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Cell fate, metabolic reprogramming and lncRNA of tumor-initiating stem-like cells induced by alcohol
Chemico-Biological Interactions 323 (2020) 109055
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Cell fate, metabolic reprogramming and lncRNA of tumor-initiating stemlike cells induced by alcohol
T
Keigo Machidaa,b,∗∗ a b
Southern California Research Center for ALPD and Cirrhosis, Los Angeles, CA, USA Department of Molecular Microbiology and Immunology, Los Angeles, CA, USA
A R T I C LE I N FO
A B S T R A C T
Keywords: Alcohol HCC Cancer stem cells Tumor-initiating stem-like cells (TICs) lncRNA NUMB
Alcoholism synergizes the development of the hepatocellular carcinoma (HCC) in patients infected with hepatitis B or C virus (HBV or HCV). Tumor-initiating stem-like cells (TICs) are refractory to therapy and have expression of stemness transcription factors. Leaky-gut-derived endotoxin stimulates TLR4-NANOG pathway that skews asymmetric cell division and that metabolically reprograms hepatocytes/liver progenitor cells, leading to selfrenewal. TICs isolated from mouse HCC models or human HCCs are tumorigenic and have p53 degradation via phosphorylation of the protective protein NUMB and its dissociation from p53 by the oncofetal protein TBC1D15. Furthermore, dysregulation of lncRNA promotes genesis of TICs, leading to HCC development. This review describes roles of cell fate decision, metabolic reprogramming and lncRNA for TIC genesis and liver oncogenesis. This project was supported by NIH grants 1R01AA018857-01, 5R21AA025470, P50AA11999 (Animal Core, Morphology Core, and Pilot Project Program), R24AA012885 (Non-Parenchymal Liver Cell Core) and pilot project funding (5P30DK048522-13).
1. Introduction Alcoholic liver disease (ALD), obesity and viral hepatitis (HBV and HCV) are associated with development of hepatocellular carcinoma (HCC) [12,32,33]. HCV/HBV infection, ALD, and non-alcoholic fatty liver disease (NAFLD) synergizes liver oncogenesis through leaky-gut derived endotoxinemia, oxidant stress, organelle stress, and metabolic dysregulation. Tumor-initiating stem cell-like cells (TICs) or so-called cancer stem cells (CSCs) are rare, highly malignant cells that are present in diverse tumor types and refractory to chemotherapy, radiation therapy and surgical resection. There is still controversy as to what defines a tumorinitiating cell in HCC. In studies for blood cancer, Dr. Morrison's group successfully isolated cancer stem cells., Many other studies showed multipotent tumor-initiating stem-like cells, but not pluripotent cancer stem cells, HCV NS5A Tg mice fed alcohol western diet are specific models by which TICs are defined in the studies described and potential caveats to these model systems. Findings are listed, with deeper analysis and scrutiny in our published papers. Normal stem cells and TICs share three major characteristics, (i) self-renewal, (ii) clonality, and (iii) plasticity [27,37]. A multi-kinase inhibitor Sorafenib is the commonly used and FDA-approved monotherapy agent for the treatment of HCC; however, resistance to sorafenib eventually occurs in patients [43]. ∗
Forty percent of HCC has clonality that originates from hepatocytes/ progenitor cells [1,36,41,51]. TICs are chemo-resistant [37], survive during an initial therapy, clonally expand, leading to tumor recurrence [24,25,44]. 2. Co-morbidity of life-style factors for disease burdens (alcoholism and obesity) with viral hepatitis (HBV and HCV) synergizes HCC development Alcohol and obesity are leading life-style factors for disease burdens around the world and synergistically elevate HCC incidence with infection of hepatitis viruses. More than 70 million people worldwide are infected with HCV [32,33,48]. Co-existence of alcohol abuse or obesity synergizes HCV risk of developing HCC by additional 8-fold, culminating to an overall 45-55-fold increase in the risk as compared to normal subjects [50]. Therefore, co-morbidity of lifestyle factors with viral hepatitis is an urgent unmet need for fundamental understanding mechanisms and discovering new therapeutic targets. Obesity and alcoholism increase gut permeability leading to endotoxemia, which in turn activates Toll-like receptor 4 (TLR4) in the liver with production of cytokines and an inflammatory response, leading to subsequent development of obesity/alcohol-related liver disease [13]. Circulating endotoxin binds CD14-TLR4 complex, activates hepatocytes/hepatoblasts
Southern California Research Center for ALPD and Cirrhosis, Los Angeles, CA, USA. E-mail address: [email protected].
https://doi.org/10.1016/j.cbi.2020.109055 Received 1 August 2019; Received in revised form 13 December 2019; Accepted 10 March 2020 Available online 11 March 2020 0009-2797/ © 2020 Elsevier B.V. All rights reserved.
Chemico-Biological Interactions 323 (2020) 109055
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and induces the stem cell marker NANOG. This process generates TLR4/NANOG-dependent, chemo-resistant tumor-initiating stem-like cells (TICs; CD133+), which can induce HCC in mice [11]. Tumors contain double-positive cells for NANOG and CD133 or CD49f (24). Liver-specific expression of the HCV NS5A protein in mice fed alcohol for 12 months develop liver tumors in a TLR4-dependent manner [11]. Treatment with sorafenib made TICs more susceptible to tumor growth retardation, with a decrease in tumor size by ~55% when combined with knockdown of NANOG-inducible proto-oncogenes (including YAP1, which induces antioxidant gene programs) [11]. However, the underlying mechanism of chemo-resistance and self-renewal of TICs remains incompletely understood. 3. TLR4-NANOG pathway metabolically reprograms drug resistance TICs via suppression of mitochondrial OXPHOS and activation of FAO Toll-like receptor 4 (TLR4) is a putative proto-oncogene involved in the genesis/maintenance of TICs and liver tumor in HCV transgenic models. TLR4 silencing reduces heightened expression of stemness genes and cell proliferation [10]. CD133+/CD49f + cells are TLR4/ NANOG-dependent TICs. Hepatoblastic HCC subtype with poor prognosis has a gene expression profile with markers of hepatic oval cells [3,8,21,47]. HCC often recurs after chemotherapy due presumably to the presence of chemo-resistant TICs [35]. Endotoxin ligand-TLR4-CD14 complex formation triggers E2F1 phosphorylation to transactivate NANOG [11]. Complementary NANOG ChIP-seq and metabolomics studies of TICs demonstrated that NANOG induced by TLR4 suppressed mitochondrial OXPHOS and activated fatty acid oxidation (FAO), thus inhibiting OCR and ROS production. NANOG ChIP-seq identified genes associated with NANOGdependent mitochondrial metabolic pathways to maintain TICs. The causal roles of NANOG in mitochondrial metabolic reprogramming occurred through the inhibition of oxidative phosphorylation (OXPHOS) with decreased production of mitochondrial ROS and activation of FAO, which was required for self-renewal and drug resistance [11]. Restoration of OXPHOS activity and inhibition of FAO rendered TICs susceptible to a standard care chemotherapy drug, sorafenib [11]. NANOG-dependent downstream effect on mitochondrial function contributed to the chemotherapy resistance [11]. These metabolic reprogramming promoted self-renewal/oncogenesis, and explained how NANOG activation inhibited therapy-mediated apoptosis by quenching ROS production. Restoration of OXPHOS or decreased FAO reduces tumorigenic capacity of TICs and increases susceptibility to chemotherapy [11] (Fig. 1). The metabolic bases of altered cell functions and cell fate in TICs define potentially new approaches for chemosensitization and elimination of TICs for more efficacious HCC therapies. As TICs rely on active FAO for their maintenance and function, FAO inhibitor suppresses self-renewal of TICs [38]. The FAO gene silencing reduces TIC phenotype while overexpression of FAO genes restores the original TIC phenotype. Thus FAO metabolically switches the fate of stem cells [14]. Potential mechanisms by which elevation of FAO maintains self-renewal ability include: (i) shunting of long-chain FA away from lipid and cell membrane synthesis; (ii) downregulation of ROS through production of NADPH to avoid loss of TICs; and (iii) reduction of metabolic resistance to chemotherapy. By these criteria, NANOG function could be construed to serve as a gatekeeper for FAO activity. Activation of fatty acid oxidation (FAO) metabolically switches the fate of stem cells and TICs. Therefore, to develop better therapeutics, the underlying molecular mechanisms regulating obesity/alcohol/HCV-induced hepatocarcinogenesis should be elucidated. NANOG maintains chemotherapy resistance of TICs involving not only the direct activation of self-renewal via stemness genes, but also the subsequent metabolic reprogramming in these cells leading to amplification of TIC oncogenic activity and their overall survival. Our
Fig. 1. Environmental factors (alcohol exposure and obesity) and virus infection (HBV and HCV) generates TIC through metabolic reprogramming and resistance to therapy-mediated ROS burst (Top) Endotoxinemia leads to TLR4-NANOG activation and promotes self-renewal. (Bottom) Drug resistant TICs acquires resistance to therapy-induced ROS burst and survive via reprogramming of metabolism.
data showed that NANOG reprogramming of mitochondrial metabolism was indeed responsible for human TIC oncogenicity and chemo-resistance. These studies have led to a paradigm shift in our understanding the underlying basis of alcohol/HCV-associated cancer, thus facilitating future development of new personalized treatment strategies targeted towards NANOG + TICs arising from obesity, alcohol, or HCV-related HCC. The studies provide insights into the mechanisms of NANOG-mediated generation of TICs, tumorigenesis and chemo-resistance due to metabolic reprograming of mitochondrial functions. 4. HCV-mediated mechanisms also promote genesis of TICs to synergize oncogenesis through multiple direct oncogenic functions HCV proteins (nucleocapside Core and NS5A) are linked to transformation through overproduction of reactive oxygen species which may cause mitochondrial or nuclear DNA damage [20,30,33]. The Core protein inhibits microsomal triglyceride transfer protein activity and VLDL (very-low-density lipoprotein) secretion [34], which contributes the genesis of fatty liver. The core 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 SOCS3 [17] in a manner dependent on PA28γ 73, or via IRS serine phosphorylation [5]. These mechanisms promote oncogenesis. 5. TICs have the heightened expression and activation of a pluripotency-associated transcription factor network TICs maintain expression and activation of a pluripotency (stemness)-associated transcription factor (TF) network [15], such as Wnt/βcatenin, Notch, Hedgehog/SMO, and Oct3/4 [6,9,42]. TICs also have dysregulated signaling and gene expression, including PI3K/AKT [26], signal transducer and activator of transcription 3 (STAT3) [46,49], and hedgehog [39,40] while defective tumor suppressor transforming growth factor-beta (TGF-β) pathway is also implicated [19,31]. 6. Inactivation of cell fate determinant NUMB skews asymmetric cell division to generate TICs, leading to oncogenesis via p53 degradation, NUMB inactivation and stemness activation Self-renewing divisions maintained stem cell populations since one 2
Chemico-Biological Interactions 323 (2020) 109055
K. Machida
Table 1 LncRNAs linked to HCC. lncRNA
Classification
Size (kb)
Tissues
Expression
Function
Reference
BC017743 BC043430 LINC01152 aHIF PWAR5 (PAR5) AF070632
Unknown Unknown Unknown Unknown Unknown Unknown
2.3 1.9 3.1 1.0 ~3.6 ~1.9
Liver Liver Liver Liver Liver Liver
Up Up Down Down Down Down
[53] [53] [53] [53] [53] [52]
AK021443 LINC01419 UCA1
Unknown Unknown Unknown
~1.6 ~5.1 ~7.3
Up Up Up
WRAP53 Linc-p21 H19 LET HULK HOTAIR HOTTIP
Antisense Unknown Unknown Unknown Unknown Unknown Unknown
~1.8
Liver Liver Liver, bladder, gastric, ovary, esophagus Liver Liver Liver Liver Liver Liver Liver
Tumor suppressor region Tumor suppressor region Unknown Poor prognostic outcomes Poor prognostic outcomes LncRNA-protein interaction, supress angiogenesis, potential biomarker and therapeutic target Cell cycle regulation, Proliferation Cell cycle regulation, Proliferation LncRNA-miRNA interaction, Proliferation
Up Up Up Up Up Up Up
Unknown, Biomarker Unknown, Biomarker Unknown, Biomarker Unknown, Biomarker IGF2BP1 regulation, Biomarker Guide of epigenetic repressors Guide of epigenetic activators
[16] [23] [23] [23] [23] [45] [45]
[52] [52] [16]
human HCCs [45]. LncRNAs guide epigenetic activators/repressors (i.e, HOTAIR, HOTTIP, H19, ANRIL, UCA1, TUG1), block transcription factor binding sites (i.e, lncRNA-DILC), sequester miRs (i.e, DANCR, ICR, PCNA-AS1) or proteins (i.e, MALAT1, lnc-PRAL, GASS, lncBRM), serve as protein scaffold (i.e, lnc-β-Catm, MEG3, HULC, lncRNA-MVIH) [45]. Therefore, more thorough investigation on lncRNA’s roles in cancer biology are required. LncRNAs expression (LINC01419, AK021443, UCA1 and WRAP53) are increased in HCCs compared to those of non-cancerous tissues (dysplasia) while lncRNA AF070632 is decreased in advanced HCC samples compared with early HCC. These lncRNAs are associated with Child-Pugh score. Therefore, these association suggests roles of lncRNA contribution to liver oncogenesis. LINC01419 and AK021443 were mostly involved in cell cycle progression, whereas AF070632 regulates cofactor binding, oxidation-reduction and carboxylic acid catabolic process [52]. Two lncRNAs, including urothelial carcinoma associated1 (UCA1) and WD repeat containing, antisense to TP53 (WRAP53) are upregulated in serum. UCA1 and WRAP53 (+) HCC patients had a decreased recurrence-free survival (RFS) and increased cumulative hazards. WRAP53 was an independent prognostic factor of RFS [16]. Some of these lncRNAs were dysregulated predominantly in one specific hepatitis virus-related HCC, including PCAT-29 in HBV-related HCC, aHIF and PAR5 in HCV-related HCC, and Y3 in HDV-related HCC. DBH-AS1, hDREH and hPVT1 were differentially expressed in HCC of different viral etiology [53]. Unknown mechanism of alcohol-mediated oncogenesis through lncRNA pathways warrants further investigation to elucidate mechanisms of TIC genesis through dysregulation of lncRNAs (See Table 1).
daughter cell commits to a particular fate while the other retains the multipotent characteristics of its parent. Disruption of p53 reprograms cells to the pluripotent state [4,18,29] while p53 represses the expression of pluripotency-associated transcription factors in proliferating stem cells [22], interacts with the polarity determinant NUMB protein to maintain asymmetry and cell polarity. A tumor suppressor NUMB is asymmetrically distributed in dividing stem cells and is segregated into the daughter cell which undergoes differentiation. The association with NUMB stabilizes p53 [7,28] and protects deregulated expansion of TICs. Interaction between NUMB and p53 maintain the homeostasis of both stem cells and differentiated cells. NUMB-associated oncoproteins MDM2 E3 ubiquitin ligase destabilize the NUMB-p53 complex and promote proteolysis of p53 [2]. We identified that phosphorylation of NUMB destabilize p53 and promotes self-renewal of TICs by pluripotency-associated transcription factor NANOG dependent manner. Phosphorylated NUMB is dissociated with binding partners and lose asymmetric functions, NANOG phosphorylates NUMB via aPKCζ, through the direct induction of Aurora A kinase (AURKA) and the repression of an aPKCζ inhibitor, LGL-2. Phosphorylation of NUMB by aPKCζ destabilizes the NUMB-p53 interaction, p53 proteolysis and to deregulate self-renewal in TICs. Furthermore, non-phosphorylatable mutant of NUMB expression reduced HCC incidence in HVC NS5A transgenic mice fed alcohol western diet (unpublished observation). These results indicate that NUMB phosphorylation is therapeutic targets for HCC treatment. 7. Dysregulated LncRNAs generates TICs to promote HCC development
8. Conclusions and discussions
Non-coding RNAs [LncRNA, microRNA (miRNA) and etc.] are dysregulated in liver carcinogenesis via regulation of gene expression. The roles of LncRNAs are very broad both in physiology and in cancer. Several LncRNAs regulate cancer biology, including MALAT and etc. LncRNAs have pleiotropic effects on miRNAs, mRNAs, and proteins and alter miRNA and mRNA expression and stability, affect protein expression, degradation, structure, or interactions with transcriptional regulators. Therefore, novel lncRNAs serve as biomarkers to achieve precision therapy for HCC [23]. The function of the 98% of non-coding sequences in the human genome is elusive [45]. Many microRNAs function as 'oncomiRs' to repress tumor-suppressor genes or, serve as a tumor suppressor to counteract oncogenes [45]. LncRNAs regulate gene expression and other molecular functions through interacting with DNA, RNA and proteins [45]. LncRNAs regulate liver carcinogenesis of diverse etiologies, liver TIC formation, epigenetic reprogramming and metastasis. LncRNAs serve as biomarkers and therapeutic targets of
TICs were identified in HCC sections of alcoholic patients by immunostaining and isolated from such patients to validate induction of TLR4-dependent stemness genes and transformation. Nearly 40% of HCCs are derived from clonal expansion of TICs, indicating that targeting TICs are more strategic ways to eliminate HCC and inhibit recurrence issues. NANOG-mediated p53 degradation disengages from the protective NUMB protein via TBC1D15 interaction. NANOGAURKA-aPKCζ pathway post-translationally modifies NUMB in TICs self-renewal and tumorigenesis. As the NANOG-NUMB-p53 signaling regulates TICs self-renewal and liver tumorigenesis, targeting NUMBphosphorylation is a therapeutic strategy. However, further in depth in vivo and clinical studies are warranted to verify this suggestion. These studies are now exploring potential mechanistic connections to metabolic programming known to occur in cancer cells and TICs in 3
Chemico-Biological Interactions 323 (2020) 109055
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promoting and maintaining “stem cell fate” via molecular, genetic, and epigenetic mechanisms via dysregulation of lncRNA. [14]
Declaration of competing interest [15]
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
[16]
Acknowledgments
[17]
This project was supported by NIH grants 1R01AA018857-01, 5R21AA025470, P50AA11999, R01 AA025204-01A1, R21 AA02547001A1 (Animal Core, Morphology Core, and Pilot Project Program), R24AA012885 (Non-Parenchymal Liver Cell Core) and pilot project funding (5P30DK048522-13). This research is also supported by a Research Scholar Grant, RSG-12-177-01-MPC and pilot funding (IRG58-007-48) from American Cancer Society. Microscopy was performed by the Cell and Tissue Imaging Core of the USC Research Center for Liver Diseases (P30 DK048522). Liver tissues were obtained from The Liver Tissue Cell Distribution System (LTCDS) in University of Minnesota. We also acknowledge the Animal and Morphology Core facilities of the NIAAA-supported Southern California Research Center for ALPD and Cirrhosis (P50 AA011999) for providing a mouse model of intragastric ethanol infusion and morphological analyses of liver tissues.
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Appendix A. Supplementary data [23]
Supplementary data to this article can be found online at https:// doi.org/10.1016/j.cbi.2020.109055.
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Cell fate, metabolic reprogramming and lncRNA of tumor-initiating stemlike cells induced by alcohol
T
Keigo Machidaa,b,∗∗ a b
Southern California Research Center for ALPD and Cirrhosis, Los Angeles, CA, USA Department of Molecular Microbiology and Immunology, Los Angeles, CA, USA
A R T I C LE I N FO
A B S T R A C T
Keywords: Alcohol HCC Cancer stem cells Tumor-initiating stem-like cells (TICs) lncRNA NUMB
Alcoholism synergizes the development of the hepatocellular carcinoma (HCC) in patients infected with hepatitis B or C virus (HBV or HCV). Tumor-initiating stem-like cells (TICs) are refractory to therapy and have expression of stemness transcription factors. Leaky-gut-derived endotoxin stimulates TLR4-NANOG pathway that skews asymmetric cell division and that metabolically reprograms hepatocytes/liver progenitor cells, leading to selfrenewal. TICs isolated from mouse HCC models or human HCCs are tumorigenic and have p53 degradation via phosphorylation of the protective protein NUMB and its dissociation from p53 by the oncofetal protein TBC1D15. Furthermore, dysregulation of lncRNA promotes genesis of TICs, leading to HCC development. This review describes roles of cell fate decision, metabolic reprogramming and lncRNA for TIC genesis and liver oncogenesis. This project was supported by NIH grants 1R01AA018857-01, 5R21AA025470, P50AA11999 (Animal Core, Morphology Core, and Pilot Project Program), R24AA012885 (Non-Parenchymal Liver Cell Core) and pilot project funding (5P30DK048522-13).
1. Introduction Alcoholic liver disease (ALD), obesity and viral hepatitis (HBV and HCV) are associated with development of hepatocellular carcinoma (HCC) [12,32,33]. HCV/HBV infection, ALD, and non-alcoholic fatty liver disease (NAFLD) synergizes liver oncogenesis through leaky-gut derived endotoxinemia, oxidant stress, organelle stress, and metabolic dysregulation. Tumor-initiating stem cell-like cells (TICs) or so-called cancer stem cells (CSCs) are rare, highly malignant cells that are present in diverse tumor types and refractory to chemotherapy, radiation therapy and surgical resection. There is still controversy as to what defines a tumorinitiating cell in HCC. In studies for blood cancer, Dr. Morrison's group successfully isolated cancer stem cells., Many other studies showed multipotent tumor-initiating stem-like cells, but not pluripotent cancer stem cells, HCV NS5A Tg mice fed alcohol western diet are specific models by which TICs are defined in the studies described and potential caveats to these model systems. Findings are listed, with deeper analysis and scrutiny in our published papers. Normal stem cells and TICs share three major characteristics, (i) self-renewal, (ii) clonality, and (iii) plasticity [27,37]. A multi-kinase inhibitor Sorafenib is the commonly used and FDA-approved monotherapy agent for the treatment of HCC; however, resistance to sorafenib eventually occurs in patients [43]. ∗
Forty percent of HCC has clonality that originates from hepatocytes/ progenitor cells [1,36,41,51]. TICs are chemo-resistant [37], survive during an initial therapy, clonally expand, leading to tumor recurrence [24,25,44]. 2. Co-morbidity of life-style factors for disease burdens (alcoholism and obesity) with viral hepatitis (HBV and HCV) synergizes HCC development Alcohol and obesity are leading life-style factors for disease burdens around the world and synergistically elevate HCC incidence with infection of hepatitis viruses. More than 70 million people worldwide are infected with HCV [32,33,48]. Co-existence of alcohol abuse or obesity synergizes HCV risk of developing HCC by additional 8-fold, culminating to an overall 45-55-fold increase in the risk as compared to normal subjects [50]. Therefore, co-morbidity of lifestyle factors with viral hepatitis is an urgent unmet need for fundamental understanding mechanisms and discovering new therapeutic targets. Obesity and alcoholism increase gut permeability leading to endotoxemia, which in turn activates Toll-like receptor 4 (TLR4) in the liver with production of cytokines and an inflammatory response, leading to subsequent development of obesity/alcohol-related liver disease [13]. Circulating endotoxin binds CD14-TLR4 complex, activates hepatocytes/hepatoblasts
Southern California Research Center for ALPD and Cirrhosis, Los Angeles, CA, USA. E-mail address: [email protected].
https://doi.org/10.1016/j.cbi.2020.109055 Received 1 August 2019; Received in revised form 13 December 2019; Accepted 10 March 2020 Available online 11 March 2020 0009-2797/ © 2020 Elsevier B.V. All rights reserved.
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and induces the stem cell marker NANOG. This process generates TLR4/NANOG-dependent, chemo-resistant tumor-initiating stem-like cells (TICs; CD133+), which can induce HCC in mice [11]. Tumors contain double-positive cells for NANOG and CD133 or CD49f (24). Liver-specific expression of the HCV NS5A protein in mice fed alcohol for 12 months develop liver tumors in a TLR4-dependent manner [11]. Treatment with sorafenib made TICs more susceptible to tumor growth retardation, with a decrease in tumor size by ~55% when combined with knockdown of NANOG-inducible proto-oncogenes (including YAP1, which induces antioxidant gene programs) [11]. However, the underlying mechanism of chemo-resistance and self-renewal of TICs remains incompletely understood. 3. TLR4-NANOG pathway metabolically reprograms drug resistance TICs via suppression of mitochondrial OXPHOS and activation of FAO Toll-like receptor 4 (TLR4) is a putative proto-oncogene involved in the genesis/maintenance of TICs and liver tumor in HCV transgenic models. TLR4 silencing reduces heightened expression of stemness genes and cell proliferation [10]. CD133+/CD49f + cells are TLR4/ NANOG-dependent TICs. Hepatoblastic HCC subtype with poor prognosis has a gene expression profile with markers of hepatic oval cells [3,8,21,47]. HCC often recurs after chemotherapy due presumably to the presence of chemo-resistant TICs [35]. Endotoxin ligand-TLR4-CD14 complex formation triggers E2F1 phosphorylation to transactivate NANOG [11]. Complementary NANOG ChIP-seq and metabolomics studies of TICs demonstrated that NANOG induced by TLR4 suppressed mitochondrial OXPHOS and activated fatty acid oxidation (FAO), thus inhibiting OCR and ROS production. NANOG ChIP-seq identified genes associated with NANOGdependent mitochondrial metabolic pathways to maintain TICs. The causal roles of NANOG in mitochondrial metabolic reprogramming occurred through the inhibition of oxidative phosphorylation (OXPHOS) with decreased production of mitochondrial ROS and activation of FAO, which was required for self-renewal and drug resistance [11]. Restoration of OXPHOS activity and inhibition of FAO rendered TICs susceptible to a standard care chemotherapy drug, sorafenib [11]. NANOG-dependent downstream effect on mitochondrial function contributed to the chemotherapy resistance [11]. These metabolic reprogramming promoted self-renewal/oncogenesis, and explained how NANOG activation inhibited therapy-mediated apoptosis by quenching ROS production. Restoration of OXPHOS or decreased FAO reduces tumorigenic capacity of TICs and increases susceptibility to chemotherapy [11] (Fig. 1). The metabolic bases of altered cell functions and cell fate in TICs define potentially new approaches for chemosensitization and elimination of TICs for more efficacious HCC therapies. As TICs rely on active FAO for their maintenance and function, FAO inhibitor suppresses self-renewal of TICs [38]. The FAO gene silencing reduces TIC phenotype while overexpression of FAO genes restores the original TIC phenotype. Thus FAO metabolically switches the fate of stem cells [14]. Potential mechanisms by which elevation of FAO maintains self-renewal ability include: (i) shunting of long-chain FA away from lipid and cell membrane synthesis; (ii) downregulation of ROS through production of NADPH to avoid loss of TICs; and (iii) reduction of metabolic resistance to chemotherapy. By these criteria, NANOG function could be construed to serve as a gatekeeper for FAO activity. Activation of fatty acid oxidation (FAO) metabolically switches the fate of stem cells and TICs. Therefore, to develop better therapeutics, the underlying molecular mechanisms regulating obesity/alcohol/HCV-induced hepatocarcinogenesis should be elucidated. NANOG maintains chemotherapy resistance of TICs involving not only the direct activation of self-renewal via stemness genes, but also the subsequent metabolic reprogramming in these cells leading to amplification of TIC oncogenic activity and their overall survival. Our
Fig. 1. Environmental factors (alcohol exposure and obesity) and virus infection (HBV and HCV) generates TIC through metabolic reprogramming and resistance to therapy-mediated ROS burst (Top) Endotoxinemia leads to TLR4-NANOG activation and promotes self-renewal. (Bottom) Drug resistant TICs acquires resistance to therapy-induced ROS burst and survive via reprogramming of metabolism.
data showed that NANOG reprogramming of mitochondrial metabolism was indeed responsible for human TIC oncogenicity and chemo-resistance. These studies have led to a paradigm shift in our understanding the underlying basis of alcohol/HCV-associated cancer, thus facilitating future development of new personalized treatment strategies targeted towards NANOG + TICs arising from obesity, alcohol, or HCV-related HCC. The studies provide insights into the mechanisms of NANOG-mediated generation of TICs, tumorigenesis and chemo-resistance due to metabolic reprograming of mitochondrial functions. 4. HCV-mediated mechanisms also promote genesis of TICs to synergize oncogenesis through multiple direct oncogenic functions HCV proteins (nucleocapside Core and NS5A) are linked to transformation through overproduction of reactive oxygen species which may cause mitochondrial or nuclear DNA damage [20,30,33]. The Core protein inhibits microsomal triglyceride transfer protein activity and VLDL (very-low-density lipoprotein) secretion [34], which contributes the genesis of fatty liver. The core 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 SOCS3 [17] in a manner dependent on PA28γ 73, or via IRS serine phosphorylation [5]. These mechanisms promote oncogenesis. 5. TICs have the heightened expression and activation of a pluripotency-associated transcription factor network TICs maintain expression and activation of a pluripotency (stemness)-associated transcription factor (TF) network [15], such as Wnt/βcatenin, Notch, Hedgehog/SMO, and Oct3/4 [6,9,42]. TICs also have dysregulated signaling and gene expression, including PI3K/AKT [26], signal transducer and activator of transcription 3 (STAT3) [46,49], and hedgehog [39,40] while defective tumor suppressor transforming growth factor-beta (TGF-β) pathway is also implicated [19,31]. 6. Inactivation of cell fate determinant NUMB skews asymmetric cell division to generate TICs, leading to oncogenesis via p53 degradation, NUMB inactivation and stemness activation Self-renewing divisions maintained stem cell populations since one 2
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K. Machida
Table 1 LncRNAs linked to HCC. lncRNA
Classification
Size (kb)
Tissues
Expression
Function
Reference
BC017743 BC043430 LINC01152 aHIF PWAR5 (PAR5) AF070632
Unknown Unknown Unknown Unknown Unknown Unknown
2.3 1.9 3.1 1.0 ~3.6 ~1.9
Liver Liver Liver Liver Liver Liver
Up Up Down Down Down Down
[53] [53] [53] [53] [53] [52]
AK021443 LINC01419 UCA1
Unknown Unknown Unknown
~1.6 ~5.1 ~7.3
Up Up Up
WRAP53 Linc-p21 H19 LET HULK HOTAIR HOTTIP
Antisense Unknown Unknown Unknown Unknown Unknown Unknown
~1.8
Liver Liver Liver, bladder, gastric, ovary, esophagus Liver Liver Liver Liver Liver Liver Liver
Tumor suppressor region Tumor suppressor region Unknown Poor prognostic outcomes Poor prognostic outcomes LncRNA-protein interaction, supress angiogenesis, potential biomarker and therapeutic target Cell cycle regulation, Proliferation Cell cycle regulation, Proliferation LncRNA-miRNA interaction, Proliferation
Up Up Up Up Up Up Up
Unknown, Biomarker Unknown, Biomarker Unknown, Biomarker Unknown, Biomarker IGF2BP1 regulation, Biomarker Guide of epigenetic repressors Guide of epigenetic activators
[16] [23] [23] [23] [23] [45] [45]
[52] [52] [16]
human HCCs [45]. LncRNAs guide epigenetic activators/repressors (i.e, HOTAIR, HOTTIP, H19, ANRIL, UCA1, TUG1), block transcription factor binding sites (i.e, lncRNA-DILC), sequester miRs (i.e, DANCR, ICR, PCNA-AS1) or proteins (i.e, MALAT1, lnc-PRAL, GASS, lncBRM), serve as protein scaffold (i.e, lnc-β-Catm, MEG3, HULC, lncRNA-MVIH) [45]. Therefore, more thorough investigation on lncRNA’s roles in cancer biology are required. LncRNAs expression (LINC01419, AK021443, UCA1 and WRAP53) are increased in HCCs compared to those of non-cancerous tissues (dysplasia) while lncRNA AF070632 is decreased in advanced HCC samples compared with early HCC. These lncRNAs are associated with Child-Pugh score. Therefore, these association suggests roles of lncRNA contribution to liver oncogenesis. LINC01419 and AK021443 were mostly involved in cell cycle progression, whereas AF070632 regulates cofactor binding, oxidation-reduction and carboxylic acid catabolic process [52]. Two lncRNAs, including urothelial carcinoma associated1 (UCA1) and WD repeat containing, antisense to TP53 (WRAP53) are upregulated in serum. UCA1 and WRAP53 (+) HCC patients had a decreased recurrence-free survival (RFS) and increased cumulative hazards. WRAP53 was an independent prognostic factor of RFS [16]. Some of these lncRNAs were dysregulated predominantly in one specific hepatitis virus-related HCC, including PCAT-29 in HBV-related HCC, aHIF and PAR5 in HCV-related HCC, and Y3 in HDV-related HCC. DBH-AS1, hDREH and hPVT1 were differentially expressed in HCC of different viral etiology [53]. Unknown mechanism of alcohol-mediated oncogenesis through lncRNA pathways warrants further investigation to elucidate mechanisms of TIC genesis through dysregulation of lncRNAs (See Table 1).
daughter cell commits to a particular fate while the other retains the multipotent characteristics of its parent. Disruption of p53 reprograms cells to the pluripotent state [4,18,29] while p53 represses the expression of pluripotency-associated transcription factors in proliferating stem cells [22], interacts with the polarity determinant NUMB protein to maintain asymmetry and cell polarity. A tumor suppressor NUMB is asymmetrically distributed in dividing stem cells and is segregated into the daughter cell which undergoes differentiation. The association with NUMB stabilizes p53 [7,28] and protects deregulated expansion of TICs. Interaction between NUMB and p53 maintain the homeostasis of both stem cells and differentiated cells. NUMB-associated oncoproteins MDM2 E3 ubiquitin ligase destabilize the NUMB-p53 complex and promote proteolysis of p53 [2]. We identified that phosphorylation of NUMB destabilize p53 and promotes self-renewal of TICs by pluripotency-associated transcription factor NANOG dependent manner. Phosphorylated NUMB is dissociated with binding partners and lose asymmetric functions, NANOG phosphorylates NUMB via aPKCζ, through the direct induction of Aurora A kinase (AURKA) and the repression of an aPKCζ inhibitor, LGL-2. Phosphorylation of NUMB by aPKCζ destabilizes the NUMB-p53 interaction, p53 proteolysis and to deregulate self-renewal in TICs. Furthermore, non-phosphorylatable mutant of NUMB expression reduced HCC incidence in HVC NS5A transgenic mice fed alcohol western diet (unpublished observation). These results indicate that NUMB phosphorylation is therapeutic targets for HCC treatment. 7. Dysregulated LncRNAs generates TICs to promote HCC development
8. Conclusions and discussions
Non-coding RNAs [LncRNA, microRNA (miRNA) and etc.] are dysregulated in liver carcinogenesis via regulation of gene expression. The roles of LncRNAs are very broad both in physiology and in cancer. Several LncRNAs regulate cancer biology, including MALAT and etc. LncRNAs have pleiotropic effects on miRNAs, mRNAs, and proteins and alter miRNA and mRNA expression and stability, affect protein expression, degradation, structure, or interactions with transcriptional regulators. Therefore, novel lncRNAs serve as biomarkers to achieve precision therapy for HCC [23]. The function of the 98% of non-coding sequences in the human genome is elusive [45]. Many microRNAs function as 'oncomiRs' to repress tumor-suppressor genes or, serve as a tumor suppressor to counteract oncogenes [45]. LncRNAs regulate gene expression and other molecular functions through interacting with DNA, RNA and proteins [45]. LncRNAs regulate liver carcinogenesis of diverse etiologies, liver TIC formation, epigenetic reprogramming and metastasis. LncRNAs serve as biomarkers and therapeutic targets of
TICs were identified in HCC sections of alcoholic patients by immunostaining and isolated from such patients to validate induction of TLR4-dependent stemness genes and transformation. Nearly 40% of HCCs are derived from clonal expansion of TICs, indicating that targeting TICs are more strategic ways to eliminate HCC and inhibit recurrence issues. NANOG-mediated p53 degradation disengages from the protective NUMB protein via TBC1D15 interaction. NANOGAURKA-aPKCζ pathway post-translationally modifies NUMB in TICs self-renewal and tumorigenesis. As the NANOG-NUMB-p53 signaling regulates TICs self-renewal and liver tumorigenesis, targeting NUMBphosphorylation is a therapeutic strategy. However, further in depth in vivo and clinical studies are warranted to verify this suggestion. These studies are now exploring potential mechanistic connections to metabolic programming known to occur in cancer cells and TICs in 3
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promoting and maintaining “stem cell fate” via molecular, genetic, and epigenetic mechanisms via dysregulation of lncRNA. [14]
Declaration of competing interest [15]
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
[16]
Acknowledgments
[17]
This project was supported by NIH grants 1R01AA018857-01, 5R21AA025470, P50AA11999, R01 AA025204-01A1, R21 AA02547001A1 (Animal Core, Morphology Core, and Pilot Project Program), R24AA012885 (Non-Parenchymal Liver Cell Core) and pilot project funding (5P30DK048522-13). This research is also supported by a Research Scholar Grant, RSG-12-177-01-MPC and pilot funding (IRG58-007-48) from American Cancer Society. Microscopy was performed by the Cell and Tissue Imaging Core of the USC Research Center for Liver Diseases (P30 DK048522). Liver tissues were obtained from The Liver Tissue Cell Distribution System (LTCDS) in University of Minnesota. We also acknowledge the Animal and Morphology Core facilities of the NIAAA-supported Southern California Research Center for ALPD and Cirrhosis (P50 AA011999) for providing a mouse model of intragastric ethanol infusion and morphological analyses of liver tissues.
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Appendix A. Supplementary data [23]
Supplementary data to this article can be found online at https:// doi.org/10.1016/j.cbi.2020.109055.
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