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. 2016 Dec 1;76(23):6964-6974.
doi: 10.1158/0008-5472.CAN-16-0258. Epub 2016 Sep 26.

Tumor-Intrinsic PD-L1 Signals Regulate Cell Growth, Pathogenesis, and Autophagy in Ovarian Cancer and Melanoma

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Tumor-Intrinsic PD-L1 Signals Regulate Cell Growth, Pathogenesis, and Autophagy in Ovarian Cancer and Melanoma

Curtis A Clark et al. Cancer Res. .
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Abstract

PD-L1 antibodies produce efficacious clinical responses in diverse human cancers, but the basis for their effects remains unclear, leaving a gap in the understanding of how to rationally leverage therapeutic activity. PD-L1 is widely expressed in tumor cells, but its contributions to tumor pathogenicity are incompletely understood. In this study, we evaluated the hypothesis that PD-L1 exerts tumor cell-intrinsic signals that are critical for pathogenesis. Using RNAi methodology, we attenuated PD-L1 in the murine ovarian cell line ID8agg and the melanoma cell line B16 (termed PD-L1lo cells), which express basal PD-L1. We observed that PD-L1lo cells proliferated more weakly than control cells in vitro As expected, PD-L1lo cells formed tumors in immunocompetent mice relatively more slowly, but unexpectedly, they also formed tumors more slowly in immunodeficient NSG mice. RNA sequencing analysis identified a number of genes involved in autophagy and mTOR signaling that were affected by PD-L1 expression. In support of a functional role, PD-L1 attenuation augmented autophagy and blunted the ability of autophagy inhibitors to limit proliferation in vitro and in vivo in NSG mice. PD-L1 attenuation also reduced mTORC1 activity and augmented the antiproliferative effects of the mTORC1 inhibitor rapamycin. PD-L1lo cells were also relatively deficient in metastasis to the lung, and we found that anti-PD-L1 administration could block tumor cell growth and metastasis in NSG mice. This therapeutic effect was observed with B16 cells but not ID8agg cells, illustrating tumor- or compartmental-specific effects in the therapeutic setting. Overall, our findings extend understanding of PD-L1 functions, illustrate nonimmune effects of anti-PD-L1 immunotherapy, and suggest broader uses for PD-L1 as a biomarker for assessing cancer therapeutic responses. Cancer Res; 76(23); 6964-74. ©2016 AACR.

Conflict of interest statement

The authors have no conflicting financial interests to declare.

Figures

Figure 1
Figure 1. Tumor-intrinsic PD-L1 controls immune-independent growth and metastatic spread
Flow cytometry for PD-L1 expression of in vitro cultured cells. Interferon (IFN)-γ 0.1 ng/mL added for 48 h as indicated for (A) ID8agg or (B) B16. C. Proliferation in vitro of B16 cells determined by MTT versus control (ctrl, set at 100%). p-value, unpaired t test. D. NSG mice challenged subcutaneously with indicated B16 cells. P values for tumor size by two-way ANOVA and for survival by log-rank test. E. NSG mice challenged with indicated B16 cells and sacrificed on day 18. Genes in whole lung lysates by qPCR. p-value, unpaired t test. *, p < 0.05, **, p < 0.01. F. Proliferation in vitro of ID8agg cells as in panel C and survival in vivo (G) as in panel D after intraperitoneal ID8agg challenge.
Figure 2
Figure 2. αPD-L1 reduces B16 growth and metastatic spread in NSG mice
A. PD-1 and PD-L1 expression in B16 melanoma and ID8agg ovarian cells measured by flow cytometry. B. Proliferation in vitro of B16 cells ± αPD-L1 or αPD-1 (50 μg/mL each) determined by MTT versus control (ctrl, set at 100%). p-value, unpaired t test. C. NSG mice challenged with indicated B16 cells and treated with αPD-L1 200 μg every other day starting one day following challenge. p-value, two-way ANOVA. D. qPCR for indicated genes from whole lung lysates from mouse challenged as in C, given αPD-L1 or αPD-1 200 μg every other day starting on day following challenge, day 18. Unpaired t test. *, p < 0.05, **, p < 0.01. E. Proliferation in vitro of ID8agg cells treated as in B. NSG mice challenged with ID8agg-luciferase and treated with αPD-L1 200 μg every other day starting one day following challenge. P values for average luciferase radiance (F) by two-way ANOVA and for survival (G) by log-rank test.
Figure 3
Figure 3. PD-L1 regulates tumor autophagy genes and functions
A. RNA was isolated from in vitro cultured control or PD-L1lo ID8agg cells and global genes were assessed using David Bioinformatics. 5.8E-10, 5.8 x 10−10, etc. B. Western blot of lysed ID8agg cells from basal (+) or serum starved (−) (24 h) conditions (left). Right, summary data of three independent experiments. p-values, unpaired t test. C. Confocal images of autophagosome formation by LC-3 aggregation (red) in control versus PD-L1lo ID8agg under basal or serum starved (24 h) conditions. Blue, DAPI for nuclei. D. Analyses of B16 cells as in B, (med + or − for basal and serum starved conditions, respectively) treated with rapamycin (R) for 16 h, chloroquine (C) for 6 h or both. E. Confocal images of autophagosome formation for control versus PD-L1lo B16 as in C.
Figure 4
Figure 4. Tumor PD-L1 regulates response to pharmacologic autophagy inhibitors
A. Indicated cells were cultured with 50 μM chloroquine and proliferation inhibition (100%-% proliferation by MTT, with control set at 0%) assessed 72 h later. P values from unpaired t test. B-D, wild type mice challenged with indicated cells and treated with chloroquine (CQ) or 3-methyladenine (3MA) as described in Materials and Methods. p-values, two-way ANOVA. PBS, phosphate buffered saline control. E-F. βδ TCR KO mice challenged and treated as in B-D. G-H. NSG mice challenged and treated as in B-D. I. Indicated B16 cells from basal (+) or serum starved (−) (24 h) conditions and cell viability normalized to basal controls assessed on a Vi-Cell. P values from unpaired t test. J. WT females challenged with indicated ID8agg, 4 x 106 cells intraperitoneally, and treated with chloroquine or 3MA as in panels B-D. No differences are significant.
Fig. 5
Fig. 5. Tumor PD-L1 regulates mTOR distinctly and blunts rapamycin proliferation inhibition
Summary data of Western blots for P70S6KT389 and AktS473 phosphorylation as ratios of phospho-protein to total protein under basal or serum starved (24 h) conditions for ID8agg (A) and B16 (B) cells. Statistical analyses from average of three independent experiments. C. Indicated cells were cultured with 5 nM rapamycin and proliferation by MTT, with control set at 100% assessed 72 h later. D. Representative Western blots for treatments with rapamycin (R) for 16 h, chloroquine (C) for 6 h or both (R+C) under basal (+) conditions for under serum-starved (−) conditions for ID8agg and B16. Summary data for these blots are in Supplemental Fig. 6.
Fig. 6
Fig. 6. Tumor cell-intrinsic PD-L1 regulates proliferation, mTOR signaling, and autophagy in human ovarian cancer cells
A. Flow cytometry for PD-L1 expression of in vitro cultured ES2 human ovarian cancer cells showing PD-L1 knock-down by shRNA. B. PD-1 and PD-L1 expression by flow cytometry. C. Proliferation in vitro of ES2 cells determined by MTT versus control (ctrl, set at 100%). p-value, unpaired t test. D. Western blot for LC3I/II in ES2 cell lysates from basal conditions. E. Confocal images of autophagosome formation by LC-3 aggregation (red) in control versus PD-L1lo ES2 under basal or serum starved (24 h) conditions. Blue, DAPI for nuclei. F. Western blot for P70S6KT389 and AktS473 phosphorylation in ES2 cells under basal conditions. G. Control and PD-L1lo ES2 cells were cultured with 50 μM chloroquine and proliferation inhibition (100%-% proliferation by MTT, with control set at 0%) assessed 72 h later. P-values, unpaired t test.

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