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Supplementary Figure 8 from Chemerin Reactivates PTEN and Suppresses PD-L1 in Tumor Cells via Modulation of a Novel CMKLR1-mediated Signaling Cascade

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posted on 2023-03-31, 21:42 authored by Keith Rennier, Woo Jae Shin, Ethan Krug, Gurpal Virdi, Russell K. Pachynski

Supplementary Figure 8. Knockdown of PTEN and PD-L1 Expression in DU145 Cells. A. Western blot analysis for PTEN expression after transfection with siRNA. Loading control bands are probed with anti-GAPDH antibody on the same blot. DU145 cells were transfected with either control siRNA or siRNA against PTEN. Relative band intensity was quantified and analyzed based on results from three independent experiments and presented as a ratio to the baseline PTEN expression in mock transfected cells (no siRNA) (n = 3). B. Western blot analysis for PTEN expression. Transfection of PTEN siRNA, but not Control siRNA, resulted in a significant decrease in PTEN expression. **p < 0.01 compared to Mock - No siRNA cells. C. Western blot analysis for PD-L1 expression after transfection with siRNA. Loading control bands are probed with anti-GAPDH antibody on the same blot. DU145 cells were transfected with either no siRNA (Mock), control siRNA or siRNA against PD-L1. Relative band intensity was quantified and analyzed based on results from three independent experiments and presented as a ratio to the baseline PD-L1 expression in mock transfected cells (n = 3). D. Quantified results for PD-L1 siRNA transfection Western blot (n = 3). Transfection of PD-L1 siRNA at the siRNA:transfection reagent ratio of both 4:10 and 5:10, resulted in a significant knockdown in PD-L1 expression, **p < 0.05 compared to Mock - No siRNA cells. E. Western blot analysis for PD-L1 expression after transfection with siRNA. Loading control bands are probed with anti-GAPDH antibody on the same blot. U2OS cells were transfected with either no siRNA (Mock), control siRNA or siRNA against PD-L1. Relative band intensity was quantified and analyzed based on results from three independent replicates and presented as a ratio to the baseline PD-L1 expression in mock transfected cells (n = 3). F. Quantified results for PD-L1 siRNA transfection Western blot (n = 3). Transfection of PD-L1 siRNA at the siRNA:transfection reagent ratio of 4:10 resulted in a significant knockdown of PD-L1 expression. **p < 0.05 compared to Mock - No siRNA cells. (n = 3).

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American Cancer Society

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ARTICLE ABSTRACT

Chemerin (retinoic acid receptor responder 2, RARRES2) is an endogenous leukocyte chemoattractant that recruits innate immune cells through its receptor, ChemR23. RARRES2 is widely expressed in nonhematopoietic tissues and often downregulated across multiple tumor types compared with 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. 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. We show for the first time that human tumors exposed to exogenous chemerin significantly upregulate PTEN expression/activity, and concomitantly suppress programmed death ligand-1 (PD-L1) expression. CMKLR1 knockdown abrogated chemerin-induced PTEN and PD-L1 modulation, exposing a novel CMKLR1/PTEN/PD-L1 signaling cascade. Targeted inhibitors suggested signaling was 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. Collectively, our data show a novel link between chemerin, PTEN, and PD-L1 in human tumor lines, which may have a role in improving T-cell–mediated immunotherapies.