Advanced therapeutic modalities in hepatocellular carcinoma: Novel insights

Abstract Hepatocellular carcinoma (HCC), the most common type of liver cancer, is usually a latent and asymptomatic malignancy caused by different aetiologies, which is a result of various aberrant molecular heterogeneity and often diagnosed at advanced stages. The incidence and prevalence have significantly increased because of sedentary lifestyle, diabetes, chronic infection with hepatotropic viruses and exposure to aflatoxins. Due to advanced intra‐ or extrahepatic metastasis, recurrence is very common even after radical resection. In this paper, we highlighted novel therapeutic modalities, such as molecular‐targeted therapies, targeted radionuclide therapies and epigenetic modification‐based therapies. These topics are trending headlines and their combination with cell‐based immunotherapies, and gene therapy has provided promising prospects for the future of HCC treatment. Moreover, a comprehensive overview of current and advanced therapeutic approaches is discussed and the advantages and limitations of each strategy are described. Finally, very recent and approved novel combined therapies and their promising results in HCC treatment have been introduced.


| BACKG ROU N D
According to the GLOBOCAN 2020 estimates of cancer incidence and mortality, liver cancer with approximately 905,677 (4.7%) new cases and 830,180 (8.3%) deaths annually is the sixth most commonly diagnosed cancer and the third leading cause of neoplastic disease-related deaths worldwide. Moreover, in 2020, the American Cancer Society estimated 42,810 new cases and 30,160 deaths for liver and intrahepatic bile duct cancer in the US. 1 Histologically, primary liver cancer can be divided into several subtypes based on cellular origin, including hepatocellular carcinoma (HCC) (comprising 75-85% of cases), intrahepatic cholangiocarcinoma (ICC) (comprising 10-15% of cases) and other rare forms. Therefore, considering the complexity of HCC due to its heterogeneity and the involvement of various signalling pathways, adopting a similar treatment regimen for all patients would not be effective and could even exacerbate symptoms, a fact that led to the introduction of the term 'personalized medicine'. However, because of the difficulties in allocating a unique treatment plan for each individual suffering from the 'same' disease, researchers have tried to classify patients into subpopulations based on their susceptibility to a particular disease or response to a specific treatment. This patient stratification has helped to tailor the treatment modalities for each individual. Such an approach has led to the US National Research Council's preference for the term 'precision medicine' rather than 'personalized medicine' in 2016. 4,5 Accordingly, different therapeutic approaches based on both molecular and cellular therapies have been developed. Molecular-targeted therapy, targeted radionuclide therapy and epigenetic modification-based therapies are therapeutic strategies at the molecular level that, in combination with cellbased treatments including immunotherapy and gene therapy, and have provided promising results. This review discusses recent advances in the development of novel therapeutic approaches in the treatment of HCC.

| MOLECUL AR-TARG E TEDTHER APYIN H CC(MULTI -KINA S EINHIB ITOR S)
It is often observed that targeting a central biomolecule in a molecular network can affect the entire network and restore a large number of disrupted pathways and functions. This target can be a key transcription factor, a crucial receptor, or a central enzyme, such as a kinase ( Figure 1A). However, targeting a single molecule (as what mono-kinase inhibitors do) faces obstacles such as drug resistance development and weak potency. On the other hand, multi-kinase inhibitors, which target several intracellular and cell surface kinases, can result in the pervasive inhibition of tumour proliferation, angiogenesis, metastasis and invasion. The approved medications in this context include sorafenib, 6 and lenvatinib 7 as the first-line and regorafenib, 8 cabozantinib 9 and ramucirumab 10 as the second line (Table S1).

| TARG E TEDR ADIONUCLIDETHER APY IN H CC
The concept of targeted radionuclide therapy (TRT) relies on the use of injectable therapeutic radioisotopes designed to specifically target cancerous tissue at the cell or molecular level. The first application of radionuclides as therapeutic agents was demonstrated in the 1940s, when iodine-131 ( 131 I) was prescribed for treating thyroid diseases. Recent advances in radionuclide production and labelling as well as improvements in the identification of appropriate and specific molecular targets make the TRT an attractive approach for cancer treatment. may be caused by hydroxyl free radical ( • OH) attack (indirect effect via water radiolysis) or by one-electron oxidation (direct effect). 12 The incidence of DNA damage is proportional to the absorbed dose and is quantified per grey (Gy) per cell. After exposure to radionuclides, DNA breaks can lead to apoptosis or cell cycle arrest in cancer cells. This destructive effect can be directed specifically towards the targeted cancer cells by conjugating a tumour-specific ligand or antibody to the radionuclide, thus minimizing off-target damage to the healthy tissues surrounding the tumour 13,14 ( Figure 1B).
It is noteworthy that primary and metastatic liver lesions are highly vascularized and receive a preferential arterial supply via the hepatic artery, while normal liver cells are supplied at 80% by the portal vein. Accordingly, the hepatic artery is the appropriate route of administration for the delivery of targeted radionuclides. 15,16
Radioimmunotherapy is another approach that represents an advanced therapeutic modality for HCC using a combination of tumour-specific antibodies with potent radiopharmaceuticals. This approach provides targeted radiation limited to the tumour cells with reduced side effects. HCC-specific antigens such as PD-1, PD-L1, CTLA-4, CD147 and endoglin (CD105) are potential targets for radionuclide antibody conjugates [20][21][22][23] (Table S2). HBV and HCV, as the leading causes of HCC, recruit DNA methyltransferases (DNMT1, DNMT3a and DNMT3b) to promote hyper-methylation-induced repression of tumour suppressor genes including CDKN2A, SFRP1, SFRP5 and CDH1. 24,25 DNMT inhibitors can be divided into two categories based on their structure and function: nucleoside analogues and non-nucleoside agents. Nucleosides, particularly cytosine analogues, such as 5-azacytidine, 5-aza-2′-deoxycytidine (decitabine) 26 and zebularine, 27 interact with, and block the activity of DNMTs, and decrease overall DNA methylation levels. Despite the promising preclinical data, these DNMT inhibitors have certain drawbacks; for example, cytidine deaminase, which is highly expressed in the liver, inactivates some cytosine analogues and reduces their half-lives and efficacy in vivo. 28 To overcome such limitations, next-generation DNMT inhibitors such as guadecitabine (SGI-110) that are resistant to cytidine deaminase degradation have been developed. Structurally, guadecitabine consists of decitabine and deoxy-guanosine linked by a phosphodiester bond that can be cleaved gradually resulting in sustained demethylation. 29 Moreover, different types of non-nucleoside compounds, such as SGI-1027, 30 procaine, 31 procainamide, 32 hydralazine 33 and EGCG, 34 have been identified that act either through direct binding to the catalytic or cofactor site of DNMTs or by targeting their regulatory mRNA sequences.

| EPI G ENE TI CALTER ATI ON -BA S ED THER APIE SINH CC
The amino acid residues on the histone N-terminal tails that protrude from the nucleosome cores may be subject to modifications Although the trial ended early due to unexpected severe immunemediated toxicities, dose-dependent regulation of downstream genes could provide proof-of-concept for miRNA-based cancer therapy 42 (Table S3).

| IMMUNOTHER APYINH CC
The chronic inflammatory state provides an opportunity for im- CTLA-4, a CD28 homolog, is present on cytotoxic T cells and recognizes the same ligands (B7-1 and B7-2) as CD28 but has a higher binding affinity towards them. It prevents co-stimulation, which would normally be provided via the CD28-B7 interaction, by outcompeting CD28. In the early phase of tumorigenesis, CTLA-4 can attenuate the immune response by producing inhibitory signals. 46 Tremelimumab (CP-675,206), a fully human IgG2 antibody against CTLA-4, was the first molecule to be clinically tested for safety and efficacy in HCC. 47 Twenty patients with advanced HCC developed from HCV-induced liver cirrhosis, who were not eligible for surgery or locoregional therapy, were treated with a suboptimal dose of the tremelimumab in a phase II clinical trial (NCT01008358).
In general, the treatment was well-tolerated and not only showed anti-tumour activity (partial response rate of 17.6% and disease con- Notably, the tumour response to durvalumab occurred early and was durable.

| Immunecell-basedtherapies
In addition to the immune checkpoint blockade, different types of

cell-based therapies involving cytokine-induced killer (CIK) cells, chimeric antigen receptor T cells (CAR-T) or NK cells (CAR-NK), and
DCs, are also being considered and evaluated for the treatment of HCC 62 (Figure 2A).  Exosomes, derived from AFP-expressing DCs (DEX AFP ) in mouse models, revealed an efficient triggering of antigen-specific immune responses, substantial tumour growth retardation and increased survival rates. In addition, the TME underwent extensive alterations, including an increase in the number of IL-2 and IFNγ-expressing CD8 + T lymphocytes and a decrease in IL-10, TGFβ and regulatory T cells. 84 Despite the current challenges and novelty of the field, the inadequate number of studies in DC-based HCC immunotherapy indicates that this area still needs further investigation.

| G ENETHER APYINH CC
Gene therapy is the delivery of therapeutic genetic materials into affected cells in order to correct a genetic abnormality or restore a missing function. In this context, scientists have faced two main challenges: an optimal delivery method and the manipulation strategy. 85 Among the delivery methods, utilizing replication-deficient modified viruses is the most widely used technique in the clinical trials (Table S4) (Table S5).
HSVtk/GCV is the first and most commonly used suicide gene therapy system. HSVtk phosphorylates GCV (a guanosine analogue) to GCV monophosphate, which is followed by two more phosphor- can also be repaired by homology-directed repair (HDR), which needs a donor or template DNA and enables the precise insertion of new sequences that could be used to correct mutated genes. 91 For example, in order to block angiogenesis after trans-arterial embolization (TAE), hypoxia-inducible factor-1α (HIF-1α) knockout was generated using a lentivirus-mediated CRISPR/Cas9 system.
The HIF-1α knockout in SMMC 7721 cells significantly inhibited cell invasiveness and migration and prolonged the survival of HCCbearing mice. 92 In another report, it was shown that a CRISPR/ Cas9-mediated NSD1 knockout suppressed HCC cell proliferation, migration and invasion via the Wnt/β-catenin signalling pathway 93 ( Figure 2B).

| Differentiation therapy
Evaluation of the transcriptome of cancer cells has revealed the re-expression of some embryonic genes that were downregulated or silenced during the differentiation/maturation process. Such alterations in gene expression patterns are gradually reflected in the morphological and proliferative features of cells, a phenomenon known as the epithelial-mesenchymal transition (EMT). Epithelial cells, by losing their polarized polygonal shapes along with a reduction in cell-cell and cell-extracellular matrix (ECM) interactions, transform into a mesenchymal-like phenotype, characterized by their spindle shape and the ability to migrate and invade. 94,95 EMT and its reverse process, mesenchymal-epithelial transition (MET), are the cornerstone of a hypothesis which suggests that neoplasia is a cell differentiation disorder. 96 Accordingly, the differentiation therapy hypothesis was proposed, suggesting that instead of ablating malignant cells, the previous well-differentiated status can be restored and normal function reactivated. 97 Therefore, all agents that can induce cell differentiation are theoretical treatment options, such as all-trans-retinoic acid (ATRA), which is often prescribed to treat acute promyelocytic leukaemia (APL) with excellent results. 98 There are several approaches in HCC differentiation therapy, On the other hand, regulation of miR-122, the most abundant liver-specific miRNA, adjusted the liver-enriched transcription factor network and re-established the expression of hepatocytic genes. 102,103 The microRNA, miR-148a, was identified as a hepatocyte differentiation inducer through inhibition of the NOTCH signalling pathway. 104 Moreover, it has been shown that certain macromolecules can inhibit EMT or even induce MET by targeting cancer stem cell-related pathways. 105,106 Altogether, it is clear that differentiation therapy as a noninvasive treatment or a complementary approach alongside conventional therapies needs more consideration in clinical studies ( Figure 2B).

| HOWFARWEHAVECOME ,HOWFAR WES TILLHAVETOG O?
HCC is typically a latent and asymptomatic malignancy with different aetiologies, which result in a wide molecular heterogeneity and is often diagnosed at advanced stages. Due to advanced intra-or extrahepatic metastasis, conventional treatments are inefficient and the implementation of multidisciplinary treatments is strongly recommended.
In this review, the advantages and limitations of five novel therapeutic approaches in HCC treatment, including molecular-targeted therapy, targeted radionuclide therapy, epigenetic modificationbased therapy, immunotherapy and gene therapy, have been discussed. Many researchers have tried to push forward current treatment protocols through combination of different approaches.
However, despite promising technological advances, novel modalities have only managed to increase the overall survival rate and progression-free survival rate by a few months. This reflects the fact that only the tip of the iceberg has been exposed, and we need to extend our knowledge in order to widen our horizons with respect to treatment modalities, perhaps before starting a new clinical trial.
It is clear that improving our understanding of the molecular mechanisms of (i) liver carcinogenesis, (ii) drug resistance (iii) and tumour metastasis should be the priority of future studies. With the advent of bioinformatics and computational tools, the molecular mechanisms underlying these areas of concern can be pinpointed with greater ease and accuracy than ever before. Bioinformatics, such as mapping genetic polymorphisms, analysing the interactions between gene products and predicting biochemical alterations, could provide precise and comprehensive insights into finding new target genes. In addition, computational methods could facilitate the design of advanced recombinant antibodies with higher affinities and reduced off-target binding as novel bio-therapeutics.
A crucial bottleneck to progress could occur in the polymorphic differences between species and differences in metabolic pathways and immune systems between human and animal models that prevent accurate interpretation of preclinical models. This could result in the failure of many drugs during their early development and clinical trials despite promising results in the preclinical phase. However, in recent years, various liver-derived in vitro models such as organoids, microfluidic liver biochips, bio-printed micro-patterned co-cultures and multi-organ-on-a-chip systems have been developed. 107 These technologies are going towards bridging the gap between preclinical and clinical studies. As shown in Figure 3 May 29, 2020). Given the positive feedback from such combination therapies, this approach seems to be a promising prospect for HCC treatment in the future.

CO N FLI C TO FI NTE R E S T
No conflict of interest can be disclosed. The authors declare that they have no competing interests.

DATAAVA I L A B I L I T YS TAT E M E N T
Data openly available in a public repository that issues datasets with