chemerin reactivates pten and suppresses pd-l1 in tumor cells … · 2020-07-01 · chemerin...

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

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Favorable PTEN/PD-L1 modulation by chemerin in tumors 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.





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.





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.





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





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-





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).



https://www.ncbi.nlm.nih.gov/tools/primer-blast/



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





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





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.





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





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





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





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.





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





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





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





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





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





(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





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





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





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-





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





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





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





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





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





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.

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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).





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).




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

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Chem

erin

Contr

ol PBS

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Chem

erin

0.0

1.0

2.0

3.0

4.0

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EN

mR

NA

Exp

ressio

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Contr

ol PBS

3nM

Chem

erin

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EN

Pro

tein

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ressio

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(-) (+)PBS

6nM c

hemerin (-) (+

)PBS

6nM c

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tive A

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igel N

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++++++++++++++ ++++++++++++++++++++++++++++++++++++ +++++++++++++++ + ++++++++++++++++ ++++++++++++++ ++++ + + + + +++ +++ +++ + + + + + +

++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++ ++++++++++++++++++++++++++++++++++++++++++++++++++ + +++++++

++++ +++++ ++ + ++ +

+

++ +

p = 0.26

0.00

0.25

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0.75

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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)

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High RARRES2n=65

Low RARRES2n=194Su

rviv

al(%

)

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50

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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).




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

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2.5

*

*

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1 2 3 4 5 6

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- - - - + +

- + - + - +

1 2 3 4 5 6

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+ + + + + +

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- + - + - +

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ctivity

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osp

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n (p

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l

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Transfection

Control siRNA

CMKLR1 siRNA

6nM Chemerin

+ + + + + +

- - + + - -

- - - - + +

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ncen

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alized

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ntr

ol

* *

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Transfection

Control siRNA

CMKLR1 siRNA

6nM Chemerin

+ + + + + +

- - + + - -

- - - - + +

- + - + - +

SKES U2OS DU145

qP

CR

W

B

Re

lati

ve P

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m

RN

A E

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ssio

n

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lati

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lati

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ncen

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ntr

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* *

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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




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.




Figure 3.

A B

- + 6nM

Chemerin

SRF EGR1 PTEN

Re

lati

ve m

RN

A E

xpre

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n

- + - + - + - - + + - - - - - - + +

6nM Chemerin

10𝜇M CCG-1423

1𝜇M RAD001

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BS

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Control P

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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.




Control PBS Chemerin

A B

C

Figure 4.

1 2 3 4 5 6

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1.0

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Exp

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PTEN

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chem

erin

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erin

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erin

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chem

erin

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chem

erin

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1.5

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ressio

n

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rmalized

to

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* *

Contr

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qP

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PD

-L1

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FAC

S * *

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PD-L1

(-) (+)0.0

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1.5

2.0

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(-)

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lati

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+ + + + + +

- - + + - -

- - - - + + - + - + - +

Transfection

Control siRNA

CMKLR1 siRNA

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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).




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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).




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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.




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

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chemerin reactivates pten and suppresses pd-l1 in tumor cells … · 2020-07-01 · chemerin...

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