Cancer Stem Cell and Hepatic Stellate Cells in Hepatocellular Carcinoma

Abstract

Hepatocellular carcinoma (HCC) is the most common liver cancer. It is highly lethal and has high recurrence. Death among HCC patients occur mainly due to tumor progression, recurrence, metastasis, and chemoresistance. Cancer stem cells (CSCs) are cell subpopulations within the tumor that promote invasion, recurrence, metastasis, and drug resistance. Hepatic stellate cells (HSCs) are important components of the tumor microenvironment (TME) responsible for primary secretory ECM proteins during liver injury and inflammation. These cells promote fibrogenesis, infiltrate the tumor stroma, and contribute to HCC development. Interactions between HSC and CSC and their microenvironment help promote carcinogenesis through different mechanisms. This review summarizes the roles of CSCs and HSCs in establishing the TME in primary liver tumors and describes their involvement in HCC chemoresistance.

Introduction

Primary liver cancer is the sixth most diagnosed cancer and the third leading cause of cancer mortality worldwide. Primary liver cancer is lethal and more common in men than in women. The major histological type is hepatocellular carcinoma (HCC), which comprises around 75% of all liver cancer cases.¹ Despite recent advances in the prevention, surveillance, diagnosis, treatment, and multidisciplinary collaboration of HCC, it remains highly lethal and with high recurrence.² More than 50% of newly diagnosed patients have advanced or unresectable disease. Death among HCC patients occurs mainly due to tumor progression, recurrence, metastasis, and chemoresistance.³

The liver has the greatest regenerative capacity of any other organ in the body, however, chronic liver damage caused by chronic hepatitis or hepatocyte death resulting from a state of liver fibrosis, alcohol abuse, non-alcoholic steatohepatitis, primary biliary cholangitis, and autoimmune hepatitis have been identified as risk factors associated with HCC.⁴ In recent years, the increase in non-viral causes of HCC such as non-alcoholic fatty liver disease has been associated with risk factors such as metabolic syndrome, insulin resistance, microbiota-altered bowel, and persistent inflammation.⁵ However, the main cause of liver damage is related to chronic viral infections caused mainly by the hepatitis C virus (HCV)^5,6 which is an RNA virus that lacks the ability for integration into the host genome and yet the infection could end in cancer. It is estimated that around 30% to 50% of hepatocytes are infected in a chronically infected individual; this leads to immune response activation (innate and adaptive) to counteract the infection, leading to the activation of hepatic stellate cells (HSCs), which are key in the fibrogenesis.⁷

HCC involves a multistep pathological process that is characterized by chronic inflammation and hepatocyte damage; however, this mechanism is not completely elucidated. Several hypotheses are being explored as an alteration in immune cell surveillance, alterations in apoptosis signaling, upregulated by HCV proteins on HSC activation, micro-RNAs, and the role of epithelial to mesenchymal transition (EMT) for induction of stem-like cells by cells in the tumor microenvironment (TME). In this review, we will explore the role of cancer stem cells (CSCs) have in HCV-related carcinogenesis and interaction of CSCs and HSCs associated as mechanisms of chemoresistance in HCC.

Cancer Stem Cells

CSCs, which have been suggested to represent the clonogenic core of various cancers, are characterized as a distinct subpopulation within the tumor bulk that is capable of high-efficiency tumorigenesis, invasiveness, self-renewal, metastasis, and drug resistance.^8–12 It is significant that these cell subpopulations display innate flexibility and dormancy, reflecting many traits of somatic stem cells but with features similar to normal stem cells.¹³ They can originate from stem cells transformation or de-differentiation of progenitor cells by EMT, which is crucial for the growth and heterogeneity of the tumor.¹⁴ CSC pool can be replenished by de-differentiation of “bulk cells” via a process referred to as transdifferentiation. Reprogramming and de-differentiation of non-CSCs have also been cited as a major factor in tumor cells acquiring CSC-like characteristics.^15,16 HCC cells known as liver cancer stem cells (LCSCs) possess stem cell characteristics and control a hierarchical architecture which are considered the source cells of initiation, progression, and recurrence of liver cancer. LCSC can develop from mature hepatocytes, hepatoblasts, and biliary cells due to liver damage, regeneration, or oncogenic de-differentiation.¹⁷

In 2006, CD133 (PROM1) was first suggested as a particular LCSC marker.¹⁸ Then, additional proteins, such as CD90 (THY1), epithelial cell adhesion molecules (EpCAM), CD24, CD13 (ANPEP), CD34, sex-determining region Y-box 9 (SOX9), ATP-binding cassette, subfamily G, member 2 (ABCG2), CD44, aldehyde dehydrogenase (ALDH), CK19 (KRT19), c-Kit+, and FMS-like tyrosine kinase 3, and OV6, which are cell CSC markers, were discovered.¹⁹ Currently, no one marker is sufficient to characterize the phenotype of CSCs. Using existing HCC cell lines and animal models, more research is required to pinpoint specific markers and signal pathways that are crucial to understanding the characteristics of CSCs.

Liver cancer is continuously encouraged by the growth of stem/progenitor cells, accumulation of genetic and/or epigenetic alterations, and modification of the microenvironment.²⁰ However, a “stem cell niche” is a particular microenvironment crucial for controlling stem cell maintenance and self-renewal by secreting diverse substances. These niches sustain the core characteristics of CSCs, maintain their phenotypic flexibility, shield them from the immune system, and promote their capacity for metastatic spread.³ Even though the LCSC niche is still unknown, some evidence points out the niche's possible role in the LCSC regulation. The most significant support cells detected in the CSC niche are fibroblasts, mesenchymal stem cells, and immune cells. Interaction between cellular and acellular components such as the extracellular matrix (ECM), signaling molecules, intrinsic factors, hypoxia, blood vessels, and exosomes are typically found in the CSC niche. The importance of the niche comes from it provides protection from chemotherapy, reduced cell death, and keep them stemness.²¹

Cellular Components Within Tumor Microenvironment and Maintenance of CSCs-Like Phenotype

The main component in HCC TME are cancer-associated fibroblasts (CAFs), tumor-associated neutrophils (TAN), lymphatic endothelial cells (LECs), tumor-associated macrophages (TAMs), and HSCs. CAFs secrete cytokines, chemokines, and growth factors, that improve CSC self-renewal. IL-6 and hepatocyte growth factor (HGF) produced by CAFs leads to CSC stemness by activation of STAT3 signaling.²² In addition, HGF drives CSC by activation of FRA1 in an Erk 1/2-dependent and c-Met/FRA1/HEY1 signaling. Other pathways activated by CAFs are NF-kB, NOTCH, and Wnt. Notch3 activation promotes LSD1 deacetylation, which keeps CSC stemness.²³

TAMs are cytokine secreting cells able to encourage CSC-like characteristics through EMT induction. Among cytokines, Wan et al have noted that TAM-secreting interleukin 6 (IL-6) and TGF-1 encourages carcinogenesis and the growth of CD44+ LCSCs. In specific, the overactivation of signaling pathways as JAK/STAT in HCC leads to cell survival, increased angiogenesis, stemness, immune-tolerance, invasion, and metastasis.²⁴ It was described that STAT3 signaling is activated by IL-6 released by HCC TAMs, which improves LCSC proliferation. Additionally, non-CSC-secreted IL-17E stimulates CSC proliferation and self-renewal in HCC via activating the JAK/STAT3 and NF-kB pathways.²⁵ Moreover, STAT3 upregulates expression of metalloproteinases (MMPs), MMP-2 and MMP-9, which degrades of ECM barrier,^26,27 and increase expression of EMT proteins Slug and Twist, increasing CSC invasiveness in HCC tumors.²⁸

Several studies support a pro-tumorigenic role of neutrophils in HCC in the peritumoral region after expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor (TNF). Recent studies have shown that TAN can promote the phenotypic transformation of HCC cells into stem lineage-like phenotype by EMT promoting expression of CD44, EpCAM, N-cadherin, and vimentin. Moreover, TAN-secreted BMP2 and transforming growth factor beta (TGF)-β2, belonging to the TGF-β superfamily are responsible for TAN-induced miR-301b-3p expression in HCC cells with a line-like phenotype.²⁹ Increased miR-301b-3p expression subsequently inhibited limbic system-associated membrane protein and CYLD lysine-63 deubiquitinase (CYLD) gene expression, resulting in hyperactivation of NF-κB signaling, C-X-C Motive chemokine 5 (CXCL5) secretion increases, and leads to an increase TAN infiltration.^30,31 LECs are components of lymphatic capillaries and blood vessels and represent one of the most important components in the TME, promote tumor cell proliferation by secreting lymphoangiocrine factors.³² It was reported that LECs interact preferentially with CD133(+) CSCs through direct interactions between high-mannose N-glycans and mannose receptors. This interaction upregulates IL-17A signaling in LECs, helping CSCs self-renew and evade immune attack. Strikingly, neutralization of IL-17A signaling inhibits CSC self-renewal.³³ Therefore, IL-17A may be a promising therapeutic target for liver cancer. During liver injury and inflammation, HSC are a crucial part of the TME and are in charge of producing primary secretory ECM proteins that support fibrogenesis, infiltrate the tumor stroma, and aid in the growth of HCC.

Activation of HSCs

HSCs are liver pericytes-like cells from the Disse gap that represent 5% to 10% of the total cell population.³⁴ In healthy liver, HSCs keep a non-proliferative phenotype (quiescent state), and store approximately 80% of vitamin A in the form of cytoplasmatic lipid drops as its normal activity. In addition, HSCs are involved in the process of liver regeneration, however, exhibit functions on liver carcinogenesis by the secretion of cytokines such as HGF and IL-6.³⁵ Hepatic fibrosis is one of the main risk factors for hepatocarcinogenesis. Fibrosis is a result of chronic hepatic damage characterized by the production of ECM components, proinflammatory cytokines, and growth factors.³⁶ Upon liver damage or extracellular stimuli (profibrogenic factors, pro-inflammatory cytokines, DAMPs, or PAMPs), the HSCs become activated, lose the storage of vitamin A, and transdifferentiate into myofibroblast-like cells as shown in Figure 1.³⁷

Figure 1.

The main components of the tumor microenvironment can activate HSC in HCC. TME-induced signals leading to HSC activation can promote the state of fibrosis and trigger tumor progression.

Activated HSCs are characterized by increased proliferation, migration, contractility, excessive synthesis of ECM molecules, and the release of pro-inflammatory and pro-fibrogenic factors, α-smooth muscle actin (α-SMA), collagen type I and III, and fibronectin for ECM deposition. Some markers include the expression of αSMA, TIMP1, PDGFRβ, and S100a6. However, HSCs also regulate the production of these components through the expression of degrading enzymes such as MMPs and tissue inhibitors of metalloproteinases (TIMPs).³⁸ All these factors are produced to form a scar in the space of Disse to protect the liver from further damage. However, continued activation of HSCs only leads to excessive scar formation in the liver that develops into fibrosis and cirrhosis and ultimately leads to a liver TME.^39,40

Certain components of TME participate in the activation of HSCs as TGF-β, platelet-derived growth factor (PDGF), vasoactive substances such thrombin, angiotensin II, and endothelin-1, cytokines as monocyte chemotactic protein-1, and adipokines as leptin, connective tissue growth factor (CCN2, previously CTGF), vascular endothelial growth factor (VEGF), and viral infections. For example, in HCV-infected hepatocytes, miR-192 is up-regulated and causes the increase of TGF-β1 mRNA expression.⁴¹ In addition, a well-known inductor of EMT in hepatocytes and promoter of HSCs transdifferentiation, miR-19a, directly regulates SOCS3 in the recipient HSCs, leading to STAT3 mediated TGF-β signaling pathway activation, enhancing fibrosis marker gene expression.⁴² The activation is mediated by focal adhesion kinase (FAK)-MMP9 signaling, p53/21, phosphatidylinositol 3-kinase/protein kinase B (MAPK/ERK), IL-6/ signal transducer and activator of transcription 3 (IL-6/STAT3) signaling pathways, and tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN), peroxisome proliferator-activated receptor gamma (PPARγ), nuclear factor kappa-light-chain-enhancer of activated B cells, and toll-like receptors, and recently was associated with the pituitary tumor transforming gene 1 (PTTG1)/delta like non-canonical notch ligand 1 (DLK1) signaling pathway.^43–48 When activated, HSCs promote migration, proliferation, and resistance of HCC cells though modulation of TGF-β signaling and MMP9 upregulation.^35,49

Autophagy, an intracellular degradation system and protective mechanism, has recently been shown to be involved in the activation of HSCs.⁵⁰ The autophagy of HSCs is accelerated by stimuli, like cytokines, released from hepatocytes or immune cells to activate HSCs and contribute to fibrosis. For example, during HSCs activation, exposure to autocrine factors can provoke cells to kick off anti-fibrotic miRNAs to avoid the repression of ECM production. The anti-fibrotic miR-29a is a miRNA repressed during fibrosis in response to TGF-β and PDGF-β signaling by promoting miRNA secretion. The secreted miR-29a has no pro-fibrogenic effect but leads to reduction of intracellular miR-29a levels.⁵¹ The process was dependent on the enhanced (and abnormal) autophagic activity and when autophagy was inhibited, the secretion stopped and the miR-29a accumulated and repressed its direct targets, such as collagen 1A1 (COL1A1). Consistently, expression loss of miR-29a on hepatic tissues and increased miR-29a levels in serum samples were observed in murine carbon tetrachloride (CCl4) fibrosis model and clinical samples from HCV-chronic liver disease patients.⁵²

Besides changes in secretion patterns, HSCs activation involves change to a myofibroblast-like phenotype and production of ECM proteins, as α-SMA,⁵³ and infiltration to HCC stroma.⁵⁴ Also, Mrp-type transporters are highly expressed in activated HSCs, such Mrp1, which promotes its survival.⁵⁵ Thus, HSCs activation facilitates the development of the accurate TME that favors the maintenance of cancer cells. It is still unclear how HSCs and CSCs communicate with one another, however according to studies, liver damage can cause HSCs to release paracrine substances, which in turn increase the development of progenitor cells, which may result in either hepatic regeneration or hepatocarcinogenesis. It is interesting to note that other research has demonstrated, based on several factors, that HSCs can directly transdifferentiate into progenitor cells or promote CSCs.

In addition to the well-documented role of miRNAs in tumor cells, several studies have revealed that various miRNAs are abnormally expressed during liver fibrosis regulating numerous cell-signaling pathways.⁵⁶ MiRNA regulation functions in 2 levels: (a) intracellularly, a stress/injury signal turn-on/turns off the miRNA transcription, for example, the miR-200 family, miR-203, and miR-216/217 that are suppressed by transcriptional regulators downstream of TGF-β signaling,⁵⁷ or (b) as cell-to-cell regulatory molecules by horizontal transfer (here reviewed). MiRNAs, lipids, and proteins can be transferred by lipid bilayer-delimited particle bodies that are naturally released from almost all cell types and are referred to as extracellular vesicles (EVs).⁵⁸ EVs deliver their cargoes to other cells via receptor interaction, membrane fusion, and or endocytosis/phagocytosis leading to functional alterations of the recipient cells and contributing to TME formation.^59–61

Crosstalk between the TME cellular compounds mediated by exosome-delivered miRNAs can directly impact the maintenance and activation of quiescent HSCs (Figure 1). The exposure of HSCs to a hepatocytes primed-medium caused a decrease in α-SMA and COL1α mRNA expression levels. When EVs production is inhibited, the effect of EVs over HSCs activation is loosed and fibrotic markers mRNA levels increase in the recipient HSCs. RNA sequencing of EVs content revealed that hepatocytes express 103 miRNAs in higher concentrations compared to EVs from other analyzed cell types. Among them, the miR-423-5p directly targets COL1α mRNA.⁶² This protective effect was mirrored in vivo using C57BL6 mice and CCl4 hepatic injury model, in which treatment with intravenous injection of HepG2-EVs prevented an elevation of the hepatic enzymes AST and ALT, the expression of the fibrosis markers αSMA, COL1α, PDGF, TGF-β, TIMP1, and TIMP2, and reduce the fibrotic tissue in the mouse liver. Transfection of pHSCs with a miR-423-5p mimic reduced the target genes, reproducing the data obtained with HepG2-EV treatment.

miR-214 and miR-199a-5p are highly expressed in qHSC-EVs in contrast to activated HSC-EVs and act as anti-fibrotic miRNAs by direct targeting of CCN2^63,64 a related marker of the malignant characteristics of tumor cells.⁶⁵ When the miR-124 gets into activated HSC, it suppresses CCN2 expression and downregulates a-SMA and collagen expression downstream of CCN2.⁶⁴ Decreased levels of circulating miR-124 were also observed in a fibrosis model. In line, miR-199a-5p downregulation was associated with increased CCN2 in fibrotic hepatic tissue and activated HSCs. As observed with miR-124, EVs form quiescent cells that can bind to the membrane of activated HSCs in vivo, and inhibit the CCN2, α-SMA, or collagen in activated HSCs in-vitro in a miR-199a-5p-dependent manner.⁶³ Circulating levels of miR-574-5p positively correlated with HSCs activation, collagen deposition, and the miR-574-5p expression in liver tissues.⁶⁶

During TME development, immune cell regulation plays a pivotal role in tumor immune evasion.⁶⁷ HSCs activation can lead to influence immune cells to promote a profibrotic state. A recent study showed that trans-differentiation of HSCs alters the production and content of EVs. When modified EVs reach the macrophage cell membranes, they stimulate cytokine synthesis-release and macrophage migration.⁶⁸ Using a biliary duct ligation rat model and following HSCs isolation, the authors found that HSCs increased EVs production upon activation. EVs changed in size (from 125-250 nm size range to 50-100 nm after activation) and miRNA content compared to non-activated HSCs. miRNA profiling showed that miRs-19b, miR-141, miR-145, miR-150, and miR-200 expression were elevated in active HSCs in mouse, rat, and human-derived HSC-EVs, being miR-19b and miR-200 the most abundant. Also, the exposition of RAW 264.7 macrophages to activated rat HSC-EVs, caused an increase in both mRNA and protein levels of IL-6 and TNF-α, 2 important HCC drivers by impacting in macrophage polarization process, angiogenesis and hepatocyte proliferation, and apoptosis.⁶⁹ When HCC has established, HCC cells directly participate in sustaining the myofibroblastic phenotype of the HSCs.⁷⁰ The authors used cell lines to demonstrate that HCC cells produce more EVs than normal cells. EVs from HCC cells could bind to the LX2 cells membrane and caused an increase in the expression of α-SMA expression, promoting cell growth, cell proliferation, and migration of activated HSCs in-vitro. In addition, intravenous exposition to HCC cells derived-EVs potentiated the growth of subcutaneously implanted tumors in nude mice. MiRNA profiling showed that miR-21, miR-27, miR-34, miR-122, and miR-126 were highly expressed in tumor cell lines with miR-21 being the most abundant. Further analysis by miRNA mimics demonstrated that miR-21 directly mediates the HSCs activation and transdifferentiation through the PTEN/PDK1/Akt pathway. Unfortunately, high levels of miR-21 in serum EVs from cancer patients correlated with low survival prognosis.

HSCs Contribution, Cytokines Involved, and the Relationship Between HSC and CSC in HCC Development

As detailed above, CSCs need a microenvironment that allows them to survive. This microenvironment is generated with the presence of other cell lines, components of the ECM, and some soluble factors. Nevertheless, the communication between HSC and CSC has not yet complete elucidated. Some studies have demonstrated that factors produced by activated HSCs induce the expression of stemness markers (CD44⁺, ALDH⁺, EpCAM⁺, CD133⁺) in CSC as shown in Figure 2 and confer chemoresistance upon LCSC.⁷¹

Figure 2.

A schematic relationship between HSC and LCSC. Factors released by activated HSC promote the maintenance or proliferation and invasion of LCSC in HCC.

The interplay between HSCs and CSC demonstrates a characteristic chemoresistance niche and highlights additional potential targets to interrupt this communication for HCC treatments. For example, studies identified an enzyme called Stearoyl-CoA desaturase 1 (SCD1) responsible for synthesizing a critical player in mediating CSC properties. SCD1 is upregulated by HSCs and induces Wnt/β-catenin signaling. Also, SCD1 contributes to sorafenib resistance by inhibiting the unfolded protein response. Therefore, suppression of SCD1 can be used to suppress liver fibrosis and tumorigenesis and sensitize the cells to sorafenib treatment.^72,73

Diverse studies have shown that crosstalk between HSCs and CSC and non-cellular factors induce the development of TME through molecules production and triggering molecular pathways that are elicited after HSC activation (Table 1).

Table 1.

Summary of Main Regulator Factors Produced in Liver Tumor Microenvironment Related to HCC Development.

Main regulator factor	Effect on tumor microenvironment	Refs
MMP2 and MMP9	Produced from activated HSC, they induce migration and the invasive potential of HCC tumors by collagen degradation in the ECM through FAK-MMP signaling.	74,75
PD-L1, VEGF, GM-CSF, SDF-1and IFN-γ	Products of activated HSC, they can induce an immunosuppressive microenvironment, and promote angiogenesis and tumor proliferation.	76–78
CCN2 and IL-6	Hepatocyte derived CCN2 activates HSCs and cytokine production promoting tumor cell proliferation.	65
Ang-1, Gli-1 and IL-8	Activated HSCs secrete Ang-1, Gli-1, and IL-8 promoting HCC angiogenesis. Some angiogenic effects can be blocked with IL8 neutralization.	79–81
SCD1	SCD1 is upregulated by HSC and promotes liver cancer stemness through MUFAs synthesis as a critical player in CSC properties.	72,73
HGF and IL-6	HSCs secrete HGF and IL-6 that induce expression of stemness markers in CSCs mediated STAT3 signaling and confer chemoresistance.	34
FOXM1	FOXM1 is a main player in HSC activation and stemness in CSCs in-vitro.	82

Abbreviations: MMP, matrix metalloproteinase; PD-L1, programmed death-ligand 1; VEGF, vascular endothelial growth factor; GM-CSF, granulocyte macrophage colony-stimulating factor; SDF-1, stromal cell-derived factor 1; IFN, interferon; CCN2, connective tissue growth factor; IL, interleukin; Ang-1, angiopoietin-1; Gli-1, glioma-associated oncogene 1; SCD1, stearoyl-CoA desaturase 1; MUFAs, mono-unsaturated fatty acids; HGF, hepatocyte growth factor; FOXM1, forkhead box M1.

Mechanism Associated With Resistance by CSC Activated With HSC in HCC

Inherent and external mechanisms can mediate multidrug resistance of CSC: DNA damage repair pathway activation, self-renewal-related genes, quiescence pro-survival or antiapoptotic signals, activation of signaling pathways related to EMT stimulation of hypoxia, and aberrant angiogenesis. In general, the resistance mechanisms are related to TME elements and the interaction between them and CSC activates survivor and proliferating pathways. Next, a brief overview of those CSC resistance mechanisms brought on by the activation of HSC in HCC will be given. The following mechanisms will be described in Figure 3.

Figure 3.

Activated HSCs improve multidrug resistance mechanisms. HSCs can improve CSC chemoresistance by secretion of factors, activation of molecular pathways, and metabolic changes in tumor cells.

DNA Damage Repair Pathway Activation

DNA repair mechanisms are highly controlled to avoid malignant transformation in normal cells, however, in cancer DNA repair mechanisms are dysregulated allowing proliferation. In HCC high DNA repair was related to worse survival, intratumor heterogeneity of cells and mutations.⁸³ In nucleotide excision repair (NER) process, overexpression of ERCC1 and XPC are related to liver fibrosis and chemoresistance.⁸⁴ Moreover, it was demonstrated that overexpression of ERCC-1 provides resistance to platinum-based anti-cancer agents by eliminating platinum/DNA adducts.⁸⁵ Furthermore, loss of expression of mismatch repair genes is related to chemoresistance to agents as 6-thioguanine, methylating agents, cisplatin, and carboplatin. Subexpression in HCC was reported in hMSH2, hMLH1, GTBP, hPMS2, and hPMS1.⁸⁶ Also, apurinic/apyrimidinic endonuclease (APE1) overexpression in HCC cells provides radiotherapy resistance through base excision repair.⁸⁷ Thus, DNA repairing genes could be an attractive marker of chemotherapy resistance that needs more research.

Returning to HSCs, activated HSCs can also inhibit p53 activation which protects cancer cells to chemotherapy induced apoptosis. It has been reported that HSCs in coculture with Huh-7 cells confers resistance to sorafenib by the HGF/c-Met/Akt and JAK/STAT3 pathways.⁸⁸ LX-2 cells coculture with Hep3B cells reduce the cisplatin activity on growth inhibition.³⁴ However, cancer cells do not remain passive because, in retribution, they promote HSCs activation and proliferation by a TGF-β1 mediated pathway and secretion of growth differentiation factor 15 (GDF15).⁸⁹ This mechanism involves the generation of a loop allowing the activation of both cell types to develop the TME.

DNA damage checkpoint kinases (CHKs) activated by genotoxic stress are one of the main regulators of CSC resistance to chemotherapy. It has been demonstrated that the phosphorylated form of CHK2 was presented in HCC patients with paclitaxel resistance.⁹⁰14-3-3ζ is a CHK that regulates the cell cycle, differentiation, and apoptosis, and was observed that it promotes resistance to radiation in CD133+ Huh7 CSC. Resistance is mediated by activating the pro-survival AKT/PKB and Bcl-2 signaling pathways, that inhibit apoptosis activation.¹⁹ On the other hand, it has been shown that susceptibility to sorafenib can be increased in hepatoma cell lines through RNA inhibitors (siRNA) against ataxia telangiectasia mutated (ATM) by decreasing AKT phosphorylation. Aberrant AKT activation contributes to CSC features in HCC; however, the effect of siRNA against ATM in CSC has not been explored.⁹¹

Self-Renewal-Related Genes

Self-renovation is one of the main characteristics of CSC that allows maintenance of the stemness state. Epithelial cellular adhesion molecule (EpCAM) has been proposed as a marker of CSC in various cancers such as HCC, due to its relation to proliferation and metastasis.⁹² EpCAM-positive liver CSC activate Wingless (Wnt) signaling and resistance to sorafenib; CD90 + or CD105+ CSC present resistance to 5-Fluorouracil.⁹³

Another pathway involved in proliferation and stemness is Wnt/β-catenin. Among its activators, protein tyrosine kinase 2 (PTK2) promotes signaling and nuclear translocation of β-catenin, which enhances the viability and survival of HCC CSC and induces sorafenib resistance. Another activator is phosphatidylinositol 4-phosphate adaptor protein 2 (FAPP2). Its inhibition reduced HCC progression.⁹⁴ This finding demonstrates that activation of survivor pathways provides CSC immunity to chemotherapy.

HCC chemoresistance is also related to enhance expression of the Hedgehog signaling pathway. As an inhibitor of this signaling, cyclopamine, inhibits CSC proliferation.⁹⁵

Yes-associated protein 1 (YAP1) is a core component of the Hippo pathway, that requires coactivation with the PDZ-binding motif (TAZ) and functions as a transcription factor. The expression of YAP1 in HCC CSC is very important for self-renewal and cell-fate determination.¹⁴ Moreover, the levels of YAP1 in CSC are correlated with stemness markers such as NANOG, OCT-3/4, and CD133, and consequently with the severity of HCC.⁹³ In addition, TME participates in stemness regulation. Models have been developed for TME as the multicellular tumor spheroid, which includes mixed patient-derived HCC cells, HSCs, and epithelial cells, and explore YAP/TAZ overexpression. In this model, the inhibition of YAP1 increases the sensitivity to sorafenib.⁹⁶ Thus, this leads to the conclusion that HSC could contribute to the overexpression of YAP1 in the CSC microenvironment.

Another mechanism is the suppression of TGF-β signaling, which induce the activation of Toll-like receptor 4/ NANOG and regulates the immune and inflammatory response and increase tumor growth and chemoresistance in HCC CSC.⁹⁵ However, this finding is contrary to the fact that CSC generate TGF-β, indicating a change in secretion pattern of factors to increase survivor.⁸⁹

HGF receptor is encoded by a c-MET oncogene, thus is related to proliferation in HCC cells. Cells positive to c-MET present CSC characteristics such as chemoresistance, tumor sphere formation, and increased expression of CSC markers such as CD44 and ABCG2.⁹⁵ As mentioned before, HGF is one of the products released by activated HSCs.³⁵ Thus, it is another way in which HSCs can lead to increase of CSC chemoresistance.

EPHB2 is part of the Eph receptors of tyrosine kinase transmembrane glycoproteins that is overexpressed in HCC and promotes stemness. CSC overexpressing EPHB2 presents more stemness and chemoresistance, this mediated by SRC/AKT/GSK3/β-catenin pathway. In murine models, the inhibition of EPHB2 dramatically increases HCC cells sensitivity to sorafenib and inhibits tumor growth.⁹⁷

Quiescence

CSCs regulate the cell cycle to improve its survival. CSCs commonly proliferate at a low rate, a characteristic that improves resistance, as chemotherapeutic drugs and radiation eliminate high proliferating tumor cells. Thus, quiescent cells can later proliferate and are related with tumor relapse.⁹⁸ In quiescence, the signaling pathways remain in a poised state, avoiding metabolic stress and preserves genomic integrity. In CSC cells of HCC overexpression of molecules that promotes quiescence and chemoresistance has been demonstrated. In turn, laminin-332 expression decreases cell mitosis and sorafenib resistance, by the α3β1/Ln-332 axis.^99–101 Ubiquitin-specific protease 22 (UPS22) stabilizes HIF-1α, and forms a HIF-1α/USP22 positive feedback loop that promotes glycolysis and stemness on TP53 inactivated cells, which control quiescence in sorafenib resistance HCC.¹⁰² CD13+ CSC in HCC also are reported as semiquiescent, and present 5-fluorouracil resistance. The inhibition of CD13 suppresses self-renovation and tumor growth.¹⁰³

Prosurvival or Antiapoptotic Signals

Among prosurvival signaling pathways, PI3K/AKT highlights the CSC promoting chemoresistance. CD133+/EpCAM + hepatic CSC present resistance to rapamycin and sorafenib through the PI3K/AKT/mTOR pathway. In addition to chemotherapeutic agents, PI3K/AKT/Bab signaling is responsible for inhibiting other pro-apoptotic activators proposed as alternative cancer treatments. For example, this pathway blocks apoptosis activation induced by TNF-related apoptosis-inducing ligand in CSC from hepatic cancer cell lines. Another target for cancer therapies is nuclear factor kappa B (NF-κβ), an antiapoptotic signal transcription factor on CSC that can be activated with drugs such as sorafenib, contributing to enhanced chemoresistance in CD133 + hepatic CSC.¹⁰⁴ Moreover, micro RNAs also participate in resistance as they can modulate survival pathways of CSC such as Wnt/β-catenin, TGF-β, and JAK/STAT signaling.¹⁹

Activation of Signaling Pathways Related to the EMT Stimulation of Hypoxia

Activated HSCs products induce the EMT of HCC cells and allows metastasis.¹⁰⁵ HSCs allows EMT in cancer cells by reducing E-cadherin expression and pronounced upregulating of N-cadherin and Vimentin. Mesenchymal phenotypes are driven by the E-Cadherin/β-Catenin complex and nuclear translocation of β-Catenin, and β-Catenin expression allows EMT and CSC proliferation.³⁴

Other TME factors also affect the response to therapies. In turn, hypoxia activates the expression of hypoxia-induced factor-1 (HIF-1), which has been proved that triggers proliferation, angiogenesis, metastasis, and most importantly chemo- and radio-resistance of HCC cells.¹⁰⁶ One accumulation mechanism of its accumulation in TME is by transglutaminase 2 (TGM2), a protein upregulated in HCC cells by activated HSC. TGM2 depletes von Hippel-Lindau protein, which degrades HIF-1a, leading to its accumulation and EMT development.¹⁰⁷ Hypoxia also can induce the expression of DLK1 from HSC during fibrosis, which helps maintenance of CSC by Wnt/β-catenin signaling. Moreover, DLK1 suppresses PPARγ, which inhibits tumor growth, invasion, and EMT.⁴⁶

Multidrug resistance can also be mediated by overexpression of ATP-binding cassette transporters (ABC transporters), which extrude a variety of toxic substances, thus increasing cancer stemness, EMT, and chemoresistance.^108–111 As a result, ABC transports could be used in research as CSC and chemotherapy markers. EMT is highly related to CSCs, and greatly impacts in chemoresistance as poorly differentiated liver cancers present with high levels of mesenchymal markers with resistance to cisplatin, doxorubicin, and sorafenib. In addition, the CSC marker, CD44, is presented in cancer cells resistant to sorafenib.¹¹² The expression of EMT-related genes and higher expression of TGF-β is associated with paclitaxel resistance.⁸⁸

HSCs cells can also increase CSC proliferation by HGF secretion, since CD133 positive cell population was enriched in Hep3B cells in coculture with LX-2 cells which is mediated by HGF receptor tyrosine kinase Met signaling. In addition, the stemness of this population was also demonstrated by the expression of Bmi1 and Klf4 transcripts.³⁴

Besides promoting metastasis, EMT also seems to be associated with increased survival pathways involving resistance to chemotherapies. HSC enhances these properties by bringing the necessary factors and chemokines for CSC maintenance and tumor development.

Aberrant Angiogenesis

Angiogenesis is an important step during metastasis development, and HCC is described as a highly vascularized tumor. The angiogenesis process requires VEGF and VEGFR and endothelial cells, which are targets for therapy. High VEGF expression is related to invasiveness, a shorter survival time, and poor prognosis.¹¹³ Moreover, endothelial cells secrete proangiogenic factors that promote CSC expansion, survival, and dissemination. CSC by itself can also transdifferentiate into endothelial cells, contributing to TME establishment. Sorafenib agents can promote angiogenesis in HCC tumors with high CSC number.¹¹⁴ In addition, there is a high expression of E-cadherin and CK19, which are CSC markers in HCC.

Another proangiogenic factor is secretory clusterin (sCLU), which also has antiapoptotic and pro-metastasis activity in HCC. This factor activates the PI3K/AKT signaling pathway, leading to phosphorylation and inactivation of GSK3β, stabilizing β-catenin, and upregulation of the Wnt/β-catenin pathway.¹¹⁵ As being relatedly new, sCLU could represent another target for cancer therapies.

Promotion of HCC Chemoresistance

In HCC, ECM components increase as collagens and cytokines are produced mainly by activated HSCs, contributing to tissue fibrosis and chemoresistance.⁸⁹ Among mechanisms related to increased chemoresistance, it has been demonstrated that HSC leads to resistance through the production of COL1A1, TGF-β1, and CCN2.⁸⁹ The increase in collagen production by HSC results in liver localized fibrosis. This change in ECM reduces nutrients availability and increases hypoxia. Reduced oxygen affects chemotherapeutic treatments such as the drug transporters p-glycoprotein (MDR1, multidrug resistance 1), drug targets (topoisomerase II), or by initiating drug-induced apoptosis.¹¹⁶ Moreover, in-vitro experiments show that this ECM compaction increases resistance to sorafenib and cisplatin.⁸⁹ Calponin 2 (CNN2) expression increases chemoresistance since its inhibition sensitizes Hep3B cells to doxorubicin. The mechanism associated with CNN2 is the overactivation of the drug efflux pumps ABCC1 and ABCC2.¹¹⁷

Laminin (Ln)-332 is also highly secreted by HSCs. Activated HSC produces large amounts of laminin (Ln)-332, a ligand of α3β1 and α6β4 integrins expressed in HCC cells by the presence of TGF-β1.¹⁰¹ When it binds to α3β1 integrin on cancer cells, it inhibits fibroblast activation protein from ubiquitination, and thus, apoptosis by drugs such as sorafenib.^89,101 The pathways activated by these integrins are mediated by FAK, and modulate cellular functions including adhesion, migration, and invasion. Moreover, Ln-332 also promotes CSC maintenance.¹⁰¹

As part of TME, HSC also modulate immune activation, upregulating B7 homolog 1 (B7H1/CD274/PD-L1) on activated HSC, reducing T cell activation, apoptosis, infiltration, and cytotoxicity, which drives tumor cell escape.⁸⁹ All this indicates the important role of HSC in improving CSC chemoresistance, even as secretion of ECM components increase fibrosis impeding the diffusion of agents into TME and improving immunotolerance or the activation of specific pathways in CSC that promote survival.

Conclusion

HCV chronic infection triggers changes in liver related with damage and potentially leads into cancer. However, the process of cancer development requires the settle of TME elements and the improvement of EMT. As reviewed here, activated HSCs are the main participants in TME as their activation is one of the central events of liver fibrosis that promotes hepatocarcinogenesis. They promote miRNA secretion, ECM remodeling, and LCSCs maintenance that triggers CSC survivor and cancer metastasis. Moreover, LCSCs improve the constant activation of HSCs, leading to an activation loop. Thus, chemoresistance seen in HCC is highly related to metabolic and molecular changes induced by HSC. This paper focuses on the role of activated HSCs and the mechanisms involved in promoting LCSC proliferation and resistance. However, it must be considered that within the microenvironment, HCV and its proteins also play a central role in carcinogenesis and how they modulate the microenvironment to regulate the expression of some microRNAs or liver damage markers. It is necessary to study the progression of the infection into the fibrogenic and tumorigenic process as well as the links between activated HSCs at all stages of fibrosis and cirrhosis needs to be studied to understand HCC tumorigenesis better. This knowledge is key to identifying markers or therapeutic targets that could be used to change the course of this disease.

Abbreviations

ABCG2

ATP-binding cassette subfamily G member 2

ALDH

Aldehyde dehydrogenase

ALT

Alanine transaminase

α- SMA

Alfa-smooth muscle actin

Ang-1

Angiopoietin-1

APEI

Apurinic/apyrimidinic endonuclease

AST

Aspartate transaminase

ATM

Against ataxia telangiectasia mutated

CSC

Cancer stem cells

CAF

Cancerassociated fibroblasts

CCl4

Carbon tetrachloride

COL1A1

Collagen 1A1

CCN2

Connective tissue growth factor

CXCL5

C-X-C Motive chemokine 5

CHKs

DNA damage checkpoint kinases

DLK1

Delta like non-canonical notch ligand 1

ECM

Extracellular matrix

EMT

Epithelial to mesenchymal transition

EpCAM

Epithelial cell adhesion molecules

EVs

Extracellular vesicles

FAPP2

Phosphatidylinositol 4-phosphate adaptor protein 2

FOXM1

Forkhead box M1

GDF15

Growth differentiation factor 15

GM-CSF

Granulocyte macrophage colony-stimulating factor

Gli-1

Glioma-associated oncogene 1

HGF

Hepatocyte growth factor

HCC

Hepatocellular carcinoma

HSCs

Hepatic stellate cells

HGF

Hepatocyte growth factor

HCV

Hepatitis C virus

IFN

Interferon

IL

Interleukin

IL-6

Interleukin 6

LCSCs

Liver cancer stem cells

Ln

Laminin

LECs

Lymphatic endothelial cells

NF-kB

Nuclear factor kappa B

MMP

Matrix metalloproteinase

MUFAs

Mono-unsaturated fatty acids

PDGFRβ

Platelet derived growth factor receptor-beta

PDGF

Plateletderived growth factor

PPARγ

Peroxisome proliferator-activated receptor gamma

PTTG1

Pituitary tumor transforming gene 1

PD-L1

Programmed death-ligand 1

siRNA

RNA inhibitors

SDF-1

Stromal cell-derived factor 1

SCD1

Stearoyl-CoA desaturase 1

SOX-9

Sex-determining region Y-box 9

TAMs

Tumor-associated macrophages

TAN

Tumor-associated neutrophils

TGFβ-1

Transforming growth factor beta-1

TIMP1

Inhibitors of metalloproteinases

TME

Tumor microenvironment

TNF

Tumor necrosis factor

UPS22

Ubiquitin-specific protease 22

VEGF

vascular endothelial growth factor

WNT

Wingless

YAP1

Yes-associated protein 1.