Differentiation-inducing therapy: A potential therapeutic strategy that targets liver cancer stem cells

Abstract

Hepatocellular carcinoma (HCC) remains a major therapeutic challenges, largely owing to its high recurrence and metastasis rates. According to the liver cancer stem cells (LCSCs) theory, HCC can arise through two distinct pathways: Blocked differentiation of liver stem/progenitor cells (LSPCs) or dedifferentiation of hepatocytes. LCSCs constitute a core subpopulation of cells that drive tumor growth, drug resistance, and metastasis. Differentiation-inducing therapy is first applied to treat leukemia and generally extended to solid tumors, including HCC. In this review, we first focus on the potential therapeutic role of differentiation-inducing therapy in HCC via the blocked differentiation of LSPCs and the dedifferentiation of hepatocytes. We then survey a range of agents with differentiation-inducing therapeutic potential and discuss their underlying mechanism. Finally, we propose a novel strategy for differentiation-inducing therapy that may complement traditional treatment and potentially address the root drivers of malignant progression. This approach may offer new theoretical insights and identify actionable targets for precision medicine in HCC.

Key Words: Differentiation-inducing therapy; Liver cancer; Liver stem/progenitor cells; Liver cancer stem cells; Malignant transformation

Core Tip: Differentiation-inducing therapy may be a potential strategy for hepatocellular carcinoma (HCC), as it can target both the arrested differentiation of liver stem/progenitor cells and the dedifferentiation of mature hepatocytes. We systematically retrieved and analyzed recent relevant research on liver cancer stem cells (LCSCs) origin and differentiation therapy, and we summarized how differentiation-inducing therapy plays therapeutic role based on the theory of LCSCs. In addition, we explored the potential molecules and agents with differentiation-inducing therapeutic effect, and assessed translational prospects and clinical applicability of these candidate agents for future HCC treatment.

INTRODUCTION

High rates of drug resistance, recurrence, and metastasis make hepatocellular carcinoma (HCC) treatment highly challenging[1]. According to the liver cancer stem cells (LCSCs) theory LCSCs originate either from the arrested differentiation of liver stem/progenitor cells (LSPCs) or the dedifferentiation of mature hepatocytes[2]. Although this explanation provides breadcrumbs for an HCC treatment strategy, there is no standard management based on this theory.

All-trans retinoic acid (ATRA) is used in differentiation-inducing therapy for leukemia. Studies have shown that all-trans-retinoic acid has a differentiation-inducing effect in HCC cases[3]. Differentiation-inducing therapy may thus be a potential strategy for HCC, as it can target both the arrested differentiation of LSPCs and the dedifferentiation of mature hepatocytes. For one thing, differentiation-inducing therapy can induce LCSCs transformation into LSPCs or converting them into non-LCSCs that are sensitive to anti-cancer therapeutic agents; for another, differentiation-inducing therapy can reverse the progressive dedifferentiation of HCC and decrease the malignancy of cancer or inhibit aggressiveness[4].

In this review, we systematically retrieved and analyzed recent relevant research on LCSCs origin and differentiation therapy, summarized how differentiation-inducing therapy plays therapeutic role based on LCSCS theory. Furthermore, we explored the potential molecules and agents with differentiation-inducing therapeutic effect, and assessed translational prospects and clinical applicability of these agents. We searched Web of Science and PubMed for studies on differentiation-inducing therapy of HCC published through January 28, 2000. All four authors screen the literature in references independently and excluded other unrelated articles.

THEORIES ON THE ORIGIN OF LCSCS

Determining the origin of LCSCs is the starting point for understanding the characteristics of HCC. Current mainstream theories posit that LCSCs arise from the aberrant transformation of two types of precursor cells, with the core concept being the acquisition and dysregulation of “stemness”. LSPCs constitute the first type of precursor cells and mature hepatocytes was the second one (Figure 1). As LSPCs differentiate into mature hepatocytes, arrested differentiation may lead to the malignant transformation of LSPCs, causing LCSCs to appear[5]. In contrast, mature hepatocytes can undergo “reverse” reprogramming or dedifferentiation, resulting in a stem cell-like phenotype[6,7].

Figure 1 Molecular mechanism of differentiation-inducing therapy in hepatocellular carcinoma. Based on the theory of liver cancer stem cells origination, differentiation-inducing therapy can target both differentiation arrested of liver stem/progenitor cells and dedifferentiation of mature hepatocytes. LCSCs: Liver cancer stem cells; HNF: Hepatocyte nuclear factor; EpCAM: Epithelial cell adhesion molecule; CXCR4: C-X-C receptor 4; TGF-β: Transforming growth factor β; LSPCs: Liver stem/progenitor cells. This figure was created on www.figdraw.com (Supplementary material).

DIFFERENTIATION-INDUCING THERAPY TO REVERSE THE DIFFERENTIATION ARREST OF LSPCS

Differentiation-inducing therapy that targets the arrested differentiation of liver stem cells is aimed at restoring the normal differentiation process of liver stem cells, reversing their malignant transformation, and eliminating LCSCS. This strategy has shown promising results in preclinical studies and may be a suitable new approach for HCC treatment.

Malignant transformation of LSPCs

The malignant transformation of liver stem cells due to differentiation arrest is regulated by multiple signaling pathways and molecular mechanisms. Understanding these mechanisms is crucial for developing effective induced differentiation therapeutic strategies. Overexpression of the HOX transcript antisense RNA and miR-10b induces epithelial-mesenchymal transition (EMT) via the downregulation of E-cadherin, which induces cancer stem cell traits such as enhanced proliferation, invasiveness, and tumorigenicity[8]. The Wnt/β-catenin signaling pathway plays a key role in regulating the self-renewal and differentiation of liver stem cells[9]. Abnormal activation of this pathway in liver stem cells leads to differentiation arrest and enhanced stemness[10]. The Notch signaling pathway is essential for maintaining the balance between self-renewal and differentiation of liver stem cells. Zhu et al[11] demonstrated that hepatocyte nuclear factor (HNF)-1β promotes HCC stemness by activating Notch, leading to the upregulation of progenitor markers and EMT.

Differentiation-inducing therapy to reverse the malignant transformation of LSPCs

Based on the molecular mechanisms underlying the malignant transformation of LSPCs, several therapeutic strategies that induce differentiation have been developed; these reverse differentiation arrest and eliminate LCSCs. The reexpression of specific hepatic transcription factors in arrested liver stem cells can restore their differentiation capacity. Inhibiting the Wnt/β-catenin pathway has been shown to restore the differentiation of arrested cells. Chen et al[12] demonstrated that transforming the growth factor (TGF)-β adaptor β2 spectrin repressed the properties of LCSCs by inducing differentiation. Further, Zhang et al[13] found that sophocarpine can reduce HCC cell viability, eliminate HCC, and reverse the malignant phenotype of hepatoma cells, as well as reduce the ratio of LCSCs and inhibit the sphere formation of LCSCs in vitro. Reexpression of HNF-4α has been shown to induce the differentiation of hepatoma cells into hepatocytes and thus dramatically decrease “stemness” gene expression and the percentage of CD133+ and CD90+ cells[14]. These findings indicate that differentiation-inducing therapy that reverses liver stem cell differentiation arrest holds great potential for HCC treatment.

DIFFERENTIATION-INDUCING THERAPY TO REVERSE MATURE HEPATOCYTES DEDIFFERENTIATION

In addition to targeting liver stem cell differentiation arrest, reversing the dedifferentiation of mature hepatocytes is an important approach for differentiation-inducing therapy of HCC. This strategy is aimed at restoring the differentiated characteristics of dedifferentiated hepatocytes, which eliminates the LCSCs derived from this pathway.

Molecular mechanisms for hepatocyte dedifferentiation

The dedifferentiation of mature hepatocytes is a complex process regulated by multiple molecular mechanisms. Understanding these mechanisms is essential for developing effective therapeutic strategies to reverse hepatocyte dedifferentiation.

First, the HNF family plays variable roles in hepatocyte dedifferentiation. HNF-4α is the most critical factor[15]. Gankyrin-mediated dedifferentiation of hepatocytes and hepatoma cells occurs via the downregulation of HNF-4α[16]. Further, high HNF-1β expression promotes the dedifferentiation of HCC cells into LCSCs by activating the Notch pathway[11]. Hepatocyte dedifferentiation involves the coordinated loss of liver-enriched transcription factors and the gain of stemness-associated programs[17]. Meanwhile, HNF-3γ expression decreases in dedifferentiated HCC cells[18].

Second, multiple signaling pathways participate in hepatocyte dedifferentiation. As key factors of EMT, β-catenin overexpression[19] and E-cadherin downregulation[20] may result in hepatocytes dedifferentiating into malignant, immature hepatocyte progenitors. In other solid tumors such as pancreatic cancer, it has been proven that ICG-001, a specific antagonist of CREB-binding protein/β-catenin, can eliminate drug-resistant cancer stem cells or tumor-initiating cells via differentiation-inducing therapeutic effects[21].

Third, microRNAs (miRNAs) play important roles in regulating hepatocyte differentiation and dedifferentiation. Downregulation of miR-122, a hepatocyte-specific miRNA, is frequently observed in dedifferentiated hepatocytes, and miR-122 deficiency in mature hepatocytes leads to the downregulation of hepatocyte-specific genes and the upregulation of stem cell-related genes, resulting in dedifferentiation[22]. Jung et al[23] demonstrated that miR-148a deficiency in liver progenitor cells leads to differentiation arrest and promotes their transformation into LCSCs by upregulating the expression of stem cell markers such as CD133 and epithelial cell adhesion molecule (EpCAM).

Fourth, cellular stress, such as oxidative stress and metabolic stress, can induce hepatocyte dedifferentiation. Aguilar-Bravo et al[24] identified C-X-C receptor 4 as a driver of hepatocyte dedifferentiation profiling in alcohol-related liver disease. Therefore, metabolic dysfunction in mature hepatocytes, such as impaired fatty acid oxidation, also contributes to dedifferentiation.

Molecular mechanism by which differentiation-inducing therapy reverses hepatocytes dedifferentiation

Differentiation-inducing therapy that reverses hepatocyte dedifferentiation allows for restoring the normal differentiated state of dedifferentiated hepatocytes by targeting the molecular mechanisms for dedifferentiation. Several therapeutic strategies have been developed and evaluated in preclinical studies. Epigenetic modifiers that reverse DNA methylation or histone deacetylation have shown promising results in reversing hepatocyte dedifferentiation[25]. Nishida and Kudo[26] demonstrated that TP53 and CNTTB1 mutations are associated with the disturbance of epigenetic integrity, and this epigenetic alteration is reversible. Based on this theory, differentiation therapy is a potential approach for HCC with advanced epigenetic alterations.

MEDICINES WITH DIFFERENTIATION-INDUCING POTENTIAL IN HCC TREATMENT

Based on the above mechanisms, a variety of drugs and molecules with induced differentiation potential targeting LCSCs have been identified and evaluated in preclinical and clinical studies (Table 1). These agents can be classified into the following categories based on their sources and mechanisms of action.

Table 1 Molecules and agents with differentiation-inducing effects in hepatocellular carcinoma.

Agent/molecule	Target/mechanism	Key effects on HCC	Ref.
HNF-4α (gene delivery)	Master hepatocyte transcription factor	Induces differentiation, reduces stemness (CD133, CD90), abolishes tumorigenicity, inhibits metastasis	[16]
HNF-3γ (gene delivery)	HNF	Promotes differentiation, sensitizes to sorafenib via OATP1B1/1B3	[18]
miR-148a mimic	IKKα/NUMB/Notch inhibitor	Induces hepatocytic differentiation, suppresses tumor growth, reduces fibrosis	[23]
ATRA	Nuclear receptor	Induces differentiation of tumor-initiating cells, reverses EMT, potentiates cisplatin, eradicates LCSCs	[3,27]
Sophocarpine	AKT/GSK-3β/β-catenin, TGF-β/EMT	Reduces LCSCs ratio, inhibits sphere formation, reverses malignant phenotype	[13]
ICG-001	CBP/β-catenin interaction	Eliminates drug-resistant, upregulates let-7a (in pancreatic cancer)	[21]
P-T3H2 peptide	TRIB3-HNF-4α interaction	Stabilizes HNF-4α, restores hepatic functions, induces senescence	[15]
5-azacytidine	CpG demethylation	Inhibiting HCC cell tumorigenicity	[25]
Trichostatin A	Histone deacetylase inhibitor	Cell growth inhibition and the mature hepatic gene expression	[28]
Oxygen species	Activation of Akt/IKK signaling pathway	Trans-differentiation into endothelial	[31]
Small molecule cocktail	TGF-β, GSK-3β, G9a, ATRA	Induces differentiation via Akt/mTOR/HIF1α, suppresses Snail	[34]

HCC: Hepatocellular carcinoma; HNF: Hepatocyte nuclear factor; IKKα: Inhibitor of kappa kinase α; NUMB: NUMB endocytic adaptor protein; ATRA: All-trans retinoic acid; EMT: Epithelial-mesenchymal transition; LCSCs: Liver cancer stem cells; AKT: Protein kinase B; GSK-3β: Glycogen synthase kinase-3 beta; TGF-β: Transforming growth factor β; CBP: CREB-binding protein; TRIB3: Tribbles homolog 3; mTOR: Mammalian target of rapamycin; HIF1α: Hypoxia-inducible factor 1 alpha.

Chemical agents

Retinoids: Retinoids, including ATRA and 13-cis retinoic acid, are derivatives of vitamin A that play important roles in regulating cell differentiation. ATRA has been extensively studied for its differentiation-inducing effect on LCSCs[27]. Zhang et al[3] demonstrated that ATRA treatment promotes the differentiation of LCSCs by upregulating the expression of hepatocyte-specific genes and downregulating stem cell markers. In addition, ATRA enhances the sensitivity of LCSCs to chemotherapy by reducing their chemo resistance. However, the clinical application of ATRA is limited by side effects such as skin toxicity and teratogenicity. Excluding in vitro data and focusing on clinical practice, ATRA remains one of the limited agents validated to have a differentiation therapy effect against HCC. Nevertheless, given the marked heterogeneity of liver cancer, a good clinical response is not observed in every patient treated with ATRA.

DNA methyltransferase inhibitors: As mentioned earlier, DNA methyltransferase inhibitors can reverse the epigenetic silencing of hepatocyte-specific genes and thereby promote LCSCs differentiation. Using an in vivo tumor growth model, Gailhouste et al[25] proved that 5-azacytidine treatment increases the efficacy of HCC differentiation therapy via epigenetic reconditioning. Although 5-azacytidine as a differentiation inducer offers a mechanistically attractive, epigenetically-targeted strategy for HCC, lack of clinical trial data in liver cancer make it a doubleedged sword.

Histone deacetylase inhibitors: Trichostatin A[28] and other histone deacetylase inhibitors, such as hydroxamic acid-based hybrid polar compounds[29], and MS275 have been reported to induce cell growth arrest, apoptosis, and differentiation in tumor cells[30].

Signaling pathway inhibitors: Certain inhibitors target signaling pathways involved in LCSCs maintenance and differentiation effects. Zhao et al[31] found that reactive oxygen species promote the endothelial differentiation of cancer stem-like sphere cells enriched from the human HCC cell line Hep G2 by activating the protein kinase B/inhibitor of kappa kinase signaling pathway. Meanwhile, cyclopamine (hedgehog inhibitor) has been related to LCSCs differentiation via mesenchymal-epithelial transition[32]. Liu et al[33] pointed out that inhibiting acetylation of HNF-4α on lysine 458 might provide a more promising candidate for differential therapy of HCC. At present, the use of these inhibitors in HCC remains experimental, and careful patient selection as well as rational combination regimens is essential if this approach is to be translated into safe clinical practice.

Biological agents

miRNAs that regulate LCSCs differentiation, such as miR-122 and miR-148a, have exhibited the potential to serve as induced differentiation agents. For instance, miR-148a delivery using viral vectors or lipid nanoparticles has been shown to promote LCSCs differentiation and inhibit tumor growth in a preclinical model[23].

Combination therapy

Combination therapy using two or more differentiation-inducing agents with different mechanisms of action has been shown to exhibit synergistic effects in promoting LCSC differentiation and inhibiting tumor growth. Similar to the cocktail treatment for acquired immune deficiency syndrome, Zhang et al[34] reported that a small molecule cocktail consisting of SB431542 (TGF-β inhibitor), CHIR99021 (glycogen synthase kinase-3 beta inhibitor), BIX01294 (H3K9 methyltransferase/G9a inhibitor), and ATRA can induce the differentiation of liver cancer cells, including cell lines, primary cancer cells, cancer stem cells, and drug-resistant cells.

CONTROVERSIES AND FUTURE PERSPECTIVES

Current controversies

Despite significant progress in differentiation-inducing therapy that targets LCSCs, several controversies and unresolved questions remain. First, the relative contribution of LCSCs differentiation arrest and mature hepatocyte dedifferentiation to LCSCs formation is context-dependent and remains debatable. Some studies reported that differentiation arrest of LSPCs is the main origin of LCSCs in early-stage HCC, whereas dedifferentiation of mature hepatocyte appears to play an important role in advanced-stage HCC[35]. However, these findings are not universally consistent, and the interplay between these two mechanisms across different HCC subtypes is still poorly understood[36]. Further studies using lineage tracing techniques are needed to clarify this issue.

Second, the identification and characterization of LCSCs rely on specific markers, but there is no universal marker for LCSCs. Different studies have used different markers, such as CD133, CD44, CD90, EpCAM, and aldehyde dehydrogenase, to isolate LCSCs[32,37]. The heterogeneity of LCSCs markers may reflect the varying origins and biological characteristics of LCSCs, but it also makes the development of targeted therapies challenging.

Third, the safety of differentiation-inducing therapeutic agents is an important consideration for clinical translation. As in other solid tumors, HCC tumorigenesis involves multiple oncogenic pathways, making the diversion to normal cells complex[38]. The clinical application of such therapies is further challenged by the fact that several key differentiation pathways, including Wnt and Notch signaling, are also essential for normal adult tissue homeostasis; therefore, drug safety may limit the therapeutic window[39]. In addition, the long-term effects of these agents on normal liver stem cells and mature hepatocytes are not fully understood.

Future perspectives

Despite the current challenges, differentiation-inducing therapy that targets LCSCs may offer a promising avenue for HCC treatment. Several key directions are available to overcome the current limitations and improve the efficacy of this therapy.

First, the development of agents that specifically target LCSCs, without affecting normal cells, is crucial for improving the safety and efficacy of differentiation-inducing therapy[40]. Targeting specific markers or signaling pathways that are uniquely expressed or activated in LCSCs may help achieve this goal. Second, considering the heterogeneity of LCSCs and HCC, personalized therapy strategies are urgently needed. Identifying biomarkers that predict the tumor response to differentiation-inducing therapy can facilitate patient selection and improve therapeutic outcomes[41]. Third, the development of rational combination therapy strategies involving differentiation-inducing agents and other therapeutic modalities, such as chemotherapy, radiotherapy, targeted therapy, and immunotherapy, may lead to synergistic effects and overcome drug resistance[34]. For example, combining differentiation-inducing therapy with immune checkpoint inhibitors may reduce the immunosuppressive microenvironment created by LCSCs and thereby enhance the antitumor immune response. Finally, large-scale, well-controlled clinical trials are critically needed to definitively establish the safety and efficacy of these approaches in HCC patients.

CONCLUSION

LCSCs-targeted differentiation-inducing therapy is a promising new approach for HCC treatment. Despite the challenges and controversies that currently exist, significant progress has been made in understanding the mechanisms underlying LCSCs formation and in developing differentiation-inducing strategies. With continued research and development, this therapeutic approach may become an important part of comprehensive HCC treatment.