects of PTEN knockdown
Research. on September 20, 2020. © 2020 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 30, 2020; DOI: 10.1158/1078-0432.CCR-19-4245
Favorable PTEN/PD-L1 modulation by chemerin in tumors Page 22 of 33
via siRNA transfection (supplemental figure 8). In control-treated cells, knockdown of
PTEN resulted in significantly less killing by activated T cells (figure 5D). PTEN siRNA
knockdown in chemerin-treated cells resulted in the complete abrogation of the increase
in T cell killing seen with chemerin-treated/control siRNA cells, to the level of PBS-
treated/control siRNA tumor cells (figure 5D). PTEN knockdown significantly reduced
the effect of chemerin treatment – thus, the difference in cytotoxicity seen between
control and chemerin-treated cells using control siRNA was significantly greater than the
increase seen using PTEN siRNA. This strongly supports a mechanistic role for PTEN
in chemerin-augmented T cell cytotoxicity. Together, these data support roles for both
PTEN and PD-L1 in how chemerin augments sensitivity to T cell mediated cytotoxicity.
Chemerin treatment is as effective as atezolizumab at augmenting T cell-mediated
cytotoxicity
While statistically significant increases in T cell cytotoxicity were seen with chemerin, we
compared this result to a clinically validated and approved checkpoint inhibitor, anti-PD-
L1 antibody atezolizumab, in our cytotoxicity assays. The addition of isotype antibody to
the cytotoxicity assay did not impact the established beneficial effect of chemerin
treatment. The addition of atezolizumab significantly increased activated T cell-
mediated cytotoxicity in the control-treated DU145 cells (figure 5E), consistent with
studies showing the effects of blocking PD-L1 in in vitro cytotoxicity assays 45. There
was no statistically significant difference between chemerin-treated DU145 cells with the
addition of atezolizumab antibody compared to isotype control, while there was a
significant difference with the addition of atezolizumab to PBS control treated tumor
cells compared to isotype control. Blockade of PD-L1 with atezolizumab negated the
Research. on September 20, 2020. © 2020 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 30, 2020; DOI: 10.1158/1078-0432.CCR-19-4245
Favorable PTEN/PD-L1 modulation by chemerin in tumors Page 23 of 33
significant difference in lysis between control and chemerin-treated DU145s (figure 5E).
Together, these suggest that atezolizumab is effective in control-treated cells, with basal
PD-L1 expression, but added no additional significant impact in chemerin-treated
DU145 cells, where chemerin pre-treatment suppresses PD-L1 expression. With
effective PD-L1 blockade by atezolizumab, chemerin treatment did not further augment
T cell cytotoxicity, highlighting PD-L1 as a key downstream pathway component in
mediating chemerin’s effects on tumor cells.
We then set out to compare the effect of chemerin treatment on T cell cytotoxic directly
to both PD-L1 siRNA knockdown as well as atezolizumab blockade. Using experimental
conditions as above, we applied these conditions in parallel, independently repeating
with comparable results in both DU145 and U2OS tumor cells. No impact was seen in
the cytotoxicity using naïve T cells (data not shown). Using activated T cells, we again
found that chemerin treatment significantly increased T cell-mediated cytotoxicity of
DU145 cells (figure 5F). Similarly, chemerin treatment of U2OS cells lead to
significantly increased T cell-mediated cytotoxicity (figure 5G). Importantly, chemerin
treatment was as effective at augmenting T cell mediated tumor cell lysis in comparison
to PD-L1 siRNA or atezolizumab blockade, with no significant differences between the
three conditions for both DU145 and U2OS cells (figure 5F, 5G). We looked at
treatment of the activated effector T cells, which lack CMLKR1, and saw no impact of
chemerin treatment on immune cell PTEN expression, or cytolytic ability (supplemental
figure 6), suggesting the effects seen were due to tumor-intrinsic changes after
chemerin treatment.
Collectively, these data show that in two different tumor cell lines chemerin, via
CMKLR1, can induce the upregulation of PTEN and concurrent downregulation of PD-
Research. on September 20, 2020. © 2020 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 30, 2020; DOI: 10.1158/1078-0432.CCR-19-4245
Favorable PTEN/PD-L1 modulation by chemerin in tumors Page 24 of 33
L1 expression in tumor cells. This results in a significant increase in T cell-mediated
cytotoxicity, comparable – and not statistically different – in our assays to siRNA
knockdown of PD-L1 or the clinically approved atezolizumab.
Expression of chemerin in the tumor microenvironment leads to decreased in vivo tumor
growth
In order to test our hypothesis that forced overexpression of chemerin by tumor cells
would act to suppress tumor growth in part by modulation of PTEN and PD-L1, we used
plasmid transfection to introduce the human RARRES2 gene into the DU145 tumor cells.
Both wild type and vector control DU145 cell lines showed no detectable chemerin by
ELISA, while the RARRES2-transfected line showed significant production of secreted
chemerin, with no differences in in vitro proliferation seen (supplemental figure 2E, F). In
order to determine if the tumor-secreted chemerin was functional and active, we utilized
conditioned media from both control and chemerin-expressing tumor lines in a
chemotaxis assay and found that only the chemerin-expressing media was able to
mediate chemotaxis of CMKLR1-positive cells (supplemental figure 2G). Tumor cells
were inoculated subcutaneously in NSG mice and growth was monitored. Chemerin
expression in the TME resulted in significantly reduced tumor growth compared to
control tumors (figure 6A). While chemerin can recruit immune effector cells into the
TME 10,46, the immunodeficiencies in NSG mice (absent NK/T/B and defective
macrophage and dendritic cell) suggested the differences in tumor growth seen could
be due in part to tumor-intrinsic factors. We analyzed ex vivo tumors and found that
chemerin-expressing tumors had significantly higher PTEN and significantly lower PD-
L1 levels compared to controls (figure 6B). This is consistent with our in vitro data, and
Research. on September 20, 2020. © 2020 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 30, 2020; DOI: 10.1158/1078-0432.CCR-19-4245
Favorable PTEN/PD-L1 modulation by chemerin in tumors Page 25 of 33
suggests chemerin may play a role at modulating tumor PTEN and PD-L1, favorably, in
vivo as well.
While tumor line studies are informative, they have limited applicability to the clinical
setting. In order to investigate the effects of chemerin on human primary prostate
tumors, we collected primary tumors from 4 patients (2 local, 2 metastatic tumors) under
an IRB-approved protocol. One patient had enough tumor collected that allowed us to
perform several experiments, while the other 3 had limited tumor cell content. Primary
tumor cells were cultured in the presence or absence of recombinant human chemerin
and assessed for changes in PTEN and PD-L1 expression by qPCR. Compared to
controls, there was a significant increase in PTEN and decrease in PD-L1 (figure 6C,
6D) in primary human prostate tumor cells treated with chemerin. While limited by the
amount of tumor collected from patients, we were able to analyze tumor cells from one
patient and found detectable surface expression of CMKLR1 on these tumor cells (not
shown), suggesting, as in our tumor cell lines, that chemerin may be acting through
CMKLR1 on tumor cells to modulate PTEN and PD-L1.
Lastly, we looked at human clinical trial data from metastatic prostate cancer patients
treated with ipilimumab (anti-CTLA-4) on a single institution clinical trial (NCT02113657)
47. Published RNA expression data was used to look at RARRES2, PTEN, and PD-L1
(CD274) in these patients, and evaluate clinical outcomes. Comparison of patients with
the highest and lowest quartile RARRES2 expression showed almost 3-fold increase in
PTEN expression in those patients whose tumors had the highest RARRES2
expression (figure 6E). Tumor PD-L1 RNA was low and not different between groups
(not shown) and mostly undetectable by immunohistochemistry (IHC). However,
evaluable TME immune cell PD-L1 and CD8 by IHC was available. >50% reduction in
Research. on September 20, 2020. © 2020 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 30, 2020; DOI: 10.1158/1078-0432.CCR-19-4245
Favorable PTEN/PD-L1 modulation by chemerin in tumors Page 26 of 33
PD-L1 and ~4-fold increase in CD8 expression was seen in the highest RARRES2
quartile compared to the lowest (figure 6E). Clinical outcomes for patients above and
below the median RARRES2 expression were analyzed; the RARRES2 high group had
a median OS of 40.3 months compared to 5.8 months for low RARRES2 (HR, 0.83;
95% CI, 0.27-2.6; P =.39). Median PSA PFS was also increased in the RARRES2 high
compared to the low group, 11.2 v 0.7 mos (HR, 0.49; 95% CI, 0.16-1.5; P =.12) (figure
6F). Relative abundancies of indicated immune populations (based on RNAseq
signatures) in both high and low RARRES2 expression groups showed significant
increases in immune effector populations in the RARRES2 high group compared to the
low group (figure 6G). While limited in sample size, these data suggest that high
RARRES2 in the TME is correlated with increased PTEN, decreased PD-L1, and
increased immune effector populations. Thus, a strategy for increasing expression of
chemerin within the TME in humans may be beneficial in the clinical setting.
Discussion
Tumors have developed various suppressive mechanisms to evade anti-tumor immune
responses and regulatory signaling that may limit their growth. As both are altered in the
TME, further study of the interplay between tumor cell-intrinsic oncogenic signaling and
extrinsic anti-tumor immuno-surveillance is necessary to improve current
immunotherapies.
The link between PD-L1 (cell-extrinsic immune responses) and PTEN (cell-intrinsic
responses) expression has been described, with several examples of PTEN loss or
suppression resulting in increased PD-L1 expression in tumors 28,34,35. Other studies
Research. on September 20, 2020. © 2020 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 30, 2020; DOI: 10.1158/1078-0432.CCR-19-4245
Favorable PTEN/PD-L1 modulation by chemerin in tumors Page 27 of 33
suggest PD-L1 expression in prostate, breast, and lung carcinoma may be dependent
on PI3K, commonly regulated by PTEN 48. However, this association is likely context
dependent, as the regulation of PD-L1 expression is controlled by many factors and
pathways (reviewed in 49). PD-L1 expression correlates with tumor aggressiveness and
poor clinical outcomes 50-53, as does loss of PTEN
52,54-56, in several datasets, further
supporting the clinical impact of alterations in these two key proteins. In prostate, PD-L1
expression has been reported on up to ~47% of de novo metastatic prostate cancers 57,
and has been found to correlate with poorer prognosis and risk of disease recurrence 52
53, while PTEN loss has been correlated with both risk of recurrence in localized disease
and lethal progression 55, 56, suggesting a therapeutic strategy to augment PTEN
expression may reduce prostate cancer lethality.
Recent studies describe functional consequences of modulating PTEN signaling and its
impact on immunoresistance. Toso et al used a conditional PTEN-null mouse model to
study the impact of PTEN loss within prostate tumors. They found loss of PTEN resulted
in a significant increase in several immunosuppressive cytokines, as well as infiltration
by granulocytic myeloid-derived suppressor cells (MDSCs) 58. Furthermore, Peng et al
showed that the PTEN loss led to inhibited T cell-mediated tumor killing and decreased
T cell trafficking into the TME. Importantly, they showed that metastatic melanoma
patients with PTEN-positive tumors treated with anti-PD-1 antibodies had significantly
better responses than otherwise matched patients with PTEN-negative tumors. They
show that PI3Kβ inhibition – part of the PI3K/Akt pathway activated with PTEN loss -
enhanced the activity of T cell–mediated immunotherapy in mice bearing PTEN-
deficient tumors 31. Additional evidence recently elucidated the importance of PTEN loss
in developed resistance to anti-PD-1 immunotherapy in human sarcoma 59, supporting
Research. on September 20, 2020. © 2020 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 30, 2020; DOI: 10.1158/1078-0432.CCR-19-4245
Favorable PTEN/PD-L1 modulation by chemerin in tumors Page 28 of 33
the clinical relevance of this mechanism. Thus, PTEN alterations that impact
immunotherapy efficacy are key mechanisms to consider in optimization of these
therapies.
It is important to recognize, however, the variety of PTEN alterations that exist across
cancers. In addition to deletion, expression can be altered by DNA methylation,
transcriptional repression, and translational disorder, reducing PTEN expression in
numerous tumor types60. Deletion can be bi- or mono-allelic, with approximately 42% of
prostate cancer patients having monoallelic PTEN loss 61. The various modes of PTEN
loss in malignancy can lead to distinctive signaling modulations that are not always
equivalently regulated. Thus, the exact type of PTEN loss in tumors would potentially
dictate the relevance of chemerin modulation in humans. For example, complete allelic
loss of PTEN (as in our PC3 cells) in tumors might suggest that modulating tumor
chemerin levels in these patients would not result in changes in tumor PD-L1; however,
the ability of chemerin to recruit immune effector cells into the TME may still have
beneficial outcomes. In those tumors with intact- but decreased- PTEN expression,
chemerin modulation may then act to increase its expression and potentially decrease
PD-L1, suppressing tumor growth and improving responses to immunotherapies.
Our study is the first to show that chemerin, an innate immunocyte chemoattractant, can
reactivate PTEN in human prostate and sarcoma tumor lines, while concomitantly
suppressing PD-L1 expression. We describe a novel mechanistic link between
chemerin/CMKLR1, PTEN, and PD-L1 in tumor cells, and identify key signaling pathway
components. We show a beneficial, functional impact of chemerin treatment, with
reduced tumor cell migration/invasion and increased T cell-mediated cytotoxicity, on par
with the clinically approved anti-PD-L1 antibody atezolizumab. In vivo tumor studies
Research. on September 20, 2020. © 2020 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 30, 2020; DOI: 10.1158/1078-0432.CCR-19-4245
Favorable PTEN/PD-L1 modulation by chemerin in tumors Page 29 of 33
showed chemerin expression in the TME significantly reduced tumor growth, with an
increase in tumor PTEN and decrease in tumor PD-L1 seen. Primary human prostate
tumor cultures recapitulated our cell line studies, again showing chemerin treatment
resulting in favorable modulation of PTEN and PD-L1.
A recent study showed that chemerin could suppress hepatocellular carcinoma (HCC)
growth and metastases via the PTEN-Akt signaling axis in a mouse model 30. Using
human HCC cell lines, Li et al showed that chemerin overexpression resulted in PTEN
upregulation and suppression of the PI3K/Akt pathway. As in our studies, they also saw
a significant decrease in tumor cell migration/invasion with exposure to chemerin. Their
data is supportive of our initial findings with PTEN, but our study extends this
mechanistically and elucidates a novel signaling cascade in tumors linking
chemerin/CMKLR1 to PD-L1. Independent validation of findings across labs and tumor
types suggests this axis may be biologically and clinically relevant.
In conclusion, we report a previously unidentified signaling cascade linking
chemerin/CMKLR1, PTEN , and PD-L1 in human tumor cell lines, resulting in a
significant decrease in tumor migration/invasion and increase in T cell-mediated killing,
with significant suppression of in vivo tumor growth. In addition to its already described
role of favorably modulating anti-tumor immune responses by recruitment of immune
effector cells into the TME, this data now shows a new tumor cell-intrinsic mechanism of
chemerin treatment. Ongoing and future studies will further investigate biologic
consequences of modulating this axis, with the goal of clinical translation.
References
Research. on September 20, 2020. © 2020 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 30, 2020; DOI: 10.1158/1078-0432.CCR-19-4245
Favorable PTEN/PD-L1 modulation by chemerin in tumors Page 30 of 33
1 Shin, W. J. & Pachynski, R. K. Chemerin modulation of tumor growth: potential clinical applications in cancer. Discov Med 26, 31-37 (2018).
2 Zabel, B. A. et al. Chemoattractants, extracellular proteases, and the integrated host defense response. Exp Hematol 34, 1021-1032, doi:S0301-472X(06)00314-6 [pii]
10.1016/j.exphem.2006.05.003 (2006). 3 Parolini, S. et al. The role of chemerin in the colocalization of NK and dendritic cell
subsets into inflamed tissues. Blood 109, 3625-3632, doi:10.1182/blood-2006-08-038844 (2007).
4 Wittamer, V. et al. Specific recruitment of antigen-presenting cells by chemerin, a novel processed ligand from human inflammatory fluids. J Exp Med 198, 977-985, doi:10.1084/jem.20030382 (2003).
5 Zabel, B. A., Silverio, A. M. & Butcher, E. C. Chemokine-like receptor 1 expression and chemerin-directed chemotaxis distinguish plasmacytoid from myeloid dendritic cells in human blood. J Immunol 174, 244-251 (2005).
6 Zabel, B. A. et al. Chemokine-like receptor 1 expression by macrophages in vivo: regulation by TGF-beta and TLR ligands. Exp Hematol 34, 1106-1114, doi:10.1016/j.exphem.2006.03.011 (2006).
7 Tummler, C. et al. Inhibition of chemerin/CMKLR1 axis in neuroblastoma cells reduces clonogenicity and cell viability in vitro and impairs tumor growth in vivo. Oncotarget 8, 95135-95151, doi:10.18632/oncotarget.19619 (2017).
8 Hutchinson JM, V. M., Loadman P, Nicolaou A, Hull M. CHEMR23 AND BLT1 RECEPTOR EXPRESSION IN COLORECTAL CANCER. Gut 62, A306, doi:10.1136/gutjnl-2013-304907.451 (2013).
9 Lazennec, G. & Richmond, A. Chemokines and chemokine receptors: new insights into cancer-related inflammation. Trends Mol Med 16, 133-144, doi:10.1016/j.molmed.2010.01.003 (2010).
10 Pachynski, R. K. et al. The chemoattractant chemerin suppresses melanoma by recruiting natural killer cell antitumor defenses. J Exp Med 209, 1427-1435, doi:10.1084/jem.20112124 (2012).
11 Dillon, L. M. & Miller, T. W. Therapeutic targeting of cancers with loss of PTEN function. Curr Drug Targets 15, 65-79 (2014).
12 Wise, H. M., Hermida, M. A. & Leslie, N. R. Prostate cancer, PI3K, PTEN and prognosis. Clinical science 131, 197-210, doi:10.1042/CS20160026 (2017).
13 Robinson, D. et al. Integrative clinical genomics of advanced prostate cancer. Cell 161, 1215-1228, doi:10.1016/j.cell.2015.05.001 (2015).
14 Yin, L. et al. Analysis of PTEN methylation patterns in soft tissue sarcomas by MassARRAY spectrometry. PLoS One 8, e62971, doi:10.1371/journal.pone.0062971 (2013).
15 Gao, P., Seebacher, N. A., Hornicek, F., Guo, Z. & Duan, Z. Advances in sarcoma gene mutations and therapeutic targets. Cancer Treat Rev 62, 98-109, doi:10.1016/j.ctrv.2017.11.001 (2018).
16 Shin, W. J., Zabel, B. A. & Pachynski, R. K. Mechanisms and Functions of Chemerin in Cancer: Potential Roles in Therapeutic Intervention. Frontiers in immunology 9, 2772, doi:10.3389/fimmu.2018.02772 (2018).
17 Hanash, S. M. et al. Integrating cancer genomics and proteomics in the post-genome era. Proteomics 2, 69-75 (2002).
18 Tamura, M., Gu, J., Takino, T. & Yamada, K. M. Tumor suppressor PTEN inhibition of cell invasion, migration, and growth: differential involvement of focal adhesion kinase and p130Cas. Cancer Res 59, 442-449 (1999).
19 Hu, Y., Xu, S., Jin, W., Yi, Q. & Wei, W. Effect of the PTEN gene on adhesion, invasion and metastasis of osteosarcoma cells. Oncol Rep 32, 1741-1747, doi:10.3892/or.2014.3362 (2014).
Research. on September 20, 2020. © 2020 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 30, 2020; DOI: 10.1158/1078-0432.CCR-19-4245
Favorable PTEN/PD-L1 modulation by chemerin in tumors Page 31 of 33
20 Zabel, B. A. et al. Mast cell-expressed orphan receptor CCRL2 binds chemerin and is required for optimal induction of IgE-mediated passive cutaneous anaphylaxis. J Exp Med 205, 2207-2220, doi:10.1084/jem.20080300 (2008).
21 Liu, P., Cheng, H., Roberts, T. M. & Zhao, J. J. Targeting the phosphoinositide 3-kinase pathway in cancer. Nature reviews. Drug discovery 8, 627-644, doi:10.1038/nrd2926 (2009).
22 Rourke, J. L., Dranse, H. J. & Sinal, C. J. CMKLR1 and GPR1 mediate chemerin signaling through the RhoA/ROCK pathway. Mol Cell Endocrinol 417, 36-51, doi:10.1016/j.mce.2015.09.002 (2015).
23 Virolle, T. et al. The Egr-1 transcription factor directly activates PTEN during irradiation-induced signalling. Nat Cell Biol 3, 1124-1128, doi:10.1038/ncb1201-1124 (2001).
24 Horita, H. N. et al. Serum response factor regulates expression of phosphatase and tensin homolog through a microRNA network in vascular smooth muscle cells. Arterioscler Thromb Vasc Biol 31, 2909-2919, doi:10.1161/ATVBAHA.111.233585 (2011).
25 Shin, S. Y. et al. Suppression of Egr-1 transcription through targeting of the serum response factor by oncogenic H-Ras. EMBO J 25, 1093-1103, doi:10.1038/sj.emboj.7600987 (2006).
26 Lee, Y. R., Chen, M. & Pandolfi, P. P. The functions and regulation of the PTEN tumour suppressor: new modes and prospects. Nature reviews. Molecular cell biology 19, 547-562, doi:10.1038/s41580-018-0015-0 (2018).
27 Trotman, L. C. et al. Pten dose dictates cancer progression in the prostate. PLoS Biol 1, E59, doi:10.1371/journal.pbio.0000059 (2003).
28 Parsa, A. T. et al. Loss of tumor suppressor PTEN function increases B7-H1 expression and immunoresistance in glioma. Nat Med 13, 84-88, doi:10.1038/nm1517 (2007).
29 Lastwika, K. J. et al. Control of PD-L1 Expression by Oncogenic Activation of the AKT-mTOR Pathway in Non-Small Cell Lung Cancer. Cancer Res 76, 227-238, doi:10.1158/0008-5472.CAN-14-3362 (2016).
30 Li, J. J. et al. Chemerin suppresses hepatocellular carcinoma metastasis through CMKLR1-PTEN-Akt axis. Br J Cancer 118, 1337-1348, doi:10.1038/s41416-018-0077-y (2018).
31 Peng, W. et al. Loss of PTEN Promotes Resistance to T Cell-Mediated Immunotherapy. Cancer Discov 6, 202-216, doi:10.1158/2159-8290.CD-15-0283 (2016).
32 Yasumizu, Y. et al. PKM2 under hypoxic environment causes resistance to mTOR inhibitor in human castration resistant prostate cancer. Oncotarget 9, 27698-27707, doi:10.18632/oncotarget.25498 (2018).
33 Graham, K. L. et al. A novel CMKLR1 small molecule antagonist suppresses CNS autoimmune inflammatory disease. PLoS One 9, e112925, doi:10.1371/journal.pone.0112925 (2014).
34 Zhang, Y. et al. PTEN/PI3K/mTOR/B7-H1 signaling pathway regulates cell progression and immuno-resistance in pancreatic cancer. Hepatogastroenterology 60, 1766-1772 (2013).
35 Song, M. et al. PTEN loss increases PD-L1 protein expression and affects the correlation between PD-L1 expression and clinical parameters in colorectal cancer. PLoS One 8, e65821, doi:10.1371/journal.pone.0065821 (2013).
36 Han, S. J. et al. Gamma interferon-mediated superinduction of B7-H1 in PTEN-deficient glioblastoma: a paradoxical mechanism of immune evasion. Neuroreport 20, 1597-1602, doi:10.1097/WNR.0b013e32833188f7 (2009).
37 Gajewski, T. F., Meng, Y. & Harlin, H. Immune suppression in the tumor microenvironment. J Immunother 29, 233-240, doi:10.1097/01.cji.0000199193.29048.56
00002371-200605000-00001 [pii] (2006). 38 Frost, P., Ng, C. P., Belldegrun, A. & Bonavida, B. Immunosensitization of prostate
carcinoma cell lines for lymphocytes (CTL, TIL, LAK)-mediated apoptosis via the Fas-
Research. on September 20, 2020. © 2020 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 30, 2020; DOI: 10.1158/1078-0432.CCR-19-4245
Favorable PTEN/PD-L1 modulation by chemerin in tumors Page 32 of 33
Fas-ligand pathway of cytotoxicity. Cell Immunol 180, 70-83, doi:10.1006/cimm.1997.1169 (1997).
39 Carlsson, B., Forsberg, O., Bengtsson, M., Totterman, T. H. & Essand, M. Characterization of human prostate and breast cancer cell lines for experimental T cell-based immunotherapy. Prostate 67, 389-395, doi:10.1002/pros.20498 (2007).
40 Frost, P. J., Butterfield, L. H., Dissette, V. B., Economou, J. S. & Bonavida, B. Immunosensitization of melanoma tumor cells to non-MHC Fas-mediated killing by MART-1-specific CTL cultures. J Immunol 166, 3564-3573, doi:10.4049/jimmunol.166.5.3564 (2001).
41 Sanchez, C. et al. Combining T-cell immunotherapy and anti-androgen therapy for prostate cancer. Prostate Cancer Prostatic Dis 16, 123-131, S121, doi:10.1038/pcan.2012.49 (2013).
42 Li, B. et al. Comprehensive analyses of tumor immunity: implications for cancer immunotherapy. Genome Biol 17, 174, doi:10.1186/s13059-016-1028-7 (2016).
43 Sfanos, K. S. et al. Human prostate-infiltrating CD8+ T lymphocytes are oligoclonal and PD-1+. Prostate 69, 1694-1703, doi:10.1002/pros.21020 (2009).
44 Antonarakis, E. S. et al. Pembrolizumab for Treatment-Refractory Metastatic Castration-Resistant Prostate Cancer: Multicohort, Open-Label Phase II KEYNOTE-199 Study. J Clin Oncol, JCO1901638, doi:10.1200/JCO.19.01638 (2019).
45 Donahue, R. N. et al. Analyses of the peripheral immunome following multiple administrations of avelumab, a human IgG1 anti-PD-L1 monoclonal antibody. Journal for immunotherapy of cancer 5, 20, doi:10.1186/s40425-017-0220-y (2017).
46 Pachynski RK, W. P., Salazar N, Yayue Zheng, Leona Nease, Jesse Rosalez, Weng-in Leong, Gurpal Virdi2, Keith Rennier, Woo Jae Shin, Viet Nguyen, Eugene Butcher, Zabel BA. Chemerin suppresses breast cancer growth by recruiting immune effector cells into the tumor microenvironment. Frontiers in immunology, doi:doi: 10.3389/fimmu.2019.00983 (2019).
47 Subudhi, S. K. et al. Neoantigen responses, immune correlates, and favorable outcomes after ipilimumab treatment of patients with prostate cancer. Sci Transl Med 12, doi:10.1126/scitranslmed.aaz3577 (2020).
48 Cretella, D., Digiacomo, G., Giovannetti, E. & Cavazzoni, A. PTEN Alterations as a Potential Mechanism for Tumor Cell Escape from PD-1/PD-L1 Inhibition. Cancers (Basel) 11, doi:10.3390/cancers11091318 (2019).
49 Kumar, S. & Sharawat, S. K. Epigenetic regulators of programmed death-ligand 1 expression in human cancers. Transl Res 202, 129-145, doi:10.1016/j.trsl.2018.05.011 (2018).
50 Zhang, M. et al. Expression of PD-L1 and prognosis in breast cancer: a meta-analysis. Oncotarget 8, 31347-31354, doi:10.18632/oncotarget.15532 (2017).
51 Que, Y. et al. PD-L1 Expression Is Associated with FOXP3+ Regulatory T-Cell Infiltration of Soft Tissue Sarcoma and Poor Patient Prognosis. Journal of Cancer 8, 2018-2025, doi:10.7150/jca.18683 (2017).
52 Gevensleben, H. et al. The Immune Checkpoint Regulator PD-L1 Is Highly Expressed in Aggressive Primary Prostate Cancer. Clin Cancer Res 22, 1969-1977, doi:10.1158/1078-0432.CCR-15-2042 (2016).
53 Ness, N. et al. The prognostic role of immune checkpoint markers programmed cell death protein 1 (PD-1) and programmed death ligand 1 (PD-L1) in a large, multicenter prostate cancer cohort. Oncotarget 8, 26789-26801, doi:10.18632/oncotarget.15817 (2017).
54 Lahdensuo, K. et al. Loss of PTEN expression in ERG-negative prostate cancer predicts secondary therapies and leads to shorter disease-specific survival time after radical prostatectomy. Mod Pathol 29, 1565-1574, doi:10.1038/modpathol.2016.154 (2016).
55 Chaux, A. et al. Loss of PTEN expression is associated with increased risk of recurrence after prostatectomy for clinically localized prostate cancer. Mod Pathol 25, 1543-1549, doi:10.1038/modpathol.2012.104 (2012).
Research. on September 20, 2020. © 2020 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 30, 2020; DOI: 10.1158/1078-0432.CCR-19-4245
Favorable PTEN/PD-L1 modulation by chemerin in tumors Page 33 of 33
56 Ahearn, T. U. et al. A Prospective Investigation of PTEN Loss and ERG Expression in Lethal Prostate Cancer. J Natl Cancer Inst 108, doi:10.1093/jnci/djv346 (2016).
57 Iacovelli, R. et al. PD-L1 Expression in De Novo Metastatic Castration-sensitive Prostate Cancer. J Immunother 42, 269-273, doi:10.1097/CJI.0000000000000287 (2019).
58 Toso, A. et al. Enhancing chemotherapy efficacy in Pten-deficient prostate tumors by activating the senescence-associated antitumor immunity. Cell reports 9, 75-89, doi:10.1016/j.celrep.2014.08.044 (2014).
59 George, S. et al. Loss of PTEN Is Associated with Resistance to Anti-PD-1 Checkpoint Blockade Therapy in Metastatic Uterine Leiomyosarcoma. Immunity 46, 197-204, doi:10.1016/j.immuni.2017.02.001 (2017).
60 Jamaspishvili, T. et al. Clinical implications of PTEN loss in prostate cancer. Nat Rev Urol 15, 222-234, doi:10.1038/nrurol.2018.9 (2018).
61 Keniry, M. & Parsons, R. The role of PTEN signaling perturbations in cancer and in targeted therapy. Oncogene 27, 5477-5485, doi:10.1038/onc.2008.248 (2008).
Research. on September 20, 2020. © 2020 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 30, 2020; DOI: 10.1158/1078-0432.CCR-19-4245
C
E
Figure 1.
F
D
G H I
PTEN
GAPDH
Control PBS
6nMChem
DU145
(+)
PC3
Control PBS
6nMChem
3nMChem
GAPDH
PTEN
(-)
SKES U2OS
Control PBS
6nMChem
3nMChem
Control PBS
6nMChem
3nMChem
Contr
ol PBS
6nM
Chem
erin
Contr
ol PBS
6nM
Chem
erin
0.0
0.5
1.0
1.5
2.0
2.5
PT
EN
mR
NA
Exp
ressio
n *
PC3 DU145
PC3
(-)
Contr
ol PBS
6nM
Chem
erin
Contr
ol PBS
6nM
Chem
erin
0.0
1.0
2.0
3.0
4.0
PT
EN
mR
NA
Exp
ressio
n
**
SKES U2OS
Contr
ol PBS
3nM
Chem
erin
6nM
Chem
erin
0.0
0.5
1.0
1.5
2.0
PT
EN
Pro
tein
Exp
ressio
n
*
*
SKES
DU145 U2OS SKES
(-) (+)PBS
6nM c
hemerin (-) (+
)PBS
6nM c
hemerin (-) (+
)PBS
6nM c
hemerin
0.0
1.0
2.0
3.0
4.0
Rela
tive A
bso
rban
ce a
t 590n
m
No
rmalized
to
No
Matr
igel N
o F
BS
**
*
DU145 U2OS SKESJ K
++++++++++++++ ++++++++++++++++++++++++++++++++++++ +++++++++++++++ + ++++++++++++++++ ++++++++++++++ ++++ + + + + +++ +++ +++ + + + + + +
++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++ ++++++++++++++++++++++++++++++++++++++++++++++++++ + +++++++
++++ +++++ ++ + ++ +
+
++ +
p = 0.26
0.00
0.25
0.50
0.75
1.00
0 1000 2000 3000 4000 5000Time in days
Su
rviv
al p
rob
ability
Expression Level+
+High expression (n=125)Low/Medium−expression (n=372)
Effect of RARRES2 expression level on PRAD patient survival
High RARRES2n=125
Low RARRES2n=372
Surv
ival
(%)
100
50
0Time (days)
Prostate Cancer SarcomaA
High RARRES2n=65
Low RARRES2n=194Su
rviv
al(%
)
100
50
0Time (days)
B
Contr
ol PBS
6nM
Chem
erin
Contr
ol PBS
3nM
Chem
erin
6nM
Chem
erin
0.5
1.0
1.5
2.0
PT
EN
Pro
tein
Exp
ressio
n
*
*
PC3 DU145
Contr
ol PBS
6nM
Chem
erin
Contr
ol PBS
3nM
Chem
erin
6nM
Chem
erin
0.5
1.0
1.5
2.0
PT
EN
Pro
tein
Exp
ressio
n
*
*
PC3 DU145
Contr
ol PBS
6nM
Chem
erin
Contr
ol PBS
3nM
Chem
erin
6nM
Chem
erin
0.5
1.0
1.5
2.0
PT
EN
Pro
tein
Exp
ressio
n
*
*
PC3 DU145
Research. on September 20, 2020. © 2020 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 30, 2020; DOI: 10.1158/1078-0432.CCR-19-4245
Figure 1. Recombinant chemerin upregulates PTEN expression in tumor cells. Survival for patients with high and low RARRES2 from TCGA datasets for both prostate A. and sarcoma B. was analyzed using UALCAN (ualcan.path.uab.edu). C. RT-qPCR results of PTEN mRNA expression in prostate cancer cells treated with vehicle control (PBS) or 6nM recombinant chemerin (6nM Chem). PTEN Expression is normalized to GAPDH loading control for each sample and normalized to control PBS across the dataset (*p < 0.01, n = 4 independent experiments). D. RT-qPCR results of PTEN mRNA expression in Ewing sarcoma (SKES) and osteosarcoma (U2OS) cells treated with PBS (Control) or 6nM recombinant chemerin. PTEN Expression is normalized to GAPDH loading control for each sample and normalized to control PBS across the dataset (*p < 0.01, n = 4). E. Representative Western blots for PTEN protein expression in Normal Prostate - RWPE1 (+), PC3 , and DU145 cells treated with vehicle (control PBS) or 6nM Chem (6nM chemerin) for 48h. F. Representative Western blot for PTEN protein expression in PC3 (-), SKES, and U2OS cells treated with vehicle (Control PBS) or 3nM or 6nM Chem (6nM chemerin) for 48h. G. Quantified Western blot results showing PTEN protein expression in control or chemerin treated PCa cells. Normalized to GAPDH loading control for each respective sample and each dataset is normalize to Control PBS (*p < 0.05, n = 3). H-I. Quantified Western blot results for PTEN protein expression in PBS (Control) or Chemerin treated sarcoma cells (H. SKES, I. U2OS). Each sample is normalized to GAPDH loading control and the dataset is normalized to Control PBS. Each sample set was repeated three times in independent experiments (*p < 0.05, n = 3). J. Representative 4X images showing tumor cell invasion were normalized to baseline cell migration, No matrigel matrix and No FBS. The following groups were compared: No matrigel – No FBS, No matrigel – CM + 10% FBS, 1mg/mL matrigel + cells treated with 48h PBS, or 1mg/mL matrigel + cells treated with 48h 6nM recombinant human chemerin (6nM chemerin). Scale bar = 100μm. K. Quantified tumor cell invasion results for each respective tumor cell line comparing matrigel invasion in cells treated with PBS (vehicle) or 6nM Chemerin for 48h (*p < 0.05, n = 4 independent experiments).
Research. on September 20, 2020. © 2020 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 30, 2020; DOI: 10.1158/1078-0432.CCR-19-4245
GAPDH
PTEN
Transfection
Control siRNA
CMKLR1 siRNA
6nM Chemerin
C
+ + + + + +
- - + + - -
- - - - + +
- + - + - +
B
Transfection
Control siRNA
CMKLR1 siRNA
6nM Chemerin
+ + + + + +
- - + + - -
- - - - + +
- + - + - +
Figure 2.
SKES U2OS DU145
1 2 3 4 5 6
0.0
0.5
1.0
1.5
2.0
2.5
*
*
NS
1 2 3 4 5 6
0.0
0.5
1.0
1.5
2.0
2.5
**
NS
+ + + + + +
- - + + - -
- - - - + +
- + - + - +
1 2 3 4 5 6
0.0
0.5
1.0
1.5
2.0
2.5
**
NS
+ + + + + +
- - + + - -
- - - - + +
- + - + - +
1 2 3 4 5 6
0.5
1.0
1.5*
*
NS
(+) (-)0.5
1.0
1.5
2.0
PT
EN
P
ho
sp
hatase A
ctivity
Ph
osp
hate C
on
cen
tratio
n (p
Mo
l)
No
rm
alized
to
C
on
tro
l
* *NS
Transfection
Control siRNA
CMKLR1 siRNA
6nM Chemerin
+ + + + + +
- - + + - -
- - - - + +
- + - + - +
(+) (-)0.5
1.0
1.5
2.0
PT
EN
Ph
osp
hata
se A
cti
vit
yP
ho
sp
hate
Co
ncen
trati
on
(p
Mo
l)N
orm
alized
to
Co
ntr
ol
* *
NS
Transfection
Control siRNA
CMKLR1 siRNA
6nM Chemerin
+ + + + + +
- - + + - -
- - - - + +
- + - + - +
SKES U2OS DU145
qP
CR
W
B
Re
lati
ve P
TEN
m
RN
A E
xpre
ssio
n
Re
lati
ve P
TEN
P
rote
in E
xpre
ssio
n
Re
lati
ve P
TEN
P
ho
sph
atas
e A
ctiv
ity
(+) (-)0.5
1.0
1.5
2.0
PT
EN
Ph
osp
hata
se A
cti
vit
yP
ho
sp
hate
Co
ncen
trati
on
(p
Mo
l)N
orm
alized
to
Co
ntr
ol
* *
NS
DU145 A SKES U2OS
GAPDH
CMKLR1
NT CMKLR1
siRNA Control siRNA
GAPDH
CMKLR1
NT CMKLR1
siRNA Control siRNA
GAPDH
CMKLR1
NT Control siRNA
CMKLR1 siRNA
3:9 4:10
Research. on September 20, 2020. © 2020 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 30, 2020; DOI: 10.1158/1078-0432.CCR-19-4245
Figure 2. Chemerin induces PTEN expression and activity via CMKLR1. A. Representative Western blot for CMKLR1 expression after transfection with siRNA, using either 3:9 or 4:10 siRNA to transfection reagent ratio. Loading control bands were probed with anti-GAPDH antibody on the same blot. (Left) DU145 (Middle) SKES and (Right) U2OS cells transfected with CMKLR1 siRNA as indicated. B (Top) RT-qPCR results of PTEN mRNA expression in DU145 (Left), SKES (Middle), U2OS (Right) cancer cells transfected with the following groups: Mock (no siRNA), Control siRNA (non-specific sequence), or CMKLR1 siRNA. Following transfection, each respective group was treated with PBS (Control) or 6nM recombinant chemerin. PTEN expression is normalized to GAPDH loading control for each sample and each pair was normalized to Control PBS, respectively (*p < 0.01, n = 4 independent experiments. NS = No significant difference). (Middle) Representative Western blot for PTEN protein expression in the transfected DU145, SKES, U2OS cell subsets treated with PBS or 6nM chemerin for 48h. (Bottom) Quantified Western blot results showing PTEN protein expression in PBS or Chemerin treated DU145, SKES, U2OS cells following transfection. Sample expression was normalized to GAPDH loading control and each pair was normalized to each Control PBS, respectively (*p < 0.05, n = 3). C. Cells were treated with either PBS or 6nM chemerin for 48h PBS or Chemerin DU145 cells transfected with Mock (no siRNA), Control siRNA, or CMKLR1 siRNA. Each sample set and condition were repeated in three independent experiments (n = 3). Positive control (+) corresponds to 3pM PIP3 + recombinant human PTEN protein and negative control (-) was incubation with 3pM PIP3 only.
Research. on September 20, 2020. © 2020 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 30, 2020; DOI: 10.1158/1078-0432.CCR-19-4245
Figure 3.
A B
- + 6nM
Chemerin
SRF EGR1 PTEN
Re
lati
ve m
RN
A E
xpre
ssio
n
- + - + - + - - + + - - - - - - + +
6nM Chemerin
10𝜇M CCG-1423
1𝜇M RAD001
Control P
BS
6nM C
hemerin
Control P
BS
6nM C
hemerin
Control P
BS
6nM C
hemerin
0.0
0.5
1.0
1.5
2.0
Rel
ativ
e P
rote
in E
xpre
ssio
n
**
*Contr
ol PBS
6nM
Chem
erin
Contr
ol PBS
6nM
Chem
erin
Contr
ol PBS
6nM
Chem
erin
0.0
0.5
1.0
1.5
2.0
2.5
Rela
tive P
rote
in E
xp
ressio
n
**
*
GAPDH
- + - +
pAKTser473
AKT
GAPDH
PD-L1
Control PBS
6nM Chem
PD-L1
pS6ser235/6
S6
pAKT/AKT pS6/S6 q
PC
R
Re
lati
ve P
rote
in E
xpre
ssio
n
WB
+ - + - - - + + - + - +
Control PBS
𝛼-NETA
6nM Chemerin
Total S6
GAPDH
pAKTser473
AKT
pS6ser235/6
PD-L1
PTEN E
- + 6nM
Chemerin - + - +
Re
lati
ve P
rote
in
Exp
ress
ion
GAPDH
PD-L1
Contr
ol PBS
6nM
Chem
erin
Contr
ol + 1
0uM
CCG
-142
3
Chem
erin
+ 1
0uM
CCG
-142
3
Contr
ol + 1
uM R
AD00
1
Chem
erin
+ 1
uM R
AD00
10.0
0.5
1.0
1.5
2.0
Rela
tive P
rote
in E
xp
ressio
n
*
NSNS
PTE
N
PD
-L1
Re
lati
ve P
rote
in E
xpre
ssio
n
PTEN Contr
ol PBS
6nM
Chem
erin
Contr
ol + 1
0uM
CCG
-142
3
Chem
erin
+ 1
0uM
CCG
-142
3
Contr
ol + 1
uM R
AD00
1
Chem
erin
+ 1
uM R
AD00
10.0
0.5
1.0
1.5
Rela
tive P
rote
in E
xp
ressio
n
*
NS
NS
D
Re
lati
ve P
rote
in E
xpre
ssio
n
Contr
ol PBS
6nM
Chem
erin
Contr
ol + α
NETA
Chem
erin
+ α
NETA
0.0
0.5
1.0
1.5
2.0
Rela
tive P
TE
N
Pro
tein
Exp
ressio
n
*
NS
NS
Contr
ol PBS
6nM
Chem
erin
Contr
ol + α
NETA
Chem
erin
+ α
NETA
0.0
0.5
1.0
1.5
Rela
tive P
D-L
1
Pro
tein
Exp
ressio
n * NS
NS
PD-L1 PTEN pS6/S6 pAKT/AKT
Contr
ol PBS
6nM
Chem
erin
Contr
ol + α
NETA
Chem
erin
+ α
NETA
0.0
0.5
1.0
1.5
Rela
tive p
AK
T/A
KT
Pro
tein
Exp
ressio
n *NS
NS
+ - + - - - + + - + - +
Control PBS
BEZ235
6nM Chemerin
Total S6
GAPDH
pAKTser473
AKT
pS6ser235/6
PD-L1
PTEN C
Contr
ol PBS
6nM
Chem
erin
Contr
ol + B
EZ23
5
Chem
erin
+ B
EZ23
50.0
0.5
1.0
1.5
Rela
tive P
D-L
1
Pro
tein
Exp
ressio
n
*
NS
Contr
ol PBS
6nM
Chem
erin
Contr
ol + B
EZ23
5
Chem
erin
+ B
EZ23
50.0
0.5
1.0
1.5
Rela
tive p
AK
T/A
KT
Pro
tein
Exp
ressio
n
NS
*
PD-L1
PTEN
pS6/S6
pAKT/AKT
Contr
ol PBS
6nM
Chem
erin
Contr
ol + B
EZ235
Chem
erin
+ B
EZ235
0.0
0.5
1.0
1.5
2.0
Rela
tive P
TE
N
Pro
tein
Exp
ressio
n
*
NS NS
*
Re
lati
ve P
rote
in E
xpre
ssio
n
Contr
ol PBS
6nM
Chem
erin
Contr
ol + α
NETA
Chem
erin
+ α
NETA
0.0
0.5
1.0
1.5
2.0
Rela
tive P
TE
N
Pro
tein
Exp
ressio
n
*
NS
NS
Contr
ol PBS
6nM
Chem
erin
Contr
ol + α
NETA
Chem
erin
+ α
NETA
0.0
0.5
1.0
1.5
Rela
tive P
D-L
1
Pro
tein
Exp
ressio
n * NS
NS
Contr
ol PBS
6nM
Chem
erin
Contr
ol + α
NETA
Chem
erin
+ α
NETA
0.0
0.5
1.0
1.5
Rela
tive p
AK
T/A
KT
Pro
tein
Exp
ressio
n *NS
NS
Contr
ol PBS
6nM
Chem
erin
Contr
ol + α
NETA
Chem
erin
+ α
NETA
0.0
0.5
1.0
1.5
Rela
tive p
S6/S
6
Pro
tein
Exp
ressio
n *NS
NS
Contr
ol PBS
6nM
Chem
erin
0.0
0.5
1.0
1.5
Rela
tive p
S6/t
ota
l S
6
Pro
tein
Exp
ressio
n *
Contr
ol PBS
6nM
Chem
erin
0.0
0.5
1.0
1.5
Rela
tive P
D-L
1
Pro
tein
Exp
ressio
n *
Contr
ol PBS
6nM
Chem
erin
0.0
0.5
1.0
1.5
Rela
tive p
AK
T/t
ota
l A
KT
Pro
tein
Exp
ressio
n
*
Contr
ol PBS
6nM
Chem
erin
0.0
0.5
1.0
1.5
Rela
tive p
AK
T/t
ota
l A
KT
Pro
tein
Exp
ressio
n
*
Research. on September 20, 2020. © 2020 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 30, 2020; DOI: 10.1158/1078-0432.CCR-19-4245
Figure 3. Chemerin modulates PTEN/AKT/PD-L1 and its signaling constituents. A. (Top) RT-qPCR results of transcriptional regulators, SRF and EGR-1, including PTEN mRNA expression in DU145 cells treated for 48hrs using either vehicle control (control PBS) or 6nM chemerin (*p < 0.05, n = 3). (Bottom) Quantified Western blot (WB) results showing SRF, EGR-1, and PTEN protein expression in control or chemerin treated DU145 cells. All samples were normalized to GAPDH loading control for each respective sample and each dataset is normalized to each respective Control PBS (*p < 0.05, n = 3). Representative Western blots for SRF, EGR-1, and PTEN are shown below the quantified graph. B. pAKT (ser473) vs total AKT, pS6 (ser235/6) vs total S6 and PD-L1 protein expression in PBS (Control PBS) or 6nM chemerin treated DU145 cells. Representative blots showing pAKT, total AKT, pS6, total S6, and PD-L1 expression, each set including GAPDH loading control for DU145 cells treated with vehicle (Control PBS) or 6nM Chem (6nM chemerin) for 48h. (*p < 0.05, n = 4 independent experiments) with quantification shown. C. Representative blots and quantified graphs for PD-L1 and pS6 (ser235/6) vs total S6 for DU145 cells +/- 6nM chemerin and +/- RAD001 (mTOR inhibitor, everolimus, 1𝜇M). D. Quantified protein expression for PTEN and PD-L1 in control PBS vs 6nM chemerin treated DU145 cells with or without CCG-1423 (RhoA/SRF inhibitor, 10𝜇M) or with RAD001 (1𝜇M). Below the graph are representative blots for each sample set (*p < 0.05, n = 3 independent sample sets). E. Utilizing a CMKLR1 antagonist, 𝛼-NETA (10𝜇M), we show a downstream signaling expression: PTEN, PD-L1, pAKT, total AKT, pS6, total S6 in +/- chemerin treated DU145. Blot images representative of each target (left) and quantified graphical data (right) are presented to show the role of CMKLR1 in the Chemerin/PTEN/PD-L1 signaling axis.
Research. on September 20, 2020. © 2020 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 30, 2020; DOI: 10.1158/1078-0432.CCR-19-4245
Control PBS Chemerin
A B
C
Figure 4.
1 2 3 4 5 6
0.5
1.0
1.5
2.0
mR
NA
Exp
ressio
n
No
rmalized
to
Co
ntr
ol P
BS *
**
PTEN
Contr
ol PBS
3nM
chem
erin
6nM
chem
erin
12nM
chem
erin
36nM
chem
erin
62nM
chem
erin
0.0
0.5
1.0
1.5
mR
NA
Exp
ressio
n
No
rmalized
to
Co
ntr
ol P
BS
* *
Contr
ol PBS
3nM
Chem
erin
6nM
Chem
erin
12nM
Chem
erin
36nM
Chem
erin
62nM
Chem
erin
0.5
1.0
1.5
2.0
mR
NA
Exp
ressio
n
No
rmalized
to
Co
ntr
ol P
BS *
**
PD-L1
1 2
0.0
0.5
1.0
1.5
2.0
RN
A E
xp
ressio
n
RN
A/C
op
ies p
er
Nu
cle
i
No
rmalized
to
Co
ntr
ol P
BS
*
Contr
ol PBS
Chem
erin
0.0
0.5
1.0
1.5
RN
A E
xp
ressio
n
RN
A/C
op
ies p
er
Nu
cle
i
No
rmalized
to
Co
ntr
ol P
BS
*
Re
lati
ve m
RN
A E
xpre
ssio
n (
qP
CR
)
MFI Percent R
ela
tive
PD
-L1
Exp
ress
ion
by
FAC
S * *
NS
PD-L1
(-) (+)0.0
0.5
1.0
1.5
2.0
Rela
tive A
bso
rban
ce a
t 590n
m
No
rmalized
to
(-)
No
Matr
igel/F
BS
* * NS
Re
lati
ve m
RN
A E
xpre
ssio
n
+ + + + + +
- - + + - -
- - - - + + - + - + - +
Transfection
Control siRNA
CMKLR1 siRNA
6nM Chemerin
D
GAPDH
PD-L1 1 2 3 4 5 6
0.0
0.5
1.0
1.5
Rela
tive P
rote
in E
xp
ressio
n
* *
NS
In situ hybridization (ISH)
PTE
N
PD
-L1
Re
lati
ve E
xpre
ssio
n
PTEN
PD-L1
WB
q
PC
R
E
Con
trol
+ IF
Nγ
Che
mer
in ->
IFNγ
Con
trol
+ IF
Nγ
Che
mer
in ->
IFNγ
0
1
2
3
4
5
PD
-L1 E
xp
ressio
n
No
rmalized
to
Co
ntr
ol P
BS
#
#*
*
MFI Percent
#
#
Research. on September 20, 2020. © 2020 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 30, 2020; DOI: 10.1158/1078-0432.CCR-19-4245
Figure 4. Chemerin upregulates PTEN with simultaneous decrease in PD-L1 via CMKLR1. A. (Top) RT-qPCR results of PTEN mRNA expression in DU145 cells treated for 48hrs using either vehicle control (control PBS) or a varying doses of chemerin (3nM to 62nM). (Bottom) RT-qPCR results of PD-L1 mRNA expression. mRNA expression is normalized to GAPDH control for each sample (*p < 0.05, n = 3 independent sample sets). B. Representative RNA in situ hybridization (ISH) images for PTEN (top) and PD-L1 (bottom) expression in DU145 cells treated with PBS or Chemerin (6nM), as indicated. (Right) Quantified PTEN and PD-L1 RNA expression using HALO image analysis software for PBS vs 6nM Chemerin treated DU145 cells (*p < 0.05, n = 4 independent experiments). C. (Top) Compiled qPCR data showing PTEN mRNA expression in mock, control siRNA or CMKLR1 siRNA transfected cells treated with PBS or chemerin. (Bottom) Using the same samples, PD-L1 protein expression by Western blot (WB) is quantified in each of the transfected cell subsets (*p < 0.05, n = 4 individual sample sets. NS = no significant difference). D. FACS expression data (percent positive and mean fluorescence intensity, MFI) showing PD-L1 cell surface expression for PBS vs 6nM Chemerin treated DU145 cells (*p < 0.05 compared to control PBS, n = 4 independent experiments). PD-L1 PE-conjugated antibody was used to measure PD-L1 expression compared to a PE-conjugated IgG Isotype stained and unstained DU145 cells. E. FACS expression data showing PD-L1 surface expression in IFN-ɣ treated DU145 cells pretreated 48h with PBS or 6nM chemerin, normalized to the control IFN-ɣ sample set (*p < 0.05 compared to control PBS in F., #p < 0.05 compared to control IFN-ɣ, n = 4 independent experiments).
Research. on September 20, 2020. © 2020 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 30, 2020; DOI: 10.1158/1078-0432.CCR-19-4245
C D
B
Figure 5.
CMKLR1 Knockdown A
PTEN Knockdown
Naive T cells Active T cells
0
10
20
30
40
% L
ysis
via
FA
CS
Control siRNA
PTEN siRNA
*
D
NS
Chemerin - - + + - - + +Naive T cells Active T cells
0
10
20
30
40
% L
ysis
via
FA
CS
Control siRNA
PTEN siRNA
*
D
NS
Chemerin - - + + - - + +
PD-L1 Knockdown
Naive T cells Active T cells
0
10
20
30
40
% L
ysis
via
FA
CS
Control siRNA
PD-L1 siRNA
*
NS
Chemerin - - + + - - + +
NS
3:1 T cells to DU145
Naive T cells Active T cells0
10
20
30
40%
Lysis
via
FA
CS
Control PBS6nM Chemerin
*
NS
Naive T cells Active T cells0
10
20
30
40
% L
ysis
via
FA
CS
Control PBS6nM Chemerin
*
NS
Naive T cells Active T cells
0
10
20
30
40
% L
ysis
via
FA
CS
Control siRNA
PD-L1 siRNA
*
NS
Chemerin - - + + - - + +
NS
* * *
* *
◆
Naïve T cells Activated T cells
: :
:
Naïve T cells Activated T cells
Naïve T cells Activated T cells
NS
E Activated T cells
% L
ysis
by
FAC
S
Naive T cells Active T cells0
10
20
30
40
% Lysis via FACS
Control PBS6nM Chemerin
*
NS
Control PBS 6nM Chemerin
NS
* * *
G
Con
trol P
BS
6nM
Chem
erin
Con
trol s
iRNA -
PBS
Contr
ol siR
NA -
Che
mer
in
PD-L
1 si
RNA -
PBS
PD-L
1 si
RNA -
Che
mer
in
IgG Is
otype
Ate
zoliz
umab
0
10
20
30
40
% L
ysis
* **NS
NS
% L
ysis
by
FAC
S
Con
trol
PBS
6nM
Che
mer
in
Con
trol s
iRNA -
PBS
Cont
rol s
iRNA -
Chem
erin
PD-L
1 si
RNA -
PBS
PD-L
1 si
RNA -
Che
mer
in
IgG Is
otype
Ate
zoliz
umab
0
10
20
30
40
% L
ysis
* **NS
NS
Con
trol P
BS
6nM
Chem
erin
Con
trol s
iRNA -
PBS
Contr
ol siR
NA -
Chem
erin
PD-L
1 si
RNA -
PBS
PD-L
1 si
RNA -
Che
mer
in
IgG Is
otyp
e
Ate
zoliz
umab
0
10
20
30
40
% L
ysis
* **NS
NS
Control Treament
Con
trol
PBS
Con
trol s
iRNA
Isoty
pe Ig
G
Chem
erin
PD-L
1 si
RNA
Ate
zoliz
umab
0
10
20
30
40
% L
ysis
via
FA
CS
NS
NS
*
Controls Treatment
Con
trol P
BS
6nM
Che
mer
in
Con
trol
siR
NA -
PBS
Cont
rol s
iRNA -
Che
mer
in
PD-L
1 si
RNA -
PBS
PD-L
1 si
RNA -
Che
mer
in
IgG Is
otype
Ate
zoliz
umab
0
10
20
30
40
% L
ysis
* **NS
NS
Con
trol
PBS
6nM
Che
mer
in
Con
trol
siR
NA -
PBS
Cont
rol s
iRNA -
Chem
erin
PD-L
1 si
RNA -
PBS
PD-L
1 si
RNA -
Che
mer
in
IgG Is
otype
Ate
zoliz
umab
0
10
20
30
40
% L
ysis
* **NS
NS
Con
trol P
BS
6nM
Chem
erin
Con
trol s
iRNA -
PBS
Contr
ol siR
NA -
Chem
erin
PD-L
1 si
RNA -
PBS
PD-L
1 si
RNA -
Che
mer
in
IgG Is
otyp
e
Ate
zoliz
umab
0
10
20
30
40
% L
ysis
* **NS
NS
U2OS F
Con
trol
PBS
Con
trol s
iRNA
Isoty
pe Ig
G
Chem
erin
PD-L
1 si
RNA
Ate
zoliz
umab
0
10
20
30
40
% L
ysis
via
FA
CS
NS
NS
*
Controls TreatmentDU145
% L
ysis
by
FAC
S
Control Treament
Naïve T cells Activated T cells
Research. on September 20, 2020. © 2020 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 30, 2020; DOI: 10.1158/1078-0432.CCR-19-4245
Figure 5. Chemerin improves T cell mediated cell cytotoxicity in tumor cells, mediated in part by PTEN and PD-L1. A. Naïve and CD3/CD28/CD2 tetramer activated T cell mediated cytotoxicity in PBS vs 6nM chemerin treated DU145s using the most effective ratio, 3:1 E:T. Tumor cells were incubated with recombinant chemerin, then washed prior to co-culture with T cells in order to ensure no chemerin was present during the cytotoxicity assay itself (*p < 0.01, using triplicate samples for each experiment and repeated for n = 3 independent experiments). B-D. DU145 cells were transfected with either Control or indicated specific siRNA target for 48h. Transfected cells were treated with control (PBS) or chemerin prior to T cell mediated cytotoxicity. B. The effect of 6nM chemerin on cytotoxicity is abrogated following CMKLR1 knockdown (*p < 0.05, n = 3 individual, repeated experiments). PD-L1 knockdown (C.) increased T cell mediated cytotoxicity compared to control siRNA cells (*p < 0.05, n = 3 independent experiments). D. PTEN knockdown significantly decreases cytotoxicity compared to control siRNA cells. Chemerin is able to recover cytotoxicity in PTEN siRNA cells to the level of PBS treated control siRNA cells. In the presence of chemerin, however, PTEN knockdown significantly abrogates T cell cytotoxicity (◆) to the level of PBS/control siRNA treated cells (*p < 0.05 compared to control siRNA + PBS, △p < 0.05 compared to PTEN siRNA + PBS, ◆p < 0.05 compared to control siRNA + chemerin, n = 3 independent experiments). E. T cell mediated cytotoxicity against PBS vs chemerin treated DU145s, with either control IgG Isotype or atezolizumab (anti-PD-L1, 10ug/mL)(E:T ratio at 3:1; *p < 0.05, n = 3). F. Cytotoxicity vs. DU145s treated with the following: PBS, control siRNA, IgG Isotype, chemerin, PD-L1 siRNA, or atezolizumab. (E:T ratio at 3:1; *p < 0.01, n = 3 independent experiments). G. Cytotoxicity using activated T cells vs. U2OSs treated with the following: PBS, 6nM chemerin, control siRNA, PD-L1 siRNA, IgG Isotype, or atezolizumab. (E:T ratio at 3:1; *p < 0.05, n = 3 individual, repeated experiments).
Research. on September 20, 2020. © 2020 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 30, 2020; DOI: 10.1158/1078-0432.CCR-19-4245
Control
Chemerin-expressing
B
Ex vivo qPCR
A
Figure 6.
E F
Hig
h RARRES
2
Low R
ARRES
2
T Cells
Cytotoxic T Cells
Treg
Helper T Cells
TH17
B Cells
NK Cells
Dendritic Cells
Memory T Cells
Macrophages
Data 1
ColumnLabelsTitle
-0.4
-0.2
0
0.2
Hig
h RARRES
2
Low R
ARRES
2
T Cells
Cytotoxic T Cells
Treg
Helper T Cells
TH17
B Cells
NK Cells
Dendritic Cells
Memory T Cells
Macrophages
Data 1
ColumnLabelsTitle
-0.4
-0.2
0
0.2
Hig
h RARRES
2
Low R
ARRES
2
T Cells
Cytotoxic T Cells
Treg
Helper T Cells
TH17
B Cells
NK Cells
Dendritic Cells
Memory T Cells
Macrophages
Data 1
ColumnLabelsTitle
-0.4
-0.2
0
0.2
0.003
0.007
0.374
0.560
0.033
0.159
0.000
0.001
0.006
0.145
p-value
HIGH RARRES2
LOW RARRES2
OS
rPFS
PSA P
FS
0
5
10
15
30
40
Median
Med
ian
Su
rviv
al
(mo
nth
s)
Above and below Median for RR2
RARRES2 Low
RARRES2 High
G
OS
rPFS
PSA P
FS
0
5
10
15
30
40
Median
Med
ian
Su
rviv
al
(mo
nth
s)
Above and below Median for RR2
RARRES2 Low
RARRES2 High
0 12 24 36 48 60
0
25
50
75
100
Months
Pro
bab
ilit
y o
f S
urv
ival
mCRPC patients treated with ipilimumab
Stratified by RARRES2
RARRES2 High
RARRES2 Low
Median OS (mos)
0 12 24
0
25
50
75
100
Months
Pro
bab
ilit
y o
f P
SA
PF
S
PSA PFS
RARRES2 High
RARRES2 Low
OS
PSA PFS
Intratumoral Leukocytes PTEN
Low High
0
1
2
3
4
5
6
7
Fo
ld C
han
ge
Transform of CD8
Low High
0.0
0.5
1.0
1.5
Transform of PDL1
PD-L1 (density)
Fold
Ch
ange
CD8 (density)
C D
1.0
3.2
1.0
2.3
1.0
1.7
PB33
5 Contr
ol PBS
PB33
5 + 6
nM C
hemer
in
PB37
5 Contr
ol PBS
PB37
5 +
6nM
Chem
erin
PB06
4 Contr
ol PBS
PB06
4 + 6
nM C
hemer
in0
1
2
3
4
PT
EN
mR
NA
Exp
ressio
nPD-L1 PTEN
OS
0 20 40 60
12345678
12345678
Overall Survival (months)
Pati
en
ts
OS
RARRES2 Low
RARRES2 High> 12 mos OS :
25% 63%
OS
rPFS
PSA P
FS
0
5
10
15
30
40
Median
Med
ian
Su
rviv
al
(mo
nth
s)
Above and below Median for RR2
RARRES2 Low
RARRES2 High
Fold
Ch
ange
Contr
ol PBS
6nM
Chem
erin
12nM
Chem
erin
0.0
0.5
1.0
Rela
tive m
RN
A E
xp
ressio
n *
NS
PD-L1
DU14
5-Vc
DU14
5-hTIG
2
0
1
2
3
4
PT
EN
mR
NA
Exp
ressio
n *
DU14
5-Vc
DU14
5-hTIG
2
0
20
40
60
80
Rela
tive m
RN
A E
xp
ressio
n *
RARRES2 PTEN PD-L1
Control Chemerin -expressing
DU14
5-Vc
DU14
5-hTIG
2
0.0
0.5
1.0
1.5
2.0
PD
-L1 m
RN
A E
xp
ressio
n
*
Control Chemerin -expressing
Control Chemerin -expressing
DU14
5-Vc
DU14
5-hTIG
2
0
20
40
60
80
Rela
tive m
RN
A E
xp
ressio
n *
DU14
5-Vc
DU14
5-hTIG
2
0
20
40
60
80
Rela
tive m
RN
A E
xp
ressio
n *
DU14
5-Vc
DU14
5-hTIG
2
0
20
40
60
80
Rela
tive m
RN
A E
xp
ressio
n *
NS
PTEN
DU14
5-Vc
DU14
5-hTIG
2
0
20
40
60
80
Rela
tive m
RN
A E
xp
ressio
n *
Contr
ol PBS
6nM
Chem
erin
12nM
Chem
erin
0.0
0.5
1.0
1.5
2.0
Rela
tive m
RN
A E
xp
ressio
n
*
NS
DU14
5-Vc
DU14
5-hTIG
2
0
20
40
60
80
Rela
tive m
RN
A E
xp
ressio
n *
Rela
tive m
RN
A E
xp
ressio
n
NS
Rela
tive m
RN
A E
xp
ressio
n
Research. on September 20, 2020. © 2020 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 30, 2020; DOI: 10.1158/1078-0432.CCR-19-4245
Figure 6. Chemerin overexpression significantly suppresses tumor growth in vivo. A. Chemerin-expressing or vector control–transfected DU145 cells were implanted subcutaneously into NOD/SCID/IL2R gamma (null) (NSG) mice, and growth was measured over time. Graphs are from a representative experiment of three performed with two independently derived chemerin-expressing transfectant pools. Tumor size is represented as mean ± SEM, with cohorts of 3-5 mice per group. *p < 0.05 comparing control vs. chemerin-expressing tumors by two-tailed Student’s t test. (B). Following tumor resection, cells suspensions were created out of the collected vector control and chemerin-expressing tumor tissue. Ex vivo analysis of both control and chemerin-expressing tumor cell expression was investigated via RT-qPCR from 3 independent in vivo experiments. (Left) RARRES2 expression, (Middle) PTEN expression, (Right) PD-L1 expression in control and chemerin-expressing DU145 tumors ex vivo. Pooled normalized data sets were compiled from 3 independent in vivo experiments and SEM shown. *p < 0.05 comparing control vs. chemerin-expressing cells, n = 11 per cohort. C) Primary tumor cells from Patient PB284 showed an increase in PTEN and decrease of PD-L1, after chemerin treatment compared to control treated cells; n = 3 replicate experiments; *p < 0.05 by student’s t-test. D) Primary tumor cultures from 3 additional patients (PB335, PB375, PB064) treated with chemerin, showing increases in PTEN and decreases in PD-L1 expression as assessed by qPCR;. Triplicate samples analyzed, normalized to control with mean/SD shown. (E-G) Ipilimumab in metastatic prostate cancer patients: public data from patients treated with ipilimumab on trial NCT02113657 (Subudhi et al 2020) were analyzed E. RNAseq data (RPKM normalized) shows a comparison of patients with the highest and lowest quartile RARRES2 expression; fold change in tumor PTEN RNA expression, associated PD-L1 density (immune cells/mm2), and intratumoral CD8 T cells (cells/mm2) are shown, with data normalized to “RARRES2 Low” group, F. Clinical outcomes for patients above (RARRES2 High) and below (RARRES2 Low) the median RARRES2 expression were analyzed; the RARRES2 high group had a median OS of 40.3 months compared to 5.8 months for low RARRES2 (HR, 0.83; 95% CI, 0.27-2.6; P = .39). Median PSA PFS was also increased in the RARRES2 high compared to the low group, 11.2 v 0.7 mos (HR, 0.49; 95% CI, 0.16-1.5; P = .12). G. Relative abundancies of indicated immune populations (based on RNAseq signatures) in both high and low RARRES2 expression groups. Markers for immune cells and transformation of RNAseq data described in Subudhi et al. Significant differences between groups (p-values by unpaired t-test) are highlighted in bold.
Research. on September 20, 2020. © 2020 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 30, 2020; DOI: 10.1158/1078-0432.CCR-19-4245
Published OnlineFirst June 30, 2020.Clin Cancer Res Keith R Rennier, Woo Jae Shin, Ethan Krug, et al. cascadecells via modulation of a novel CMKLR1-mediated signaling Chemerin reactivates PTEN and suppresses PD-L1 in tumor
Updated version
10.1158/1078-0432.CCR-19-4245doi:
Access the most recent version of this article at:
Material
Supplementary
http://clincancerres.aacrjournals.org/content/suppl/2020/07/02/1078-0432.CCR-19-4245.DC2
http://clincancerres.aacrjournals.org/content/suppl/2020/07/02/1078-0432.CCR-19-4245.DC1Access the most recent supplemental material at:
Manuscript
Authoredited. Author manuscripts have been peer reviewed and accepted for publication but have not yet been
E-mail alerts related to this article or journal.Sign up to receive free email-alerts
Subscriptions
Reprints and
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Permissions
Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)
.http://clincancerres.aacrjournals.org/content/early/2020/07/01/1078-0432.CCR-19-4245To request permission to re-use all or part of this article, use this link
Research. on September 20, 2020. © 2020 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 30, 2020; DOI: 10.1158/1078-0432.CCR-19-4245