Bidirectional regulation of the cyclic guanosine monophosphate-adenosine monophosphate synthase-stimulator of interferon gene pathway and its impact on hepatocellular carcinoma

Abstract

BACKGROUND

Hepatocellular carcinoma (HCC) ranks as the fourth leading cause of cancer-related deaths in China, and the treatment options are limited. The cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS) activates the stimulator of interferon gene (STING) signaling pathway as a crucial immune response pathway in the cytoplasm, which detects cytoplasmic DNA to regulate innate and adaptive immune responses. As a potential therapeutic target, cGAS-STING pathway markedly inhibits tumor cell proliferation and metastasis, with its activation being particularly relevant in HCC. However, prolonged pathway activation may lead to an immunosuppressive tumor microenvironment, which fostering the invasion or metastasis of liver tumor cells.

AIM

To investigate the dual-regulation mechanism of cGAS-STING in HCC.

METHODS

This review was conducted according to the PRISMA guidelines. The study conducted a comprehensive search for articles related to HCC on PubMed and Web of Science databases. Through rigorous screening and meticulous analysis of the retrieved literature, the research aimed to summarize and elucidate the impact of the cGAS-STING pathway on HCC tumors.

RESULTS

All authors collaboratively selected studies for inclusion, extracted data, and the initial search of online databases yielded 1445 studies. After removing duplicates, the remaining 964 records were screened. Ultimately, 55 articles met the inclusion criteria and were included in this review.

CONCLUSION

Acute inflammation can have a few inhibitory effects on cancer, while chronic inflammation generally promotes its progression. Extended cGAS-STING pathway activation will result in a suppressive tumor microenvironment.

Key Words: Hepatocellular carcinoma; Cyclic guanosine monophosphate-adenosine monophosphate synthase-stimulator of interferon gene; Interferon genes; The metastasis of a tumor; Immunology

Core Tip: As a crucial signaling pathway for both innate and adaptive immune responses, studies have demonstrated that cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS)-stimulator of interferon gene (STING) pathway can be triggered by tumor DNA and genomic instability, thereby promoting or inhibiting tumor development and metastasis. A considerable amount of evidence indicates that the cGAS-STING pathway, as a potential therapeutic target, markedly inhibits tumor cell proliferation and metastasis, with its activation being particularly relevant in hepatocellular carcinoma. But at the same time, prolonged pathway activation may lead to an immunosuppressive tumor microenvironment, fostering liver damage, fibrosis, carcinogenesis, and the invasion or metastasis of liver tumor cells.

INTRODUCTION

Cancer remains a leading significant public health issue worldwide, and it ranks among the most common causes of death. Specifically, liver cancer, especially hepatocellular carcinoma (HCC), ranks fourth for its cause of death in China[1]. On the global scale, the death rate from this disease has been on the rise, having reached the third-highest by 2018 and second-highest by 2020[2]. HCC is one of the most common types of liver cancers, found all over the world[3]. Identifying HCC at an early stage enables patients to access a range of treatments, both curative and non-curative, that can be beneficial. Performing surgical resection for early-stage HCC can lead to significant improvements in survival rates, typically ranging from 50% to 70% over a 5-year period. Nevertheless, the risk of tumor recurrence and metastasis remains as high as 70%[4]. In addition, HCC can be quite elusive and has a tendency to rapidly spread. When patients are diagnosed, a significant number of them have already reached an advanced stage where surgery is no longer an option. Until recently, treatment options for individuals with advanced HCC were very limited. There are various factors at play here, such as HCC’s strong resistance to standard treatment plan and its limited tolerance for this treatment due to potential liver damage[5].

In 2007, sorafenib, a multikinase inhibitor that targets the vascular endothelial growth factor receptor family, became the first drug to demonstrate improved overall survival in advanced HCC, which was a major breakthrough in liver cancer treatment[6]. However, from 2007 to 2017, the range and effectiveness of available drugs remained limited. Indeed, the success of several clinical trials that have investigated targeted therapy in the management of a range of cancers has not even emulated or reached that of sorafenib in terms of its success at improving the prognosis or lengthening survival among HCC patients[7-10]. In 2018, Lenvatinib was approved as the first-line therapy for advanced HCC[11]. It has shown to be more effective compared to sorafenib[12].

An enormous work advancement has been observed in the understanding of the crossroads of immunology with oncology in recent years; many studies have been documented and concluded that immunology apparently plays an important role in defense mechanisms with the major purpose of preventing or protection from the process of tumorigenesis[13]. Some researchers have noted that when given to those cancer patients who do not respond to it in other ways, immunotherapy shows some of the best results, and that it does work the best for those patients who already have quite advanced stages of the disease[14]. This introduces a new perspective of cancer immunotherapy. More recent studies underline the opportunity to make the immune response of the body towards DNA, through the cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS)-stimulator of interferon gene (STING) pathway, effective and have high relevance for cancer as well[15]. Recognition of aberrant DNA species in the cytosol activates the STING signaling pathway through sensing of cytoplasmic DNA by cGAS[16]. Such activation further enhances the expression of type 1 interferon (IFN) genes[17], which is very essential for the immune response.

MATERIALS AND METHODS

This review was conducted according to the PRISMA guidelines[18].

Search strategy and databases

The study searched PubMed and Web of Science, employing combinations of the following search terms in titles, abstracts, and keywords: “cGAS-STING”, “hepatocellular carcinoma”, “cancer”, “HCC”, “liver”, and “tumor metastasis”. Relevant studies published from June 1, 2013, to May 1, 2024, and accessible online in full text were included. This review did not impose restrictions on research designs and included studies without language or publication date limits. In addition to automated searches, we manually examined the reference lists of selected articles to identify additional relevant studies.

Screening process and eligibility criteria

The articles were imported into EndNote, which intelligently removed duplicates. We then alphabetically organized the titles and manually removed duplicates to ensure completeness.

Articles that met the eligibility criteria proceeded to the next phase of screening, where Xiao ZH and Deng JL assessed them for: The regulatory, promotional, and inhibitory effects of cGAS-STING on tumors; The correlation between HCC and cGAS-STING; and the impact of inhibitors or agonists on HCC metastasis and invasion. only studies published in English were considered for the meta-analysis.

RESULTS

All authors collaboratively selected studies for inclusion, extracted data, and the initial search of online databases yielded 1445 studies. After removing duplicates, the remaining 964 records were screened. Ultimately, 55 articles met the inclusion criteria and were included in this review. Figure 1 displays the PRISMA 2020 flowchart.

Figure 1 Selection process diagram.

Understanding the structure and signal transduction of the cGAS-STING pathway

cGAS is a cytosolic sensor for diverse forms of double-strand DNA (dsDNA)[19]. Direct binding with dsDNA activates cGAS, which adopts a conformational change[20] that permits the catalysis of cGAS to produce cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) using adenosine triphosphate (ATP) and guanosine triphosphate (GTP)[21,22]. This way, it can identify exogenous DNA from pathogens and host endogenous DNA[23], such as mitochondrial DNA (mtDNA), micronucleus DNA, and cytoplasmic chromatin[24-27]. In its capacity as a second messenger, cGAMP interacts with the STING protein in the endoplasmic reticulum (ER)[28], inducing the oligomerization of STING into a tetramer and, eventually, a more complex structure[29]. This interaction also results in the movement of STING to the Golgi intermediate compartment and the Golgi apparatus[30]. The C-terminal tails of the STING dimer engage with cGAMP upon binding, leading to enhanced TANK-binding kinase 1 (TBK1) phosphorylation and activation of TBK1[29-33]. With the support of TBK1, STING is able to activate interferon regulatory factor 3 (IRF3) after it has been phosphorylated[34,35]. Once activated, IRF3 is involved in phosphorylation, dimer formation, and nuclear translocation to initiate IFN-I transcription[32,36]. Meanwhile, STING causes IκB kinase (IKK) to be activated, leading to the phosphorylation[37], ubiquitination[38] , and proteasomal destruction of proteins belonging to the IκB family[39,40]. When IκB degrades, nuclear factor kappa-B (NF-κB) is liberated and relocates to the nucleus. The process of transcribing different cytokines, like IFN-β, interleukin (IL)-6, and tumor necrosis factor (TNF), begins when it binds to the κB site on DNA[41-44]. Figure 2 shows the cGAS-STING pathway.

Figure 2 Molecular mechanism of cyclic guanosine monophosphate-adenosine monophosphate synthase-stimulator of interferon gene signalling. Cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS) is an integral protein responsible for detecting a diverse array of cytosolic double-stranded DNA (dsDNA). Upon binding to dsDNA, cGAS utilizes adenosine triphosphate and guanosine triphosphate as substrates to catalyze the synthesis of cyclic guanosine monophosphate-adenosine monophosphate, serving as a crucial second messenger that activates stimulator of interferon gene (STING). Subsequently, STING undergoes translocation from the endoplasmic reticulum to Golgi compartments, where it orchestrates the recruitment of kinases such as TANK-binding kinase 1 (TBK1) and IκB kinase. These kinases, in turn, catalyze the phosphorylation of interferon regulatory factor 3 (IRF3) and the nuclear factor-κB (NF-κB) inhibitor, IκBα. Phosphorylated IRF3 transits to the nucleus, prompting the transcription of type I interferon genes. Concurrently, phosphorylated IκBα engages NF-κB, initiating the transcription of genes that encode proinflammatory cytokines. dsDNA: Double-stranded DNA; cGAS: Cyclic guanosine monophosphate-adenosine monophosphate synthase; cGAMP: Cyclic guanosine monophosphate-adenosine monophosphate; ATP: Adenosine triphosphate; GTP: Guanosine triphosphate; STING: Stimulator of interferon gene; NF-κB: Nuclear factor-κB; TKB1: TANK binding kinase 1; IRF3: I nterferon regulatory factor 3; ER: Endoplasmic reticulum; IFN: Interferon; IL: Interleukin; TNF: Tumor necrosis factor.

The dual-regulation mechanism of cGAS-STING in cancer

Antitumor mechanisms: Tumor cells, unlike normal cells, generally harbor abundant cytoplasmic dsDNA. Chromosomal instability (CIN) is widely recognized as a key contributor to cytoplasmic dsDNA, correlating with tumor progression, distant metastasis, and resistance to therapy[45]. When chromosomes are improperly segregated, chromosomal breaks may occur, potentially leading to oncogenic mutations. These lagging chromosomes often produce micronuclei in a cell cycle-dependent manner[46]. Approximately half of these micronuclei, with compromised membranes, rupture and release their genomic content into the cytoplasmic sol, thereby activating the cGAS-STING pathway[47]. Additionally, external factors like chemotherapy and radiotherapy can also induce DNA damage, which results in mtDNA leakage and further promotes a cGAS-STING-mediated antitumor response[48].

DNA damage can prompt acute STING signal transduction, resulting in cellular senescence[49]. Cellular senescence is characterized by a state of permanent growth arrest in damaged or aging cells and represents an irreversible halt in the cell cycle[50]. Activation of STING signaling impacts the release of pro-inflammatory cytokines, chemokines, proteases, and growth factors, collectively referred to as the senescence-associated secretory phenotype (SASP), which restricts tumor progression. SASP also promotes immune cell recruitment and the clearance of senescent cells[51]. The cGAS-STING pathway mainly regulates cellular aging through the secretion of soluble factors and other autocrine/paracrine signal transduction mechanisms[52]. Additionally, the downregulation of nuclear lamina protein B1 in senescent cells can lead to nuclear envelope rupture and the formation of cytoplasmic chromatin fragments, which further activates acute STING signaling. This evidence suggests that the cGAS-STING pathway plays a central role in regulating cellular senescence and activating SASP through endogenous DNA recognition[53,54]. Moreover, SASP signals to the immune system, regulating the tissue microenvironment. In a liver cancer model, senescent hepatocytes attract various immune cells through SASP, especially natural killer (NK) cells and neutrophils, which help eliminate tumor cells. The cGAS-STING pathway is critical for the antitumor effects mediated by SASP[55]. In STING-deficient mouse models, immune cell infiltration is reduced compared to controls, with observed reductions in the clearance of early senescent cells and substantial tumor growth in later stages in the experimental group[24].

The STING signaling pathway in cancer cells also promotes their own apoptosis. Activation of the cGAS-STING pathway generates substantial amounts of type I IFN, which plays a critical role in antitumor activity. Type I IFN recruits cytotoxic T cells to cancer cells and initiates a type I T helper cell response[56]. Furthermore, type I IFNs aid in the maturation of dendritic cells, which present tumor-specific antigens to cluster of differentiation (CD) 4 + and CD8 + T cells for protective immune responses[57]. CD4 + T cells serve not only as helper cells but also exhibit direct cytolytic activity[58], playing a crucial role in antiviral and antitumor immunity. Additionally, as the primary executors of acquired cellular immunity, CD8 + T cells possess significant cytotoxic potential when encountering cancer cells[59]. Upon phagocytosis of tumor cells by antigen-presenting cells, dendritic cells uptake tumor-associated antigens and migrate to tumor-draining lymph nodes via lymphatic vessels, where they present these antigens to T cells. This presentation activates CD8 + T cells, which then proliferate within the tumor-draining lymph nodes and subsequently travel through the bloodstream to target and eliminate cancer cells[60]. These findings suggest that type I IFN generation is closely linked to T cell activation and the antigenic response of cancer cells. Activation of the cGAS-STING pathway in cancer cells may inhibit tumor progression by upregulating various inflammatory genes, including type I IFN.

Tumor-promoting mechanism: While substantial evidence demonstrates that SASP functions as an antitumor component, it is crucial to recognize its potential role in promoting tumorigenesis[61]. Prolonged SASP activity can drive epithelial-mesenchymal transition and increase invasiveness, mainly through a paracrine mechanism involving SASP factors like IL-6 and IL-8. Simultaneously, it accelerates and intensifies the loss of function of the tumor suppressor protein p53 and increases expression of the oncogene RAS, contributing to genotoxic stress and cellular aging in normal cells[62]. RAS, a driver of cell proliferation in various cancers, can further exacerbate genotoxic stress and aging, causing senescent cells to exhibit unintended tumor-promoting effects[63]. Genotoxic stress activates both p53 and SASP; activation of p53 can halt cellular aging, thereby exerting antitumor effects. However, p53 also acts to inhibit SASP. When p53 function is compromised alongside cellular damage, SASP expansion is promoted. Furthermore, cells lacking p53 that escape senescence in response to genotoxic stress pose a significant threat, as they not only proliferate but also drive further SASP expansion, exacerbating tissue harm[62,64].

Additionally, senescent cells release cytokine receptors, which can assist neighboring precancerous or malignant cells in evading immune surveillance[65]. The persistent presence of senescent cells within tissues may create a pro-inflammatory environment, ultimately promoting tumor growth and metastasis[66].

Although the cancer surveillance function of the cGAS-STING pathway is well-documented, growing evidence suggests that cancer cells may deactivate cytoplasmic DNA-sensing pathways to evade immune detection. The chronic release of DNA activates the cGAS-STING-mediated IFN signaling pathway, posing a threat to cancer cells. Consequently, to counteract this inhibitory effect, cancer cells suppress this pathway to support tumor growth and metastasis. Studies have shown that in certain advanced cancer cells, the expression levels of cGAS and STING are downregulated[67,68]. This observation implies that cancer cells can downregulate cGAS and STING, promoting immune evasion in a manner dependent on the tumor microenvironment[69]. Micronuclei rupture, resulting from chromosomal segregation errors, releases genomic DNA into the cytoplasmic sol. This activates the cGAS-STING pathway and downstream non-canonical NF-κB signaling, with NF-κB known to drive inflammation, immune diseases, and tumor development and progression[70]. In highly aneuploid tumor models, genetic suppression of CIN significantly delays metastasis, while the induction of persistent chromosomal segregation errors promotes cell invasion and metastasis in a STING-dependent manner[71,72].

Autophagy in tumor: Autophagy is a cellular process in which the cytoplasmic sol and organelles are enclosed within double-membrane vesicles[73]. cGAMP-induced autophagy is crucial for the clearance of DNA or viruses within the cytoplasmic sol. Recent studies indicate that STING can induce autophagy in response to cGAMP stimulation without triggering IFN production. STING is also capable of activating non-canonical autophagy independently of IFN induction[74]. This activation of autophagy is vital for cell death and plays a central role in preventing cancer progression; hence, loss of autophagy is considered necessary for tumor development. Research by Nassour et al[75] has demonstrated that the terminal response to replication crisis in early cancer cells results in mitotic delay, unprotected telomere elongation, and cell death. Knocking out cellular cGAS and STING allows these cells to bypass crisis proliferation, showing decreased LC3-II levels and p62 accumulation. Accumulation of p62 leads to overactivation of the tumor necrosis factor receptor-associated factor 6-NF-κB pathway, promoting caspase-8 aggregation and interaction with the Cullin 3-type ubiquitin ligase Kelch like ECH associated protein 1 associated with nuclear factor-erythroid 2-related factor 2. This process facilitates the formation of intracellular inclusion bodies, potentially leading to pro-tumorigenic signaling[76]. Multiple preclinical studies suggest that autophagy supports late-stage tumor growth by activating various oncogenes and/or inactivating tumor suppressor genes, sustaining metabolic pathways within cancer cells, and promoting survival in challenging tumor microenvironments. Additionally, autophagy contributes to the effectiveness of various anticancer therapies[77-79].

Effect on HCC: The close association between DNA damage and cancer is well established. The initiation of the cGAS-STING signaling pathway, triggered by the recognition of abnormal dsDNA, leads to antigen presentation by dendritic cells, culminating in the activation and differentiation of T cells and the production of IFN-I with antitumor effects, significantly enhancing the host’s resistance to tumor cells. Interactions between dendritic (DC)-T cells also occur in the liver[80]. Targeted strategies to enhance the function of DC cells, such as the injection of DC vaccines, have been used to bolster antitumor responses[81]. DC-based immunotherapies can yield better results, improving the CD4 + T/CD8 + T cell ratio and ensuring patient safety. In HCC, CD8 + and CD4 + T cells are enriched within the tumor and the peritumoral area[82]. Flecken et al[83] found that the occurrence of tumor-related antigen-specific CD8 + T cell responses in HCC patients is associated with improved progression-free survival, observable in over 50% of patients. It is hypothesized that tumor-related antigen-specific CD8 + T cell responses may inhibit tumor growth and prolong the duration of early-stage disease, thereby benefiting patient survival. In addition, experiments have shown that lipid metabolism disorders in nonalcoholic fatty liver disease lead to a selective loss of CD4 + T cells in the liver, where CD4 + T lymphocytes have a greater mitochondrial mass than CD8 + T lymphocytes and produce higher levels of mitochondrial reactive oxygen species (ROS). Reversing lipid metabolism disorder-induced reductions in hepatic CD4 + T lymphocytes can also delay HCC[84]. Treatment of HCC with tetrathiomolybdate has shown that it induces the accumulation of ROS and activates various cell death pathways, including necroptosis and ferroptosis. This results in mitochondrial damage and the release of mtDNA into the cytoplasm, which subsequently activates the cGAS-STING-IFN axis, promoting the infiltration and activation of CD8 + T cells[85].

However, the impact of cGAS-STING on HCC has both benefits and drawbacks. Trials involving the treatment of HCC with tetrathiomolybdate have also found that the IFN pathway induces the expression of Programmed cell death 1 ligand 1 (PD-L1) in liver cancer cells, facilitating immune evasion[85]. Chromosome instability and the presence of tumor byproducts in the form of self-DNA can activate the cGAS-STING pathway, thereby either promoting or inhibiting tumor development. Ma et al’s team demonstrated that disruption of the BRCA1 and PALB2 interaction in mice leads to persistent high levels of DNA damage in HCC cells, activating the cGAS-STING pathway in malignant hepatocytes and M1 macrophages in the tumor microenvironment[86]. This activation, through the STING-IRF3-signal transducer and activator of transcription 1 pathway, induces PD-L1 expression, causing immunosuppression and promoting tumor occurrence and progression while recruiting T lymphocytes, leading to their infiltration into the tumor[86]. Consequently, blocking the PD-L1 pathway has shown significant antitumor effects in advanced cancer patients. Activating the cGAS pathway promotes IFN expression and downregulates PD-L1 expression, effectively preventing the progression and metastasis of HCC[87]. Numerous studies have confirmed that blocking PD-L1 enhances the anti-HCC properties[88-90]. Figure 3 shows the role of the cGAS-STING signaling pathway in HCC.

Figure 3 The role of the cyclic guanosine monophosphate-adenosine monophosphate synthase-stimulator of interferon gene signaling pathway in hepatocellular carcinoma. The pivotal role of the cyclic guanosine monophosphate-adenosine monophosphate synthase-stimulator of interferon gene (cGAS-STING) signaling pathway in hepatocellular carcinoma (HCC) is profound. When DNA damage occurs within HCC cells, it triggers the formation of double-stranded DNA. This, in turn, stimulates and activates the cGAS-STING signaling cascade, ultimately inducing autophagy and activating the apoptotic process. Furthermore, this pathway facilitates the release of cytokines, which subsequently elicits a robust systemic antitumor immune response. This immune response is instrumental in controlling both local and distant metastasis, thereby mitigating the progression of tumor growth.

The impact of the cGAS-STING pathway on tumor development

Facilitate tumor growth: Ongoing cGAS-STING pathway activation has the ability to enhance tumor development and metastasis. By maintaining an immune-suppressing tumor microenvironment, persistent activation of this signaling pathway can aid tumor development, metastasis, and treatment resistance[15,91]. In fact, higher STING expression levels have been associated with a poor outcome in certain colorectal cancer cases[60], again hinting that this pathway may actually be taking part in driving tumor growth and thus preventing its elimination by the immune system. In fact, it has been demonstrated that the stimulation of STING has the power to inhibit the immune response against the tumor in NK cells because it produces IL-35, which further potentiates the Breg cell function[92]. In addition, the activity of STING within the tumor microenvironment enhances indoleamine 2,3-dioxygenase (IDO) activity, which stimulates the advancement of Lewis lung cancer[93]. In this line, the observation by Cheng et al[94] showed that enhanced levels of mitochondrial Lon resulted in an increase in the production of ROS and oxidative damage to mtDNA. mtDNA, when damaged and released in the cytosol, stimulates an IFN signaling pathway in cancer cells via the STING-TBK1 pathway, followed by the upregulation of the expression of PD-L1 and IDO-1. This upregulation process inhibits the T-cell activation, thus resulting in weak T-cell immunity within the tumor microenvironment, an environment that ultimately may contribute to promoting the tumor.

Facilitate tumor spread: Finally, the STING signaling pathway has been found to be upregulated by IDO activity, known to assist in facilitating tumor growth[93], as it catalyzes the first, rate-limiting enzymatic step in the conversion of L-tryptophan to N-formylkynurenine in an effort to evade immune detection and T-cell growth[95]. Studies implicate that sustained activation of the cGAS-STING pathway and type I IFN responses could be a mechanism of immune evasion of the cancer cells from being eliminated[96,97]. Moreover, the presence of CIN is very likely to bring about continuous cGAS-STING activation that would otherwise modify signaling pathways in cancer cells, supporting the building of a metastatic tumor microenvironment[98]. When cells have unstable chromosomes, most of the DNA fragments from the cells are emitted to the cytoplasm through micronuclei. This, in turn, triggers the cGAS-STING pathway not reliant on the conventional NF-κB pathway. Aneuploidy, the appearance of a variety of karyotypes, and the occurrence of micronuclei can all be grouped under the genetic abnormalities, among many others, which may thus be expected to arise out of such instability[69]. This causes a rise in the chromosomal content in these micronuclei, and when the membrane is disrupted[71], the chromosomes are released into the cytoplasm. This leads to the activation of the cGAS-STING pathway, among other signaling pathways[99,100], and raises the expression level of the p52 gene. This, in turn, causes the cells to be killed by T-cells[101].

Effect on tumor growth: Extensive research has been done on the cGAS-STING pathway in tumor cells and its potential to boost the antitumor effect[102-106]. Several clinical trials have demonstrated their promising potential in enhancing the body’s ability to fight against tumors through this pathway. Its activation is very crucial in breaking immunological tolerance and enhancing tumor-specific immunity by increasing tumor cell death, autophagy, and the IFN response. Activation of the cGAS-STING pathway leads to the induction of the type I IFN response and T cell activation to cause the regression of the tumor[69,101]. In their study, Ka et al[107], they found that nuclear receptor NR1D1 can be activated by NR1D1. This activation leads to enhanced body immunity against tumors, with decreased growth of breast cancer and lung metastasis. In a colon cancer study, the higher levels of cGAS and STING were associated with a higher expression of effector CD8 and mature CD4 gene markers compared to the lower ones[108]. In addition, research in living organisms and laboratory settings has shown considerable effects of Disitamab Vedotin, the new antibody-drug conjugate, on the reduction of human epidermal growth factor receptor 2-positive colon cancer[109]. Furthermore, it can enhance the efficacy of anti-PD-1 therapy in tumors through the cGAS-STING pathway[109]. The research by the team of Shen et al[110] reported that metformin mediates activation of the cGAS-STING signaling axis in gastric cancer by repression of SOX2/protein kinase B and further augments its antitumor effects. Further investigation revealed that Raddeanin A directly interacted with transactive responsive DNA-binding protein 43 and caused mitochondrial instability, which allowed the release of mtDNA[111]. Such processes, therefore, allowed Raddeanin A to activate cGAS and type I IFN signaling. These processes increase the antigen-presenting ability of dendritic cells and also activate T cells. These processes have the result of tumor suppression[111].

Activation of the cGAS-STING pathway by cytoplasmic DNA results in the induction of cell senescence. Pathways activated by cytoplasmic DNA promote autophagy, induced by inflammatory cytokines[75,112], and have an important role in tumor suppression. This identifies DNA in cells, activates the production of type I IFNs and SASP factors[113], together with the increase in the release of a number of cytokines and chemokines. These attract immune cells to the site, where senescent cells have also produced cytokines, leading to a permanent halt in cell division[52]. All of these processes driven by oncogene activation may normally lead to the promotion of cell senescence and, through this action, reduce cancer risk.

The damage to the tumor cells created is triggering the production of dsDNA, following which their death will also be triggered[15,114]. Similar to normal proliferative cells, mostly, if not exclusively, cancerous cells will execute aerobic glycolysis as the mode of glucose catabolism[115,116]. This change in metabolic activity during aerobic glycolysis increases ROS production in glycolytically active tumor cells. This causes cell death, which in turn releases mtDNA[117,118]. Upon detecting either endogenous or foreign DNA, immune cells initiate the cGAS-STING signaling cascade. Other recent findings suggest that cGAS can increase the body’s immune response to cancers by targeting free DNA produced from dying tumor cells[119-121].

Furthermore, the cGAS-STING pathway has been shown to activate a number of cellular events, such as apoptosis[122], autophagy[123], pyroptosis[124], ferroptosis[125], and necroptosis[126]. Therefore, new cancer treatments that target the immunopathology of the cGAS-STING pathway have a lot of promise.

The cGAS-STING signaling pathway in HCC

The suppressive impact of cGAS-STING on HCC: Liver cancer progression is aided by the cGAS-STING pathway, which allows for tumor immunosuppression and T lymphocyte infiltration in HCC[86,127]. Pu et al[128] highlighted the significance of specific genes in the cGAS-STING pathway in relation to HCC in their investigation. Researchers discovered that individuals with HCC had a worse prognosis when tumor tissues had lower levels of STING expression. Furthermore, research in animal models of HCC has shown that teniposide can improve the efficacy of anti-cancer therapies[129]. In order to accomplish this, certain pathways within the immune response are activated, and dendritic cells, which are essential for delivering antigens to T cells, are encouraged to become activated[129,130]. Also, cancer cells can be made to die when STING agonists are given to them directly. Tang et al[131] discovered that Eμ-TCL1 mice with chronic lymphocytic leukemia undergo apoptosis when exposed to the STING agonist 3’3’-cGAMP. Researchers Song et al[132] found that injecting SHR1032 directly into MC38 mice syngeneic tumors significantly reduced tumor growth. Vitamin C enhances the efficacy of immunotherapy by activating a particular route in tumors, which in turn normalizes blood vessel function. Furthermore, tumor cells are little affected by Sting deficiency caused by its ablation, but cells with high cGAS expression are unaffected[133]. Improving combination immunotherapy for liver cancer may be as simple as encouraging tumor vascular normalization, as these results highlight the tumor immune environment’s interaction with vascular endothelial cells. According to research on HCC models, cyclic dinucleotides (CDN) STING agonists can activate the TBK1/IRF3 and NF-κB signaling pathways, which in turn cause the release of type I IFN and pro-inflammatory cytokines. This, in turn, stimulates an immune response and successfully decreases the size of tumors[134,135]. By administering the DNA damage response inhibitor AZD6738 to HCC mice, Sheng et al[136] found that cGAS, phosphorylated STING (p-STING), and p-TBK1 levels were elevated. A notable impact on PD-L1 expression in HCC cells was the elevation of p-STING levels[136]. Additionally, it enhanced the CD8/CD3 T lymphocyte ratio, increased the population of activated CD8 T lymphocytes via the cGAS-STING pathway, and led to an upregulation of regulatory T cells. This led to its beneficial effect of reducing tumor growth[96]. What is more, injectable hydrogels integrated with cleaved OK-432 and doxorubicin have shown good promise for the treatment of remaining liver cancer. These hydrogels reactivate the cGAS-STING-IFN-I signaling pathway, leading to good outcomes[137]. These findings have pointed out the cGAS-STING axis as a potential therapeutic target for HCC. This pathway has been described to be capable of inducing tumor cell regression and, at the same time, to activate an immune response in diverse tumor models[131,135,138,139]. This vast collection of experimental evidence highlights promising molecular targets for further advancing HCC immunotherapy.

The role of cGAS-STING in promoting HCC: Study has found a correlation between high expression of cGAS in patients and poor survival outcomes[140,141]. Neutrophil extracellular traps (NETs) derived from diabetic patients might support the spread and invasiveness of HCC[142]. Inhibiting cGAS would, therefore, result in effectively suppressing the activation of the non-classical NF-κB pathway caused by NETs DNA and thus result in inhibiting the translocation of NF-κB RELB that causes a reduction in invasiveness into HCC cells. This study therefore uncovers the mechanism by which cGAS activation leads to the translocation of STING near the nucleus in HCC cells and thereby facilitates local invasion or metastasis. In addition, DNA damage caused by hepatocyte ferroptosis can result in liver damage, fibrosis, and the progression of cancer. On the other hand, the inhibitor C-176 has been shown to reduce tumor burden in TAK1 models by suppressing STING activity[143]. The cytoplasmic dsDNA sensing cascade of the cGAS-STING pathway plays a crucial role in initiating innate immune responses, which can lead to liver damage and autoimmune disorders[45,144]. In a study conducted by Du et al[141], it was found that the liver experiences significant DNA accumulation, leading to the production of IFN-I and activation of cGAS-STING. This, in turn, exacerbates liver cell damage. It should be appreciated that an interdependent IFN regulation in the tumors is implicated. First, the IFN signaling augments the anti-tumor activity[111,145]. Secondly, the mechanism discussed above may enhance tumor cell resistance to the immune checkpoint inhibition effect[146]. This emphasizes a subtle role of the cGAS-STING pathway in HCC.

STING agonists and inhibitors

STING agonists: Activation of the cGAS-STING pathway within the tumor microenvironment can induce effective cross-priming of tumor-specific antigens and promote effector T cell infiltration. Consequently, targeting the cGAS-STING pathway has become a high priority in drug development. To date, various STING agonists have been identified, primarily classified into the following categories: CDNs and their derivatives, 5,6-dimethylxanthenone-4-acetic acid (DMXAA) and its analogs, and small-molecule agonists.

CDNs are the first class of STING agonists to enter drug development, serving as natural ligands of STING by directly binding to and activating it. This class includes cyclic di-GMP (c-di-GMP), cyclic di-AMP, and cGAMP[147], which have shown considerable potential in cancer therapy[148,149]. CDN agonists efficiently activate the STING pathway by mimicking the structure of natural ligands, thereby triggering immune responses. The antitumor effect of this activation was first observed with c-di-GMP[150]. In a melanoma mouse model, peptide nanotubes loaded with c-di-GMP promoted the expression of INF-β, TNF-α, IL-6, and IL-1β, upregulating the activity of CD4 and CD8 T cells to kill tumor cells and intensify the immune response within tumor tissues, significantly inhibiting tumor growth in tumor-bearing mice[151]. In a mouse liver cancer model, intratumoral injection of the STING agonist c-di-GMP significantly increased the expression of TNF-α and IFN-γ in CD4 + and CD8 + T cells, along with a notable rise in the percentage of IFN-γ-producing NK cells. These findings suggest that intratumoral administration of c-di-GMP supports a pro-inflammatory environment conducive to T and NK cell activation[134]. In malignant B cell tumors, 3’3’-cGAMP selectively triggers mitochondria-mediated apoptosis in both normal and malignant B cells. It induces the prolonged presence of STING within the ER or Golgi, forming protein complexes that activate apoptosis. This indicates that beyond their established role in promoting antitumor immune responses, STING agonists can directly eradicate malignant B cells, potentially providing therapeutic benefits for B cell malignancies such as chronic lymphocytic leukemia and multiple myeloma[131]. When cGAMP was used to treat mice with colorectal adenocarcinoma, data indicated that the cGAMP-cGAS-STING-IRF3 pathway stimulates and strengthens innate immune responses, increases antitumor cytokine production, and activates DCs to inhibit tumor growth. Additionally, cGAMP may activate other STING-independent pathways to suppress colorectal cancer tumor growth[152].

In cancer treatment, DMXAA primarily exerts antitumor effects by activating the body’s immune response, particularly through the activation and recruitment of immune cells such as DCs, macrophages, and NK cells at the tumor site[153]. DMXAA can also inhibit tumor angiogenesis, thereby reducing the blood supply and nutrient flow to the tumor. This restriction of resources limits tumor growth and spread while making it easier for immune cells to reach the tumor site[154]. In mesothelioma treatment, DMXAA has been observed to promote tumor antigen presentation in tumor-draining lymph nodes and reduce the size of tumor blood vessels. Despite its effectiveness in treating mesothelioma, it led to a decrease in the proportion of CD4 + and CD8 + T cells in tumors, with no sustained T cell response, which may ultimately allow tumor cells to evade immune attack and resume growth. Notably, combining DMXAA with agonistic anti-CD40 antibodies or IL-2 reduced antitumor efficacy[155]. DMXAA treatment also showed significant antitumor effects and a tumor-specific CD8 + T cell immune response against TC-1 tumors, though it did not enhance the antigen-specific immune response in tumor-bearing mice[156]. Positive results were also achieved in melanoma treatment[157]. However, clinical studies on DMXAA in patients did not confirm the expected outcomes, likely due to structural differences in the STING protein between mice and humans. After 7 days of DMXAA administration, upregulation of hypoxia inducible factor (HIF)-1α levels and an increase in the number of blood vessels in the tumor were observed, leading to tumor regrowth. Tumor regeneration occurred at the periphery, while the central area was largely necrotic, with an increased density of blood vessels in the marginal zone[158]. This outcome is thought to result from DMXAA-induced necrosis around damaged vessels and hypoxic regions within the tumor, leading to activation of the HIF-1α transcription factor. Additionally, it stimulates new blood vessel formation, enhancing survival, proliferative potential, and invasiveness of tumor cells, and ultimately triggering immune suppression[159,160]. While the antitumor effect of DMXAA is undeniable, further research is required to understand its impact on the tumor microenvironment and its combined use with immunotherapies.

Currently, small molecule-based cancer immunotherapy represents a breakthrough in cancer treatment. These small molecule compounds can activate STING by binding to different sites on the protein or by influencing conformational changes in STING[161]. Amide benzoimidazole is a novel STING agonist, and intravenous delivery of this small molecule in immunocompetent mice has resulted in pronounced tumor regression. Ramanjulu et al[162] have demonstrated that this new compound has significantly enhanced binding affinity, leading to an adaptive CD8 + T cell response in vivo, showing substantial potential for advancing immunotherapy in human cancers.

STING inhibitors: With the deepening understanding of the cGAS-STING pathway, it has become clear that excessive activation of this pathway can prolong the expression and secretion of inflammatory cytokines, promoting tumor growth and metastasis. Therefore, the development of STING inhibitors to intervene in such disease processes is also necessary. Existing inhibitors can be classified by their inhibition mechanisms into palmitoylation inhibitors and competitive antagonists targeting the CDN binding site[163]. STING activation requires palmitoylation at the Golgi apparatus[164]. Blocking STING palmitoylation is an effective strategy for inhibiting STING activation. STING inhibitors act by covalently binding to the Cys88 or Cys91 residues within the transmembrane domain of the STING protein, thereby disrupting palmitoylation. The 3-acylamino indole derivative indole ureas (H-151) can form a covalent bond with Cys91. In a mouse model of STING activation, administration of H-151 achieved effective systemic levels and significantly reduced serum levels of type I IFN and IL-6. This indicates that H-151 is an effective small molecule antagonist of STING, capable of inhibiting type I IFN response, TBK1 phosphorylation, and STING palmitoylation[165]. In human cells, nitro-fatty acids (NO2-FAs), effective inhibitors of STING signaling, are formed after herpes simplex virus-2 infection and mediate anti-inflammatory, antioxidative, and cytoprotective effects. Derivatives of NO2-FAs also covalently modify the Cys88 and Cys91 residues of STING, inhibiting its palmitoylation and thereby suppressing IFN-I production in fibroblasts associated with STING-related vascular diseases that onset in infancy[166].

Competitive antagonists targeting the CDN binding site interact with the C-terminal ligand-binding domain of STING, competing with endogenous STING ligands. Additionally, they promote STING protein degradation and downregulate the STING pathway. Before binding with cGAMP, the STING apoprotein adopts an “open” conformation, which shifts to a “closed” conformation upon binding. Tetrahydroisoquinolone compounds can bind to this “open” site, resulting in protein activation. By taking advantage of the natural symmetry of the STING protein and using a 2:1 binding stoichiometry, these compounds occupy the binding site fully, effectively competing with cGAMP, while maintaining favorable physicochemical properties for oral administration[167]. Astin C, a cyclic peptide isolated from the medicinal plant Aster tataricus, binds to the C-terminal of STING, blocking IRF3 recruitment and inhibiting STING pathway activation. Studies indicate that Astin C can significantly reduce autoinflammatory responses, displaying low cytotoxic side effects in cellular and animal models. This suggests that Astin C may be applicable in treating STING-mediated cancers and autoimmune diseases[168].

DISCUSSION

Innate immunity plays a pivotal role in the pathogenesis of cancer. Beyond the canonical role of cGAS-STING, recent evidence has emerged that expands the functional roles of cGAS-STING to cancer[15]. The cGAS-STING pathway serves as a direct sensor for cytoplasmic DNA within the innate immune system[19,20]. While the self-DNA sensor cGAS can detect cellular or tissue damage, excessive activation of the cGAS-STING pathway can incite inflammation and subsequent complications[61].

Investigating the cGAS-STING pathway in relation to HCC enhances our understanding of how innate immune responses contribute to the development of liver inflammation and injury. Recent discoveries regarding the regulation of this pathway will facilitate the identification of critical molecules that could serve as potential therapeutic targets for liver disease.

DNA damage leads to the formation of dsDNA in cancer cells, and under its stimulation, the cGAS STING signaling pathway is activated and promotes the release of type I IFN, which is crucial for DC maturation[56,57]. The activation of the cGAS STING signaling pathway in DCs is a core step in the entire cancer immune cycle, which can be initiated through phagocytosis of dead or damaged cancer cells, exosome metastasis, and cGAMP gap junctions. Then, with the help of type I IFN, DCs migrate to tumor draining lymph nodes and cross activate tumor specific CD8 +T cells, inducing systemic anti-tumor immunity to control local and distant tumor growth[60].

However, sustained activation of the cGAS-STING pathway may facilitate inflammation-driven tumorigenesis[61]. CIN within tumor cells can lead to cGAS-STING-dependent DNA sensing of micronuclei, which has the potential to stimulate tumor metastasis[71,72]. Although STING agonists and inhibitors have demonstrated efficacy in tumor treatment, the clinical and molecular guidelines for their application are still being established. Recognizing the dual, or dichotomous, roles of cGAS-STING in cancer progression is of utmost importance for the advancement of cancer therapeutic strategies.

CONCLUSION

In aggregate, these studies suggest the cGAS-STING pathway as a critical therapeutic target for tumors. The cGAS-STING pathway has been shown to potently inhibit the proliferation and metastasis of tumor cells, including HCC. The cGAS-STING pathway activation within HCC augments the body’s immunity against the tumor by provoking cellular senescence and inducing cell death. The combined effects additively result in an effective inhibition of tumor growth. Research in this pathway opens an exciting opportunity for the development of HCC therapeutics. On the other hand, the cGAS-STING pathway also contributes to HCC development and progression. If acute inflammation can have a few inhibitory effects on cancer, chronic inflammation generally promotes its progression. Extended pathway activation results in a suppressive tumor microenvironment; this may be contributory to the promotion of liver damage, fibrosis, and the growth of cancer and the outspread of tumor cells to other portions of the liver. Prior to using this pathway for HCC treatment, it is essential to evaluate the possible adverse effects, taking into account the different effects on tumors. The dosing and timing of STING agonists and inhibitors in cancer treatment must be therefore carefully considered. An innovative therapeutic strategy that has the ability to significantly improve HCC treatments is the cGAS-STING pathway.

ACKNOWLEDGEMENTS

The authors are grateful to the dedicated and committed participants in the study.