Identification of tumor antigens and anoikis-based molecular subtypes in the hepatocellular carcinoma immune microenvironment: implications for mRNA vaccine development and precision treatment

Hepatocellular carcinoma (HCC), the most common primary liver malignancy, ranks as the seventh most prevalent cancer worldwide, with 905,677 newly diagnosed cases (4.7%) and 830,180 deaths (8.3%) [1]. Patients with early-stage HCC may qualify for curative treatments such as surgery and liver transplantation, while those with advanced HCC, comprising approximately 70% to 80% of patients, generally experience a survival rate of less than 12% over a 5-year period. This is primarily attributed to the scarcity of treatment options available and the suboptimal response to initial therapy [2, 3]. Therefore, identifying innovative approaches for earlier detection is essential for better prediction of therapy response and survival in patients with HCC [4]. Since 2017, immune checkpoint inhibitors (ICIs) and combinations based on ICIs have emerged as the most promising approach for improving the prognosis of patients with unresectable HCC. However, accumulating evidence has shown that the poor pharmacokinetic properties of antibodies can induce treatment-related adverse events, while off-target immunological effects on other organs can trigger immune-related adverse events, which can lead to treatment cessation and drug-related deaths, thereby limiting the application of this therapy [5]. Randomized trials assessing the efficacy of anti-PD-1 monotherapy in both the first-line and second-line setting showed no improvement in overall survival (OS). Consequently, the development of new combination therapy strategies aimed at activating the suppressive immune microenvironment in HCC is necessary [6].

Tumor-associated antigens (TAAs) are crucial targets for activating immunotherapy, as they are normal self-proteins that are overexpressed or re-expressed by tumors. Developing effective, minimally invasive, and long-lasting treatments targeting TAAs has been a long-standing goal in cancer therapy. Cancer vaccines aim to stimulate tumor-specific immune responses by targeting TAAs and increasing cytotoxic CD8 + T cells. Additionally, effective immunotherapy responses have been associated with neoantigens, which are novel immunogenic sequences resulting from tumor mutations. Neoantigens are also being considered as potential targets for cancer immunotherapies, such as tumor vaccines, adoptive cell therapy (ACT), and antibody-based treatments, and as predictors for immune checkpoint blockers (ICBs). Despite the fact that HCC tumor cells can elicit significant immune responses against a range of mutant antigens, no clinically developed HCC vaccine targeting a tumor antigen currently exists. Therefore, there is an urgent need to identify novel antigens in HCC and develop HCC vaccines.

Apoptosis is a programmed cell death mechanism that helps maintain tissue homeostasis and prevent the development of cancer. When cells lose contact with neighboring cells or their extracellular matrix (ECM), they undergo a process called anoikis, which is a type of apoptosis that is triggered by detachment from the ECM [7]. Cancer cells detaching from primary sites acquire anoikis resistance and can colonize target organs or tissues via the circulatory and lymphatic systems [8]. Therefore, anoikis, a new hallmark of cancer metastasis, has attracted the attention of oncologists [9]. In HCC, strategies that reverse the anoikis resistance phenotype of HCC cells to inhibit cancer metastasis may be promising for the treatment of patients with local metastasis and potential vascular invasion. Some studies have elucidated the relationship between tumor resistance to anoikis and immunity, revealing that anoikis affects the tumor immune microenvironment and anoikis resistance of cancer cells [10, 11]. Zhang et al. discovered that the deactivation of IL1RAP promotes anoikis and suppresses Ewing sarcoma cell metastasis, revealing the promising potential of anoikis-targeted therapy in cancer [11]. Thus, exploring the role of anoikis in HCC will provide valuable insights into patient prognostic stratification and the development of novel therapeutic targets, which also has the potential to enhance combination therapy with cancer vaccines and immunotherapy in HCC.

LncRNAs have been discovered to modulate the expression of genes associated with anoikis, either by facilitating or impeding anoikis resistance in cancer. Previous studies have shown that silencing lncRNA ANRIL promotes caspase activity, leading to anoikis-induced cell death in glioma cells. Additionally, lncRNA-HOX antisense intergenic RNA (lncRNA-HOTAIR) is highly expressed in various cancers and has been shown to contribute to cell survival and epithelial–mesenchymal transition (EMT) [12]. However, the molecular role and clinical implications of anoikis in HCC remain unknown. Hence, deciphering the molecular underpinnings of the relationship between the anoikis phenotype and lncRNAs in HCC based on datasets from multiple clinical RNA sequencing sample cohorts may illuminate the intricate mechanisms underlying HCC progression and metastasis. Additionally, such investigations may uncover promising therapeutic targets, paving the way for effective interventions to impede the development of HCC.

In this study, we aimed to identify tumor antigens that could be utilized in the development of an mRNA vaccine for HCC. Furthermore, we constructed a prognostic risk score model based on anoikis-related lncRNAs, which has the potential to predict the prognosis of HCC patients as well as provide insights into the immunological tumor microenvironment (TME) of HCC. The risk model can be further employed to facilitate precision HCC treatment, such as chemoimmunotherapy, and the development of HCC vaccines.

mRNA vaccines had an impressive impact during the COVID-19 pandemic, leading to a surge in preclinical and clinical research in both oncology and infectious diseases [32]. Hepatocellular carcinoma (HCC) is a particularly lethal cancer with a complex molecular profile and limited treatment options once diagnosed. Although the development of mRNA vaccines has revolutionized the therapeutic treatment of HCC, the effects of mRNA cancer vaccines in HCC patients are still suboptimal and are not yet fully understood. Malignant cells produce both tumor-specific and non-tumor-specific antigens, making mRNA cancer vaccines a potential immunotherapy for the treatment of malignancies. Both types of antigens could be targeted with mRNA vaccines to induce tumor regression in preclinical models and humans [33]. However, most cancer vaccines under investigation are based on peptides representing only a single tumor-associated antigen, which could lead to the selection of T cells with low-affinity T-cell receptors (TCRs), or severe autoimmune toxicities can result when antigens are targeted with high-affinity engineered TCRs. On the other hand, tumor-specific neoantigens can arise from nonsynonymous somatic mutations that result in the presentation of mutated peptides on the cell surface, where they can be recognized by T cells. Nevertheless, since neoantigen-specific T cells recognize peptides unique to tumors, they are not influenced by central tolerance and should not cause autoimmunity. Therefore, developing neoantigen strategies is a promising goal for immune cell-based cancer therapy.

The discovery of possible tumor-associated antigens (TAAs) and tumor-specific antigens (TSAs) is essential for the development of mRNA vaccines. Huang et al. examined prospective tumor antigens for the development of an mRNA vaccine by identifying genes that were mutated, amplified, or overexpressed in pancreatic adenocarcinoma and cholangiocarcinoma [34, 35]. Gui et al. identified potential antigens expressed in bladder cancer that could be used to develop mRNA vaccines for bladder cancer treatment and constructed a prognostic risk score model based on anoikis, which could be vital for developing personalized oncology therapeutic strategies, and these results enhanced the understanding of tumor mutation, tumor death mechanisms, and the tumor microenvironment [36]. In our study, we integrated the aforementioned methods to discover eight candidate tumor antigens (AURKA, CCNB1, CDK1, DNASE1L3, KPNA2, PRC1, PTTG1, and UBE2S) by identifying important genes that were amplified, mutated, and overexpressed in HCC. The oncogenesis and prognosis of HCC were also found to be intimately linked to these seven antigens.

It is worth mentioning that not only was there a significant correlation between high expression of tumor antigens and poor prognosis in HCC, but this expression could also cause the recruitment of APCs, which further supports their viability as mRNA vaccine antigen candidates. Du et al. cited significant evidence supporting the potential of AURKA as a therapeutic target for cancer [37]. The combination of AURKA-targeting inhibitors and immunotherapy reduced Myc expression in tumor cells, enhanced HLDA1 and p53 protein levels, increased autophagy, caused apoptosis (neurofibroma), and significantly inhibited the growth of melanoma [38‐40]. In a study conducted by another research team, alisertib promoted the development of an anticancer immune microenvironment by reducing the number of myeloid-derived suppressor cells and increasing the activity of CD8 + and CD4 + T lymphocytes, which showed alisertib combined with an anti-PD-L1 antibody exhibited strong synergistic effects [41]. According to a bioinformatics study by Si et al., CCNB1 is a marker for variant genes in hepatocellular carcinoma and is linked to variation in p53. The expression of CCNB1 is associated with a higher proportion of T effector cells, such as CD8 + T cells, which include cytotoxic T cells, natural killer cells, and other antitumor immune cells in the periphery [38, 42]. Reports have shown a correlation between CDK1 activity and patient prognosis, and CDK1 expression is increased in numerous human malignant tumor tissues, making it a crucial molecular target. Xiao and Li et al. demonstrated the impact of DNASE1L3 on HCC development, apoptosis, and glucose metabolism reprogramming. Targeting DNASE1L3 may be a viable therapeutic option for HCC, and detecting elevated levels of DNASE1L3 in the blood may help doctors diagnose hepatocellular carcinoma caused by the hepatitis B virus [43]. KPNA2 plays a critical role in HCC cells, and an inability of KPNA2 to import PLAG1 into the nucleus is a strong predictor of poor survival in HCC patients after hepatectomy [44]. Chen et al. discovered that PRC1 is markedly elevated in HCC tumors and highly related to early HCC recurrence [45]. In 46 HCC tumor samples, Huang et al. found that PTTG1 was commonly upregulated and strongly associated with PTTG3P [46]. This research revealed that UBE2S was significantly expressed in HCC, particularly in the nucleus, and was linked to HCC patients' clinical prognoses. Through its nuclear localization signal (NLS), UBE2S enters the nucleus, interacts with TRIM28, and promotes HCC development by ubiquitinating p27 [47]. Anoikis is an inherent protective mechanism in organisms that occurs when the connection between epithelial cells and the extracellular matrix (ECM) is broken, preventing the readhesion of dead or dying cells to an inappropriate location [48]. This mechanism is essential for the organism to respond to harmful intracellular or extracellular stimuli, such as viral infections, DNA damage, exposure to toxins, metabolic, oxidative, or hypoxic stressors, or loss of anchoring [49]. However, cancer cells may exhibit abnormal execution of anoikis, leading to tumor invasion, migration, distant organ metastases, and treatment resistance. In certain pathological circumstances, such as malignancies, cells may develop resistance to anoikis. Multiple studies have shown that tumor cell resistance to anoikis enables them to metastasize away from the primary tumor location via the lymphatic and circulatory systems, where they may continue to proliferate [50]. This resistance may also facilitate immune evasion and contribute to the development of treatment resistance. Although it has been shown that anoikis is involved in the invasion and metastasis of various solid tumors, few studies have thoroughly investigated the role of anoikis-related genes and lncRNAs in HCC. Additionally, the TME infiltration mediated by the integrated effects of distinct ARGs and anoikis-related lncRNAs has been underrecognized in the HCC TME. Determining the significance of specific anoikis regulatory patterns in TME cell invasion will enhance our knowledge of the TME antitumor immune response and guide the development of more efficient immunotherapy approaches.

Using an unsupervised consistency clustering algorithm, patients with HCC were categorized, and 28 ARGs were selected to identify two distinct regulatory patterns of anoikis. The two clusters were then studied and assessed, revealing a difference in survival time between the two clusters of HCC patients. Differences in immune cell infiltration and immunological targets were examined between the subgroups, and the two groups with different patterns of anoikis and cellular infiltration into the tumor microenvironment displayed considerable differences. The results showed that ARG cluster A had a poor survival time, and anoikis-related genes were highly expressed in this cluster. In comparison to cluster B, cluster A exhibited a greater degree of immune cell infiltration into the tumor stroma, including infiltration of activated CD4 T cells, activated dendritic cells, CD56⁺ natural killer cells, immature dendritic cells, MDSCs, natural killer T cells, natural killer cells, plasmacytoid dendritic cells, regulatory T cells, T follicular helper cells, and type 2 T helper cells.

Extensive research has shown that programmed cell death serves two roles in modulating the TME. In addition to its antitumor action, programmed cell death also indirectly facilitates immune escape by generating an immunosuppressive microenvironment [51]. Consistent with previous results, KEGG analysis showed higher enrichment of cell cycle pathways in group A (high expression of anoikis genes) than in group B, but the activity of metabolic pathways was lower in group A than group B, suggesting that group A had a worse prognosis. Although group A had a high level of immune cell infiltration, its survival was worse than that of group B, and we can reasonably speculate that the high expression of anoikis-resistance genes in cluster A created a special immunosuppressive microenvironment that made the infiltrating immune cells dysfunctional in the stroma. Previous research has shown that the immunosuppressive features of the tumor microenvironment facilitate cytotoxic immune cell exhaustion and death, promoting the development of protumoral immune cells such as Tregs, M2 macrophages, and myeloid-derived suppressor cells (MDSCs) [51]. This supports our findings, and it was therefore not surprising that a comprehensive exploration of the cellular infiltration patterns in the tumor microenvironment defined by the anoikis regulatory model revealed that cluster A had a higher level of active immune cells but poorer survival. This again demonstrates the complexity and heterogeneity of the role of types of apoptosis, including anoikis, in the tumor microenvironment, but further exploration of the role in the hepatocellular carcinoma microenvironment is needed.

Notably, recent research has focused on understanding how anoikis affects the activation of the immune microenvironment. There is now evidence that altered cancer metabolism is due, in part, to anoikis resistance. Alteration of kynurenine (Kyn, a key metabolic component) triggered a pathway that was increased by TNHCC in suspension culture. In general, Kyn can reduce immune surveillance, but in glioma, it reduces antitumor immune responses and enhances tumor cell survival and motility by acting in both a paracrine and autocrine mode. Kyn has been shown to act as an endogenous ligand to activate AhR in both immune cells and tumor cells [52]. Previous studies have suggested that HCC tumors with a hypermetabolic phenotype generally have a favorable prognosis. Research conducted by Yang on the metabolic subtypes of HCC found that HCCs with high metabolic activity had distinct metabolic signatures and were similar to differentiated nonproliferative HCCs with low AFP expression and a favorable prognosis. The subtype with low metabolic activity, which was related to immunology profiles and high expression of immune checkpoint genes, showed pharmacological sensitivity to CTLA4 inhibitors and cabozantinib. This class played a minor role in the formation of metabolic signatures. The intermediate metabolic activity subtype, which was associated with a higher level of AFP and a poorer prognosis, had lower enrichment of metabolic signatures than the high metabolic activity subtype but higher enrichment than the low metabolic activity subtype [53]. Similar results were also found in the study by Huo et al. C1 exhibited the highest metabolic activity, the best prognosis, and the most eosinophils and natural killer cells. C2 had the lowest metabolic activity, the worst prognosis, the highest TP53 mutation rate, the highest immune checkpoint expression, and substantial regulatory T-cell infiltration. C3 displayed strong neutrophil and macrophage infiltration, moderate metabolic activity, and the highest LRP1B mutation rate [54].

Given that mRNA vaccines have shown benefits in only a relatively specialized group of cancer patients, we used the expression profiles of anoikis-lncRNAs to divide HCC patients into high-risk and low-risk subtypes to select the most promising candidates for the vaccine. Additionally, the ARG-based signature contained numerous genes, and there is a high degree of heterogeneity in tumor microenvironments, thus limiting the role of the signature in predicting liver cancer prognosis. Therefore, we constructed a prognostic model based on the ARI. Among the ARGs related to prognosis that we identified, accumulating evidence has shown that the ARGs used to build the ARI risk model play a crucial role in cancer. For instance, Zhang et al. found that the expression of PMSB8-AS1 was elevated in PC tissues and cell lines, and it was inversely correlated with survival in people with PC. Studies of the mechanism of PMSB8-AS1 found that the lncRNA promotes pancreatic cancer progression via STAT1 by sponging miR-382–3p and is involved in the regulation of PD-L1 [55]. As an upregulated lncRNA in liver cancer, ZFPM2-AS1 was identified as a competing endogenous RNA (ceRNA), competitively binding to miR-139 and regulating GDF10 expression. The authors suggested that enhancement of the malignant phenotype in liver cancer occurs via the ZFPM2-AS1/miR-139/GDF10 pathway, which has potential as a target for treatment in HCC [56]. A similar result has been found in gastric cancer: Kong et al. analyzed ZFPM2-AS1 and found that it controls the progression of gastric cancer and uncovered a new ZFPM2-AS1/MIF/p53 signaling pathway, providing insight into the molecular processes underlying the tumorigenicity of certain malignant gastric cells [57]. In gastric cancer, exosome-transferred LINC01559 stimulated the phosphatidylinositol 3-kinase/AKT serine/threonine kinase (PI3K/AKT) pathway by upregulating PGK1 and downregulating PTEN. Other studies have shown that LINC01559 recruits insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2) to stabilize ZEB1 mRNA in GC cells, thus upregulating ZEB1 [58, 59]. Other studies have shown that LINC01559 accelerates pancreatic cancer progression by relying on YAP [60]. Our bioinformatics analysis also identified these lncRNAs as anoikis-related prognostic markers; these results deepened the understanding of the tumor microenvironment related to anoikis in HCC and supported the practical significance of the ARI model.

To verify the model, we conducted KM analysis, ROC curve analysis, random sampling validation, and subgroup analysis in the studied cohorts. These analyses demonstrated that the novel signature had strong prognostic power in predicting the outcome of HCC. Additionally, a statistically significant correlation between the ARI risk score and immune cell infiltration and immunotherapeutic efficacy was observed. The ARI risk score was a valid and practical factor that could be used to characterize the heterogeneity of tumor anoikis regulation patterns and to identify TME infiltration patterns and tumor immune phenotypes. Furthermore, integrated analysis confirmed that the risk score was an effective predictive indicator for liver cancer. Patients with a lower risk score had a longer overall survival rate, as assessed in the overall, test, and validation groups. The ROC curve analysis showed that the gene signature based on the lncRNAs had high sensitivity and specificity, demonstrating the excellent predictive efficiency of our model.

To further support the practical application and predictive validity of the model in clinical practice, we created a nomogram jointly considering the ARG and ARI scores as well as clinicopathological factors, including pathological stage, clinical stage, age, and sex. The combined ROC analysis demonstrated that the predictive validity of the combined nomogram was better than that of the two separate scoring systems alone. As a result, the combined nomogram provided a more accurate prediction of 1-, 2-, and 3-year survival in hepatocellular cancer, with an AUC of 0.723–0.757.

Next, we analyzed the expression of infiltrating immune cells in the ARI high-risk and low-risk groups. We found that the types and quantities of infiltrating immune cells in the low-risk group were significantly higher than those in the high-risk group, indicating that the low-risk group had an immunologically hot phenotype; the low-risk group was also associated with improved survival. The low-risk group was characterized by elevated levels of various immune-related factors, including APC coinhibitory molecules, checkpoint molecules, cytolytic molecules, HLA molecules, proinflammatory molecules, MHC class I molecules, parainflammatory markers, T-cell coinhibitory molecules, T-cell costimulatory molecules, and indicators of type II IFN responses. These findings further supported the hypothesis that the low-risk group possessed a robust immune profile. Moreover, we found high expression levels of HLA receptors in the low-risk group, further emphasizing the strong immune activation properties of this group. Finally, our results revealed a significant correlation between the ARI score and tumor mutation load: the low-risk ARI group had low TMB levels and high levels of immune cell infiltration, a characteristic of an immune hot phenotype, resulting in a more favorable survival prognosis than the high ARI score group. Tumor response to immune checkpoint therapy has been observed to vary based on the degree of immune cell infiltration and the mutational burden of the tumor. We investigated the relationship between the ARI and immune checkpoint signaling, and the results suggested that anoikis can influence the efficacy of immune checkpoint-based treatments. Generally, cancers classified as “hot”, such as melanoma and lung cancer, exhibit greater responsiveness to therapy than “cold” tumors, such as pancreatic and prostate cancers. Tumors with a high mutational burden are believed to have more favorable outcomes with ICD therapy. However, our work showed that patients in the low-ARI group had higher expression of PD1 and PD-L1, suggesting that ICD treatment may have better outcomes in this group.

Combining immune therapy with the exploration of tumor neoantigens to develop tumor antigen vaccines may enhance the efficacy of existing treatments in ARI high-risk populations if ICD is ineffective due to very immune-suppressive microenvironments or matrix barriers expressed in HCC. For instance, in a study using DCs pulsed with autologous tumor lysates in 31 treated patients, 12.9% had a partial response, 54.8% had stable disease, and the 1-year survival rate was 10.7% (± 9.4) compared to 63.3% (± 12.0) with maintenance therapy. In addition, the administration of monoclonal antibodies, adoptive T-cell therapy, or vaccinations combined with the administration of gene therapy vectors encoding monoclonal antibodies and/or immunostimulatory cytokines are effective methods for treating HCC. Combining existing tumor therapy models and ARI-based stratification in the development of tumor antigen vaccines may be a promising strategy for achieving a synergistic immunostimulatory effect, leading to significant clinical benefits of vaccine therapies.

In summary, this study analyzed the expression of antigens in liver cancer, providing a new perspective for developing liver cancer vaccines. Unsupervised clustering of ARGs related to anoikis and prognosis and construction of the ARI model revealed innovative ideas for enhancing the clinical response of patients to immunotherapy and identified distinct tumor anoikis phenotypes that can promote precision cancer immunotherapy. This study has some limitations; for example, the feasibility and effectiveness of antigen vaccine development has yet to be verified through experiments, and further follow-up studies are needed to validate its potential due to the absence of available RNA sequencing data of HCC immunotherapy cohorts and clinical data. In addition, machine learning and feature selection algorithms can be further employed to optimize models, enabling better development of liver cancer vaccines and more accurate predictions of the prognosis of liver cancer patients [61, 62].

Our study identified anoikis-related regulators, including CCNB1, CDK1, DNASE1L3, KPNA2, PRC1, PTTG, and UBE2S, as promising tumor antigens for the development of mRNA vaccines for HCC. Moreover, we identified four distinct HCC subtypes related to anoikis and suggested that these subtypes could guide the selection of appropriate patients for immunotherapies. We also developed a novel lncRNA scoring model related to anoikis that could help estimate immunotherapy response, design mRNA vaccines for HCC therapy, and identify the most suitable patients for immunization. These findings have significant implications for the development of targeted immunotherapies for HCC patients.