Autophagy inhibition enhances PD-L1 expression in gastric cancer

Gastric cancer is a prevalent malignant tumor of the digestive tract and remains the third leading cause of cancer-related death worldwide. An estimated 951,600 newly diagnosed cases and 723,100 deaths of gastric cancer occurred in 2012 [1]. The 5-year survival rate for patients with advanced gastric cancer is only 5–20% with 10 months of median overall survival [1]. Therefore, new molecular targets and therapeutic approaches are urgently needed.

Autophagy is a highly conserved homeostatic process that involves the formation of a double-membrane structure, autophagosome, which subsequently fuses with the lysosome to generate autolysosome leading to the degradation of the cellular proteins and damaged organelles. This catabolic pathway plays a pivotal role in cell survival, cellular metabolism and immune responses. Growing evidence reveals that the role of autophagy in tumorigenesis is complex and context-dependent [2]. On one hand, autophagy can inhibit tumor formation by reducing oxidative stress and DNA damage in normal tissues [3]. On the other hand, autophagy can promote tumor cell survival by providing cells with energy and vital compounds upon various stress stimuli in developed cancers [2, 4]. Also, autophagy can be activated in response to cytotoxic chemotherapeutics, acting as a mechanism of drug resistance [5‐7]. Thus, modulating autophagy is an attractive option, which allows us to harness this process for improving the disease management in cancers.

Programmed cell death-1 (PD-1) with its ligand (PD-L1) are important immune checkpoint proteins. Elevated expression of PD-L1 receptors on cancer cell membranes has been observed in many cancer types. PD-L1 can interact with PD-1 and CD80 on the surface of T-cells, thus protecting cancer cells from immune-mediated rejection by inhibition of T effector functions [8, 9]. PD-L1 expression can be induced by inflammatory cytokines, such as interferon (IFN)-γ [10] secreted by infiltrating lymphocytes or induced by tumor-cell intrinsic signaling, including nuclear factor (NF)-κB, mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K), mammalian target of rapamycin (mTOR) and Janus kinase/signal transducers and activators of transcription (JAK/STAT) [11]. In addition, PD-L1 is regulated by the tumor suppressor genes PTEN and LKB1 as well as epithelial-mesenchymal transition-related molecules [12, 13]. More recently, evidences suggest that PD1 receptor and its ligand PD-L1 can have crosstalk with autophagy in cancer cells. In mouse melanoma and human ovarian cancer, tumor cell-intrinsic PD-L1 upregulates mTOR complex 1 signaling to inhibit autophagy and sensitizes tumor cells to clinically available autophagy inhibitors [14]. Recent work shows that CMTM6 co-localizes with PD-L1 at the cell membrane and in endosome, where it protects PD-L1 from lysosome-mediated degradation in a broad range of cancer cells [15]. Defective autophagy has also been shown to promote PD-L1 expression in cerulein-treated Atg5^L/L mice with pancreatitis [16]. The link between autophagy and PD-L1 in gastric cancer is unclear. Here, we investigated if tumor-intrinsic PD-L1 could be regulated by autophagy in gastric cancer. To test our hypothesis, we determined if inhibition of autophagy could increase PD-L1 levels in human gastric cancer cells.

Autophagy has opposing, context-dependent roles in cancer and perturbations in autophagy are found in gastric cancer [24]. As one of the most important survival mechanisms, autophagy helps tumor cells to adjust and adapt to an unfavourable environment, to escape from immune surveillance, and hence to promote tumor growth. Recent studies have delineated the mechanism underlying autophagy and the intricate involvement of PD-L1/PD1 axis in cancer cells. A study by Clark et al. found that tumor-intrinsic PD-L1 signals regulate cell proliferation and autophagy in ovarian cancer and melanoma. Tumor cells with high levels of PD-L1 expression are more sensitive to autophagy inhibitors than cells with lower PD-L1 levels in murine melanoma cells and human ovarian cancer cells [14]. Melanoma cell-intrinsic PD-1 cooperates with PD-L1 to promote tumorigenesis and modulates downstream effectors of mTOR signaling [17]. Blockade of PD-L1 in sarcoma cells inhibits mTOR activity and dampens glycolysis, thereby restoring glucose in tumor microenvironment [25]. Depletion of glucose also induces autophagy through the mTOR complex 1 pathway [26]. Until now, the link between autophagy and the immune checkpoint molecule PD-L1 is not quite well understood in gastric cancer. Here, we demonstrated that inhibition of autophagy by pharmacological or RNA interference approach could induce the expression of PD-L1, unveiling the unreported intrinsic regulation of PD-L1 by autophagy.

As a ligand of PD-1, PD-L1 is a transmembrane protein that is expressed on a wide variety of cells including tumor cells to inhibit CD8⁺ T cell activities and suppress antitumor immunity. So, the PD-L1 protein on cell membrane mainly exerts its antitumor effect. Thus, we detected the expression of surface PD-L1 by flow cytometry in accordance with many of the published papers studying PD-L1 to determine its functional proportion [14, 15, 27‐29]. Also, we evaluated the total expression of PD-L1 protein to demonstrate the upregulation of PD-L1 by autophagy inhibition through Western blots [30]. We demonstrated that blockade of autophagy increased the mRNA levels of PD-L1 as well as protein expression in gastric cancer cells. Accordantly, Yang et al. found that defective autophagy with deletion of Atg5 by using a mouse model of cerulein-induced pancreatitis, activated the IκB kinase-related kinase TBK1 and promoted PD-L1 upregulation. These findings hinted at novel beneficial effects of autophagy inhibitors and their possible synergy with drugs targeting the PD-L1/PD-1 axis [16].

We noticed that the basal protein levels of PD-L1 are higher in NCI-N87 and AGS cells than other gastric cancer cells as shown in Fig. 1a, which may attribute to the specific genomic mutations harbored by the cells with SMAD4 and TP53 mutations in NCI-N87 cells and CDH1, CTNNB1, KRAS and PIK3CA mutations in AGS cells. In this regard, PD-L1 expression was significantly higher in tumors with TP53 mutation in lung cancer while KRAS mutation could induce PD-L1 expression in lung adenocarcinoma [31, 32]. Also, the oncogenic activation of the AKT-mTOR pathway could upregulate the expression of PD-L1 in non-small cell lung cancer [33]. Both pharmacological agents and siRNA targeting non-lysosomal components of autophagy could up-regulate PD-L1 expression in gastric cancer cell lines, and the induction of IFN-γ further increased PD-L1 levels as shown in Figs. 2 and 3. 3-MA could effectively block an early stage of autophagy by inhibiting the class III PtdIns3K, but also non-selectively inhibit the class I PI3K and affect cell survival through AKT and other kinases which may in turn inhibit PD-L1 expression in particular settings. It is therefore likely that the overall upregulation of PD-L1 expression by autophagy inhibition is alleviated upon treatment by 3-MA compared to other autophagy inhibitors in AGS cells with PIK3CA mutations (Fig. 2b). Recently, the precise mechanism of how CQ blocks autophagy was firmly demonstrated – CQ mainly inhibits autophagy by impairing autophagosome fusion with lysosome but not affecting the acidity of this organelle [34]. The mutant p53 proteins was reported to counteract the formation of autophagic vesicles and the fusion with lysosomes via the repression of autophagy-related proteins and enzymes in pancreas and breast cancer cells [35]. Concordantly, we found that CQ induced a lower increase on PD-L1 expression compared to 3-MA and bafilomycin A1 group in NCI-N87 cells with TP53 mutation (Fig. 2b). Thus, in studies where the effect of autophagy inhibition is being investigated, it is important to confirm results by inhibiting autophagy at different stages with several pharmacological inhibitors. We found that IFN-γ significantly induced PD-L1 expression through activation of STAT1 signaling independent of autophagy levels in AGS and NCI-n87 cells (Additional file 1: Figure S5A), which is in accordance with others in several types of cancer [36‐38]. Upon autophagy inhibition, the levels of p-p65 was upregulated in AGS and NCI-N87 cells treated with or without IFN-γ (Additional file 1: Figure S5B). These results indicated that autophagy inhibition upregulated the levels of PD-L1 protein via NF-κB signaling whereas the IFN-γ induced PD-L1 expression through STAT1 signaling.

Clinical interventions to manipulate autophagy mainly by pharmacological inhibitors, including chloroquine and hydroxychloroquine, with other chemotherapeutics in search of synergistic interactions in cancer are already underway [39]. Due to the lack of our understanding of the interplay between autophagy and the immune response, a study has sought to elucidate their relationship and demonstrated that the antitumor adaptive immunity is not adversely impaired by autophagy inhibition in immune-competent mouse models of melanoma and mammary cancer [40]. Such findings are corroborated by our findings that autophagy inhibition had minimal effect on T cell function and PD-L1 levels were still upregulated in the cocultures of gastric cancer cells and PBMC (Fig. 5b). The increase in basal levels of PD-L1 expression in co-cultures makes the fold change reduced (Fig. 5b) compared to the gastric cancer cells alone group (Fig. 2a). The reduced fold change could be explained that the co-culture with lymphocytes itself already had an inducing effect on the expression of PD-L1 than cells without lymphocytes. In this respect, an inducing effect of lymphocytes on the expression of PD-L1 was found in cocultures with melanoma cells [29]. In addition, the pharmacological inhibitors may also have some effects on the lymphocytes, which has to be evaluated in our future study. In our study, we did not observe the inducing effect of IFN-γ on the levels of PD-L1 in co-cultured condition (Fig. 5b). The cytokines including IFN-γ secreted by the lymphocytes may have inducing effect on the levels of PD-L1 in co-culture conditions, which therefore attenuated the effect of exogeneous IFN-γ added to the co-cultures [41].

Our work suggests that autophagy inhibition plus anti-PD-L1 is an attractive combination for further investigation, particularly for tumors with high levels of autophagy, and provides potential biomarkers and mechanisms to assess clinical efficacy. However, contradictory evidence also exists in the literature. Peng et al. reported that the loss of PTEN decreased T cell infiltration in tumors, inhibited autophagy and was correlated with inferior outcomes with PD-1 inhibitor therapy [42]. PD-L1 expression can be induced by inflammatory cytokines or tumor-cell intrinsic signaling, including NF-κB, MAPK, PI3K, mTOR and JAK/STAT. Pearson correlation analysis as shown in Additional file 1: Figure S6C and Fig. 6f suggests that the expression of PD-L1 in gastric cancer is in part correlated with high levels of LC3 and p62/SQSTM1. These findings indicate that additional factors must be considered to discern the various scenarios in which blockade of autophagy would be beneficial in cancer therapy.

Out data highlighted the existence of an additional, important signalling on the regulation of tumour intrinsic PD-L1. We found that inhibition of autophagy upregulated the expression of PD-L1 in gastric cancer cells in vitro and in vivo. As our understanding of tumor cell-intrinsic signals on the regulation of PD-L1 increases, our ability to predict treatment responses to various agents and combine them effectively will improve. We anticipate that our study will inform the development of autophagy inhibitors combined with immune checkpoint inhibitors in gastric cancer. Given the competing and context-dependent effects of autophagy, the best strategy would be to decide which patients would benefit from autophagy inhibition therapy.