PD-1/PD-L1 pathway: Current research in breast cancer

4.1.Cancer immune system

To effectively eliminate cancer cells, the immune response must include a series of sequential and repetitive processes that establish a cycle of cancer immunity (Fig. 3). Cancer cells initially produce neoantigens that are subsequently released and captured by antigen-presenting cells (APCs). To elicit an anti-tumor response and mitigate peripheral resistance to the generated tumor antigen, a distinct signal is required during this phase. These immunogenic signals include stimuli such as inflammatory cytokines and chemical substances that are discharged by apoptotic tumor cells [11].

Fig. 3.

Cancer immune system [11]. Created with Biorender.com.

The processed antigen is presented to the T cells when the APCs attach to the major histocompatibility complex (MHC) molecules. The effector T cells then identify cancer-specific antigens as potentially harmful invaders, at which time the ratio between the effector and regulatory (T reg) T cells determines the type of immune response activated. Once activated, effector T cells proceed to the tumor site and interact with antigens on the tumor cells, after which they bind to these cells and kill them. As tumor cells are destroyed, the response cycle strengthens with the addition of greater numbers of tumor-specific antigens. This process is not ideal and several problems can arise with this system. For example, tumor antigens might not be detected; APCs and T cells might mistake tumor antigens for self-antigens, resulting in a T reg response rather than an anti-tumor response; and the tumor microenvironment could suppress the effector cells that are produced [11].

The cancer immunological cycle requires the coordinated action of several components, including immune stimulators that activate the immune system against cancer cells and inhibitors that regulate this activity. These factors collaborate at each step of the cycle. Stimulating factors promote immunity, while inhibitory factors monitor ongoing processes, reduce immune response activity, and/or prevent auto-immunity. Checkpoint proteins such as PD-L1, which are termed immunostats, exhibit inhibitory functions to modulate the active immune response in tumor tissues. For example, when the PD-1 receptor on the surface of T cells binds to PD-L1 expressed in cancer cells, T cell functioning is inhibited. Over-expression of PD-L1 is common in various types of cancer and is associated with a poor prognosis due to its inhibitory effect on anti-tumor responses [12].

4.2.Immunoediting

The immunoediting process elucidates the role of the immune system in the advancement and evolution of breast cancer. The immune system contributes to the prevention of cancer as well as the development, invasion, and metastasis of the disease. Currently, a number of cancer therapy approaches focus on methods to utilize the immune system more efficiently, such as the use of adaptive immunotherapy and monoclonal antibodies to prevent inhibitory signals received by T cells, such as those of anti-PD-1 [13].

Fig. 4.

Stages of the immunoediting process [13]. Created with Biorender.com.

Fig. 5.

The main stages of immune system-based metastasis cascade [14]. Created with Biorender.com.

The three primary stages of immunoediting are elimination, equilibrium, and escape (Fig. 4). The elimination phase involves the identification and eradication of cancer cells by the adaptive immune system. Perforin is released by cytolytic immune cells to trigger cancer cell lysis. The cancer cells that pass through the elimination stage transition into the equilibrium phase, where they exploit the negative regulatory dynamics of the immune system. During this phase, the immune response efficiently regulates the proliferation, spread, and programmed cell death (apoptosis) of the cancer cells. However, heterogeneity and genetic variation among cancer cells allow them to escape from this equilibrium, at which time immunosuppressive mechanisms are activated [14].

4.3.The effect of the immune response on tumor development

The growth and metastasis of circulating tumor cells (CTCs) involve the release of cancer cells, evasion of the immune system, and the production of distant metastases by adhesion to and exudation from blood vessels. The interactions of the immune system with CTCs are crucial for these processes. Primary tumors release thousands of tumor cells on average into the blood each day; however, the number of CTCs is rarely measured. This is because tumor cells that are released from the original immunosuppressive microenvironment become vulnerable to attacks by immune cells [14]. A diagram of the immunological escape of CTCs is presented in Fig. 5.

The immune response is highly dependent on the ability of the immune system to recognize the antigen that triggers the immune reaction. The immune response cells originate from pluripotent stem cells that differentiate through either the lymphoid pathway, which generates lymphocytes and their subsets, or the myeloid pathway, which forms phagocytic and other cells. The main immunocompetent cells are T lymphocytes, while the minor components include T helper, T cytotoxic, and T regulator cells, each of which serves a different function [15].

Immunoediting is an important part of tumor progression. The elimination of transformed cells can induce the production of tumor variants with low immunogenicity and more resistance to effector T cells in the equilibrium phase through methods such as the production of inhibitory signals. Furthermore, tumor-derived soluble factor (TDSF) can induce several mechanisms that allow for escape from immune cell attacks in the tumor microenvironment [16,17]. The failure of immune cells to eliminate primary tumor cells causes these cells to proliferate and undergo mitosis. Cell proliferation is generally associated with rapid tumor growth and more aggressive biological and clinical behavior [18], and this activity can be identified by performing a simple calculation of the mitotic index. The mitotic index is the gold standard for determining the aggressiveness and degree of differentiation of several tumors, including BC [19].

Increased tumor progression due to failure of the immune response is evident when an increase in tumor size is observed. A more significant amount of proliferation and mitosis of tumor cells results in an accelerated size increase, which can be evaluated through clinical examination and imaging. Solid cancer treatment involves both the removal of the primary tumor and the elimination of metastases; therefore, an understanding and control of metastasis is important in clinical practice. Metastases start from a single solid tumor that undergoes several stages of metastasis, leading to cancers that have spread and are challenging to cure. In each stage, the host immune system can identify the cancer cells. At the primary tumor stage, some immune cells act as pro-metastatic agents, such as T regs, which provide immunosuppression through the secretion of IL 10, thereby inhibiting the response of cytotoxic T cells and NK cells. Additional immune cells, including macrophages, participate in the process of angiogenesis, whereby M2 tumor-associated macrophages (TAMs) generate CCL-2 and IL-10 to encourage angiogenesis; boost immunosuppression by blocking DCs, T cells, and NK cells via expressing arginase; and promote invasion by using matrix metalloprotease to modify the stroma. Some immune cells act as anti-metastatic agents, such as cytotoxic NK cells, T cells, and M1 TAMs, which kill cancer cells. At the M2 circulation stage, TAMs and T reg cells act as pro-metastatic agents, whereby T reg cells reduce and suppress NK cells and T cells in the circulation, which allows metastatic cells to survive. While cytotoxic T cells and NK cells recognize and kill cancer cells, M1 TAM cells produce CXCL9, IL-6, and IL-12 to express iNOS and activate the immune system to directly destroy tumor cells through the generation of nitric oxide. At the metastatic-niche stage, mature NK cells promote metastatic niche growth when these cells have low immune capacities [19].

In contrast, immature NK cells inhibit metastatic niche formation. At this stage, macrophages (M2) are involved in preparing the metastatic niche by inducing the local immunosuppressive environment, while M1 stimulates local immunity. In addition, cytotoxic T cells can recognize and kill cancer cells when they enter the metastatic niche.

5.1.PD-L1 structure

When T lymphocytes recognize antigens on specific cells through the identification of the major histocompatibility complex (MHC), inflammatory cytokines are secreted into the circulatory system. Immunological tolerance is promoted by programmed death-1 (PD-1; CD279) protein activation on T cells, which leads to the stimulation of programmed death-ligand 1 (PD-L1) production in tissues via the action of a specific cytokine. The user has included a number reference, suggesting the inclusion of a citation or source [20].

The inhibitory co-receptor PD-1 is found on the surface of several cell types, including B lymphocytes, CD8+ and CD4+ T cells, NK cells, and other subtypes of tumor-infiltrating lymphocytes (TILs). The user has supplied no textual description of the number reference. Given that long-term antigen exposure may induce a selective increase in PD-1 expression, T cell PD-1 expression could indicate T cell fatigue. Academic rewriting of the user’s material is not possible due to a lack of information. The type 1 transmembrane protein PD-1 is encoded by the programmed cell death protein 1 (PDCD1) gene.

Fig. 6.

Diagrammatic representation of the protein structure domains, mRNA, and PD-L1 gene [22]. Created with Biorender.com.

The PD-1 molecule is composed of three primary structural elements: an external immunoglobulin variable (IgV) domain, a hydrophobic transmembrane region, and several intracellular regions. The intracellular tail components include the immunoreceptor tyrosine-based switch motif (ITSM) and the immunological receptor tyrosine-based inhibitory motif (ITIM), both of which have the potential to undergo phosphorylation. The activation of immune receptor PD-1 has been suggested to require the presence of active immunoreceptor tyrosine-based switch motifs (ITSMs) for the modulation of T cell responses [21].

PD-L2 (also known as B7-DC or CD273) and PD-L1 (also known as B7-H1 or CD274) are ligands that function as co-inhibitors and comprise the PD-1 receptor. PD-L1 is a protein encoded by the CD274 gene that contains 290 amino acids and has a molecular weight of 40 kDa (Fig. 6). PD-L1 is a crucial modulator of the adaptive immune response and is potentially situated on chromosome 9 p24 [21].

The promoter region of the CD274 gene contains numerous components that respond to IFN-γ; therefore, CD274 plays a crucial role in the IFN-γ-induced upregulation of PD-L1 expression [23]. PD-L1 operates as a transmembrane protein that consists of two extracellular domains (Ig-V and Ig-C) and a single transmembrane region. PD-L1 is a concise cytoplasmic domain that transmits intracellular signals; it is encoded by the CD274 gene and consists of 7 exons (Fig. 7), each of which codes for a specific part of the protein [24].

Fig. 7.

Protein structure, mRNA and PD-L1 gene [24]. Created with Biorender.com.

Exon 1 encodes the intracellular and transmembrane domains, the signal sequence, and the IgC-like and IgV-like domains, while exon 7 encodes the 3’ UTR and intracellular domain. The PD-L1 gene contains seven exons (Fig. 7) and the PD-L1 protein is composed of a transmembrane domain, two extracellular domains that are similar to immunoglobulin C and immunoglobulin V, and a solitary intracellular domain [24].

5.2.PD-L1 regulation

Various cells, including tumor cells, macrophages, and T and B lymphocytes, express CD40-ligand 1 (PD-L1) [25]. However, not all tumors or malignant cells express PD-L1, despite its presence in the cytoplasm and plasma membrane of cancer cells. Figure 8A shows the many pro-inflammatory molecules, VEFG, GM-CSF, LPS, TNF-α, types I and II IFN-γ, IL-4, and IL-10 cytokines, that initiate PD-L1 expression. Among them, activated T cells release the most effective trigger, IFN-γ [21], while TGF-β, PTEN, and p53 inhibit PD-L1 expression (Fig. 8B). In contrast to PD-L1, which can be expressed in several cell types, PD-L2 expression is limited to APCs [24].

Fig. 8.

PD-L1 regulatory signaling pathway [23]. Created with Biorender.com.

Studies have shown that PD-L1 expression is regulated by several signaling pathways, transcriptional factors, and epigenetic factors [24].

5.2.2.Hedgehog signaling pathway

Recent therapeutic advancements have focused on the utilization of small molecular inhibitors, such as SMO and GLI, to modify the Hedgehog (Hh) signaling pathway. This pathway has been deemed crucial for the proliferation of substrate cells, and deviations in its functioning can result in the development of tumors [37]. Furthermore, Hh signaling could have a role in the regulation of PD-L1 production, and the inhibition of Hh signaling may elicit anti-tumor activity in lymphocytes [38]. According to Chakrabarti et al. [39], specific patients should receive a combined treatment approach that targets both the Hh signaling and immunological checkpoints since Hh signals are involved in PD-1/PD-L1 inhibition, which leads to the reversal of Gli2-induced tolerance and the expression of PD-L1 in gastric cancer.

Fig. 9.

PD-1/PD-L1 signaling reduces proliferation, survival, and cytokine production of CD8+ T cells [25]. Created with Biorender.com.

5.3.PD-1/PD-L1 signaling

Numerous studies have reported that immunopathology and immunological tolerance inhibitory signals are conveyed through interactions between PD-L1 and PD-1. PD-1/PD-L1 signaling can hinder the immune response through the phosphoinositide 3-kinase (PI3K), T-cell receptor (TCR), and phosphatase-1 (SHP-1) signaling pathways (Fig. 9) [23]. The effect of PD-L1 on effector T cells and PD-1 inhibits TCR signal transduction, which in turn suppresses T cell cytotoxicity. The activity of immunoreceptor tyrosine-based switch motifs (ITSM) and tyrosine-based inhibitory motifs (ITIM) may be enhanced when SHP-1 is suppressed and CD8+ T cell activity is controlled by sodium stibogluconate. The ITSM motif of PD-1 may be tyrosine phosphorylated by ITSM, which then transports SHP-2 and SHP-1 to the ITSM motif. Following this absorption, the signaling pathway prevents dendritic cells and T cells from interacting and suppressing the stop signal. Lastly, blocking TCR signal transmission inhibits MAPK and PI3K/Akt signaling. In addition, the activation of Akt and inhibition of PI3K reduce B-cell lymphoma-extra-large (Bcl-xl) expression, which in turn increases T cell apoptosis [23].

PD-L1 blocks the Ras/MEK/ERK and PI3K/Akt pathways, which prevents the activation of T cells and the operation of transcription factors that are critical for T cell survival. The interaction between PD-1 and PD-L1 could inhibit the expression of T-bet and GATA-3. The GATA-3 transcription factor is involved in Th2 cell production, while the T-bet transcription factor could enhance T-cell proliferation by binding to the T-box region [23].

However, it is unclear whether all types of cancer use the exact mechanism of PD-L1 signaling since some cancer types differ in their prognoses, possibly due to the involvement of different PD-L1 mechanisms [25].

Fig. 10.

PD-1/PD-L1 inhibitors in malignancies [43]. Created with Biorender.com.

6.1.Breast cancer

PD expression has been correlated with epithelial-mesenchymal transition (EMT) in human breast cancer stem cells (BCSCs). The observed increase in PD-L1 expression in MCF-7 and BT-549 tumorspheres when compared to the cell line may be partly attributed to demethylation inside the PD-L1 promoter region. In addition, the overexpression of histone acetylase in the tumor can accelerate PD-L1 transcription. The distribution of slow histones in the PD-L1 promoter region is less evident in the tumor than it is in the cell line [40]. These results indicate that PD-L1 has an important function in BCSCs.

Recent pathological investigations have determined that multiple subtypes of estrogen receptor (ER) α-negative BC contained increased levels of PD-L1. Liu et al. revealed a significant discrepancy in the mean expression of PD-L1 mRNA between breast cancer cell lines that were negative for ERα and those that were positive for Erα. Therefore, the expression of PD-L1 in ER-negative (MDA-MB-231 and BT549) and ER-positive breast cancer cell lines (MCF7 and T47D) was examined. The protein and mRNA levels of PD-L1 in the T47D and MCF7 cells were significantly lower than those in the MDA-MB-231 and BT549 cells, according to RT-qPCR and western blot studies [41]). Recent studies have shown that PD-L1 suppresses the development of cancer cells and that doxorubicin-induced and spontaneous death in BC cells are increased when PD-L1 is silenced [42].