Liver Cancer Stem Cells Markers Research Articles

Sorted-cell sequencing on HCC specimens reveals EPS8L3 as a key player in CD24/CD13/EpCAM-triple positive, stemness-related HCC cells

Background & AimsHepatocellular carcinoma (HCC) is a heterogeneous cancer with varying levels of liver tumor initiating or cancer stem cells in the tumors. We aimed to investigate the expression of different liver cancer stem cell (LCSC) markers in human HCCs, and identify their regulatory mechanisms in stemness-related cells. MethodsWe used an unbiased, single-marker sorting approach by flow cytometry, fluorescence-activated cell sorting, and transcriptomic analyses on HCC patients’ resected specimens. Knockdown approach was employed and relevant functional assays were conducted on the identified targets of interest. ResultsFlow cytometry on a total of 60 HCC resected specimens showed significant heterogeneity in the expression of LCSC markers, with CD24, CD13, and EpCAM mainly contributing to this heterogeneity. Concomitant expression of CD24, CD13 and EpCAM was detected in 32 HCC samples, and this was associated with advanced tumor stages. Transcriptomic sequencing on the HCC cells sorted for these individual markers identified EPS8L3 as a common gene associated with the three markers and was functionally validated in HCC cells. Knocking down EPS8L3 suppressed the expression of all three markers. To search for the upstream regulation of EPS8L3, we found SP1 bound to EPS8L3 promoter to drive EPS8L3 expression. Furthermore, using Akt inhibitor MK2206, we showed that Akt-signaling-driven SP1 drove the expression of the three LCSC markers ConclusionsOur findings suggest that Akt-signaling-driven SP1 promotes EPS8L3 expression, which is critical in maintaining the downstream expression of CD24, CD13 and EpCAM. The findings provide insight into potential LCSC-targeting therapeutic strategies.

Abstract 5431: Identification of serine/threonine kinase 39 (STK39) as a driver for multidrug resistance in hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) is a common type of cancer globally and is associated with a poor prognosis due to its high recurrence rate and resistance to treatment. Solid evidence showed the existence of cancer stem cells (CSCs) that are crucial for therapeutic resistance. However, there are only a few kinase inhibitors that have been found to specifically target liver T-ICs for the HCC treatment. To understand the regulatory mechanism of liver CSCs, we employed RNA-Seq analysis to compare the gene expression profiles, with a particular focus on protein kinases, between (1) sorted CD133+ liver CSCs and CD133- differentiated cells extracted from two HCC cell lines (Huh7 and PLC8024), as well as cultured hepatospheres and adherent differentiated cells of HCC cell line PLC/PRF/5. Our analysis identified Serine/Threonine kinase 39 (STK39) as a commonly up-regulated protein kinase in both CD133+ liver CSCs and CSC-enriched hepatospheres. Based on our analysis of the TCGA data, a positive correlation has consistently been observed between STK39 mRNA expression and the expression of liver CSC markers, including CD24, CD133, and CD47. Utilizing publicly available transcriptome datasets from the NCBI Gene Expression Omnibus (GEO), including GSE14520 and GSE25097, we found that overexpression of STK39 is detected in HCC tumor tissues (p<0.001) and is significantly associated with poorer overall survival and disease-free survival. Through the use of lentiviral based overexpression and knockdown approaches, we have demonstrated the role of STK39 in regulating liver CSC properties, including self-renewal, tumorigenicity, cell invasiveness, and the expression of liver CSC markers. STK39 expression was found to be significantly elevated in in HCC cells that were resistant to both sorafenib and lenvatinib, and its inhibition resulted in increased sensitivity of HCC cells to these treatments. To identify the downstream target of STK39 for regulation of cancer stemness, tandem affinity purification coupled with mass spectrometry was employed. The analysis led to the identification of PARP1 as a novel protein binding partner of STK39. Upon suppression of STK39 in HCC cells, it was observed that the phosphorylation of PARP1 at Threonine 368 (T368) was significantly inhibited, indicating its role as a downstream effector of STK39-mediated liver CSC functions. Interestingly, STK39-mediated PARP1 phosphorylation led to development of resistance to PARP1 inhibitor, rucaparib. Additionally, STK39 plays a critical role in regulating PARP1-mediated chromatin decondensation, which enables it to open chromatin structure at the promoter regions of SOX2/OCT4. In summary, treatment of molecular targeted drugs including rucaparib and lenvatinib with STK39 inhibitor may be a potential therapeutic strategy for treatment of this deadly disease. Citation Format: Carmen Oi Ning Leung, Terence Kin-Wah Lee. Identification of serine/threonine kinase 39 (STK39) as a driver for multidrug resistance in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5431.

Aberrantly expressed HIF-1α enhances HCC stem cell-like traits via Wnt/β-catenin signaling activation after insufficient radiofrequency ablation.

Radiofrequency ablation has become a favorable treatment modality for small hepatocellular carcinoma (HCC) recently; however, insufficient radiofrequency ablation (RFA) was shown to lead to enhanced invasiveness and metastasis of HCC in our previous study, while the underlying molecular mechanism has not been understood. In order to explore the influence of the hypoxic microenvironment on residual cancer and cancer stem cell (CSC)-like characteristics of HCC cells in this process, an in vitro hypoxic model and an insufficient RFA mouse model were established with HCC cancer cell lines. Immunochemistry staining and western blot were used to examine the expression of hypoxia-inducible factor (HIF)-1α and liver CSC markers. The 3D colon formation assay, tumor cell invasion assay, and gene transfection assays were applied to test the change in liver CSC stemness and HCC cell invasion. After insufficient RFA treatment, the upregulated HIF-1α expression was associated with an increase in the CSC-like population in residual cancer. In vitro, hypoxic tumor cells showed aggressive CSC-like properties and phenotypes. Wnt/β-catenin signaling activation was shown to be necessary for the acquisition of liver CSC-like characteristics under hypoxic conditions. Overall, the aberrantly enhanced HIF-1α expression enhanced the liver CSC-like traits via abnormal Wnt/β-catenin signaling activation after insufficient RFA, and the overexpressed HIF-1α would be a vital factor and useful biomarker during the HCC recurrence and metastasis.

Abstract 2443: Serine/threonine kinase 39 (STK39) regulates cancer stemness through regulating PARP-mediated SOX2/OCT4 expression in hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) is one of the common malignancies worldwide. Poor prognosis of HCC patients is attributed to high frequency of tumor relapse and therapeutic resistance. Increasing evidence showed the existence of cancer stem cells (CSCs) which is crucial for tumor initiation and therapeutic resistance. However, there are limited kinase inhibitors available that show specific targeting against liver T-ICs for HCC treatment. To understand the mechanism for regulation of liver CSCs, we have employed RNA-Seq profiling to compare the gene expression profiles with focuses on protein kinases between (1) sorted CD133+ liver CSCs and CD133- differentiated counterparts isolated from two HCC cell lines (Huh7 and PLC8024) and cultured hepatospheres and adherent differentiated cells of HCC cell line PLC/PRF/5. This analysis revealed Serine/Threonine kinase 39 (STK39), also known as SPAK, to be the commonly up-regulated protein kinase common in both CD133+ liver CSCs and CSC enriched hepatospheres. Consistently, based on the TCGA data analysis, STK39 mRNA expression is positively correlated with the expression of liver CSC markers including CD24, CD133, and CD47. Using publicly available transcriptome datasets deposited in the NCBI Gene Expression Omnibus (GEO) including GSE14520 and GSE25097, we found that overexpression of STK39 was also observed in HCC tumor tissues (p<0.001) and correlated with poorer overall survival and disease-free survival. By lentiviral based overexpression and knockdown approaches, we demonstrated the role of STK39 in regulation of liver CSC properties, including self-renewal, tumorigenicity, cell invasiveness and expression of liver CSC markers. Specially, STK39 expression was highly elevated in sorafenib- and lenvatinib-resistant HCC cells, and its inhibition led to sensitization of HCC cells towards these treatments. In order to identify downstream target of STK39 for regulation of cancer stemness, tandem affinity purification coupled with mass spectrometry was employed. Upon analysis, PARP1, was identified to be the novel protein binding partner of STK39. Upon suppression of STK39 in HCC cells, phosphorylation of PARP1 at Threonine 368 (T368) was found to be highly inhibited, suggesting its role as downstream effector of STK39-mediated liver CSC functions. Furthermore, STK39 regulates PARP1-mediated chromatin decondensation, and thus opens chromatin structure at the promoter regions of SOX2/OCT4. In summary, STK39 regulates cancer stemness and drug resistance by directly modulating PARP1-mediated SOX2/OCT4 expression via chromatin remodelling. Targeting STK39/PARP1 signaling cascade alone or in combination with molecular targeted drugs including rucaparib and lenvatnib can be the potential therapeutic strategy for treatment of this deadly disease. Citation Format: Carmen Oi Ning Leung, Terence Kin-Wah Lee. Serine/threonine kinase 39 (STK39) regulates cancer stemness through regulating PARP-mediated SOX2/OCT4 expression in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2443.

Research progress in targeting liver cancer stem cells for the treatment of liver cancer

CSCs (tumour stem cells) have been shown to be resistant to conventional chemotherapy or irradiation, and surviving CSCs can re-proliferate and lead to tumour recurrenc and Metastasis under suitable conditions due to their strong self-renewal and multiple differentiation functions. The development of metastasis, recurrence and drug resistance caused by CSCs is now considered to be a major cause of treatment failure and patient death inmalignancies[1] .Therefore,direct targeting of LCSCs (hepatocellular carcinoma stem cells) is a new strategy for the treatment of hepatocellular carcinoma. Over the years, as research on LCSCs continues, therapies targeting LCSCs have been found to have great potential in the clinical management and prognosis of hepatocellular carcinoma. This article reviews the specific application of liver cancer stem cell markers interacting with relevant signalling pathways in the treatment of hepatocellular carcinoma.

Connexin32 regulates expansion of liver cancer stem cells via the PI3K/Akt signaling pathway.

Liver cancer stem cells (LCSCs) are responsible for liver cancer recurrence, metastasis, and drug resistance. Previous studies by the authors demonstrated that upregulated expression of connexin 32 (Cx32) reversed doxorubicin resistance and reduced invasion and metastasis of liver cancer cells. However, the role of Cx32 in expansion of LCSCs remains unclear. A total of 85 patients were enrolled in the present study and followed-up for 5 years. The expression of Cx32 in hepatocellular carcinoma (HCC) tissues and corresponding paracancerous tissues were detected by immunohistochemistry (IHC). Cx32 was silenced in HepG2 cells and overexpressed in HCCLM3 cells and the stemness of liver cells was examined by detecting the expression of LCSC markers (EpCAM, CD133, Nanog, Oct4, Sox9, c-Myc), sphere formation, and xenograft tumorigenesis. Finally, the effect of the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) pathway on Cx32-regulated LCSC expansion was investigated. Cx32 was downregulated in LCSCs and HCC tissues, and predicted poor prognosis in patients with HCC. Overexpression of Cx32 in HCCLM3 cells significantly inhibited LCSC expansion, tumorigenesis, and phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) pathway activity. By contrast, silencing of Cx32 in HepG2 cells upregulated expansion of LCSCs and PI3K/Akt pathway activity. Modulating the activity of the PI3K/Akt pathway by SC-79 and LY294002 in HepG2 and HCCLM3 cells, respectively, confirmed that Cx32 could affect the expansion of LCSCs through PI3K/Akt signaling. In conclusion, the present study demonstrated that Cx32 regulated the expansion of LCSCs, and increased expression of Cx32 significantly inhibited the expansion of LCSCs, suggesting that Cx32 may be an optimal target for intervention of HCC.

Update on Glycosphingolipids Abundance in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the most frequent type of primary liver cancer. Low numbers of HCC patients being suitable for liver resection or transplantation and multidrug resistance development during pharmacotherapy leads to high death rates for HCC patients. Understanding the molecular mechanisms of HCC etiology may contribute to the development of novel therapeutic strategies for prevention and treatment of HCC. UDP-glucose ceramide glycosyltransferase (UGCG), a key enzyme in glycosphingolipid metabolism, generates glucosylceramide (GlcCer), which is the precursor for all glycosphingolipids (GSLs). Since UGCG gene expression is altered in 0.8% of HCC tumors, GSLs may play a role in cellular processes in liver cancer cells. Here, we discuss the current literature about GSLs and their abundance in normal liver cells, Gaucher disease and HCC. Furthermore, we review the involvement of UGCG/GlcCer in multidrug resistance development, globosides as a potential prognostic marker for HCC, gangliosides as a potential liver cancer stem cell marker, and the role of sulfatides in tumor metastasis. Only a limited number of molecular mechanisms executed by GSLs in HCC are known, which we summarize here briefly. Overall, the role GSLs play in HCC progression and their ability to serve as biomarkers or prognostic indicators for HCC, requires further investigation.

Cancer stem cells in hepatocellular carcinoma - from origin to clinical implications.

Hepatocellular carcinoma (HCC) is an aggressive disease with a poor clinical outcome. The cancer stem cell (CSC) model states that tumour growth is powered by a subset of tumour stem cells within cancers. This model explains several clinical observations in HCC (as well as in other cancers), including the almost inevitable recurrence of tumours after initial successful chemotherapy and/or radiotherapy, as well as the phenomena of tumour dormancy and treatment resistance. The past two decades have seen a marked increase in research on the identification and characterization of liver CSCs, which has encouraged the design of novel diagnostic and treatment strategies for HCC. These studies revealed novel aspects of liver CSCs, including their heterogeneity and unique immunobiology, which are suggestive of opportunities for new research directions and potential therapies. In this Review, we summarize the present knowledge of liver CSC markers and the regulators of stemness in HCC. We also comprehensively describe developments in the liver CSC field with emphasis on experiments utilizing single-cell transcriptomics to understand liver CSC heterogeneity, lineage-tracing and cell-ablation studies of liver CSCs, and the influence of the CSC niche and tumour microenvironment on liver cancer stemness, including interactions between CSCs and the immune system. We also discuss the potential application of liver CSC-based therapies for treatment of HCC.

Emerging Regulatory Mechanisms Involved in Liver Cancer Stem Cell Properties in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the predominant form of primary liver cancer and one of the leading causes of cancer-related deaths worldwide. A growing body of evidence supports the hypothesis that HCC is driven by a population of cells called liver cancer stem cells (LCSCs). LCSCs have been proposed to contribute to malignant HCC progression, including promoting tumor occurrence and growth, mediating tumor metastasis, and treatment resistance, but the regulatory mechanism of LCSCs in HCC remains unclear. Understanding the signaling pathways responsible for LCSC maintenance and survival may provide opportunities to improve patient outcomes. Here, we review the current literature about the origin of LCSCs and the niche composition, describe the current evidence of signaling pathways that mediate LCSC stemness, then highlight several mechanisms that modulate LCSC properties in HCC progression, and finally, summarize the new developments in therapeutic strategies targeting LCSCs markers and regulatory pathways.

Advances in Liver Cancer Stem Cell Isolation and their Characterization.

Over the last decade research on cancer stem cells (CSC) significantly contributed to a better understanding of tumor biology. Given their similarity to normal stem cells, i.e. self-renewal and pluripotency the need arises to develop robust protocols for the isolation and characterization of CSCs. As with other malignancies, hepatic tumors are composed of a heterogeneous population of cells including liver cancer stem cells (LCSC). Yet, a precise understanding of why stem cells become cancerous is still lacking. There is unmet need to develop robust protocols for the successful isolation of LCSCs from human tissue resection material as to assist in the development of molecular targeted therapies. Here we review the research progress made in the isolation and characterization of LCSCs by considering a wide range of cell surface markers and sorting methods, as applied to side populations, microsphere cultures and the gradient centrifugation method. We emphasize the different fluorescence activated cell sorting methods and the possibility to enrich LCSCs by immunomagnetic beads. We review the specificity of functional assays by considering ABCG transporter and ALDH1 enzyme activities and evaluate the in vivo tumorigenicity of LCSCs in highly sensitive bioassays. Finally, we evaluate different LCSC markers in association with viral and non-viral liver disease and explore the potential of novel drug delivery systems targeting CD133, EpCAM, CD13 and CD90 for the development of molecular targeted therapies. Graphical Abstract.

Correlation between inflammatory cytokines and liver cancer stem cell markers in DEN-induced liver cancer rats.

To study the association of inflammatory factors and hepatocarcinoma stem cells of induced liver cancer rats. 30 SD male healthy rats were selected. 10 rats were given water as normal control group. 10 rats only were implemented laparotomy as sham operation group. The remaining 10 rats were the liver cancer model group and treated with diethylnitrosamine (DEN) to induce liver cancer. Real-time quantitative PCR was used to detect the related inflammatory factors in HCC tissues, including interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), transforming growth factor-β1 (TGF-β), human interleukin-1α (IL-1α), human interleukin 1β (IL-1β) and levels of hepatocarcinoma stem cells indicators CD90, CD133, Alpha-fetoprotein (AFP). Correlation analysis was used to analyze the correlation between inflammatory factors and hepatocarcinoma stem cells markers CD90 and CD133. The expression levels of IL-6, MCP-1 and TGF-β of HCC tissues in liver cancer model group were significantly higher than those in the control group and the sham operation group. The expression levels of CD90 and CD133 of tissues in the liver cancer model group were significantly higher than those in the control group and the sham operation group. The differences were statistically significant (p<0.001). By inhibiting related inflammatory factors, the growth, migration and invasion of liver cancer cells were significantly inhibited, and apoptosis was promoted. Correlation analysis results showed that the expression changes of IL-6, MCP-1 and TGF-β were significantly positively correlated with CD90 up-regulation (p<0.05), while the expression changes of IL-6, MCP-1 and TGF-β were significantly positively correlated with CD133 up-regulation (p<0.05). The inflammatory factors IL-6, MCP-1 and TGF-β are closely related to hepatocarcinoma stem cells, which play an important role in promoting the occurrence and deterioration of liver cancer.

NEAT1 is Required for the Expression of the Liver Cancer Stem Cell Marker CD44

CD44, a cancer stem cell (CSC) marker, is required for maintaining CSC properties in hepatocellular carcinoma (HCC). Nuclear enriched abundant transcript 1 (NEAT1), a long noncoding RNA (lncRNA), is an oncogenic driver in HCC. In the present study, we investigated the significance of the NEAT1 gene in association with CD44 expression in liver CSCs of human HCC cell lines. The CSC properties were evaluated by spheroid culture, CSC marker expression, and sensitivity to anti-cancer drugs. The expression of both NEAT1 variant 1 (NEAT1v1) and variant 2 (NEAT1v2) as well as CD44 was significantly increased in the spheroid culture, compared with that in monolayer culture. Overexpression of Neat1v1, but not Neat1v2, enhanced the CSC properties, while knockout of the NEAT1 gene suppressed them. CD44 expression was increased by the overexpression of Neat1v1 and abrogated by NEAT1 knockout. The overexpression of NEAT1v1 restored the CSC properties and CD44 expression in NEAT1-knockout cells. NEAT1v1 expression in HCC tissues was correlated with poor prognosis and CD44 expression. These results suggest that NEAT1v1 is required for CD44 expression. To our surprise, NEAT1v1 also restored the CSC properties even in CD44-deficient cells, suggesting that NEAT1v1 maintains the properties of CSCs in a CD44-independent manner.

ZBP-89 negatively regulates self-renewal of liver cancer stem cells via suppression of Notch1 signaling pathway.

Liver cancer stem cells (LCSCs) initiate hepatocellular carcinoma (HCC) and contribute to its recurrence and treatment resistance. Studies have suggested ZBP-89 as a candidate tumor suppressor in HCC. We explored the role of ZBP-89 in the regulation of LCSCs. This study was performed in liver tissue samples from 104 HCC patients, 2cell lines and mouse tumor models. We demonstrated that ZBP-89 was weakly expressed in LCSCs. Patients with high expression of LCSC markers displayed reduced survivals and higher recurrence rates after curative surgical operation. The expression of ZBP-89 was predictive for decreased recurrence. LCSC markers were negatively correlated with ZBP-89 in HCC tissues and in enriched liver tumor spheres. The exogenous expression of ZBP-89 attenuated the tumor-sphere formation and secondary colony formation capabilities of LCSCs in vitro and tumorigenicity in vivo. Furthermore, the negative effect of ZBP-89 on cancer stemness was Notch1-dependent. Localized with Notch1 intracellular domain (NICD1) in the nucleus, ZBP-89 repressed the Notch1 signaling pathway by competitive binding to NICD1 with MAML1. Collectively, ZBP-89 negatively regulates HCC stemness via inhibiting the Notch1 signaling.

Single-cell transcriptomics reveals the landscape of intra-tumoral heterogeneity and stemness-related subpopulations in liver cancer.

Hepatocellular carcinoma (HCC) is heterogeneous, rendering its current curative treatments ineffective. The emergence of single-cell genomics represents a powerful strategy in delineating the complex molecular landscapes of cancers. In this study, we demonstrated the feasibility and merit of using single-cell RNA sequencing to dissect the intra-tumoral heterogeneity and analyze the single-cell transcriptomic landscape to detect rare cell subpopulations of significance. Exploration of the inter-relationship among liver cancer stem cell markers showed two distinct major cell populations according to EPCAM expression, and the EPCAM+ cells had upregulated expression of multiple oncogenes. We also identified a CD24+/CD44+-enriched cell subpopulation within the EPCAM+ cells which had specific signature genes and might indicate a novel stemness-related cell subclone in HCC. Notably, knockdown of signature gene CTSE for CD24+/CD44+ cells significantly reduced self-renewal ability on HCC cells in vitro and the stemness-related role of CTSE was further confirmed by in vivo tumorigenicity assays in nude mice. In summary, single-cell genomics is a useful tool to delineate HCC intratumoral heterogeneity at better resolution. It can identify rare but important cell subpopulations, and may guide better precision medicine in the long run.

Aquaporin 3 maintains the stemness of CD133+ hepatocellular carcinoma cells by activating STAT3

An increasing interest in liver cancer stemness arises owing to its aggressive behavior and poor prognosis. CD133, a widely known liver cancer stem cell marker, plays critical roles in the maintenance of liver cancer stemness. Thus, exploring the regulatory mechanism of CD133 expression is significant. In the present study, we proved the carcinogenesis roles of aquaporin 3 (AQP3) in hepatocellular carcinoma (HCC) and demonstrated that AQP3 promotes the stem cell-like properties of hepatoma cells by regulating CD133 expression. In addition, AQP3 promoted the stimulation and nuclear translocation of signal transducer and activator of transcription 3 (STAT3) with a subsequent increase in the level of CD133 promoter-acetylated histone H3. This phenomenon accelerated CD133 transcription. Next, whether AQP3 acted as an oncogenic gene in HCC and maintained the stemness of CD133+ hepatoma cells were elucidated; also, a novel mechanism underlying the AQP3/STAT3/CD133 pathway in HCC was deduced.

Tobacco smoke induced hepatic cancer stem cell-like properties through IL-33/p38 pathway

BackgroundTobacco smoke (TS) critically contributes to the development of hepatocellular carcinoma. Cancer stem cells (CSCs) induced by TS is an early event in the initiation of carcinogenesis. Tumor specific microenvironment including inflammatory factors is key mediator for maintaining the stemness of CSCs through various pathways such as p38 MAPK. However, the mechanisms of inflammatory factors in TS-induced acquisition of liver CSCs properties remain undefined. The aim of this study was to investigate the role of IL-33/p38 axis in long term TS-induced acquisition of hepatic CSCs properties in mouse liver tissues and human liver cells.MethodsBALB/c mice were exposed to TS for 12 weeks, along with or without 1 mg/kg SB203580 (p38 inhibitors) treatment. Histopathological analysis, alterations in the levels of IL-33, liver CSCs markers, EMT-like changes and p38 MAPK activation in liver tissues of mice were analyzed by immunohistochemical staining, immunofluorescence assay and Western blot analysis. Moreover, LO2 immortalized human liver cells were exposed to cigarette smoke extract (CSE) and the tumorsphere formation ability was determined. LO2 cells were further treated with IL-33 or CSE and the expression of phosphorylated p38, liver CSCs markers and EMT-related proteins was examined.ResultsLong term TS exposure increased the levels of CSCs markers, induced epithelial-to mesenchymal transition (EMT) and inflammatory factor IL-33 expression. Moreover, we showed that p38 MAPK modulated TS-stimulated hepatic CSC-like properties, as evidenced by the findings that long term TS exposure activated p38, and that TS-induced stemness was abolished by p38 inhibition. In addition, data from in vitro model showed that similar to cigarette smoke extract (CSE), IL-33 treatment promoted the activation of p38, increased the levels of liver CSCs markers expression and EMT-like changes.ConclusionsCollectively, these data suggested that IL-33/p38 axis plays an important role in long term TS exposure-induced acquisition of hepatic CSC-like properties.

Abstract 162: UBE2T: A molecular regulator for cancer stemness and drug resistance in hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) is one of the common cancers worldwide. Poor prognosis of HCC patients is attributed to high frequency of tumor relapse and chemoresistant nature of this disease. Increasing evidence showed the critical role of cancer stem cells (CSCs) on tumor relapse and therapeutic resistance. Since normal stem cells and CSCs share high similarity, we would like to identify the molecules/pathways crucial in liver CSCs by determining what molecules involved in normal liver stem cells during liver regeneration. Comparison of expression profiles between early regenerating liver and intact liver revealed the upregulation of DNA Damage Response pathways in the self-renewing liver, in which Ubiquitin Conjugating Enzyme E2 T (UBE2T) was the most significantly upregulated. This, together with the publicly available dataset (GSE5975) shows upregulation of UBE2T in EpCAM-enriched liver CSC populations, suggest the potential role of UBE2T on regulation of cancer stemness. By qPCR analysis UBE2T mRNA level was overexpressed in 91% of the clinical HCC specimens (n=56) and associated with aggressive tumor behavior and poorer patients' survival. The overexpression of UBE2T was further confirmed by western blot and immunohistochemical analysis. By lentiviral based knock-down approach, we demonstrated the role of UBE2T in regulation of liver CSC properties, including self-renewal, tumorigenicity, drug resistance and expression of liver CSC markers. In orthotopic HCC model, UBE2T was found to play pivotal role in lung metastasis in vivo. Mechanistically, UBE2T is found to interact directly with E3 ligase Mule and regulate its protein expression via ubiquitination. Since Mule is found to directly degrade β-catenin protein, we are examining whether UBE2T mediates liver T-IC function through direct regulation of Mule-mediated β-catenin degradation. In conclusion, we have uncovered the novel role of UBE2T signaling cascade in regulation liver CSCs, which provides attractive therapeutic target for potential treatment of HCC. Citation Format: Pui Yu Ho, Kin Wah Lee. UBE2T: A molecular regulator for cancer stemness and drug resistance in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 162.

Insufficient radiofrequency ablation promotes hepatocellular carcinoma cell progression via autophagy and the CD133 feedback loop.

Insufficient radiofrequency ablation (iRFA) often leads to residual hepatocellular carcinoma (HCC) progression. However, the mechanism is still poorly understood. In the present study, we demonstrated that LC3B protein expression levels were significantly increased in the residual hepatocellular carcinoma cells after radiofrequency ablation (RFA) treatment in vivo. Moreover, iRFA promoted autophagy, autophagosome formation and autophagic flux in Huh-7 and SMMC7721 cell lines in vitro. In addition, iRFA induced HCC cell viability and invasion. However, blockade of autophagy by the autophagosome inhibitor 3-methyladenine (3-MA) suppressed iRFA-induced cell viability and invasion. Furthermore, we revealed that the expression of liver cancer stem cell marker CD133 was also significantly increased in the residual hepatocellular carcinoma cells after RFA treatment in vivo, and was positively correlated with LC3B protein expression. iRFA also promoted CD133 protein expression in Huh-7 and SMMC7721 cell lines in vitro. CD133 was localized to autophagosomes, and was suppressed by 3-MA or chloroquine (CQ) after iRFA treatment. CD133 downregulation also suppressed iRFA-induced cell viability, invasion and autophagy. Collectively, our results indicated that RFA may promote residual HCC cell progression by autophagy and CD133 feedback loop.

C-terminal truncated hepatitis B virus X protein regulates tumorigenicity, self-renewal and drug resistance via STAT3/Nanog signaling pathway.

Hepatitis B virus (HBV) is a major risk factor of chronic liver disease and hepatocellular carcinoma (HCC). Random integration of HBV DNA into the host genome is frequent in HCC leading to truncation of the HBV DNA, particularly at the C-terminal end of the HBV X protein (HBx). C-terminally truncated HBx (HBx-ΔC) has been implicated in playing a pro-oncogenic role in hepatocarcinogenesis. However, the mechanism whereby HBx-ΔC1 contributes to hepatocarcinogenesis remains unclear. In this study, we investigated the functional role of HBx-ΔC1 in regulating liver cancer stem cell (CSC) properties. Using Tet-on inducible system, we found that HBx-ΔC1 enhanced CSC properties including self-renewal, tumorigenicity, chemoresistance, migration and expression of liver CSC markers, when compared with the full-length HBx counterpart and vector control. Interestingly, HBx-ΔC1 conferred resistance in HCC cells towards sorafenib treatment through suppression of apoptotic cascade. In addition, HBx-ΔC1 upregulated a panel of stemness genes, in which Nanog was found to be among the most significant one in both trasnfected cell lines. Consistently, Nanog was upregulated in human HCC samples which had HBx-ΔC1 expression. Furthermore, the induction of CSC properties by HBx-ΔC1 was via the Stat3/Nanog pathway, as administration of Stat3 inhibitor abolished the HBx-ΔC1-induced self-renewing capacity. In conclusion, our data suggest that HBx-ΔC1 enhances liver CSCs properties through Stat3/Nanog cascade, and provide a new insight for the therapeutic intervention for HBV-related HCC.

FAK depletion/inhibition reduces the expression of liver cancer stem cells markers

FAK depletion/inhibition reduces the expression of liver cancer stem cells markers