The Cell-Autonomous Pro-Metastatic Activities of PD-L1 in Breast Cancer Are Regulated by N-Linked Glycosylation-Dependent Activation of STAT3 and STAT1.

PD-L1 has been characterized as an inhibitory immune checkpoint, leading to the suppression of potential anti-tumor immune activities in many cancer types. In view of the relatively limited efficacy of immune checkpoint blockades against PD-L1 in breast cancer, our recent study addressed the possibility that in addition to its immune-inhibitory functions, PD-L1 promotes the pro-metastatic potential of the cancer cells themselves. Indeed, our published findings demonstrated that PD-L1 promoted pro-metastatic functions of breast cancer cells in a cell-autonomous manner, both in vitro and in vivo. These functions fully depended on the integrity of the S283 intracellular residue of PD-L1. Here, using siRNAs and the S283A-PD-L1 variant, we demonstrate that the cell-autonomous pro-metastatic functions of PD-L1-tumor cell proliferation and invasion, and release of the pro-metastatic chemokine CXCL8-required the activation of STAT3 and STAT1 in luminal A and triple-negative breast cancer cells. The cell-autonomous pro-metastatic functions of PD-L1 were potently impaired upon inhibition of N-linked glycosylation (kifunensine). Site-specific mutants at each of the N-linked glycosylation sites of PD-L1 (N35, N192, N200, and N219) revealed that they were all required for PD-L1-induced pro-metastatic functions to occur; the N219 site was the main regulator of STAT3 and STAT1 activation, with accompanying roles for N192 and N200 (depending on the cell type). Using a T cell-independent mouse system, we found that cells expressing N35A-PD-L1 and N219A-PD-L1 had a significantly lower tumorigenic and metastatic potential than cells expressing WT-PD-L1. TCGA analyses revealed significant associations between reduced survival and high levels of α-mannosidase II (inferring on N-linked glycosylation) in breast cancer patients. These findings suggest that N-linked glycosylation of PD-L1 may be used to screen for patients who are at greater risk of disease progression, and that modalities targeting N-linked glycosylated PD-L1 may lead to the inhibition of its cell-autonomous pro-metastatic functions and to lower tumor progression in breast cancer.

Inflammation-Driven Regulation of PD-L1 and PD-L2, and Their Cross-Interactions with Protective Soluble TNFα Receptors in Human Triple-Negative Breast Cancer.

Pro-inflammatory cytokines play key roles in elevating cancer progression in triple-negative breast cancer (TNBC). We demonstrate that specific combinations between TNFα, IL-1ß and IFNγ up-regulated the proportion of human TNBC cells co-expressing the inhibitory immune checkpoints PD-L1 and PD-L2: TNFα + IL-1ß in MDA-MB-231 cells and IFNγ + IL-1ß in BT-549 cells; in the latter cells, the process depended entirely on STAT1 activation, with no involvement of p65 (CRISPR-Cas9 experiments). Highly significant associations between the pro-inflammatory cytokines and PD-L1/PD-L2 expression were revealed in the TCGA dataset of basal-like breast cancer patients. In parallel, we found that the pro-inflammatory cytokines regulated the expression of the soluble receptors of tumor necrosis factor α (TNFα), namely sTNFR1 and sTNFR2; moreover, we revealed that sTNFR1 and sTNFR2 serve as anti-metastatic and protective factors in TNBC, reducing the TNFα-induced production of inflammatory pro-metastatic chemokines (CXCL8, CXCL1, CCL5) by TNBC cells. Importantly, we found that in the context of inflammatory stimulation and also without exposure to pro-inflammatory cytokines, elevated levels of PD-L1 have down-regulated the production of anti-tumor sTNFR1 and sTNFR2. These findings suggest that in addition to its immune-suppressive activities, PD-L1 may promote disease course in TNBC by inhibiting the protective effects of sTNFR1 and sTNFR2.

Correction: Erlichman et al. Tumor Cell-Autonomous Pro-Metastatic Activities of PD-L1 in Human Breast Cancer Are Mediated by PD-L1-S283 and Chemokine Axes. Cancers 2022, 14, 1042.

The authors apologize for several typos/technical inaccuracies in the original article [...].

Tumor Cell-Autonomous Pro-Metastatic Activities of PD-L1 in Human Breast Cancer Are Mediated by PD-L1-S283 and Chemokine Axes.

Therapies targeting the PD-L1/PD-1 axis have recently been introduced to triple-negative breast cancer (TNBC) with limited efficacy, suggesting that this axis promotes tumor progression through mechanisms other than immune suppression. Here, we over-expressed WT-PD-L1 in human TNBC cells (express endogenous PD-L1) and in luminal-A breast cancer cells (no endogenous PD-L1 expression) and demonstrated that cell-autonomous PD-L1 activities lead to increased tumor cell growth, invasion and release of pro-metastatic factors (CXCL8, sICAM-1, GM-CSF). These activities were promoted by PD-1 and were inhibited by mutating S283 in PD-L1. Invasion of WT-PD-L1-cells required signaling by chemokine receptors CXCR1/2, CCR2 and CCR5 through autocrine circuits involving CXCL8, CCL2 and CCL5. Studies with T cell-deficient mice demonstrated that cell-autonomous WT-PD-L1 activities in TNBC cells increased tumor growth and metastasis compared to knock-out (KO)-PD-L1-cells, whereas S283A-PD-L1-expressing cells had minimal ability to form tumors and did not metastasize. Overall, our findings reveal autonomous and PD-1-induced tumor-promoting activities of PD-L1 that depend on S283 and on chemokine circuits. These results suggest that TNBC patients whose tumors express PD-L1 could benefit from therapies that prevent immune suppression by targeting PD-1/CTLA-4, alongside with antibodies to PD-L1, which would allow maximal impact by mainly targeting the cancer cells.

Chemotherapy Shifts the Balance in Favor of CD8+ TNFR2+ TILs in Triple-Negative Breast Tumors.

Triple-negative breast cancer (TNBC) is primarily treated via chemotherapy; in parallel, efforts are made to introduce immunotherapies into TNBC treatment. CD4+ TNFR2+ lymphocytes were reported as Tregs that contribute to tumor progression. However, our published study indicated that TNFR2+ tumor-infiltrating lymphocytes (TNFR2+ TILs) were associated with improved survival in TNBC patient tumors. Based on our analyses of the contents of CD4+ and CD8+ TILs in TNBC patient tumors, in the current study, we determined the impact of chemotherapy on CD4+ and CD8+ TIL subsets in TNBC mouse tumors. We found that chemotherapy led to (1) a reduction in CD4+ TNFR2+ FOXP3+ TILs, indicating that chemotherapy decreased the content of CD4+ TNFR2+ Tregs, and (2) an elevation in CD8+ TNFR2+ and CD8+ TNFR2+ PD-1+ TILs; high levels of these two subsets were significantly associated with reduced tumor growth. In spleens of tumor-bearing mice, chemotherapy down-regulated CD4+ TNFR2+ FOXP3+ cells but the subset of CD8+ TNFR2+ PD-1+ was not present prior to chemotherapy and was not increased by the treatment. Thus, our data suggest that chemotherapy promotes the proportion of protective CD8+ TNFR2+ TILs and that, unlike other cancer types, therapeutic strategies directed against TNFR2 may be detrimental in TNBC.

Beyond Cell Motility: The Expanding Roles of Chemokines and Their Receptors in Malignancy.

The anti-tumor activities of some members of the chemokine family are often overcome by the functions of many chemokines that are strongly and causatively linked with increased tumor progression. Being key leukocyte attractants, chemokines promote the presence of inflammatory pro-tumor myeloid cells and immune-suppressive cells in tumors and metastases. In parallel, chemokines elevate additional pro-cancerous processes that depend on cell motility: endothelial cell migration (angiogenesis), recruitment of mesenchymal stem cells (MSCs) and site-specific metastasis. However, the array of chemokine activities in cancer expands beyond such "typical" migration-related processes and includes chemokine-induced/mediated atypical functions that do not activate directly motility processes; these non-conventional chemokine functions provide the tumor cells with new sets of detrimental tools. Within this scope, this review article addresses the roles of chemokines and their receptors at atypical levels that are exerted on the cancer cell themselves: promoting tumor cell proliferation and survival; controlling tumor cell senescence; enriching tumors with cancer stem cells; inducing metastasis-related functions such as epithelial-to-mesenchymal transition (EMT) and elevated expression of matrix metalloproteinases (MMPs); and promoting resistance to chemotherapy and to endocrine therapy. The review also describes atypical effects of chemokines at the tumor microenvironment: their ability to up-regulate/stabilize the expression of inhibitory immune checkpoints and to reduce the efficacy of their blockade; to induce bone remodeling and elevate osteoclastogenesis/bone resorption; and to mediate tumor-stromal interactions that promote cancer progression. To illustrate this expanding array of atypical chemokine activities at the cancer setting, the review focuses on major metastasis-promoting inflammatory chemokines-including CXCL8 (IL-8), CCL2 (MCP-1), and CCL5 (RANTES)-and their receptors. In addition, non-conventional activities of CXCL12 which is a key regulator of tumor progression, and its CXCR4 receptor are described, alongside with the other CXCL12-binding receptor CXCR7 (RDC1). CXCR7, a member of the subgroup of atypical chemokine receptors (ACKRs) known also as ACKR3, opens the gate for discussion of atypical activities of additional ACKRs in cancer: ACKR1 (DARC, Duffy), ACKR2 (D6), and ACKR4 (CCRL1). The mechanisms involved in chemokine activities and the signals delivered by their receptors are described, and the clinical implications of these findings are discussed.