Oncogenic signaling pathways and origins of tumor-initiating stem-like cells of hepatocellular carcinomas induced by hepatitis C virus, alcohol and/or obesity

Abstract

This review article discusses the importance and oncogenic signaling pathways of tumor-initiating cells (TICs) in several etiologies of hepatocellular carcinomas (HCCs) induced by hepatitis C virus (HCV), alcohol, obesity and/or chemicals. Stem cells may be present in cancer tissue, and a hierarchy of cells is formed, as is the case for normal tissue. Tumor formation, growth and propagation are maintained by a small proportion of cells with stem cell-like properties. TICs are present in alcoholfed HCV transgenic mice, diethylnitrosamine/phenobarbital-treated mice (chemical carcinogenesis) and Spnb2 +/− mice (defective TGF-β signal). Alcohol/obesity-associated endotoxemia induces the stem cell marker Nanog through TLR4 signaling to generate TICs and liver tumors in several HCC models. The oncogenic pathway (such as the STAT3 and TLR4-NANOG pathway) and mechanism of generation of TICs of HCCs associated with HCV, alcohol and obesity are discussed. Understanding the molecular stemness signaling and cellular hierarchy and defining key TIC-specific genes will accelerate the development of novel biomarkers and treatment strategies. This review highlights recent advances in understanding the pathogenesis of liver TICs and discusses unanswered questions about the concept of liver TICs. (This project was supported by NIH grants 1R01AA018857 and P50AA11999).

Synergistic increase in the HCC odds ratio according to environmental factors (alcoholic liver diseases and obesity) with virus infection (HBV and HCV)

Alcoholic liver disease (ALD) is a major health problem affecting 15 million people [1] in the USA. The obese and NAFLD populations are also increasing alarmingly in the USA. Both alcohol and obesity increase the gut permeability and lead to increased blood levels of endotoxin [2], which in turn activates TLR4 [3] and induces the production of cytokines and the inflammatory response, leading to liver injury and the development of ALD and obesity [1]. Chronic liver damage caused by viral infection and alcohol can result in an increased risk of HCC. In particular, chronic infection with HBV or HCV represents a major risk factor for HCC [4], which is the third most deadly cancer in the world [4–6]. HCC is highly prevalent in Africa and Asia (which have a higher prevalence of HBV and HCV than other areas) and does not respond well to conventional therapy [4]. HCV affects more than 170 million people worldwide [4, 7, 8]. Ample epidemiological evidence demonstrates that HCV and alcohol consumption or obesity synergistically accelerate the development of liver damage [9]. The risk of HCC, as assessed by the odds ratio, increases from 8–12 to 48–54 if HCV patients have concomitant alcoholism or obesity [10–12]. Therefore, understanding the molecular mechanisms of the HCV-induced hepatocarcinogenesis of alcoholic or obese patients is required for the eventual development of improved therapeutic modalities [13].

Stem-cell-like signature, chemoresistance and poor prognosis markers in tumor-initiating stem-like cells (TICs) of HCCs

Forty percent of HCC is clonal and originates from progenitor/stem cells [14–17] with three characteristics, including a self-renewal ability, asymmetric/multiple cell division (clonality) and plasticity. Liver TICs are responsible for tumor relapse, metastasis and chemoresistance. Liver TICs dictate a hierarchical organization that is shared in both organogenesis and tumorigenesis. Liver TICs in tumors give rise to different types of tumor cells (HCC and cholangiocarcinomas) and express “sternness” genes, including Oct3/4/Bmi and morphogene-induced signaling pathways (Wnt/p-β-catenin, Notch and Hedgehog/SMO) [18–20]. The CD133 + cells isolated from the HCC cell lines and human HCCs with higher expression of CD44/CD34 have a self-renewal ability and repeatedly give rise to tumors when injected into immunodeficient mice [21].

The percent of TICs ranges from less than 1 % to 5–8 % in tumor tissues induced by alcohol, HCV and HCV/alcoholic animal models or patients. CD133⁺ tumor cells isolated from HCC cells possess tumor-initiating properties and have characteristics similar to those of LPCs with the expression of “sternness” genes, the ability to self-renew and the ability to differentiate into non-parenchymal cell lineages [21]. These CD133 + TICs confer resistance to chemotherapy, and this presents a major obstacle to the treatment of HCC (Fig. 1). One potential reason for this chemoresistance may lie in the plasticity of TICs with dysregulated signaling and gene expression. Among many stemness factors, Nanog is one of the master transcription factors in pluripotent embryonic stem cells (ESCs) [22] required for maintaining the self-renewal ability and pluripotency of both human and mouse embryonic stem cells [23–26]. Overexpression of Nanaog induces and maintains the self-renewal characteristics and pluripotency of ESCs [27]. Nanog expression is elevated in human neoplasms, including HCC [28], breast carcinomas [29], osteosarcoma [30] and certainly germ cell tumors [29, 31–33]. Furthermore, ectopic Nanog expression induces an oncogenic potential in NIH3T3 [34]. Therefore, stemness/morphogen genes play a pivotal role in TIC oncogenesis and self-renewal of ESC.

Fig. 1.

Hypothetical models of the development of HCCs and TICs. Left In the tumor initiation step, alcohol, obesity or HCV induces DNA mutations. Middle In the tumor promotion step, DNA mutations are fixed by inflammatory responses and growth factors. Right TICs are generated, leading to metastatic tumor development

Higher levels of CD133 and EpCAM⁺ (pancarcinoma antigen epithelial cell adhesion molecule) of HCC cell expression are correlated with increased tumor grade, advanced disease stage, shorter overall survival, tumor progression, invasiveness and higher recurrence rates compared to patients with low CD133 expression [35] (Fig. 1). EpCAM is an early biomarker of HCC [36, 37], which is a direct transcriptional target of the Wnt-β-catenin canonical signaling pathway. EpCAM⁺ α-fetoprotein (AFP)⁺ HCC subtype has features of LPCs. Furthermore, CD24(+) HCC cells are critical for the maintenance, self-renewal, differentiation and metastasis of tumors and significantly impact patient prognosis [38]. CD24 is induced in chemoresistant (cisplatin) tumors compared to bulk tumors [38]. The upregulation of Nanog, together with p53 depletion, is significantly associated with clinically late stage HCC. HCC patients who express biliary cell markers (CK7 and CK10) have poor prognoses and higher recurrence rates post surgical resection/transplantation [15].

Rapamycin and sorafenib are respectively used as single chemotherapy agents for HCC, and a recent HCC xenograft study suggests a better effect when given they are together [39]. Clinical evidence, however, reveals eventual chemoresistance to these drugs in HCC patients [40, 41]. We believe this chemoresistance is caused by TICs. Indeed, Nanog antagonism targeting TICs enhances the efficacy of these two drugs in tumor-bearing mice and achieves ~90 % tumor growth suppression [39].

Origin, target cell type and genesis of TICs

Different cell types in the hepatic lineage are considered the origin of HCC, including mature hepatocytes, cholangiocytes, ductular bipotential progenitor cells and periductular stem cells. C-kit inhibition by the tyrosine kinase inhibitor imatinib mesylate attenuates c-KIT-expressing LPC expansion and inhibits liver tumor formation in mice via antiproliferative effects [42]. Further, carcinogenic insult may begin with mature hepatocytes but alter their phenotype toward bipotential progenitor cells before causing transformation in Notch-intracellular-domain (NICD)/AKT-mediated cholangiocarcinoma [43]. Human liver progenitor cells (LPCs) may give rise to HCC and ICC [44–47] since many tumors contain a mixture of mature hepatocytes and LPCs. LPCs have also been noted in hepatoblastoma, the most common liver tumors in children, which are widely believed to be stem cell derived given there can be both epithelial and mesenchymal tissue components. These tumors can even have structures mimicking intrahepatic bile ducts and form ductal plate-like structures [48]. These stemness genes are highly expressed in LPCs, bile duct epithelium and premalignant hepatic tissues, but not in adult hepatocytes [37, 49].

Before onset of HCC nodules, HcPCs are generated in dysplastic areas. HCC progenitor cells (HcPCs), isolated and characterized from different mouse HCC models, are malignantly transformed in a manner depending on autocrine IL-6-LIN28 signaling from inflammatory cells [50]. Both liver undergoing chronic damage and compensatory proliferation are stimulated in vivo and transform HcPCs into cancer. Interestingly, bipotential hepatobiliary progenitors do not give rise to tumors [50].

Our study so far supports the notion that liver progenitor cells (LPCs) are the potential source of transformed cells in HCC [51]. In fact, rat oval cells transfected with c-Ras^H give rise to HCC in immunocompromized mice [52], and oval cells chemically transformed in vitro produce cholangiocarcinomas and HCC when transplanted in vivo [53]. While mature hepatocytes proliferate to maintain the liver homeostasis after partial hepatectomy [54], differentiation from other sources such as liver progenitor cells (LPCs) may occur after injury, particularly when hepatocyte proliferation is impaired [55]. Indeed, cells whose lineage is traced to LPCs are increased after partial hepatectomy plus 2-AAF or feeding a methionine/choline-deficient diet supplemented with 0.15 % ethionine (MCDE) [56, 57]. These LPCs with bipotential activities are restricted to a subset of biliary duct cells antigenically defined as CD45⁻/CD11b⁻/CD31⁻/MIC1–1C3⁺/CD133⁺/CD26⁻ [55].

Maelstrom promotes HCC metastasis by inducing epithelial-mesenchymal transition by way of Akt/GSK-3β/Snail signaling [58]. A novel oncogene, Maelstrom (MAEL), at 1q24, enhanced AKT activity with subsequent GSK-3β phosphorylation and Snail stabilization, finally inducing epithelial-mesenchymal transition (EMT) and promoting tumor invasion and metastasis [58].

Markers and isolation of TICs from HCCs

TICs in HCC are identified by cell surface antigens including CD133, CD49f, CD24, CD90, CD44, OV6, c-KIT and CD326 (EpCAM), or by selecting the side population (SP) cells by Hoechest dye staining. Therefore, several LPC markers, such as CD133, CD49f, EpCAM, CD24 and CD90, are used to isolate liver tumor-initiating cells (TICs) with stem cell features. The TIC marker CD133 is a predictor of the effectiveness of pegylated interferon α−2b therapy against advanced HCCs [59]. CD24 is a liver TIC marker that drives TIC genesis through STAT3-mediated Nanog regulation [38]. Furthermore, ICAM-1 is a marker of HCC stem cells in humans and mice; ICAM-1 inhibitors slow tumor formation and metastasis in mice. ICAM-1 expression is regulated by the stem cell transcription factor Nanog [60]. Insulin-like growth factor (IGF)2 and IGF receptor (IGF1R) are upregulated in Nanog + TICs [61]. The Nanog gene is also induced in metastatic human liver cancer cells and human HCC tissues (120).

Stem cells have three major characteristics, self-renewal, asymmetric and multiple cell division (clonality), and plasticity. The liver has a high regenerative potential, and hepatic small oval progenitor cells around the peripheral branches of the bile ducts, the canals of Hering, can differentiate into biliary epithelial cells and hepatocytes [62]. These oval liver progenitor cells share molecular markers with adult hepatocytes [albumin, cytokeratin 7 (CK7), CK19, oval cell markers (OV-6, A6, and OV-1), chromogranin-A, NCAM (neural cell adhesion molecule)] and fetal hepatocytes (α-fetoprotein) (Table 1) [15, 62]. Indeed, a quarter to half of HCCs (28–50 %) express LPC markers (CK7 and CK19) identified by immunophenotyping of HCCs [63]. They are also positive for more common stem cell markers such as CD34⁺, Thy-1⁺, c-Kit+ and Flt-3⁺ (FMS-like tyrosine kinase 3) [64]. Thus, it currently remains unclear whether these stem cells are derived from the bone marrow and just migrate to this niche or represent true resident liver stem/progenitor cells. Binding of stroma-derived factor-1α (SDF-1α) to its surface receptor CXCR4 activates oval hepatic cells [65]. Forty percent of HCCs have clonality and thus are considered to originate from progenitor/stem cells [14–17]. Recent studies of HCC have centered on TICs, including detection of TICs in cancer, identification of TIC markers and isolation of TICs from human HCC cell lines. TICs were identified as a CD117⁺/CD133⁺ LPC in regenerating liver tissue [66] and a CD45⁻/CD90⁺ subpopulation of tumor cells in HCC [67]. The CD90⁺ cells are not present in the normal liver and, when injected into immunodeficient mice, create tumors repeatedly. In human HCC and HCC cell lines, specifically CD133⁺ cells, not CD133⁻ cells, had the ability to self-renew, create differentiated progenies and form tumors [21].

Table 1.

Markers for liver tumor-initiating cells

Gene name	Other name	Function	Species	Organ	References
CD133	Prominin 1 (PROM1)	Glycoprotein, membrane protrusions	Human, mouse	Liver, brain	[21, 28, 73–76]
CD49f	Integrina chain a6 (ITGA6)	Cell adhesion, cell signaling	Mouse	Liver	[21, 76]
CD24	CD24		Mouse	Liver, lung	[38]
CK19	Cytokeratin 19	Biliary lineage marker	Mouse	Liver	[77, 78]
OV-6	Oval cell marker	Early progenitor cells	Human	Liver	[78]
CD34	Glycoprotein	Cell-cell adhesion factor	Mouse	Liver, leukemia	[79]
AFP	α-Fetoprotein	Fetal counterpart of serum albumin	Mouse	Liver	[72]
CD90	Thy-1	Glycophosphatidylinositol (GPI) anchor	Mouse	Liver	[21]
CD44	Hyaluronic acid receptor	Cell adhesion and migration, metastasis	Mouse	Liver, breast	[21, 68]
CD117	KIT	C-kit receptor, cytokine receptor	Mouse	Liver	[21]

When compared to CD133⁻ cells, the CD133+ cells isolated from the HCC cell lines showed higher expression of CD44 (cell adhesion molecule) and CD34, but both CD133 subpopulations displayed similar expression for CD29 (integrin β1), CD49f (integrin α6), CD90 and CD117 (c-kit: gastrointestinal stroma tumor), indicating these makers are still not definitive TIC markers [21]. Nevertheless, these makers are differentially expressed in other cancers, such as CD44⁺/CD24^−/Iow in breast cancer [68], CD34⁺/CD38⁻ in acute myeloid leukemia [69] and CD44⁺/α2b1^hi/CD133⁺ in prostate cancer [70]; for this reason, identification of other surface markers, expressed along with CD133, as a goal to better characterize liver TICs is worthwhile.

A minority of the CD133⁺ tumor cell population isolated from HCC cells possesses tumor-initiating properties and has characteristics similar to those of progenitor cells including the expression of “sternness” genes, the ability to self-renew and the ability to differentiate into nonhepatocyte-like lineages [21]. Markers of stemness genes, including CD133, CD90, CD24 and CD49f, are used for isolation of TICs. In addition to the marker-based isolation of TICs, TICs are isolated by sorting the side population (SP) from HCC cells, which have high oncogenic potential and anti-apoptotic properties compared with those of non-SP cells [71, 72].

TICs induced by interactions between HCV and alcohol

Compelling evidence identifies obesity and hepatitis C virus (HCV) as comorbidity risk factors for hepatocellular carcinoma (HCC), which contains TICs. Chronic liver damage caused by viral infection and environmental factors (such as alcohol or metabolic syndrome) can result in increased risk for HCC. Clearly, understanding the molecular mechanisms of HCV-induced hepatocarcinogenesis is required for the eventual development of improved therapeutic modalities for this disease [13]. In particular, chronic infection with HBV or HCV represents a major risk factor for HCC [4]. HCV affects more than 170 million people worldwide [4, 7, 8]. HCV may induce cellular transformation by the induction of chronic liver inflammation mediated by the immune cells [80]. This chronic liver inflammation leads to cell deaths, hepatocellular regeneration and the emergence of mutated cells that may be oncogenic. Interactions between alcohol and HCV synergize immune dysfunctions and liver damage [80].

Hepatitis C and alcohol exacerbate liver injury by suppression of FOXO3 [81]. FOXO3 functions as a protective factor preventing alcoholic liver injury. The combination of HCV and alcohol, but not either condition alone, inactivates FOXO3, causing a decrease in expression of its target genes and an increase in liver injury. Modulation of the FOXO3 pathway is a potential therapeutic approach for HCV-alcohol-induced liver injury [81], with regulation of FOXO3 by phosphorylation and methylation in HCV infection and alcohol exposure [82]. The development of this novel capillary isoelectric focusing (IEF) method for the simultaneous quantification of differently modified FOXO3 species allowed us to demonstrate how HCV and alcohol combine to modify a complex pattern of FOXO3 posttranslational modifications (PTMs) that contribute to the pathogenesis [81, 82].

The high rate of chronic HCV infection in alcoholics is due to ethanol’s effects on antiviral immune responses since ethanol inhibits the humoral and cellular immune responses to genetic immunization of HCV protein (antibody levels and the CD4 + proliferative immune response to this HCV nonstructural protein) [83, 84]. Chronic ethanol consumption inhibits cellular immune responses to HCV core proteins that are restored by genetic immunizations via DNA-based immunization approach using cytokine-expressing plasmids [83, 84]. Chronic ethanol feeding inhibits Th cells and CTL activities with reduced cytokine secretion (a switch from Th1 to Th0 subtype) in proliferating CD4 + T cells [83, 84].

Several possible mechanisms may explain the high prevalence rate of HCV among alcoholics and the increased severity of liver diseases in these patients. First, alcohol may enhance the replication of HCV and thus increase the expression of viral RNA and proteins, resulting in more severe HCV-induced liver injury, independent of the damage induced by alcohol alone. Indeed, the HCV titer has been shown to exhibit a positive correlation with the amount of alcohol consumption [85]. This enhanced effect on HCV replication could be caused directly by the metabolites of ethanol, such as acetaldehyde and free radicals, which may stimulate HCV replication and gene expression. It could also be caused indirectly through alcohol-induced inhibition of the antiviral immune response. Indeed, HCV replication is more active in immunodeficient patients, such as HIV-infected patients [86], and ethanol consumption can cause immunosuppression [87].

Our recent studies using HCV transgenic mice as a model have revealed pivotal insights into the mechanisms underlying synergism between ALD and HCV. By using mice with liver-specific expression of the HCV NS5A protein, we found that mice fed an alcohol or high-cholesterol high-fat diet for 12 months developed liver tumors in a manner dependent on TLR4, which was induced by NS5A (Fig. 2, top). Our further studies indicated that the NS5A-induced TLR4 was activated by endotoxemia associated with obesity/alcoholism, leading to accentuated TLR4 signaling, which in turn upregulated the stem cell marker Nanog to accelerate liver oncogenesis [88] (Fig. 2, middle). HCV NS5A transgenic mice have liver-specific expression of NS5A, not in monocytes or macrophages. Therefore, NS5A transgenic mice do not have enhanced expression of TLR4 in monocytes or macrophages, but in hepatocytes. Therefore, this HCV NS5A-TLR4-NANOG axis may contribute to the mechanisms of the synergism between HCV and alcohol/obesity in liver pathogenesis and carcinogenesis (Fig. 2, bottom). Mechanistic understanding of the role of TLR4-mediated TICs in HCC associated with ALD/obesity and HCV addresses an important public health problem.

Fig. 2.

Hypothetical network of HCV-TLR4-Nanog signaling. Top HCV Ns5a or alcohol feeding induces TLR4 gene expression in transgenic (Tg) mouse models (HCV Ns5a Tg mice) that do not develop liver tumors without alcohol feeding. Middle Alcohol-associated endotoxemia then activates TLR4 signaling, resulting in the induction of the stem cell marker Nanog expression and liver tumors. TLR4 is induced by viral proteins such as NS5A, but also by endotoxin, a condition common in alcoholic and non-alcoholic liver diseases [17, 98, 107], and TICs isolated from the models are indeed TLR4-dependent; TICs are isolated from the liver tumors of these mice and patients. Bottom We have also discovered that TLR4 deficiency attenuates the incidence of HCC in these models

Obesity and TICs

The interaction between obesity and liver cancer is particularly strong, and obesity promotes liver diseases such as non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH). Obesity promotes the HCC burden compared to infection with hepatitis viruses [89]. Both dietary and genetic obesity promotes liver tumors by accentuated expression of IL-6 and TNF-a, leading to liver inflammation and tumorigenesis through activation of the oncogenic transcription factor STAT3 [90]. Activation of JNK induces both the activation of transcription factor activator protein-1 and transcription-independent induction of effector molecules, leading to regulation of cell death and survival, differentiation, proliferation, insulin signaling, ROS and metabolism in the liver [91]. Both loss and hyperactivation of the JNK pathway promote inflammation, leading to metabolic diseases (obesity, steatosis, and insulin resistance), ultimately leading to fibrosis and cancer development [91].

Misregulation of a pluripotency-associated transcription factor network in adult tissues is associated with the expansion of rare, highly malignant tumor-initiating stem cells (TICs) through poorly understood mechanisms. Leptin is the most important adipose-derived hormone, and its circulating concentration is proportional to the total amount of body fat. We demonstrate that robust and selective expression of the receptor for the adipocyte-derived peptide hormone leptin (OB-R) is a characteristic feature of TICs and of a broad array of embryonic and induced pluripotent stem cells and is mediated directly by the core pluripotency-associated transcription factors OCT4 and SOX2 [92]. The induction of OCT4 and SOX2 was observed in leptin-stimulated cells. TICs exhibit sensitized responses to leptin, including the phosphorylation and activation of the pluripotency-associated oncogene STAT3 and induction of Oct4 and Sox2, thereby establishing a self-reinforcing signaling module. The induction of OB-R is also observed in human HCC. We also found that TICs were highly sensitized to leptin exposure in vitro, as judged by the phosphorylation of Stat3-Y705 [92]. Further, leptin promotes the growth of tumors from TICs implanted sub-cutaneously into immune-compromised NOG mice (NOD/Shi-scid/IL-2Rγnull). Exposure of cultured mouse embryonic stem cells to leptin sustains pluripotency in the absence of leukemia inhibitory factor. By implanting TICs into leptin-deficient ob/ob mice or into comparably over-weight Lepr^db/db mice that produce leptin, we have provided evidence of a central role for the leptin-TIC–signaling axis in promoting obesity-induced tumor growth [92]. Differential responses to extrinsic, adipocyte-derived cues may promote the expansion of tumor cell subpopulations and contribute to oncogenesis [92]. These findings provide a direct link between the adipose-derived hormone leptin (obesity) and TICs.

The mechanisms of TIC generation

Poorly differentiated and aggressive human tumors have the gene expression signature of embryonic stem cells. Several oncogenic signaling pathways in cancer stem cells of HCC have been described, including activated PI3 K/AKT [28], signal transducer and activator of transcription 3 (STAT3) [93, 94], Notch [95], hedgehog [96, 97] and transforming growth factor-beta (TGF-β) (Fig. 1) [98, 99]. One potential reason for this chemoresistance may lie in the plasticity of cancer stem cells with dysregulated signaling and gene expression. The initiation of hepatocarcinogenesis is linked to chronic inflammation, which promotes a transformed phenotype and stem cell population through a positive circuit loop involving NF-кB, Lin28B, let-7 and IL-6 [100]. The mammalian homologs of lin-28 of C. elegance, LIN28 and LIN28B are overex-pressed in primary human tumors and human cancer cell lines (overall frequency 15 %) [101]. Transduction of Lin28, OCT4, Nanog and reprogram human somatic fibroblasts to pluripotency [102]. Overexpression of Lin28, OCT4 and Nanog drives self-renewal and proliferation, leading to oncogenesis [102]. Lin28B is an oncofetal circulating TIC marker associated with recurrence, advanced disease and poor clinical outcome in HCCs [101, 103–105]. Lin28 and Lin28B bind to the terminal loop of the precursors of let-7 family miRNAs and block their processing into mature miRNAs [101, 106].

Conclusions

Taken together, a mechanistic understanding of the role of TICs in HCC associated with ALD, obesity and HCV addresses an important public health problem, including the excessive morbidity, mortality and economic burden due to uncontrolled HCC. Therefore, antagonism of oncogenic signaling pathways may become a new therapeutic modality for HCC caused by HCV, obesity and alcohol and also opens a door for the potential application of a similar therapeutic approach for HCCs.