The interaction between PD-L1 and PD1 has been widely reported to interfere with the T cell receptor (TCR) signaling transduction of T cells. PD-L1 is vital in inhibiting T-cell-mediated immune response in cytotoxic T cells, leading to immune killing escape and tumor progression in several malignancies (31).

Studies have shown that PD-L1 can regulate cancer cell growth, proliferation and suppress apoptosis without PD-1 involvement (12,32–39). A study showed that the knockdown of PD-L1 expression in GC cells can significantly suppress cell proliferation, migration, invasion, apoptosis, cell cycle, tumorigenicity and cytotoxic sensitivity to CIK therapy (32). Lotfinejad et al (33) demonstrate that PD-L1 knockdown can reduce triple-negative breast cancer (TNBC) cell proliferation and induce apoptosis via intrinsic and extrinsic apoptosis pathways. In a mouse sarcoma model, blocking PD-L1 on tumors could interrupt tumor progression and cell glycolysis; the mechanism is to suppress mTOR signals and decrease the expression of some glycolytic enzymes (34). In ovarian cancer and melanoma, Clark et al (35) observed that PD-L1^low cells proliferate more weakly than control cells in vitro and PD-L1 attenuation also reduces mTORC1 activity. Fan et al (36) found that Cbl-b could interact with STAT5a and cause its ubiquitination, which downregulates PD-L1 expression and inhibits cell proliferation, but miR-940 could target Cbl-b and then upregulate PD-L1 expression and promote gastric cancer cell proliferation. A study found that in TNBC and NSCLC, the cell surface adhesion receptor CD44 was a critical positive regulator of PD-L1; CD44 could bind to the regulatory region of PD-L1, which contains the CD44-ICD binding site and activates PD-L1 transcription through its ICD; the activated PD-L1 could promote tumor cell proliferation independent of T cell response (37). During cell division, PD-L1 is a subunit of the adhesin complex: PD-L1 could compensate for the loss of Sororin and compete with Wing Apart-Like (WAPL) for binding to PDS5B, which secures proper sister chromatid cohesion and segregation; depleting PD-L1 leads to multinuclear cells and suppresses cell proliferation in vitro and tumor growth in vivo in immunodeficient NSG mice (38). In NSCLC cells, activation of EGFR could upregulate the expression of PD-L1 through IL-6/Janus kinase (JAK)/STAT3 signal pathway and promote NSCLC cell proliferation (39). Yang et al found that PIM2-mediated phosphorylation of heat shock factor 1 (HSF1) at Thr120 enhanced the stability of HSF1 protein and phosphorylation of HSF1 could bind to the promoter of PD-L1, which strengthened PD-L1 expression and promoted breast cancer cells proliferation (12).

Although a number of basic studies have demonstrated the ability of PD-L1 to promote tumor proliferation and progression (Table I), clinicopathological data have also confirmed that high PD-L1 expression is associated with poor prognosis in most cases. However, the mechanism of PD-L1 promoting tumor proliferation and progression is not well studied. Most of the studies found that PD-L1 high expression can show the proliferative phenotypes of tumor cells, but how does PD-L1 promote tumor proliferation? Does it promote the activation of transcription factors and participate in post-transcriptional modification (PTM) of certain oncogenes or tumor suppressors? These may be the next breakthroughs for PD-L1 research.

The most common and effective cancer treatment methods include surgery, chemotherapy and radiation therapy. Chemotherapy is quite important in cancer treatment and can extend the survival time of patients with a number of cancers. Advances in biotechnology and intensive research on signaling pathways have led to the rapid development of targeted therapy, which has also become an important option for tumor treatment.

Although chemotherapy has a noticeable effect in the early period of advanced tumor treatment, but, after a while, a large proportion of chemoresistance might develop, which leads to treatment failure and metastasis occurrence (40). Studies have shown that the high expression of PD-L1 in cancer cells could cause chemotherapy resistance in cancer therapy (Table II). Following doxorubicin treatment, PD-L1 was observed to transfer from membrane to nuclear concomitant with the translocation of phosphorylated Akt and promote doxorubicin-induced drug resistance (41). The mechanism was that doxorubicin-dependent downregulation of cell surface PD-L1 was accompanied by upregulation of PD-L1 in the nucleus and this redistribution of PD-L1 occurred with a similar translocation of phosphorylated Akt to the nucleus. PD-L1 was considered an independent prognostic risk factor for osteosarcoma as patients with high PD-L1 expression were observed to have a lower five-year survival rate and knocking out PD-L1 in osteosarcoma cells could increase doxorubicin and paclitaxel sensitivities (42). A pair of studies reported that PD-L1 could bind to NBS1 to form a complex and lead to cisplatin resistance in HNSCC and knockdown of PD-L1 or NBS1 could reverse this drug resistance. PD-L1 and IL-6 were over-expressed on cisplatin-resistant HNSCC cells (43,44). In cisplatin resistant NSCLC, researchers found that decreased COP1 could promote c-Jun accumulation, inhibit HDAC3 expression and enhance PD-L1 acetylation, which would mediate or maintain the drug resistance of cancer cells (45). In ovarian cancer cells, Sp17^high (PD-L1⁺MHC-II⁻) cells showed enhanced resistance to paclitaxel-induced cell death compared with Sp17^low (PD-L1⁻MHC-II⁺) cells (46), which means Sp17 and PD-L1 are related to paclitaxel resistance. lncRNA FGD5-antisense 1 (FGD5-AS1) could negatively regulate miR-142 and promote cisplatin resistance through miR-142-5p/PD-L1 axis (47). miR3609 could specifically bind to the 3′ UTR region of PD-L1 and suppress PD-L1 expression to sensitize breast cancer cells to doxorubicin (48).

Although targeted therapy is developing rapidly, the problem of rapid drug resistance is a key obstacle to its further development. PD-L1 has also been found to play a role in the resistance of some targeted therapies. In EGFR-mutated NSCLC cells, PD-L1 was correlated with the sensitivity of tyrosine kinase inhibitors (TKIs) and PD-L1 could induce EMT by activating the TGF-β/Smad signal pathway, leading to primary resistance to gefitinib (49). PD-L1 expression was found to be increased in gefitinib-resistant CRC cells, but nano-diamino-tetras (NDAT)-induced low PD-L1 expression could reverse tumor gefitinib resistance (50). In sorafenib-resistant hepatoma cells, nuclear factor erythroid 2-related factor 2 inhibited the expression of miR-1 and loss of miR-1 contributed to the PD-L1 upregulation and drug resistance (51).

Most of these studies on the role of PD-L1 in drug resistance have also focused on the description of the phenotype, while the underlying mechanisms have not been much studied. It was hypothesized that PD-L1 may be involved in regulating the expression or PTM of certain drug resistance-related genes to cause the development of drug resistance. If the mechanism can be found, it will help find a targeted drug resistance solution.

Studies have shown that PD-L1 plays a vital role in promoting EMT and maintaining the stemness of cancer stem cells (52–57) (Table III).

PD-1 fusion protein-mediated stimulation of PD-L1 and the cytoplasmic domain of PD-L1 play a critical role in promoting the EMT phenotype of Eca-109 cells (52). Upregulation of PD-L1 in skin epithelial cells promotes EMT and accelerates carcinogenesis in squamous cell carcinoma (53). The significant association between PD-L1 expression and EMT phenotype was maintained in EGFR-mutated pADCs in lung cancer cells (54). A study found that CRC characterized by a lack of CDX2 and prominent expression of ALCAM frequently (71%) showed PD-L1 positivity, representing the relations between PD-L1 and EMT (55). It was also found that EMT is associated with the overexpression of PD-L1 in NSCLC (56). One study reports that PD-L1⁺ cancer cells show the characteristics of EMT (57). A survival analysis using The Cancer Genome Atlas database shows that PD-L1⁺/EMT⁻ patients have a better prognosis than PD-L1⁺/EMT⁺ patients in HNSCC (58). A study reports that PD-L1 expression increases in the induction of human breast EMT by activating PI3K/Akt pathway and that PD-L1 can also regulate the EMT state of breast cancer cells (59). PD-L1 can induce EMT and enhance the stemness of renal cell carcinoma by upregulating SREBP-1c (60). In NSCLC, TGF-β1 can upregulate PD-L1 expression at the transcriptional level through phosphorylation of Smad2, M7824 is a novel bifunctional agent which could target both PD-L1 and TGF-β1, using M7824 to treat NSCLC could attenuate TGF-β1 mediated EMT (61).

PD-L1 is considered essential in maintaining the stemness of breast cancer stem cells because it can upregulate the expression of Oct4 and Nanog in a PI3K/Akt-dependent pathway and directly promote BMI1 expression to affect the stemness of breast CSCs (62). CD133⁺ cells in both cell lines and CRC tissues express a high level of PD-L1 (57). Fang et al (63) found that PD-L1 can promote the proliferation of leukemia-inducing cells through PD-L1/JNK/Cyclin D2 signaling pathway and prompt leukemia stem cells to enter the cell cycle. PD-L1 can induce a stem cell-like state and interact directly with high mobility group AT-hook 1 (HMGA1) to activate PI3K/Akt and MEK/ERK pathways in CRC to maintain the self-renewal of CSCs (64).

Some studies show that non-coding RNAs and miRNAs can regulate cancer stemness and EMT by binding with PD-L1. SNHG14 can sponge miR-5590-3p to upregulate ZEB1 and ZEB1 transcriptionally activate SNHG14 and PD-L1 to promote the immune evasion of diffuse large B cell lymphoma cells (65). ZEB1, an EMT activator and transcriptional repressor of miR-200, can relieve miR-200 repression of PD-L1, leading to lung adenocarcinoma metastasis (66). In lymphoma cells, MALAT1 can sponge miR-195 to regulate the expression of PD-L1, knocking down MALAT1 also suppresses the EMT-like process via the Ras/ERK signaling pathway (67). In TNBC, miR-200c can repress a number of genes encoding immunosuppressive factors, including CD274/CD273, HMOX-1 and GDF15 to reverse the classic EMT signature (68). miR-873 can inhibit PD-L1 expression by directly binding to the 3′-UTR of CD274, which attenuates the stemness and chemoresistance of breast cancer cells through the PI3K/Akt signaling pathway (69). Circular RNA circ-CPA4 can act as an RNA sponge for let-7 miRNA and inhibit cell growth, migration and EMT by downregulating PD-L1 to promote NSCLC cell death (70).

These studies have mainly focused on the role of PD-L1 in cancer cell stemness maintenance and EMT and most of them are limited to detecting the relationship between PD-L1 expression changes and markers of stemness and EMT; however, little research has been performed on the specific mechanisms. Whether PD-L1 could regulate cancer metastasis or the mechanism behind this has not been discovered, but is worth exploring, as several clinical studies (3,15,36,70) have confirmed the association between PD-L1 and lymph node metastasis and distant metastasis of tumors.

Tumor cells can continuously adjust the metabolic pathways to meet their energy requirements and respond to the availability of nutrients. Warburg found that despite sufficient oxygen, most solid tumor cells still choose the aerobic glycolysis pathway rather than the oxidative phosphorylation pathway to adapt to their microenvironment (71). Studies show that PD-L1 can promote tumor progression by boosting the glucose metabolism of tumor cells (Table IV). A study used ¹⁸F-Fluorodeoxyglucose Positron Emission Tomography/Computed Tomography (¹⁸F FDG PET/CT) to evaluate the metabolic effects of PD-L1 protein on lung cancer and it was found that high PD-L1 expression can promote glucose metabolism in NSCLC (72). PD-L1 can promote tumor cell glycolysis through Akt/mTOR signal pathway and induce immune cells to consume glucose in the microenvironment. Inhibiting the expression of PD-L1 can cause mTOR activity inhibition to downregulate glycolytic enzymes, thereby inhibiting glycolysis (34). In cervical cancer, PD-L1 directly binds to integrin β4 and activates Akt/GSK3β signaling pathway and promotes glucose metabolism (73). Retinoic acid-related orphan receptor C (RORC) is found to negatively regulate the expression of PD-L1 by binding to the PD-L1 promoter region, which can inhibit the nuclear translocation of STAT3 and further inhibit the proliferation and glucose metabolism of bladder tumor cells (74). Ma et al (75) report that in acute myeloid leukemia (AML) cell lines, glycolysis-associated genes ALDOA, PGK1, LDHA and HK2 are highly expressed in the PD-L1^high cell line and overexpressed PD-L1 enhances glucose consumption rate, accompanied by decreased apoptosis and S phase cells. Feng et al (76) report that lactate-induced PD-L1 is mediated by its receptor GPR81 and GPR81-mediated upregulation of PD-L1 in glucose-stimulated lung cancer cells that recapitulated the enhanced glycolysis is dependent on LDHA. In patients with primary lung adenocarcinoma who received ¹⁸F-FDG PET/CT before treatment, PD-L1 expression in the tumor was positively correlated with ¹⁸F-fluorodeoxyglucose maximum standardized uptake value (SUVmax), total lesion glycolysis (TLG), HK2 and glucose transporter 1 (GLUT-1) expression (77).

These studies initially reveal the regulatory role of PD-L1 in glucose metabolism, but a number of mechanisms remain to be elucidated. Moreover, the regulation of PD-L1 in other metabolic pathways has yet to be reported. As tumor metabolism is not limited to glucose metabolism, it is hypothesized that PD-L1 may also play an important role in regulating other metabolic pathways in tumor cells, so more in-depth and extensive research is needed.

In addition to the numerous intrinsic functions previously described, PD-L1 also has a role in regulating gene stability. Tu et al (78) demonstrate that PD-L1 can act as an RNA-binding protein in cells to regulate the mRNA stability of NBS1, BRCA1 and a number of other DNA damage-related genes; intracellular PD-L1 can prevent these target RNAs from being degraded, thus increasing the resistance of cells to DNA damage. This study also found that PD-L1 has the ability to regulate whole genome RNA stability by RNA immunoprecipitation and RNA-seq assays. Thus, it provides strong evidence that PD-L1 possesses an intrinsically powerful gene regulatory function. It also predicts that PD-L1 may become a target to interfere with tumor radiotherapy resistance.

PD-L1 was previously widely considered to be localized in the cytoplasm and cell membrane, but recently some studies report the nuclear localization and role of PD-L1 in tumor cells (Table V).

The distribution of PD-L1 in different tumor specimens is diverse. Nuclear PD-L1 (nPD-L1) is expressed in RCC, lung cancer and hepatocellular carcinoma tissues and nPD-L1 in human esophageal cancer tissues is significantly correlated with tumor invasion (79,80). According to some reports, the expression of nPD-L1 is associated with a poor prognosis in some tumors. Expression of nPD-L1 in cell-surface vimentin-positive circulating tumor cells is significantly associated with the short-term survival rate of CRC and prostate cancer (81). Doxorubicin treatment can redistribute PD-L1 and increase the expression of nPD-L1 through PI3K/Akt signaling pathway (41). One study has shown that in NSCLC, KPNB1 binds to PD-L1 and promotes its entry into the nucleus (79). At the same time, nPD-L1 can integrate Sp1 to regulate the synthesis of Gas6, promote the secretion of Gas6 and activate the MER proto-oncogene tyrosine kinase signaling pathway to promote cell proliferation (79). In breast cancer, under hypoxic conditions, pSTAT3 can physically interact with PD-L1 and upgrade its nuclear translocation and enhance the transcription of the gasdermin C (GSDMC) gene, GSDMC is cleaved explicitly by caspase 8 to switch cell apoptosis to pyrolysis and induce tumor necrosis (82). This study showed a new signal pathway of nPD-L1/caspase-8/GSDMC, which is required for macrophage-derived TNFα-induced tumor necrosis. PD-L1 can be acetylated and modified by p300 acetyltransferase at Lys 263 in the cytoplasmic domain and blocking the acetylation of PD-L1 can damage its nuclear translocation, reprogram the expression of immune response-related genes and block the anti-tumor response to PD-1/PD-L1 therapy (83).

These aforementioned studies report how PD-L1 enters the nucleus and the functions it plays in the nucleus, but the exact mechanism of PD-L1 action in the nucleus remains to be elucidated. Therefore, further in-depth studies are needed to clarify the nuclear membrane transfer process and the internal effects of nPD-L1, which will help understand the non-immune checkpoint functions of PD-L1 widely.

PD-L1 has an important immune checkpoint function. Its role in tumor evasion of immune killing has been apparent, but PD-L1 is highly expressed in various types of tumor and shows some inherent non-immunological functions. PD-L1 has a number of intrinsic functions, such as promoting tumor proliferation, maintaining the stemness of cancer stem cells and EMT, regulating tumor cell metabolism and promoting drug resistance in tumors (Fig. 2). It also can perform specific functions by entering the nucleus and regulating genome stability.

At present, it is known that PD-L1 has these non-immune checkpoint functions, but not the exact mechanism. For example, the exact mechanism of PD-L1 to promote tumor progression through non-immune checkpoint-dependent pathways, the mechanism of PD-L1 to regulate the EMT process and maintain the stemness of tumor stem cells, the functions of PD-L1 in cancer metastasis, the specific role and function of PD-L1 after entering the nucleus and possible role of PD-L1 in regulating other metabolic pathways in tumors need to be explored widely and deeply. If these functions and mechanisms can be studied carefully, it will help precisely to target and intervene in the PD-L1 pathway from tumor prevention to tumor recurrence and metastasis, providing more possibilities for combining tumor immunotherapy with targeted therapy.