Tumor-initiating stem-like cells and drug resistance: carcinogenesis through Toll-like receptors, environmental factors, and virus

Abstract

Neoplasms contain distinct subpopulations of cells known as tumor-initiating stem-like cells (TICs) that have been identified as key drivers of tumor growth and malignant progression with drug resistance. Stem cells normally proliferate through self-renewing divisions in which the two daughter cells differ markedly in their proliferative potential, with one displaying the differentiation phenotypes and another retaining self-renewing activity. Therefore, understanding the molecular mechanisms of hepatocarcinogenesis will be required for the eventual development of improved therapeutic modalities for treating hepatocellular carcinoma (HCC). Hepatitis C virus (HCV) and hepatitis B virus is a major cause of HCC. Compelling epidemiologic evidence identifies obesity and alcohol as co-morbidity factors that can increase the risk of HCV patients for HCC, especially in alcoholics or obese patients. The mechanisms underlying liver oncogenesis, and how environmental factors contribute to this process, are not yet understood. The HCV–Toll-like receptor 4 (TLR4)–Nanog signaling network is established since alcohol/obesity-associated endotoxemia then activates TLR4 signaling, resulting in the induction of the stem cell marker Nanog expression and liver tumors. Liver TICs are highly sensitized to leptin and exposure of TICs to leptin increases the expression and activity of an intrinsic pluripotency-associated transcriptional network comprised of signal transducer and activator of transcription 3, SOX2, OCT4, and Nanog. Stimulation of the pluripotency network may have significant implications for hepatocellular oncogenesis via genesis and maintenance of TICs. It is important to understand how HCV induces liver cancer through genesis of TICs so that better prevention and treatment can be found. This article reviews the oncogenic pathways to generate TICs.

HCV, alcohol, and HCC:

HCC has a low five-year survival rate due to the lack of therapeutic options and is highly prevalent in the world, especially in Africa and Asia ¹. HCV affects more than 170 million people worldwide^{1, 3, 4}.

Ample epidemiological evidence suggests that there is a strong connection between hepatitis C virus (HCV) and alcoholic liver diseases (ALD). First, the prevalence of HCV is significantly higher among alcoholics than in the general population; for example, while the HCV positive rate in the general population of the U.S. is roughly 1%, it is 16% for alcoholics and nearly 30% for alcoholics with liver diseases⁵. Second, the presence of HCV infection correlates with the severity of the disease in alcoholic subjects; i.e, HCV-infected patients with ALD develop liver cirrhosis and HCC at a significantly younger age than uninfected ALD patients, suggesting that alcohol and HCV work synergistically to cause liver damage⁶. The most devastating consequence of the synergism between viral hepatitis and alcohol is HCC^7–11. Heavy alcohol consumption and viral hepatitis synergistically increase the risk for HCC among blacks and whites in the U.S. ¹⁰. HCC odds ratio increases to 48.3-fold and 47.8 from 8.1 and 8.6 by having concomitant alcohol abuse in HCV or HBV-infected patients, respectively^{9, 10}.

Recent studies with mice expressing HCV proteins have shed pivotal insights into the mechanisms underlying this synergism. The HCV core protein causes overproduction of reactive oxygen species which appears to be responsible for mitochondrial DNA damage^{3, 12, 13}. The core protein also inhibits microsomal triglyceride transfer protein activity and VLDL secretion¹⁴, which may underlie the genesis of fatty liver. The core protein also induces insulin resistance in mice and cell lines, and this effect may be mediated by degradation of insulin receptor substrates (IRS) 1 and 2 via up regulation of SOCS3¹⁵ in a manner dependent on PA28γ 73, or via IRS serine phosphorylation¹⁶. Thus, these core-induced perturbations such as oxidant stress and insulin resistance, which are also known risk factors for ALD, may underlie the synergism reproduced in alcohol-fed core transgenic mice¹⁷. TLR2 and TLR4 are markedly upregulated in hepatocytes, Kupffer cells, and peripheral monocytes of patients with chronic hepatitis C. TLR2-mediated activation by hepatitis C virus is linked to the pro-inflammatory cytokine induction¹⁸. TLR-mediated signals result in liver disease associated with hepatitis B and hepatitis C viruses, alcoholic/non-alcoholic steatohepatitis, and hepatic fibrosis¹⁹. The HCV core and NS3 proteins activate TLR2/TLR1 and TLR2/TLR6 on monocytes to produce inflammatory cytokines ¹⁹. The aforementioned effects of the core protein may contribute to the mechanisms of the synergism. However, more direct mechanistic evidence has recently been attained by our research using mice expressing the HCV non-structural protein NS5A in a hepatocyte-specific manner. These mice when fed alcohol for 12 months, develop liver tumors in a manner dependent on TLR4, induced by NS5A ²⁰. This NS5A-induced TLR4 is activated by endotoxemia associated with alcohol intake, leading to accentuated TLR4 signaling, which in turn upregulates the stem cell marker Nanog required for TLR4-dependent liver oncogenesis. This finding on the NS5A-TLR4-Nanog axis in synergistic oncogenesis is beginning to shed a novel insight into molecular mechanisms for HCC in alcoholic HCV patients²⁰.

HCV contains a 9.5-kb single-stranded positive-sense RNA genome, which encodes a polyprotein that is processed into multiple proteins by cellular and viral proteases. Nonstructural protein, NS5A, may interact with an interferon-induced, double-stranded RNA-activated protein kinase PKR²¹, thus accounting for the resistance of most HCV strains to interferon treatment. NS5A has also been shown to have a cryptic trans-acting activity for some cellular gene promoters²². The core protein deserves special mention because, in addition to being a viral structural protein, it serves multiple regulatory functions, including the activation or suppression of various cellular and viral gene promoters. Furthermore, it binds to LTβR, TNF receptor and several other cellular proteins, including apolipoprotein AII²³. The immune- and cytokine-mediating roles of the HCV core protein may play a key role in the synergistic effects of alcohol liver disease (ALD) on HCV-associated liver damage.

TLRs signaling in HCC

The TLR signaling pathway is upregulated in chronic liver diseases. Many different cell types in the liver express TLRs¹⁹. Hepatocytes express TLR1 through TLR9. Stellate cells express TLR2, 3, 4, and 9^{24, 25}. The bile duct epithelium expresses TLR2, 3, 4 and 5. Kupffer cells express TLR2, 3, and 4. Chronic alcohol consumption activates other TLRs, such as TLR1, 2 and 6–9, which further increases the TNF-α response to LPS in mice¹⁹. Human monocytes exposed to ethanol for a week develop hypersensitivity to LPS through decreased IRAK-M expression, which activates mitogen-activated protein kinase (MAPK) and NF-κB through TLR4 signaling, leading to activation of NF-κB, AP-1, and ERK²⁶. Hepatocyte-specific deletion of TAK1 in mice causes spontaneous hepatocyte death, inflammation, fibrosis, and carcinogenesis partially mediated by TNFR signaling, indicating that TAK1 is an essential component for cellular homeostasis in the liver. In a NASH mouse model, TLR9 signaling induces production of IL-1β by Kupffer cells, leading to steatohepatitis, inflammation, and fibrosis via induction of IL-1β ²⁷. Furthermore, modulation of TGF-β signaling by a TLR4-MyD88-NF-κB pathway links profibrogenic and proinflammatory signals²⁸. In vivo ethanol exposure, however, downregulates TLR2-, TLR4-, and TLR9-mediated macrophage inflammatory response by limiting p38 and ERK1/2 activation²⁹. Innate immunity is suppressed by acute ethanol administration through inhibition of TLR3 signaling ³⁰. Therefore, contribution of TLR3 and TLR9 to liver diseases is controversial.

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, HCV titer has been shown to exhibit a correlation with the amount of alcohol consumption³¹. 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 immuno-deficient patients, such as HIV-infected patients³², and ethanol consumption can cause immunosuppression ³³.

Another potential mechanism is the involvement of cytokines. Both ALD and HCV cause enhanced secretion of TNF and other cytokines, such as IL-1, IL-6 and IL-8, etc. ³⁴. TNF is particularly interesting because there is a tight correlation between the serum TNF concentration and the severity of ALD^{35, 36}, and TNFR1 deficiency ameliorates experimental ALD³⁷. TNF may cause cell death through the activation of the TRADD/FADD signal transduction pathway. Oxidative stress may also contribute to TNF cytotoxicity³⁸. On the other hand, a variety of factors can modulate the effects of TNF; for examples, NFκB^39–41, manganous superoxide dismutase (MnSOD)⁴² and GSH inhibit TNF-induced cytotoxicity⁴³. In experimental ALD, the mitochondrial pool of GSH is depleted, and the hepatocytes become hypersensitive to TNF⁴³. We have recently shown that HCV core protein binds to lymphotoxin-β receptor and TNF receptor ⁴⁴, and that the expression of this protein in several cell lines sensitize them to TNF-induced cytolysis⁴⁵; therefore, HCV-infected cells are particularly sensitive to TNF. It is interesting to note that HCV core protein also sensitizes cells to apoptosis mediated by Fas⁴⁶, which shares with TNF receptors a number of signal transduction molecules such as FADD. These observations suggest that HCV-infected hepatocytes are very sensitive to TNF and possibly other cytokines as well. This enhanced sensitivity, coupled with the increased secretion of TNF in ALD, may account for the synergistic effects of ALD on HCV.

Nanog-positive cancer stem cells induced by HCV and alcohol

Alcohol synergistically enhances the progression of liver disease and the risk for liver cancer caused by HCV. Toll-like receptor 4 (TLR4) is induced by hepatocyte-specific transgenic (Tg) expression of the HCV nonstructural protein NS5A, and this induction mediates synergistic liver damage and tumor development by alcohol-induced endotoxemia²⁰. The stem/progenitor cell marker, Nanog, is up-regulated as a novel downstream gene by TLR4 activation and the presence of CD133/Nanog-positive cells in liver tumors of alcohol-fed NS5A Tg mice²⁰. Transplantation of p53-deficient hepatic progenitor cells transduced with TLR4 results in liver tumor development in mice following repetitive lipopolysaccharide (LPS) injection, but concomitant transduction of Nanog short-hairpin RNA abrogates this outcome²⁰. Despite the common understanding that TLR4 is one of the pattern recognition receptors expressed predominantly by innate immune cells such as macrophages and lymphocytes, our study demonstrates that hepatocytes can be the primary cellular site of both TLR4 upregulation and its pathologic consequences in the context of HCV infection. Therefore, the TLR4-dependent mechanism synergizes liver disease by HCV and alcohol and is partly dependent on Nanog, a TLR4 downstream gene.

Nanog transduction alone is not as effective as TLR4 activation in liver tumorigenesis, as shown by our cell transplantation experiment²⁰. We believe that TLR4 activation induces other tumor-driver genes which cooperatively work with Nanog to cause liver oncogenesis. Thus, Nanog is still essential for TLR4-dependent oncogenesis, but it alone is poorly oncogenic.

In our previous work using a cell line, we demonstrated that TLR4 promoter up-regulation by NS5A is mediated by PU.1, Oct-1, and AP-1 elements⁴⁷. The similar transcriptional mechanism may underlie TLR4 induction in primary hepatocytes. Therefore, alcohol and HCV NS5A synergistically induce liver tumor development via induction and activation of TLR4 in mice. The importance of Nanog as a direct downstream gene of TLR4 in this oncogenesis has also been identified. Pharmacologic inhibition of TLR4 signaling may become a novel therapeutic strategy for HCV-associated liver tumors.

Cancer stem cells and HCC:

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 hepatocytes⁴⁸. 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)^{48, 49}. 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) ⁵⁰. Thus, it currently remains unclear whether these stem cells are derived from the bone marrow and just migrate to this periportal niche or whether they represent true resident liver stem/progenitor cells. Binding of stroma-derived factor-1α (SDF-1α) to its surface receptor CXCR4 activates oval hepatic cells ⁵¹. Forty percent of HCC have clonality, and thus are considered to originate from progenitor/stem cells^{49, 52–54}. Recent studies of HCC have centered on CSCs, including detection of CSCs in cancer, identification of CSCs markers, and isolation of CSCs from human HCC cell lines. CSCs were identified as CD117+/CD133+ hepatic precursors in regenerating liver tissue ⁵⁵ and as a CD45−/CD90+ subpopulation of tumor cells in HCC ⁵⁶. 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 ⁵⁷. This coincided with the expression of genes associated with stem/progenitor status, such as β-catenin, Notch, Bmi, and Oct3/4. When compared to CD133− cells, the CD133+ cells isolated from the HCC cell lines showed higher expression of CD44 and CD34, but both CD133 subpopulations displayed similar expression for CD29, CD49f (integrin α6), CD90 and CD117⁵⁷. Furthermore, CD133+/CD49f+ cells in liver tumors correlate with tumorigenesity and “stemness” genes, such as Wnt/β-catenin, Notch, Hedgehog/SMO, Bmi, and Oct3/4^58–60. CD133+/CD49f+ HCC cancer stem cells confer resistance to chemotherapy, and this presents a major obstacle for the treatment of HCC⁶¹. One potential reason for this chemoresistance may lie in the plasticity of cancer stem cells with dysregulated signaling and gene expression. Several oncogenic signaling pathways in cancer stem cells of HCC have been described including activated PI3K/AKT⁶², signal transducer and activator of transcription 3 (STAT3)^{63, 64}, NOTCH⁶⁵, Hedgehog^{66, 67} and transforming growth factor-β (TGF-β)^{68, 69}.

Table 1.

Markers for liver cancer stem cells

Gene name	Other name	Function	Species	Organ	References
CD133	Prominin 1 (PROM1)	Glycoprotein, membrane protrusions	Human, Mouse	Liver, Brain	57, 61, 62, 103–105
CD49f	Integrinα chain α6 (ITGA6)	Cell adhesion, cell signaling	Mouse	Liver	57, 61
CD90	Thy-1	Glycophosphatidylinositol (GPI) anchor	Mouse	Liver	57
CD44	Hyaluronic acid receptor	Cell adhesion and migration, metastasis	Mouse	Liver, Breast	57, 106
CD117	KIT	C-kit receptor Cytokine receptor	Mouse	Liver	57
CK19	Cytokeratin 19	Biliary lineage marker	Mouse	Liver	107, 108
OV-6	Oval cell marker	Early progenitor cells	Human	Liver	108
CD34	Glycoprotein	Cell-cell adhesion factor	Mouse	Liver, Leukemia	109
AFP	α-fetoprotein	Fetal counterpart of serum albumin	Mouse	Liver	110

Nanog is one of the core transcription factors found in pluripotent embryonic stem cells (ESCs)⁷⁰. It is essential for maintaining self-renewal and pluripotency of both human and mouse embryonic stem cells^71–74. Overexpression of Nanog induces and maintains the pluripotency and self-renewing characteristics of ESCs under what normally would be differentiation-inducing culture conditions⁷⁵. Recently, Nanog expression has been reported in human neoplasms, including germ cell tumors^76–79, breast carcinomas⁷⁹, osteosarcoma⁸⁰, and HCC⁶². Ectopic expression of Nanog induces an oncogenic potential in NIH3T3 ⁸¹.

TLR4-mediated AP1 activation and HCC

Alcoholism is associated with endotoxemia that stimulates expression of proinflammatory cytokine expression and inflammation in the liver and fat tissues ⁸². Development of liver cancer in the HCV core mice is associated with inflammation ⁸³. Expression of the proinflammatory cytokines TNF-α and IL-1β is induced by HCV-infected human B cells and by its core protein in the transgenic mouse model ^{84, 85}. Recently, we have shown that HCV infection, through NS5A protein expression, upregulates TLR4 expression and pro-inflammatory cytokines ⁴⁷, providing a potential explanation for increased inflammation in HCV-infected livers. Further accentuation of TLR4 signaling in HCV would be expected if combined with alcohol abuse. This may serve as part of the synergistc mechanism when superimposed by other key patholophysiological events common in these co-morbidities such as CYP2E1 induction. CYP2E1 induction impairs hepatic insulin signaling^{86, 87}, induces oxidative DNA damage⁸⁸, primes macrophages to increase LPS-induced TNF-α production⁸⁹, sensitizes hepatocytes to TNF-α-mediated cell death via c-JUN^{90, 91}, and more importantly, leads to marked potentiation of endotoxin-induced oxidant liver injury (35). Therefore, saturated fatty acids which are implicated in obesity and diabetes, could serve as additional ligands to enhance signaling via TLR4 which is already upregulated by HCV NS5A⁹². Polyunsaturated fats actually result in greater rather than less liver injury⁹³. These interactive and synergistic mechanisms involving TLR4 in HCV and alcohol most likely contribute to increased incidence of HCC via oxidant stress and inflammation and are the focal points of my proposed research.

Disruption of c-jun leads to embryonic lethality from massive apoptosis of hepatoblasts, erythroblasts, and other cell types^{94, 95}. Using the cre/loxP conditional knockout system, c-jun expression was shown to be essential for proper proliferation in postnatal hepatocytes⁹⁶. Moreover, the deletion of c-jun in hepatocytes compromises the ability of these cells to enter the cell cycle and undergo rapid proliferation after partial hepatectomy⁹⁶. A well-accepted model of HCC utilizes a chemical carcinogen DEN (diethylnitrosamine) as a tumor initiator and phenobarbital as a promoter. By using this model and tissue specific knockout mice, the loss of JNK1 in the liver was shown to reduce DEN-induced HCC development⁹⁷. The requirement for c-jun was restricted to an early stage of tumor development in chemically induced HCC in mice⁹⁸. In our study, c-jun knockout dramatically reduced the incidence of spontaneous and DEN-induced HCC in HCV core transgenic mice, supporting the role of c-jun in this model of hepatocarcinogenesis (Hepatology in press). Alcoholic liver disease patients have increased levels of hepatic RANTES/CCL5. Ethanol augments RANTES/CCL5 expression in rat liver sinusoidal endothelial cells and human endothelial cells via activation of NF-κB, HIF-1α, and AP-1⁹⁹. In vitro studies using liver-derived cell lines have demonstrated rapid activation of AP-1 by HBV or HCV proteins¹⁰⁰, and this mitogenic effect is implicated in hepatocytes’ susceptibility to liver cell transformation via fixation of genetic mutations caused by oxidant stress. Indeed, Further, c-Jun prevents apoptosis by antagonizing p53 activity as another contributing factor for HCC development^{98, 101}. Ectopic expression of HCV core protein constitutively activates AP-1 via JNK¹⁰². Activation of JNK is also implicated in ASH and NASH^{87, 90}. Therefore, activation of JNK and c-JUN most likely plays pivotal roles in the synergistic induction of liver cancer by HCV and alcohol.