Chemoresistance In Hepatocellular Carcinoma Research Articles

Identification of Transmembrane Protein 98 as a Novel Chemoresistance-Conferring Gene in Hepatocellular Carcinoma

Chemoresistance is one of the major obstacles in systemic chemotherapy and targeted therapy for patients with advanced hepatocellular carcinoma. To identify novel chemoresistance-associated targets in hepatocellular carcinoma, chemoresistant hepatocellular carcinoma cell lines were established. By comparing the global gene expression profiles between chemoresistant and chemosensitive cell lines, eight novel chemoresistance-associated genes were identified to be significantly associated with the commonly augmented chemoresistance of hepatocellular carcinoma cells. One upregulated candidate named transmembrane protein 98 (TMEM98) was found to be overexpressed in 80 of 118 (67.80%) of patients with hepatocellular carcinoma. TMEM98 mRNA in tumor tissues was significantly higher than nontumor tissues of patients with hepatocellular carcinoma (P < 0.0001). Upregulation of TMEM98 was significantly correlated with advanced tumor stage (P = 0.048), high incidence of early tumor recurrence (P = 0.005), poor overall survival (P = 0.029), and poor disease-free survival (P = 0.011) of patients with hepatocellular carcinoma after hepatectomy. Importantly, upregulation of TMEM98 mRNA in patients with hepatocellular carcinoma who received transarterial chemoembolization (TACE) treatment was significantly higher than in patients without TACE treatment (P = 0.046). Moreover, patients with poor response to TACE treatment had higher degree of TMEM98 upregulation than the responsive patients. In vitro and in vivo studies showed that suppression of TMEM98 in chemoresistant hepatocellular carcinoma cells restored their chemosensitivity, while forced overexpression of TMEM98 enhanced their chemoresistance. The mechanism of TMEM98 in conferring chemoresistance of hepatocellular carcinoma might be possibly through activation of the AKT pathway and deactivation of p53. In conclusion, we identified a panel of novel common chemoresistance-associated genes and demonstrated that TMEM98 is a chemoresistance-conferring gene in hepatocellular carcinoma.

Mesenchymal stem cells contribute to the chemoresistance of hepatocellular carcinoma cells in inflammatory environment by inducing autophagy.

BackgroundMesenchymal stem cells (MSCs) have been reported to play an important role in tumor growth. Inflammation is an important feature of hepatocellular carcinoma (HCC). Certain inflammatory cytokines produced in tumor microenvironment modulate functional activities of MSCs. At the present time, however, the role of MSCs in the development of HCC cell resistance to chemotherapy in the inflammatory microenvironment during tumor growth has not yet been identified.MethodsMTT and PI/Annexin V-FITC assay were employed to examine the proliferation and apoptosis of HCC cell lines. The expression of TGF-β are detected by Realtime PCR and Western blot. GFP tagged LC3 expression vector and electron microscopy are utilized to demonstrate the occurrence of autophagy.ResultsWe observed that MSCs pretreated with the combination of IFN-γ and TNF-α induced resistance to chemotherapy in HCC cell lines in both the in vitro and in vivo circumstances. Following exposure to conditioned medium of MSCs that were pre-treated with IFN-γ plus TNF-α, HCC cell line cells underwent autophagy which serves as a protective mechanism for HCC cells to resist the cell toxicity of chemotherapeutic agents. Treatment of HCC cell line cells with autophagy inhibitor effectively reversed the MSCs-induced resistance to chemotherapy in these cells. Stimulation with the combination of IFN-γ and TNF-α provoked expression of TGF-β by MSCs. MSCs-induced chemoresistance in HCC cell lines was correlated with the up-regulation of TGF-β expression by MSCs. Knockdown of TGF-β expression by MSCs with siRNA attenuated MSCs-induced chemoresistance in HCC cells.ConclusionsThese results suggest that increase in TGF-β expression by MSCs in the inflammatory microenvironment of HCC promotes the development of chemoresistance in HCC cells.

Peptidylarginine deiminase IV promotes the development of chemoresistance through inducing autophagy in hepatocellular carcinoma.

BackgroundPeptidylarginine deiminase IV (PADI4) is widely distributed in several tissues and the expression is correlated with many pathological processes. Chemotherapy remains a major treatment alternatively to surgery for a large number of patients at the advanced stage of hepatocellular carcinoma (HCC). However, the role of PADI4 in the chemoresistance of HCC has not been identified.MethodsMTT and PI/Annexin V assay were employed to examine the proliferation and apoptosis of HCC cell lines. The expression of MDR1 is detected by Realtime PCR. GFP tagged LC3 expression vector and electron microscopy are utilized to demonstrate the occurrence of autophagy.ResultsWe observed that the elevated PADI4 expression is associated with chemoresistance in HCC patients with TACE after surgery. In addition, we found that overexpression of PADI4 in HCC cell lines lead to the resistance to chemotherapeutic agents in vitro and in vivo. Interestingly, the HCC cells that overexpressed PADI4 were observed to undergo autophagy which was known as a protective mechanism for cells to resist the cell tosicity from chemotherapy. Autophagy inhibitor could effectively restore the sensitivity of HCC cells to chemotherapy in vitro and in vivo.ConclusionsThese results indicate that PADI4 may induce chemoresistance in HCC cells by leading autophagy.Electronic supplementary materialThe online version of this article (doi:10.1186/2045-3701-4-49) contains supplementary material, which is available to authorized users.

Annexin A3 promotes tumorigenesis and resistance to chemotherapy in hepatocellular carcinoma

Annexin A3 (ANXA3) has been found to play important roles in cancer progression, metastasis, and drug resistance; however, its role in hepatocellular carcinoma (HCC) remains unknown. In this study, we investigated the expression level, clinical significance and biologic function of ANXA3 in HCC. Real-time quantitative reverse transcriptase-polymerase chain reaction, western blotting and immunohistochemical staining were used to examine ANXA3 expression levels in HCC tumor tissue, and its correlation with the clinicopathological features and prognosis of HCC patients was analyzed. The biological functions of ANXA3 in cell proliferation, migration, invasion, and resistance to chemotherapy were also investigated. ANXA3 expression was significantly increased in HCC tissues as compared with adjacent non-tumorous tissues. Elevated ANXA3 expression was associated with tumor size, number of lesions, tumor stage, and poor prognosis. In hepatoma cell lines, exogenous ANXA3 transduction promoted the tumorigenic activity and metastatic potential of tumor cells. Small interfering RNA silencing of ANXA3 inhibited these processes. In addition, in vitro and in vivo experiments revealed that ANXA3 overexpression enhanced resistance to chemotherapy. Taken together, our findings reveal that ANXA3 might play an important role in HCC progression and chemoresistance, and could serve as a novel prognostic marker and therapeutic target for HCC.

Tunicamycin Potentiates Cisplatin Anticancer Efficacy through the DPAGT1/Akt/ABCG2 Pathway in Mouse Xenograft Models of Human Hepatocellular Carcinoma

Hepatocellular carcinoma is highly chemoresistant, and ATP-binding cassette subfamily G member 2 (ABCG2) is thought to play a critical role in this drug resistance. The present study aims to develop effective therapeutic strategies to decrease ABCG2 expression level and to surmount drug resistance in hepatocellular carcinoma chemotherapy. First, we verified a positive correlation between the ABCG2 protein level and the drug resistance of hepatocellular carcinoma cell lines. ABCG2 was preferentially expressed in highly chemoresistant hepatocellular carcinoma cancer stem cells (CSC) enriched with CD133. In addition, ABCG2 was N-linked glycosylated in hepatocellular carcinoma cells, and this modification was involved in sustaining its protein stability. The N-linked glycosylation (NLG) inhibitor tunicamycin dramatically reduced ABCG2 expression, altered its subcellular localization, and reversed its drug efflux effect in multiple hepatocellular carcinoma cell lines. Furthermore, tunicamycin reduced the expression levels of several CSC markers and suppressed the tumorigenicity of CD133(+) CSCs. Tunicamycin combined with cisplatin (CDDP) inhibited proliferating cell nuclear antigen (PCNA) expression and increased the cleavage of PARP; this effect was partially rescued by the overexpression of ABCG2 or Akt-myr. The combination therapy more effectively suppressed tumor growth in xenograft mice than did single-agent therapy with either drug. Finally, the CDDP treatment combined with UDP-GlcNAc-dolichol-phosphate N-acetylglucosamine-1 phosphate transferase (DPAGT1) knockdown recapitulated the effect observed when CDDP was used in combination with tunicamycin. In summary, our results suggest that tunicamycin may reverse the drug resistance and improve the efficacy of combination treatments for hepatocellular carcinomas by targeting the DPAGT1/Akt/ABCG2 pathway.

Anticancer activity of an antisense oligonucleotide targeting TRADD combined with proteasome inhibitors in chemoresistant hepatocellular carcinoma cells

Chemoresistance is a major cause of mortality of patients with advanced and metastatic hepatocellular carcinoma (HCC), the fifth most common cancer in the world. We employed a molecular approach to inhibit cell proliferation and induce apoptosis in HepG2 cells, originated from human hepatocarcinoma. TRADD gene expression was knocked down by an antisense oligonucleotide (ASO TRADD), resulting in TRADD protein decrease by 60%, coinciding with increase of apoptotic cell death of up to 30%. Combination of the ASO TRADD with the cytotoxic drugs 5-fluorouracil or paclitaxel did not improve chemosensitivity of HepG2 cells, while the combined administration of the ASO TRADD with proteasome inhibitors MG132 or ALLN inhibited cell proliferation by 80% and 93%, respectively. Taken together, these findings reveal the importance to combine proteasome inhibitors with silencing of anti-apoptotic signalling components to target HCC cells effectively and provide useful data for developing potential treatments of HCC.

Apicidin-resistant HA22T hepatocellular carcinoma cells massively promote pro-survival capability via IGF-IR/PI3K/Akt signaling pathway activation

Despite rapid advances in the diagnostic and surgical procedures, hepatocellular carcinoma (HCC) remains one of the most difficult human malignancies to treat. This may be due to the chemoresistant behaviors of HCC. It is believed that acquired resistance could be overcome and improve the overall survival of HCC patients by understanding the mechanisms of chemoresistance in HCC. A stable HA22T cancer line, which is chronically resistant to a histone deacetylase inhibitor, was established. After comparing the molecular mechanism of apicidin-R HA22T cells to parental ones by Western blotting, cell cycle-regulated proteins did not change in apicidin-R cells, but apicidin-R cells were more proliferative and had higher tumor growth (wound-healing assay and nude mice xenograft model). Moreover, apicidin-R cells displayed increased levels of p-IGF-IR, p-PI3K, p-Akt, Bcl-xL, and Bcl-2 but also significantly inhibited the tumor suppressor PTEN protein and apoptotic pathways when compared to the parental strain. Therefore, the highly proliferative effect of apicidin-R HA22T cells was blocked by Akt knockdown. For all these findings, we believe that novel strategies to attenuate IGF-IR/PI3K/Akt signaling could overcome chemoresistance toward the improvement of overall survival of HCC patients.

Hypoxia-inducible Factor-1alpha: Molecular-targeted Therapy for Hepatocellular Carcinoma

Hypoxia-inducible factor (HIF)-1α is over-expressed in hepatocellular carcinoma (HCC) and degraded by ubiquitin-proteasome pathways under normoxic conditions. Hepatocyte hypoxia enhances proliferation, angiogenesis, metastasis, chemoresistance, and radioresistance of HCC. The importance role of HIF-1α expression in HCC may improve the prognostic and therapeutic technique. This article reviews the HIF-1α expression and its gene during the rat HCC development, the level of HIF-1α expression in HCC patients, and the effect of silencing HIF-1α gene by miRNA on inhibition of HepG2 cell proliferation.

Melatonin reverses tunicamycin‐induced endoplasmic reticulum stress in human hepatocellular carcinoma cells and improves cytotoxic response to doxorubicin by increasing CHOP and decreasing Survivin

Chemoresistance in hepatocellular carcinoma (HCC) is associated with multiple cellular responses to environmental stresses, such as nutrient deprivation and hypoxia. Nevertheless, whether ER stress resulting from nutrient deprivation and tumor hypoxia contributes to drug resistance remains unclear. Melatonin increased the efficacy of chemotherapeutic drugs in hepatocellular carcinoma in our previous studies. However, the effects of melatonin on endoplasmic reticulum (ER) stress-induced resistance to chemotherapeutic agents in HCC have not been tested. The effect of the endoplasmic reticulum (ER) stress response during resistance of human hepatocellular carcinoma cells against doxorubicin was investigated in this study. Pretreatment of HepG2 and SMMC-7721 cells (two human hepatocellular carcinoma cell lines) with tunicamycin, an ER stress inducer, drastically decreased the rate of apoptosis generated by doxorubicin. Interestingly, co-pretreatment with tunicamycin and melatonin significantly increased apoptosis induced by doxorubicin. Simultaneously, the expression of phosphorylated AKT (p-AKT) was elevated in HepG2 and SMMC-7721 cells given tunicamycin but reduced in the presence of melatonin. Furthermore, consistent with inhibition of AKT activation by using the PI3K inhibitor LY294002, melatonin elevated the levels of CHOP (C/EBP-homologous protein) and reduced the levels of Survivin (a member of the inhibitor of apoptosis protein family)suggesting that inhibition of the PI3K/AKT pathway by melatonin-reversed ER stress-induced resistance to doxorubicin in human hepatocellular carcinoma cells. These results demonstrate that melatonin attenuates ER stress-induced resistance to doxorubicin in human hepatocellular carcinoma cells by down-regulating the PI3K/AKT pathway, increasing the levels of CHOP and decreasing the levels of Survivin.

Abstract 4015: Amplification of GEP at chromosome 17q21 and its overexpression in human liver cancer.

Abstract Amplification of GEP at chromosome 17q21 and its overexpression in human liver cancer Hermann M.K. Yung, Wallace C.W. Yip, Phyllis F.Y. Cheung and Siu Tim Cheung Department of Surgery, The University of Hong Kong. Introduction: Hepatocellular carcinoma (HCC) is the 3rd most lethal cancer worldwide. Curative treatments, such as surgical resection, are limited to only a small group of patients. Granulin-epithelin precursor (GEP), which is a secretory growth factor, has been shown to enhance the growth, invasion, metastasis and chemo-resistance of HCC. GEP was found to overexpress in tumors compared to the non-tumor counterpart, and the overexpression correlates to poor prognosis in HCC patients. Antibody targeting GEP inhibited tumor growth in vivo. However, the overexpression mechanisms in HCC are not clear. Hypothesis: Gene amplification may be one of the mechanisms for GEP overexpression as its locus, 17q21, is frequently amplified. Method: Quantitative Microsatellite Analysis (QuMA) was used to determine the copy number alteration of the GEP gene locus. In this method, quantitative PCR was used to quantify the GEP and microsatellite loci. The genomic stable microsatellite loci were used as reference. Copy number variations were determined by normalization of HCC data to the copy numbers of blood samples from healthy donors, which were defined as diploid 2N. GEP mRNA level was quantified by real-time RT-PCR. Correlations between GEP copy number and its mRNA expression level were analyzed. Results: GEP DNA copy numbers were quantified in a panel of clinical HCC (n=60). Amplification of GEP locus was observed in 30% (20/60) HCC, and the amplification frequency was comparable to published reports on chromosome 17q. Overall, GEP copy number correlated to the mRNA overexpression levels (n=60, r=0.267, P=0.039). For HCC with GEP gene locus amplification (n=20), tight correlation with GEP expression levels were observed (r=0.646, P=0.002). Summary: We showed that GEP gene was frequently amplified in HCC, and this amplification correlates to the expression level of GEP. Fluorescent in situ hybridization (FISH) will be performed to further investigate the amplification status. Citation Format: Man Kuen Yung, Chi Wai Yip, Phyllis Fung Yi Cheung, Siu Tim Cheung. Amplification of GEP at chromosome 17q21 and its overexpression in human liver cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4015. doi:10.1158/1538-7445.AM2013-4015

Overexpression of Yes-associated protein confers doxorubicin resistance in hepatocellullar carcinoma

Hepatocellular carcinoma (HCC) is one of the most aggressive malignancies worldwide and is highly resistant to chemotherapy. Yes-associated protein (YAP) is the downstream effector of the Hippo signaling pathway, which is frequently overexpressed in many types of cancers. Amplification of the YAP gene and overexpression of YAP in HCC have previously been reported to contribute to hepatocyte malignant transformation and tumor progression. In this study, we aimed to investigate the potential role of YAP in HCC chemoresistance. Overexpression of YAP resulted in resistance against doxorubicin-induced apoptosis in HCC cell lines, whereas suppression of the endogenous YAP expression by RNA interference demonstrated the reverse effect. Western blotting revealed that, following exposure to doxorubicin, YAP-overexpressing cells exhibited decreased cleaved PARP, increased phosphorylation of Akt and ERK1/2, and elevated Bcl-xL expression in comparison to the vector control. Inhibition of YAP expression sensitized HCC cells to doxorubicin, by exhibiting increased cleaved PARP, decreased levels of phosphorylated Akt, phosphorylated ERK1/2 and Bcl-xL expression. In addition, pretreatment with the MEK1/2 inhibitor U0126 but not the PI3-K inhibitor LY294002 significantly enhanced doxorubicin-induced apoptosis and decreased Bcl-xL expression in YAP-overexpressing HCC cells. Our data provide evidence that overexpression of YAP plays an important role in conferring doxorubicin resistance to HCC, which is at least partially mediated by YAP-induced activation of the MAP kinase pathway. Targeting YAP may be a promising adjunct for overcoming doxorubicin resistance in HCC.

Mo1950 Chemoresistance in Hepatocellular Carcinoma is Modulated by Interleukin-29 (Interferon-Lambda 1) and Rapamycin

Ten to 20 percent of patients with colorectal cancer with unresectable liver metastases meet the criteria for resection after chemotherapy, leading to improved prognosis. In this “Conversion therapy”, however, it remains unclear which patients will have long-term survival. Therefore, we examined the outcomes of such patients to identify characteristics of long-term survivors after “Conversion therapy”. Methods: Multidisciplinary treatment conferences conducted by radiology, digestive surgery and clinical oncology have evaluated patients for possible conversion therapy since 2006. Preoperative chemotherapy included: mFOLFOX6, FOLFIRI or Xelox (+Bevacizumab). Patients were evaluated for resectability every twomonths. Adjuvant chemotherapy with six months ofmFOLFOX6was administered. Results: Thirty-three patients were enrolled in this study, including 4 patients with a complete remission (CR) (12%) and 17 with a partial response (PR) (52%). Eleven patients underwent hepatic resection (33%; 11/33), including 1 with relapse after CR. Of these 11 patients (7 male, 4 female), age 48-74 years (median 57), 8 had simultaneous lesions, and 3 had metachronous lesions, 4-13 courses of chemotherapy were given before surgery (7). Ten patients underwent an R0 operation. In one patient, tumor nodules were not identified by intra-operative ultrasound, followed by hepatic re-resection after several months. Four of 11 patients are alive without recurrence. Overall survival is 18-55 months (42.5), with 13-40 months after hepatectomy (36), and re-hepatectomy in one case. Lesions disappeared on imaging after chemotherapy in all 4 cases. Pathological CR lesions in the resected specimens were present in 3 cases. Four patients are alive with recurrence. Survival is 4357 months (45.5), along with 6-27 months after relapse (16). Relapse-free survival was more than 12 months after hepatectomy in all 4 cases. Three patients died, with survival of 15-24 months (23), along with 3-8 months after relapse (6). In total, 7 of 11 patients (64%) survivedmore than 40months. All the patients originally had tubular adenocarcinoma. Conclusions: Hepatic lesions with a CR (clinically or pathologically) after chemotherapy, along with the pathology of well to moderately differentiated adenocarcinoma, may be favorable factors to predict long-term survival. Long-term relapse-free patients after first hepatectomy (> 12 months) showed long-term survival (> 40 months) even with recurrent cancer. Early recurrence after hepatic resection was associated with a poor prognosis, similar to patients receiving chemotherapy alone.

Activating transcription factor 4 increases chemotherapeutics resistance of human hepatocellular carcinoma

It has been reported that activating transcription factor 4 (ATF4) increases the processes of tumor growth, metastasis and drug resistance. However, the role played by ATF4 in chemoresistance of hepatocellular carcinoma (HCC) remains unknown. Clarification of this role of ATF4 in HCC could greatly benefit the efficacy of clinical treatment of HCC. In this study, we found that ATF4 was overexpressed in about 50.7% of HCC tissues. In fact knockdown of ATF4 significantly increased the cytotoxicity of cisplatin in both in vitro and in vivo assays, while overexpression of this molecule dramatically decreased the sensitivity of HCC cell lines to cisplatin. Additionally, we found that synthesis of glutathione was significantly reduced in HCC cell lines subjected to ATF4 knockdown. Taken together, these results demonstrate that ATF4 can increase resistance to cisplatin in HCC by increased biosynthesis of glutathione, and that this may be a potent novel target for the future development of anti-HCC drugs.

Hepatocarcinogenesis and Ceramide/Cholesterol Metabolism

Sphingolipids (SLs) and cholesterol are critical structural components of membrane bilayers. Although recent evidence has revealed an emerging role of both lipids in signaling pathways, their contribution to cancer development and treatment has been largely overlooked. Sphingolipids comprise a family of bioactive lipids with divergent roles in numerous cellular processes. In particular, ceramide is the prototype of SLs and identified as a cell death effector whose levels increase in response to apoptotic stimuli such as ionizing radiation or chemotherapy. In the liver, ceramide/cholesterol accumulation contributes to a wide range of pathologies, including the transition from steatosis to steatohepatitis, which can further progress to cirrhosis and hepatocellular carcinoma (HCC). Moreover, different studies have shown that either pharmacologic ceramide accumulation or systemic intravenous administration of liposomal ceramide is an effective approach against HCC. In addition, mitochondrial cholesterol trafficking has emerged as a novel factor regulating cell death pathways and HCC tumor growth and chemoresistance. Due to the poor efficacy of current HCC treatments, understanding the role of ceramide/cholesterol in HCC may open up novel avenues for therapy. Here we describe recent evidence indicating that ceramide-generating agents and/or pharmacological targeting of sphingolipid/cholesterol metabolism, alone or in combination with other chemotherapeutic compounds, may be a promising strategy in HCC management.

Knockdown of HURP inhibits the proliferation of hepacellular carcinoma cells via downregulation of gankyrin and accumulation of p53

We determined earlier that the hepatoma upregulated protein (HURP) is overexpressed in hepatocellular carcinoma (HCC), but the role of this protein during cancer development and progression remains unknown. Here, we observed that the overexpression of HURP in HEK293 cells promoted the ubiquitination of p53 and its degradation by the proteasome. In contrast, HURP knockdown using short-hairpin RNA reversed these effects. Knockdown of HURP promoted the accumulation of p53 in SK-Hep-1 cells (p53+/−), and these cells showed reduced proliferation, while the p53-mutant Mahlavu cells were not affected. HURP knockdown did not affect the proliferation of H1299 lung carcinoma cells and Hep3B HCC cells which lack p53. Knockdown of HURP also sensitized SK-Hep-1 cells to cisplatin. On the other hand, the expression of exogenous p53 in H1299 and Hep3B cells was decreased following overexpression of HURP, and these cells showed decreased sensitivity to cisplatin-induced apoptosis. Importantly, overexpression of HURP promoted the proliferation of HEK293 cells in an anchorage-independent manner, and inoculation of SK-Hep-1 cancer cells that expressed short-hairpin RNA to knockdown HURP resulted in smaller tumors in nude mice. Gankyrin, a positive regulator of the E3 ubiquitin ligase MDM2, was found to be upregulated following HURP expression, and gankyrin knockdown decreased the HURP-mediated downregulation of p53. Notably, we detected a positive correlation between elevated HURP and gankyrin protein levels in HCC patients (r2=0.778; N=9). Taken together, these results indicate that HURP represents an oncogene that may play a role in HCC progression and chemoresistance.

Activation of PI3 kinase/Akt/HIF-1α pathway contributes to hypoxia-induced epithelial-mesenchymal transition and chemoresistance in hepatocellular carcinoma

Hypoxia is known to promote malignant progression and to induce chemoresistance in cancer. However, the exact mechanisms driving hypoxia induced malignance remain elusive. We found that with exposure to hypoxic condition, hepatocellular carcinoma (HCC) cells experienced epithelial-mesenchymal transition (EMT), with increased cell migration and invasion, and exhibited high resistance to chemotherapy. We demonstrated that hypoxia-induced EMT and chemoresistance were accompanied by increased HIF-1α expression and activation of Akt. HIF-1α could be blocked by PI3K inhibitor LY294002, indicating HIF-1α activation was regulated by PI3K/Akt pathway. Furthermore, we showed that inhibition of PI3K/Akt and HIF-1α enhanced the therapeutic efficacy of hypoxic chemotherapy in the HCC xenograft model. Our findings indicate that the activation of PI3K/Akt/HIF-1α pathway plays a critical role in mediating hypoxia-induced EMT and drug resistance leading to unfavorable treatment outcome. Our study provides novel insights into the malignant progression triggered by hypoxic microenvironment in HCC cells.

CD24 + Liver Tumor-Initiating Cells Drive Self-Renewal and Tumor Initiation through STAT3-Mediated NANOG Regulation

Tumor-initiating cells (T-ICs) are a subpopulation of chemoresistant tumor cells that have been shown to cause tumor recurrence upon chemotherapy. Identification of T-ICs and their related pathways are therefore priorities for the development of new therapeutic paradigms. We established chemoresistant hepatocellular carcinoma (HCC) xenograft tumors in immunocompromised mice in which an enriched T-IC population was capable of tumor initiation and self-renewal. With this model, we found CD24 to be upregulated in residual chemoresistant tumors when compared with bulk tumor upon cisplatin treatment. CD24(+) HCC cells were found to be critical for the maintenance, self-renewal, differentiation, and metastasis of tumors and to significantly impact patients' clinical outcome. With a lentiviral-based knockdown approach, CD24 was found to be a functional liver T-IC marker that drives T-IC genesis through STAT3-mediated NANOG regulation. Our findings point to a CD24 cascade in liver T-ICs that may provide an attractive therapeutic target for HCC patients.

The tyrosine kinase inhibitor sorafenib sensitizes hepatocellular carcinoma cells to taxol by suppressing the HURP protein

The hepatoma upregulated protein (HURP) represents a putative oncogene that is overexpressed in many human cancers, especially hepatocellular carcinoma (HCC). HURP plays an important role during mitotic spindle formation, a process that is targeted by various anti-cancer drugs like taxol. However, the role of HURP during the establishment of taxol chemoresistance in HCC remains unclear. In this study, we observed that high HURP protein level correlates with taxol resistance in HCC cells. Following HURP knockdown, HCC cells show a more sensitive response to taxol treatment. Notably, sorafenib, a tyrosine kinase inhibitor approved for the treatment of HCC, inhibits HURP expression primarily at the transcriptional level and sensitizes HCC cells to sub-lethal doses of taxol. By using real-time PCR and chromatin immunoprecipitation assays, we observed that the NF-κB family member c-Rel represents a putative transcription factor that activates HURP gene expression. In addition, the inhibitory effect of sorafenib on HURP expression was attributed to a reduced translation and nuclear translocation of c-Rel. Accordingly, downregulation of c-Rel using short-hairpin RNA was shown to reduce HURP protein level and enhance taxol-induced cell death. Taken together, our results indicate that HURP acts as a novel survival protein that protects HCC cells against taxol-induced cell death. In addition, the regulation of HURP gene expression by NF-κB signaling appears to be critical for the response of HCC cells to taxol.

Abstract 4414: Clinical significance of CHD1L in hepatocellular carcinoma and therapeutic potentials of virus-mediated CHD1L depletion

Abstract Background: Hepatocellular carcinoma (HCC) is among the most lethal of human malignancies. It is difficult to detect early, has a high recurrence rate and is refractory to chemotherapies. Amplification of 1q21 is one of the most frequent genetic alterations in HCC. CHD1L is a newly identified oncogene responsible for 1q21 amplicon. This study aims to investigate the role of CHD1L in predicting prognosis and chemotherapy response of HCC patients, its chemo-resistant mechanism and whether virus-mediated CHD1L silencing has therapeutic potentials for HCC treatment. Methods: We assessed the clinical significance of CHD1L in a cohort of 109 HCC cases including 50 cases who received transarterial chemoembolization (TACE) treatment by clinical correlation and Kaplan-Meier analyses. CHD1L-overexpressing cell model was generated and the mechanism of CHD1L-involved chemo-resistance was investigated. Adenovirus-mediated silencing method was used to knockdown CHD1L and its effects on tumorigenicity as well as chemo-resistance were investigated in vivo and in vitro. Results: Overexpression of CHD1L was significantly associated with tumor microsatellite formation (p=0.045), advanced tumor stage (p=0.018), overall survival (OS) time (p=0.002), OS time of patients who received TACE treatment (p=0.028) and chemo-resistance (p=0.020) in HCC. Interestingly, CHD1L could inhibit apoptosis induced by 5-fluorourail (5-FU) but not doxorubicin (DOX). The mechanistic study revealed that the involvement of Nur77-mediated pathway in chemotherapeutic agent-induced apoptosis can dictate if CHD1L could confer resistance to chemotherapy. Furthermore, adenoviral vector containing shRNAs against CHD1L (CHD1L-shRNAs) could suppress cell growth, clonogenicity and chemo-resistance to 5-FU. In vivo study found that CHD1L-shRNAs could inhibit xenograft tumor growth and increase the sensitivity of tumor cells to 5-FU in nude mice. Conclusions: Our study highlighted for the first time the prognostic value of CHD1L in HCC and the potential application of virus-mediated CHD1L silencing in HCC therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4414. doi:10.1158/1538-7445.AM2011-4414

Abstract 2445: CD24+ liver tumor-initiating cells drives self-renewal and tumor initiation through Stat3-mediated Nanog regulation

Abstract Tumor-initiating cells (TICs) are a subpopulation of chemoresistant tumor cells that have been shown clinically to cause tumor recurrence upon chemotherapy. Identification of T-ICs and their related pathways are therefore priorities for the development of novel therapeutic paradigms. We established chemoresistant hepatocellular carcinoma (HCC) xenograft tumors in immunocompromised mice in which an enriched T-IC population was capable of tumor initiation and self-renewal. Using this model, we found CD24 to be up-regulated in residual chemoresistant tumor tissue when compared with bulk tumor upon cisplatin treatment. The determine whether CD24 is a candidate TIC marker for HCC, cell sorting approach was employed to separate CD24-/CD24+ populations derived from HCC cell lines and clinical samples. CD24+ HCC cells were found to be critical for the maintenance of tumor growth, self-renewal, differentiation, chemoresistance and metastasis of tumors. Using quantitative PCR, CD24 was over-expressed in HCCs when compared with their non-tumor counterparts, and CD24 expression was significantly correlated with poor patients’ survival. Using a lentiviral-based knockdown approach, CD24 was found to be a functional liver T-IC marker that drives tumor initiation and self-renewal. Notably, whether by cell sorting or gene-knockdown approach, it was shown by quantitative PCR that the CD24-enriched population consistently overexpressed Nanog. Upon transfection of Nanog cDNA into CD24-knockdown cells, self-renewal and tumor formation were functionally recovered, suggesting Nanog as the downstream effecter of CD24. By Ingenuity Pathway analysis, ‘acute phase response signaling’ was found to be the most significantly altered upon CD24 knockdown in which phosphorylation of Stat3 at the Tyrosine705 residue is critical. Using Stat3 inhibitor (S3I-201), we found that Nanog promoter and its protein were down-regulated in dose-dependent manner in CD24+ HCC cells. CD24 was found to be a functional liver T-IC marker that drives T-IC genesis through Stat3-mediated Nanog regulation. In conclusion, we identified CD24+ HCC cells within bulky tumor, and they functioned to initiate tumor growth and self-renewal through Stat3-mediated Nanog up-regulation. The identification of novel CD24 signaling pathways provides an attractive therapeutic strategy against this deadly disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2445. doi:10.1158/1538-7445.AM2011-2445