Immune checkpoint molecules signal regulatory protein alpha in the development of hepatocellular carcinoma

Abstract

Hepatocellular carcinoma (HCC) is a primary malignant tumor of the liver and one of the most common malignant tumors, as well as the third leading cause of cancer-related death. In recent years, immune checkpoint inhibitors have emerged as a key strategy in cancer treatment. However, anti-programmed cell death 1/programmed death ligand 1 therapies, one of the main immunotherapeutic approaches, only elicit a response in only approximately 20% of advanced HCC. This suggests that there may be other immune checkpoints playing important roles in HCC immunotherapy. Recent studies have highlighted Signal regulatory protein alpha (SIRPα) is a phagocytic checkpoint in macrophages and other immune cells, as a promising novel therapeutic target in tumor immunotherapy. This review summarizes current progress on SIRPα in HCC and identifies key challenges for future related research.

Key Words: Signal regulatory protein alpha; Hepatocellular carcinoma; Immunotherapy; Immune checkpoint molecules; Immune checkpoint inhibitors

Core Tip: Hepatocellular carcinoma (HCC) is a primary malignant tumor of the liver and one of the most common malignant tumors, as well as the third leading cause of cancer-related death. In recent years, immune checkpoint inhibitors have emerged as a key strategy in cancer treatment. However, anti-programmed cell death 1/programmed death ligand 1 therapies, one of the main immunotherapeutic approaches, only elicit a response in only approximately 20% of advanced HCC. This suggests that there may be other immune checkpoints playing important roles in HCC immunotherapy. Recent studies have highlighted Signal regulatory protein alpha (SIRPα) is a phagocytic checkpoint in macrophages and other immune cells, as a promising novel therapeutic target in tumor immunotherapy. This review summarizes current progress on SIRPα in HCC and identifies key challenges for future related research.

INTRODUCTION

Primary liver cancer is one of the most prevalent malignancies worldwide and ranks third in cancer-related mortality. According to global cancer statistics in 2022, liver cancer ranked sixth-highest incidence[1], with hepatocellular carcinoma (HCC) accounting for approximately 90% of cases[2-5]. In early-stage HCC, radical surgical resection remains the primary treatment option. However, HCC often develops asymptomatically, and most patients (approximately 80%) are diagnosed at intermediate or advanced stages, rendering them ineligible for curative surgery[6]. Moreover, HCC exhibits a high postoperative recurrence rate of 70%, presenting substantial challenges to achieving long-term patient survival[7].

With the completion of the Human Genome Project and advances in genomic technologies, it has become evident that the key driver genes play a crucial role in tumorigenesis. The study of these genes is of strategic importance for understanding tumor origins, predicting prognosis, and developing targeted therapies and immunotherapy strategies. Among these, cancer immunotherapies, particularly immune checkpoint inhibitors, have become an essential treatment option for various cancers[8]. Clinical trials have confirmed that blocking immunosuppressive receptor pathways is a key strategy for HCC immunotherapy, yielding significant therapeutic benefits[9]. The 2020 IMbrave150 clinical trial demonstrated that in previously untreated, unresectable patients, the combination of atezolizumab [anti-programmed death ligand 1 (PD-L1)] and bevacizumab (anti-VEGF) significantly extended overall survival and progression-free survival by 6.8 months, and improved the response rate and duration of response vs standard therapy[10]. Moreover, PD-L1-high advanced HCC patients exhibit a higher objective response rate to programmed cell death 1 (PD-1)/PD-L1 inhibitors compared to controls[11]. However, only approximately 20% of patients with advanced HCC respond to anti-PD-1/PD-L1 therapy, suggesting the involvement of alternative immune checkpoints[12,13].

Signal regulatory protein alpha (SIRPα) is one of the most extensively studied immune checkpoints and a key mediator of tumor-immune crosstalk. This review synthesizes recent advances on SIRPα in HCC, delineating its mechanistic roles in tumor initiation and progression, and discusses its potential clinical applications.

THE PROTEIN STRUCTURE OF SIRPΑ

Signal regulatory protein SIRPα (also known as CD172a, PTPNS1, SHPS1, CD172A and P84), as a member of the SIRP family (SIRPα, SIRPβ1, SIRPβ2, SIRPγ and SIRPδ), is a typical inhibitory immune receptor on myeloid cell membranes (including monocytes, macrophages, neutrophils, a subset of dendritic cells and microglia). Structurally, SIRPα is a transmembrane protein encoded on chromosome 20p13 and composed of 504 amino acids (Figure 1A). Its extracellular domain contains three immunoglobulin (Ig)-like domains, classifying it within the Ig superfamily (Figure 1B). Its N-terminal domain interacts with CD47, thereby mediating signal transduction (Figure 1C). The intracellular region contains four tyrosine residues forming two immunoreceptor tyrosine-based inhibitory motifs (ITIMs), which associate with Src homology 2 domain-containing phosphatases SHP-1 and SHP-2, underscoring its immunosuppressive function[14-16]. Notably, among SIRP family members, SIRPα has the longest intracellular domain.

Figure 1 Structural characteristics of signal regulatory protein alpha. A: The three-dimensional predicted structure of signal regulatory protein alpha (SIRPα). It contains an intracellular domain, an extracellular domain, and four tyrosine residues in the cytoplasmic region; B: The structure of SIRPα. It contains three immunoglobulin like domains in the extracellular domain. It contains four tyrosine residues in the cytoplasmic region, forming two typical tyrosine immunosuppressive motifs; C: CD47-SIRPα signaling pathway. ITIM: Immunoreceptor tyrosine-based inhibitory motif; SIRPα: Signal regulatory protein alpha. Parts of Figure 1 were created in pymol.

SIRPα is selectively expressed on the membrane surface of myeloid cells and nerve cells such as macrophages, neutrophils, dendritic cells[17,18], CD8+ T cell subsets during chronic infection[19], and brain tissue[20]. In contrast, it is absent on normal mature red blood cells, B cells, and T cells (https://www.proteinatlas.org). As an immunosuppressive receptor, SIRPα plays a critical role in immune regulation, modulating immune responses to maintain homeostasis[15] (Figure 1).

THE CLASSICAL SIGNALING PATHWAY OF SIRPΑ

The CD47-SIRPα signaling pathway is currently the most extensively characterized classical signaling pathway mediating tumor immune escape in the occurrence and development of tumors. As a ligand for SIRPα, CD47 has five transmembrane domains and a single extracellular IgSF domain[21,22]. Unlike SIRPα, which selectively expresses on myelloid cells such as macrophages, CD47 exhibits ubiqutions expression across various cell types[23], with frequent overexpression in cancer cells[24], including HCC cells[25]. After binding to CD47, SIRPα on macrophages triggers an inhibitory signaling cascade that suppresses phagocytosis[23]. This interaction enables CD47 to transmit a "don't eat me" signal to macrophages and mediates the phosphorylation of ITIMs in SIRPα’s cytoplasmic tail, promoting immune escape and protecting tumor cells[26] (Figure 1C). Beyond SIRPα, CD47 also binds to its family member SIRPγ. Multiple studies have confirmed that CD47 is highly expressed on normal red blood cells, preventing macrophage-mediated phagocytosis, whereas the loss of CD47 expression in aging red blood cells can triggers their phagocytic clearance[27].

In addition, abnormal expression of SIRPα on macrophages heightens their susceptibility to red blood cells, leading to increased clearance. Similarly, platelets and lymphocytes also inhibit macrophage phagocytosis through the CD47-SIRPα signaling pathway. These findings demonstrate that SIRPα serves as an innate immune sensor that recognizes self-antigen signals on the surface of host cells, especially CD47. Although CD47 is the main ligand for SIRPα, surfactant protein (SP)-A and SP-D have been identified as ligands for SIRPα expressed on alveolar macrophages, potentially serving as potential inhibitors of apoptotic cell phagocytosis[28].

THE ROLE AND CLINICAL SIGNIFICANCE OF SIRPΑ IN THE DEVELOPMENT OF HEPATOCELLULAR CARCINOMA

Molecular mechanisms of SIRPα in HCC progression

SIRPα primarily functions as an inhibitory immune receptor on macrophages, interacting with highly expressed CD47 on tumor cells to facilitate immune evasion[29-31]. Inhibiting SIRPα function can block the CD47-SIRPα pathway to promote tumor phagocytosis and myeloid cell clearance[32,33]. Analysis of The Cancer Genome Atlas (TCGA) data showed that both SIRPα and CD47 were highly expressed in several tumor types, including cholangiocarcinoma, esophageal cancer, head and neck squamous cell carcinoma, and gastric adenocarcinoma. This overexpression pattern suggests these tumors may utilize CD47-SIRPα signaling pathway to mediate immune suppression and promote immune escape. While SIRPα expression is at an intermediate level in HCC, TCGA data analysis shows that high expression of SIRPα is positively correlated with poor prognosis in HCC patients (Figure 2A). Further analysis of the HCCDB database indicates that SIRPα expression is higher in tumors compared to normal tissues (Figure 2B). Additionally, SIRPα expression levels showed an increasing trend in normal tissues, stromal tissues, and tumor tissues of HCC (Figure 2C) (http://Lifeome.net/database/hccdb/home.html). Although current studies support the role of SIRPα in the occurrence and development of HCC, the precise molecular mechanism underlying its function remain to be fully elucidated.

Figure 2 The expression of signal regulatory protein alpha in adjacent and tumor tissues of hepatocellular carcinoma. A: The overall survival of signal regulatory protein alpha (SIRPα) (The Cancer Genome Atlas, http://gepia.cancer-pku.cn/). High expression of SIRPα is positively correlated with poor prognosis in hepatocellular carcinoma (HCC) patients; B and C: Expression patterns of SIRPα in tumor, stromal and normal tissues of HCC. The expression of SIRPα showed an increasing trend in normal tissues, stromal tissues, and tumor tissues of HCC (http://Lifeome.net/database/hccdb/home.html). SIRPα: Signal regulatory protein alpha.

Qin et al[34] found that SIRPα was closely related to the occurrence, progression, and liver regeneration processes of HCC. This study showed that SIRPα overexpression in Sk-Hep-1 cells significantly upregulated the protein expression of nuclear factor kappa-B (NF-κB), and downregulated the protein expression of P65, P50, and cyclin D1. They proposed that SIRPα may negatively regulate the abnormal proliferation of HCC cells by modulating the protein content and localization of NF-κB, thereby affecting the expression of cyclins (particularly cyclin D1) in the signaling pathway. This is the first study to explore the mechanism of SIRPα in HCC development, providing a theoretical foundation and direction for future research. Meanwhile, Pan et al[35] reported that SIRPα expression was downregulated in monocytes/macrophages derived from the paracancerous tissues of HCC patients, with a partial recovery observed in intratumoral macrophages. Co-culture with HCC cells further suppressed SIRPα expression in macrophages. Mechanistically, SIRPα knockdown sustained the activation of NF-κB and PI3K-Akt signaling upon macrophage-tumor cell interaction, promoting macrophage migration, survival, and proinflammatory cytokine secretion. These finding suggest that SIRPα plays a key role in macrophage functional polarization within the tumor microenvironment and may contribute to tumor progression. Thus, SIRPα represents a potential therapeutic target for HCC treatment (Figure 2).

Therapeutic value of the CD47-SIRPα signaling pathway

Willingham et al[24] focused on the traditional mechanism of SIRPα in tumors: The CD47-SIRPα signaling pathway. They found that CD47 expression was 3.3 times higher in tumor cells compared to their corresponding normal counterparts. Based on the author's previous research: Blocking CD47-mediated SIRPα signaling with targeted monoclonal antibodies (mAbs) has been shown to promote phagocytosis of leukemia, lymphoma, and bladder cancer cells by macrophages[36-39]. Recent studies have expanded this approach to solid tumors, demonstrating that solid tumor cells treated with anti-CD47 blocking antibodies are efficiently phagocytosed by macrophages. Additionally, anti-CD47 treatment significantly inhibits the growth of various solid tumors, including HCC. This shows that the expression of CD47 is common mechanism used by human solid tumor cells (including HCC) to escape phagocytosis. Disrupting the CD47-SIRPα signaling pathway enables phagocytosis of solid tumor cells in vitro and inhibit tumor growth in the orthotopic xenotransplantation model through blocking CD47 mAbs. These findings are expected to apply all approaches of interfering with CD47-SIRPα interactions.

Acidic microenvironment[40], like hypoxia[41,42], inflammation or immune response, is a hallmark of the tumor microenvironment[43-45]. Jiang et al[46] compared risk scores with immune checkpoint expression and found that the high-risk group exhibited significantly higher levels of TNFSF4, SIRPα, CD276, and TNFSF15., indicating that high-risk individuals may potentially response to immunotherapy targeting these molecules. This suggests that high-risk patients may be more responsive to certain immunotherapies (such as anti-TNFSF4, anti-SIRPα, anti-CD276, and anti-TNFSF15), providing valuable clinical treatment insights for HCC patients.

Novel mechanism of SIRPα in regulating T-cell immune responses

Tomiyama et al[47] analyzed the expression of SIRPα by studying RNA sequencing data from 372 HCC tissues from the TCGA dataset and immunohistochemical staining of a cohort of 189 HCC patients. In a cohort of 189 patients, high expression of SIRPα was associated with lower recurrence free survival (RFS). High expression of SIRPα is associated with higher microvascular infiltration rates and lower serum albumin levels, as well as increased intratumoral infiltration of CD68 positive macrophages and myeloid derived suppressor cells (MDSCs). In spatial transcriptome sequencing, SIRPα expression is significantly correlated with CD163 expression. The high expression of SIRPα in HCC suggests poor prognosis and may be achieved by inhibiting macrophage phagocytosis of tumor cells, promoting MDSC infiltration, and inducing anti-tumor immunity. In HCC patients, SIRPα blockade can be considered to inhibit the development of HCC. Meanwhile, multiple factor analysis showed that patients with high expression of SIRPα, low infiltration of CD8+ T cells and high infiltration of MDSCs had significantly reduced RFS and OS rates[47]. This observation raises the question whether SIRPα exerts additional mechanisms of action involving CD8+ T cells beyond the conventional CD47-SIRPα signaling pathway. Huang et al[48] precisely confirmed this point. They found that compared to WT mice, KO-Sirpα (-/-) mice had significantly slower tumor growth and significantly prolonged overall survival after subcutaneous tumor loading with hepa 1-6 cells. At the same time, by injecting three times the number of tumor cells into SIRPα (-/-) mice whose subcutaneous tumors disappeared, they found that the tumor was completely suppressed, indicating that SIRPα (-/-) mice established strong immune memory after tumor stimulation, which usually relies on adaptive immune cells, particularly T cells. They found that SIRPα deficiency not only greatly inhibits the development of multiple solid tumors, but also is not related to CD47 expression on tumor cells. Instead, SIRPα (-/-) macrophages promote T cell recruitment into tumors through Syk/Btk dependent Ccl8 secretion[48]. This suggests that targeting SIRPα on bone marrow-derived cells may be a new approach to optimize solid tumor immunotherapy.

FUTURE CHALLENGES FOR SIRPΑ IN HEPATOCELLULAR CARCINOMA

Open up new avenues for research and treatment of tumor immunity

Although many mechanisms and functions of SIRPα in tumor immune processes remain unclear, some studies have shown that SIRPα is expressed on macrophages[49] and reduces macrophage phagocytic capacity through the CD47-SIRPα signaling pathway, leading tumor immune escape. This has been confirmed in various tumors[36-39]. This suggests that SIRPα may have important biological functions between tumors and the immune system. Consequently, SIRPα can serve not only as a biomarker, but also as a target for specific antibodies to relieve immune suppression, and enhance the anti-tumor activity of immune cells[24]. Additionally, preclinical studies demonstrate that combining SIRPα-CD47 inhibitors with antibodies that promote antibody-dependent cellular phagocytosis (ADCP), such as rituximab, enhance ADCP against cancer cells and improve anti-tumor efficacy[50-53]. According to ClinicalTrials.gov (https://clinicaltrials.gov/ct2/home), since 2016, it has been a total of 16 clinical trials related to SIRPα (Table 1), mostly focused on blocking the CD47-SIRPα signaling pathway (such as anti-SIRPα Ab or anti-CD47 Ab) to relieve tumor immune suppression, as well as combination therapy with other immune checkpoint antibodies or chemotherapy for various tumors. Currently, numerous clinical trials targeting SIRPα are underway, but there is only one clinical trial on HCC.

Table 1 Clinical trials related to signal regulatory protein alpha.

Tumor	Interventions	Phase	Number enrolled	NCT number	Study start	Sponsor/collaborators
Lymphoma	Ontorpacept	II (recruiting)	41	NCT05507541	April 19, 2023	Mayo Clinic
Advanced solid tumor; breast cancer	Drug: DS-1103a; Drug: T-DXd	I (recruiting)	78	NCT05765851	May 30, 2023	Daiichi Sankyo
Colorectal cancer	Drug: Evorpacept (ALX148); Drug: Cetuximab; Drug: Pembrolizumab	II (active, not recruiting)	80	NCT05167409	July 28, 2022	University of Colorado, Denver, CO, United States
Leukemia, myeloid, acute; myelodysplastic syndromes	Drug: CC-95251; Drug: Azacitidine; Drug: Venetoclax	I (completed)	56	NCT05168202	January 19, 2022	Bristol-Myers Squibb
Solid tumors; mycosis fungoides; melanoma	TTI-621 + PD-1/PD-L1 Inhibitor	I (terminated)	56	NCT02890368	September 2016	Pfizer
Lymphoma	Drug: CD47 Antagonist ALX148; Drug: Lenalidomide; Biological: Rituximab	I/II (recruiting)	60	NCT05025800	October 13, 2021	M.D. Anderson Cancer Center
Hepatocellular carcinoma	SIRPα Ab	Complete	30	NCT02868255	January 13, 2016	Nantes University Hospital
Acute myeloid leukemia	Drug: Evorpacept; Drug: Venetoclax; Drug: Azacitidine	I (terminated)	14	NCT04755244	May 5, 2021	ALX Oncology Inc.
Higher risk myelodysplastic syndromes	Drug: Evorpacept; Drug: Azacitidine	I/II (active, not recruiting)	65	NCT04417517	October 2, 2020	ALX Oncology Inc.
Multiple myeloma	Drug: Elranatamab; Drug: Carfilzomib; Drug: Maplirpacept	I (recruiting)	90	NCT05675449	December 14, 2022	Pfizer
Brain cancer	Drug: Magrolimab	I (completed)	13	NCT05169944	April 22, 2022	University of California, San Francisco, CA, United States
Multiple myeloma	Drug: TTI-622; Drug: Daratumumab Hyaluronidase-fihj	I (active, not recruiting)	7	NCT05139225	October 28, 2021	Memorial Sloan Kettering Cancer Center
Bladder cancer; urothelial carcinoma	Drug: Evorpacept; Drug: Enfortumab Vedotin	I (recruiting)	30	NCT05524545	November 2, 2022	ALX Oncology Inc.
Advanced solid cancers; hematologic cancers	Drug: SRF231	I (completed)	148	NCT03512340	March 13, 2018	Surface Oncology
Head and neck squamous cell carcinoma	Drug: BI 765063; Drug: Ezabenlimab	I (active, not recruiting)	48	NCT05249426	April 12, 2022	Boehringer Ingelheim
Breast cancer	Drug: ALX148; Drug: Fam-Trastuzumab Deruxtecan-Nxki; Drug: Zanidatamab	I (recruiting)	54	NCT05868226	December 22, 2022	QuantumLeap Healthcare Collaborative

PD-L1: Programmed death ligand 1; PD-1: Programmed cell death 1.

QuantumLeap Healthcare Collaborative

Since 2016, it has been a total of 16 clinical trials related to SIRPα, mostly focused on blocking the CD47-SIRPα signaling pathway, but there is relatively little research related to HCC.

The main problems about the research of SIRPα

Currently, the roles, functions, and mechanisms of SIRPα in HCC remain largely unclear, presenting several unresolved questions. Beyond its established role in reducing macrophage phagocytic capacity and promoting tumor immune escape through the CD47-SIRPα signaling pathways, it is uncertain whether SIRPα engages other signaling pathways in HCC, as observed in other cancers. For instance, studies have identified alternative pathways such as SIRPα/interleukin-6 axis, which may promote lung cancer development[54] (Figure 3A), the upregulation of SIRPα driving osteosarcoma metastasis through the "SP1 stable loop" and SLC 7A3 mediated arginine uptake[55] (Figure 3B), and SIRPα (-/-) macrophages enhancing T cell recruitment into tumors through Syk/Btk dependent Ccl8 secretion[48] (Figure 3C). These pathways have not been thoroughly investigated in HCC, especially the upstream and downstream genes directly affected by SIRPα. Studies have shown that sustained anti-tumor immune responses require stimulation from adaptive immune cells[56], suggesting that SIRPα plays important roles in other immune cells besides macrophages. The study by Yamada-Hunter et al[57] showed that enhancing macrophage phagocytosis through CD47 blockade can improve the efficacy of CAR T cell therapy. Although T cell depletion was greatly enhanced by CD47 blockade, macrophage phagocytosis limits the persistence of adoptively transferred T cells even in the absence of CD47-SIRPα blockade[57]. It is well known that CD47 blockade selectively depletes aging red blood cells. Prolonged CD47 blockade leads to increased expression of the "eat me" signal on T cells while reducing CD47 expression. This raises the possibility that the CD47-SIRPα signaling pathway may similarly regulate the clearance of aged T cells[53]. Additionally, future research should further explore the impact of SIRPα on the functional state of T cells. For example, can SIRPA directly regulate the function of T cells (such as exhaustion or apoptosis, etc.) to promote tumor progression (Figure 3)?

Figure 3 The mechanism of signal regulatory protein alpha. A: The signal regulatory protein alpha (SIRPα)/interleukin-6 axis may promote lung cancer development; B: Upregulation of SIRPα promotes osteosarcoma metastasis through the "SP1 stable loop" and SLC 7A3 mediated arginine uptake; C: SIRPα (-/-) macrophages promote T cell recruitment into tumors through Syk/Btk dependent Ccl8 secretion.

CONCLUSION

HCC remains a major global health challenge. Although immunotherapies such as PD-1/PD-L1 inhibitors have shown promise, their efficacy is limited to some patients, and there is an urgent need to explore new immune escape mechanisms. Studies have shown that the inhibitory receptor SIRPα of myeloid cells conveys the "Don't eat me" signal by binding to tumor cell CD47. SIRPα is highly expressed in HCC and is associated with poor prognosis, macrophage dysfunction and increased infiltration of MDSC. In addition to inhibiting phagocytosis, SIRPα can also regulate NF-κB signaling, T cell recruitment and cytokine production, suggesting that it has a broader immunomodulatory function in the progression of HCC. In the future, exploring the extracellular functions of SIRPα in macrophages may become an important research direction. Future research on SIRPα in HCC could focus on: (1) Elucidating its role in ICI resistance through PD-1/CTLA-4 pathway interactions and treatment response dynamics; (2) Developing targeted therapies via HCC-specific antagonists and TACE combinations; and (3) Investigating non-canonical functions using spatial transcriptomics and metabolic profiling. These studies will employ advanced techniques (CyTOF, scRNA-seq) and models (humanized mice, organoids) to expand therapeutic strategies beyond PD-1/PD-L1 inhibition.