Sorafenib-resistant Hepatocellular Carcinoma Cells Research Articles

SLC27A5 promotes sorafenib-induced ferroptosis in hepatocellular carcinoma by downregulating glutathione reductase

Sorafenib, a first-line drug for advanced hepatocellular carcinoma (HCC), shows a favorable anti-tumor effect while resistance is a barrier impeding patients from benefiting from it. Thus, more efforts are needed to lift this restriction. Herein, we first find that solute carrier family 27 member 5 (SLC27A5/FATP5), an enzyme involved in the metabolism of fatty acid and bile acid, is downregulated in sorafenib-resistant HCC. SLC27A5 deficiency facilitates the resistance towards sorafenib in HCC cells, which is mediated by suppressing ferroptosis. Further mechanism studies reveal that the loss of SLC27A5 enhances the glutathione reductase (GSR) expression in a nuclear factor erythroid 2-related factor 2 (NRF2)-dependent manner, which maintains glutathione (GSH) homeostasis and renders insensitive to sorafenib-induced ferroptosis. Notably, SLC27A5 negatively correlates with GSR, and genetic or pharmacological inhibition of GSR strengthens the efficacy of sorafenib through GSH depletion and the accumulation of lipid peroxide products in SLC27A5-knockout and sorafenib-resistant HCC cells. Based on our results, the combination of sorafenib and carmustine (BCNU), a selective inhibitor of GSR, remarkably hamper tumor growth by enhancing ferroptotic cell death in vivo. In conclusion, we describe that SLC27A5 serves as a suppressor in sorafenib resistance and promotes sorafenib-triggered ferroptosis via restraining the NRF2/GSR pathway in HCC, providing a potential therapeutic strategy for overcoming sorafenib resistance.

Targeting fatty acid synthase modulates sensitivity of hepatocellular carcinoma to sorafenib via ferroptosis

BackgroundSorafenib resistance is a key impediment to successful treatment of patients with advanced hepatocellular carcinoma (HCC) and recent studies have reported reversal of drug resistance by targeting ferroptosis. The present study aimed to explore the association of fatty acid synthase (FASN) with sorafenib resistance via regulation of ferroptosis and provide a novel treatment strategy to overcome the sorafenib resistance of HCC patients.MethodsIntracellular levels of lipid peroxides, glutathione, malondialdehyde, and Fe2+ were measured as indicators of ferroptosis status. Biological information analyses, immunofluorescence assays, western blot assays, and co-immunoprecipitation analyses were conducted to elucidate the functions of FASN in HCC. Both in vitro and in vivo studies were conducted to examine the antitumor effects of the combination of orlistat and sorafenib and CalcuSyn software was used to calculate the combination index.ResultsSolute carrier family 7 member 11 (SLC7A11) was found to play an important role in mediating sorafenib resistance. The up-regulation of FASN antagonize of SLC7A11-mediated ferroptosis and thereby promoted sorafenib resistance. Mechanistically, FASN enhanced sorafenib-induced ferroptosis resistance by binding to hypoxia-inducible factor 1-alpha (HIF1α), promoting HIF1α nuclear translocation, inhibiting ubiquitination and proteasomal degradation of HIF1α, and subsequently enhancing transcription of SLC7A11. Orlistat, an inhibitor of FASN, with sorafenib had significant synergistic antitumor effects and reversed sorafenib resistance both in vitro and in vivo.ConclusionTargeting the FASN/HIF1α/SLC7A11 pathway resensitized HCC cells to sorafenib. The combination of orlistat and sorafenib had superior synergistic antitumor effects in sorafenib-resistant HCC cells.

CDCBLD2 mediates sorafenib resistance in hepatocellular carcinoma by sponging miR-345-5p binding to the TOP2A coding sequence.

Sorafenib is a first-line chemotherapy drug for treating advanced hepatocellular carcinoma (HCC). However, its therapeutic effect has been seriously affected by the emergence of sorafenib resistance in HCC patients. The underlying mechanism of sorafenib resistance is unclear. Here, we report a circular RNA, cDCBLD2, which plays an important role in sorafenib resistance in HCC. We found that cDCBLD2 was upregulated in sorafenib-resistant (SR) HCC cells, and knocking down cDCBLD2 expression could significantly increase sorafenib-related cytotoxicity. Further evidence showed that cDCBLD2 can bind to microRNA (miR)-345-5p through a competing endogenous RNA mechanism, increase type IIA topoisomerase (TOP2A) mRNA stability through a miRNA sponge mechanism, and reduce the effects of sorafenib treatment on HCC by inhibiting apoptosis. Our findings also suggest that miR-345-5p can negatively regulate TOP2A levels by binding to the coding sequence region of its mRNA. Additionally, targeting cDCBLD2 by injecting a specific small interfering RNA (siRNA) could significantly overcome sorafenib resistance in a patient-derived xenograft (PDX) mouse model of HCC. Taken together, our study provides a proof-of-concept for a potential strategy to overcome sorafenib resistance in HCC patients by targeting cDCBLD2 or TOP2A.

HBx Modulates Drug Resistance of Sorafenib-Resistant Hepatocellular Carcinoma Cells.

Approximately 50% of hepatocellular carcinoma (HCC) arises due to the infection by hepatitis B virus X protein (HBx). Sorafenib, a unique targeted oral kinase inhibitor, is the therapeutic agent of choice for advanced HCC. The mechanism of HBx in drug resistance of sorafenib-resistant HCC cells was evaluated in this study. Employing a stepwise increase of the sorafenib content, Hep3B and HepG2 cells were iteratively induced to establish drug-resistant cell lines (Hep3B/R and HepG2/R). The survival rate of Hep3B, Hep3B/R, HepG2, and HepG2/R cells was estimated using the cell counting kit-8 (CCK-8) assay. The IC50 values of sorafenib were calculated, exploring its effects under varying concentrations. The HBx content was quantified via quantitative reverse transcription PCR (RT-qPCR) and Western Blot. HBx overexpression and interfering virus vectors were constructed and transfected into Hep3B/R and HepG2/R cells. Cell viability and metastasis were assessed by colony formation, wound healing, and transwell assays. E-cadherin, N-cadherin, Vimentin, Slug, and Snail content was evaluated via Western Blot. HBx content was significantly elevated in Hep3B/R and HepG2/R subgroups compared to Hep3B and HepG2 subgroups. The proliferation, clonogenicity, invasiveness, and migratory abilities of Hep3B/R and HepG2/R cells in the HBx subgroup were markedly enhanced; E-cadherin content was significantly reduced, whereas the content of N-cadherin, Vimentin, Slug, and Snail was significantly elevated in the HBx subgroup. Conversely, in the sh-HBx subgroup, the proliferation, clonogenicity, invasion, and migration of Hep3B/R and HepG2/R cells were significantly reduced, E-cadherin content was markedly increased, and N-cadherin, Vimentin, Slug, and Snail content was significantly reduced, compared to the sh-negative control (NC) subgroup. HBx knockout may affect the development of HCC by reducing the proliferation, invasion, and migration of Hep3B/R and HepG2/R cells through the inhibition of Epithelial-Mesenchymal Transition (EMT).

DN200434, an orally available inverse agonist of estrogen-related receptor γ, induces ferroptosis in sorafenib-resistant hepatocellular carcinoma

Sorafenib, originally identified as an inhibitor of multiple oncogenic kinases, induces ferroptosis in hepatocellular carcinoma (HCC) cells. Several pathways that mitigate sorafenib-induced ferroptosis confer drug resistance; thus strategies that enhance ferroptosis increase sorafenib efficacy. Orphan nuclear receptor estrogen-related receptor γ (ERRγ) is upregulated in human HCC tissues and plays a role in cancer cell proliferation. The aim of this study was to determine whether inhibition of ERRγ with DN200434, an orally available inverse agonist, can overcome resistance to sorafenib through induction of ferroptosis. Sorafenib-resistant HCC cells were less sensitive to sorafenibinduced ferroptosis and showed significantly higher ERRγ levels than sorafenib-sensitive HCC cells. DN200434 induced lipid peroxidation and ferroptosis in sorafenib-resistant HCC cells. Mechanistically, DN200434 increased mitochondrial ROS generation by reducing glutathione/glutathione disulfide levels, which subsequently reduced mTOR activity and GPX4 levels. DN200434 induced amplification of the antitumor effects of sorafenib was confirmed in a tumor xenograft model. The present results indicate that DN200434 may be a novel therapeutic strategy to re-sensitize HCC cells to sorafenib. [BMB Reports 2022; 55(11): 547-552].

Transcriptional regulation of NDUFA4L2 by NFIB induces sorafenib resistance by decreasing reactive oxygen species inhepatocellular carcinoma.

Sorafenib is one a first-line therapeutic drugs for advanced hepatocellular carcinoma (HCC). However, only 30% of patients benefit from sorafenib due to drug resistance. We and other groups have revealed that nuclear factor I B (NFIB) regulates liver regeneration and carcinogenesis, but its role in drug resistance is poorly known. We found that NFIB was more upregulated in sorafenib-resistant SMMC-7721 cells compared to parental cells. NFIB knockdown not only sensitized drug-resistant cells to sorafenib but also inhibited the proliferation and invasion of these cells. Meanwhile, NFIB promoted the proliferation and invasion of HCC cells in vitro and facilitated tumor growth and metastasis in vivo. Knocking down NFIB synergetically inhibited tumor growth with sorafenib. Mechanically, gene expression profiling and subsequent verification experiments proved that NFIB could bind with the promoter region of a complex I inhibitor NDUFA4L2 and promote its transcription. Transcriptional upregulation of NDUFA4L2 by NFIB could thus inhibit the sorafenib-induced reactive oxygen species accumulation. Finally, we found that NFIB was highly expressed in HCC tissues, and high NFIB expression level was associated with macrovascular invasion, advanced tumor stage, and poor prognosis of HCC patients (n=156). In summary, we demonstrated that NFIB could transcriptionally upregulate NDUFA4L2 to enhance both intrinsic and acquired sorafenib resistance of HCC cells by reducing reactive oxygen species induction.

Circular RNA ITCH increases sorafenib-sensitivity in hepatocellular carcinoma via sequestering miR-20b-5p and modulating the downstream PTEN-PI3K/Akt pathway

BackgroundsSorafenib-resistance leads to poor prognosis and high mortality in advanced hepatocellular carcinoma (HCC), and this study aims to investigate the functional role of a circular RNA ITCH (circITCH) in regulating the sorafenib-resistance of HCC and its underlying mechanisms. MethodsThe expression of circITCH in HCC tissues and cell lines were detected by performing quantitative real-time polymerase chain reaction. Sorafenib-resistant HCC cells were transfected with PLCDH-circITCH to upregulate circITCH and intervened with sorafenib, and MTT assay, flow cytometry and transwell assay were used to test the cell viability, apoptosis and migration ability, respectively. The downstream target of circITCH were explored by using bioinformatic analysis, dual luciferase reporter system and Western blot. ResultsCircITCH was significantly down-regulated in HCC tissues and cell lines, compared with their normal counterparts. Especially, in contrast with the sorafenib-sensitive HCC cells, continuous sorafenib treatment decreased the expression levels of circITCH in the sorafenib-resistant HCC cells. Overexpression of circITCH increased sorafenib-sensitivity, promoted cell apoptosis and reduced cell migration abilities in the sorafenib-resistant HCC cells. Mechanically, circITCH elevated PTEN expression to inactivate the PI3K/Akt signals through negatively regulating miR-20b-5p in HCC, and upregulating miR-20b-5p or inhibiting PTEN abolished the enhancing effect of circITCH overexpression on sorafenib-induced cytotoxicity in sorafenib-resistant HCC cells. ConclusionTaken together, this study proves that circITCH enhances sorafenib-sensitivity in sorafenib-resistant HCC cells via regulating the miR-20b-5p/PTEN/PI3K/Akt signaling cascade, which highlights the potential value of circITCH as a target for enhancing the sorafenib-sensitivity in HCC.

KIAA1429 mediates epithelial mesenchymal transition in sorafenib-resistant hepatocellular carcinoma through m6A methylation modification.

Hepatocellular carcinoma (HCC) is a primary liver cancer with high mortality. The long-term use of sorafenib, a targeted drug for hepatocellular carcinoma, will lead to drug resistance, and patients are prone to cancer metastasis, the molecular mechanism of which is still unclear. In our study, we constructed a sorafenib-resistant hepatocellular carcinoma cell line (HepG2/Sora) and validated the resistance in vivo and in vitro. Transwell assays confirmed the invasion and migration abilities of cells. Colorimetric assays confirmed that the level of m6A methylation modification in cells. RT-qPCR and Western blot assays confirmed that the expression levels of KIAA1429 in HepG2/Sora cells and tissues. The EMT related proteins were detected by Western blot assay. Transwell and Western blot assays confirmed that HepG2/Sora cells had higher invasion and migration abilities and showed EMT phenomena. Colorimetric assays confirmed that the level of m6A methylation modification was upregulated in HepG2/Sora cells. Transwell and Western blot assays confirmed that inhibiting m6A methylation in HepG2/Sora cells inhibited their invasion, migration ability and EMT phenomenon. RT-qPCR and Western blot assays confirmed that the expression levels of KIAA1429 in HepG2/Sora cells and tissues was increased. Silencing KIAA1429 in HepG2/Sora cells inhibited their invasion, migration ability and EMT phenomenon. Finally, we found that the medium supernatant of sorafenib-resistant HepG2/Sora cells induced vascular production of EA.hy926 cells, and silencing KIAA1429 inhibited this induction effect. We suggest that KIAA1429 promotes sorafenib-resistant hepatocellular carcinoma invasion, migration and EMT by mediating m6A methylation. KIAA1429 with its mediated m6A methylation may be a key factor for sorafenib-resistant patients prone to cancer cell metastasis.

Fucoidan inhibits EGFR redistribution and potentiates sorafenib to overcome sorafenib-resistant hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related deaths worldwide. Although sorafenib is a standard first-line molecule-targeted drug against advanced HCC, the drug resistance development and adverse side effects usually limit its efficacy. This study investigated the effect of fucoidan on the sorafenib sensitivity of sorafenib-resistant human HCC cell line HepG2-SR established by long-time exposure of HepG2 to sorafenib. We demonstrated fucoidan combined with sorafenib synergistically promoted apoptosis and cell cycle arrest whereas inhibited cell migration in HepG2-SR cells. This combination treatment effectively suppressed the cellular epithelial growth factor receptor (EGFR) nuclear distribution and downstream gene transcription. Interestingly, fucoidan bound the cell surface EGFR, dampening EGFR translocation to lipid raft and further nuclear distribution, restoring the sorafenib sensitivity in HepG2-SR cells. Blocking fucoidan-EGFR interaction using EGFR antibody restrained the enhanced anti-tumor effects upon the combined administration. Besides, EGFR knockdown abolished the combination treatment-improved anti-tumor efficacy. This combination also suppressed in vivo xenograft tumor growth in nude mice. Our present study uncovered that fucoidan overcame sorafenib resistance in HCC via its interaction with cell membrane EGFR and further suppression of EGFR redistribution and downstream signaling in sorafenib-resistant cells. Overall, current results suggest that simultaneous treatment of fucoidan and sorafenib might serve as a potential therapeutic strategy against sorafenib-resistant HCC.

Nalidixic acid potentiates the antitumor activity in sorafenib-resistant hepatocellular carcinoma via the tumor immune microenvironment analysis.

Sorafenib resistance is often developed and impedes the benefits of clinical therapy in hepatocellular carcinoma (HCC) patients. However, the relationship between sorafenib resistance and tumor immune environment and adjuvant drugs for sorafenib-resistant HCC are not systemically identified. This study first analyzed the expression profiles of sorafenib-resistant HCC cells to explore immune cell infiltration levels and differentially expressed immune-related genes (DEIRGs). The prognostic value of DEIRGs was analyzed using Cox regression and Kaplan–Meier analysis based on The Cancer Genome Atlas. The primary immune cells infiltrated in sorafenib-resistant HCC mice were explored using flow cytometry (FCM). Finally, small-molecule drugs for sorafenib-resistant HCC treatment were screened and validated by experiments. The CIBERSORT algorithm and mice model showed that macrophages and neutrophils are highly infiltrated, while CD8+ T cells are downregulated in sorafenib-resistant HCC. Totally, 34 DEIRGs were obtained from sorafenib-resistant and control groups, which were highly enriched in immune-associated biological processes and pathways. NR6A1, CXCL5, C3, and TGFB1 were further identified as prognostic markers for HCC patients. Finally, nalidixic acid was identified as a promising antagonist for sorafenib-resistant HCC treatment. Collectively, our study reveals the tumor immune microenvironment changes and explores a promising adjuvant drug to overcome sorafenib resistance in HCC.

FXYD5 promotes sorafenib resistance through the Akt/mTOR signaling pathway in hepatocellular carcinoma

Tumor chemoresistance is often a major cause for the failure of chemotherapy. The resistance of hepatocellular carcinoma (HCC) cells to sorafenib significantly limits its therapeutic effect in HCC patients. For the first time, we found that FXYD domain-containing ion transport regulator 5 (FXYD5) is highly expressed in sorafenib-resistant HCC cells. In addition, the protein expression level of FXYD5 was markedly higher in HCC tissues than in paracancerous tissues. Remarkably, downregulation of FXYD5 expression in Huh7/sora cells reversed their resistance to sorafenib. Moreover, overexpression of FXYD5 reduced the sensitivity of HCC cells to sorafenib, while the downregulation of its expression in HCC cells had the opposite effect. We also found abnormal activation of the Akt/mTOR signaling pathway in Huh7/sora cells. Furthermore, MK2206, an Akt inhibitor, was found to significantly increase the sensitivity of HCC cells to sorafenib. More importantly, the expression level of p-Akt was positively correlated with the expression of FXYD5 in HCC tissues. Therefore, mechanistically, FXYD5 enhances the resistance of HCC cells to sorafenib by activating the Akt/mTOR signaling pathway. In conclusion, this study showed that the activation of the FXYD5/Akt/mTOR signaling axis plays key role in the resistance of HCC cells to sorafenib, and FXYD5 may represent a new potential target for HCC therapy.

KDM5B regulates the PTEN/PI3K/Akt pathway to increase sorafenib-resistance in hepatocellular carcinoma.

Lysine-specific demethylase 5B (KDM5B) exerts its tumor-promoting functions in numerous malignancies, although the possible mechanisms by which KDM5B promotes cancer aggressiveness in hepatocellular carcinoma (HCC) have been preliminarily explored, the role of this gene in regulating sorafenib-resistance in HCC has not been studied. Thus, the present study was designed to resolve this problem, and our data suggested that KDM5B was significantly upregulated in the HCC tissues collected from patients with sorafenib treatment history. Consistently, continuous low-dose sorafenib administration increased KDM5B expression levels in the sorafenib-resistant HCC cells compared to their sorafenib-sensitive counterparts. Next, by performing the functional experiments, we found that KDM5B positively regulated sorafenib-resistance and cancer stem cell (CSC) properties in HCC cells in vitro and in vivo . Furthermore, upregulation of KDM5B-degraded phosphatase and tensin homolog (PTEN), results in the activation of the downstream oncogenic PI3K/Akt pathway. Subsequently, the rescuing experiments verified that the promoting effects of KDM5B overexpression on chemoresistance and cancer stemness in HCC cells were all abrogated by PI3K (p110) knockdown and PTEN overexpression. Collectively, those data hinted that KDM5B influenced CSC properties and sorafenib-resistance in HCC cells through modulating the PTEN/PI3K/Akt pathway, and KDM5B could be used as a potential target for the treatment of HCC in clinic.

S-palmitoylation of PCSK9 induces sorafenib resistance in liver cancer by activating the PI3K/AKT pathway.

Sorafenib is currently the first-line treatment for advanced hepatocellular carcinoma (HCC). However, sorafenib resistance remains a significant challenge. Aberrant AKT signaling activation is a crucial mechanism driving sorafenib resistance in HCC. Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a vital role in antitumor immune responses. In this study, we demonstrate that aberrant PCSK9 upregulation promotes cell proliferation and sorafenib resistance in HCC by inducing AKT-S473 phosphorylation. After palmitoylation at cysteine 600, the binding affinity between PCSK9 and tensin homolog (PTEN) is dramatically increased, inducing lysosome-mediated PTEN degradation and subsequent AKT activation. We identify zinc finger DHHC-type palmitoyltransferase 16 (ZDHHC16) as a palmitoyltransferase that promotes PCSK9 palmitoylation at cysteine 600. We also develop a biologically active PCSK9-derived peptide that competitively inhibits PCSK9 palmitoylation, suppressing AKT phosphorylation and augmenting the antitumor effects of sorafenib in HCC.

CircFMN2 Boosts Sorafenib Resistance in Hepatocellular Carcinoma Cells via Upregulating CNBP by Restraining Ubiquitination.

Purpose Noncoding RNAs exert critical biological effects in hepatocellular carcinoma (HCC). The role of circFMN2, a newly discovered functional RNA in prostate cancer and colorectal cancer, was investigated for the first time in sorafenib-resistance HCC cells. Methods The level of circFMN2 was assessed via quantitative real-time PCR (qRT-PCR). Cell proliferation was detected via CCK-8 and colony formation assay. Cell apoptosis was measured via the TUNEL assay and flow cytometry analysis. A Western blot assay was conducted to detect the CCHC-type zinc finger nucleic acid binding protein (CNBP) level and ubiquitination. RNA pull-down assay and RNA immunoprecipitation were carried out to explore the interaction between circFMN2 and CNBP. Results CircFMN2 was highly expressed in multidrug-resistant (MDR) cells. CircFMN2 overexpression exerted pro-proliferation effects in sorafenib-treated HCC cells, while depletion of circFMN2 displayed negative effect on sorafenib-treated MDR cells. Moreover, CNBP was verified as the binding protein of circFMN2. CNBP was upregulated in MDR cells, which was achieved by inhibition of ubiquitination by circFMN2. Besides, CNBP overexpression was found to boost sorafenib resistance in HCC cells. Conclusions CircFMN2 is aberrantly expressed in sorafenib-resistant HCC cells and contributes to sorafenib resistance in HCC cells via upregulation of CNBP by restraining ubiquitination.

Ets1 mediates sorafenib resistance by regulating mitochondrial ROS pathway in hepatocellular carcinoma

The incidence and mortality of hepatocellular carcinoma (HCC) are on a rise in the Western countries including US, attributed mostly to late detection. Sorafenib has been the first-line FDA-approved drug for advanced unresectable HCC for almost a decade, but with limited efficacy due to the development of resistance. More recently, several other multi-kinase inhibitors (lenvatinib, cabozantinib, regorafenib), human monoclonal antibody (ramucirumab), and immune checkpoint inhibitors (nivolumab, pembrolizumab) have been approved as systemic therapies. Despite this, the median survival of patients is not significantly increased. Understanding of the molecular mechanism(s) that govern HCC resistance is critically needed to increase efficacy of current drugs and to develop more efficacious ones in the future. Our studies with sorafenib-resistant (soraR) HCC cells using transcription factor RT2 Profiler PCR Arrays revealed an increase in E26 transformation–specific-1 (Ets-1) transcription factor in all soraR cells. HCC TMA studies showed an increase in Ets-1 expression in advanced HCC compared to the normal livers. Overexpression or knocking down Ets-1 modulated sorafenib resistance-related epithelial–mesenchymal transition (EMT), migration, and cell survival. In addition, the soraR cells showed a significant reduction of mitochondrial damage and mitochondrial reactive oxygen species (mROS) generation, which were antagonized by knocking down Ets-1 expression. More in-depth analysis identified GPX-2 as a downstream mediator of Ets-1-induced sorafenib resistance, which was down-regulated by Ets-1 knockdown while other antioxidant pathway genes were not affected. Interestingly, knocking down GPX2 expression significantly increased sorafenib sensitivity in the soraR cells. Our studies indicate the activation of a novel Ets-1–GPX2 signaling axis in soraR cells, targeting which might successfully antagonize resistance and increase efficacy.

HMOX1 Attenuates the Sensitivity of Hepatocellular Carcinoma Cells to Sorafenib via Modulating the Expression of ABC Transporters.

Hepatocellular carcinoma (HCC) represents a common malignancy, and mechanisms of acquired sorafenib resistance during the treatment of HCC patients remain elusive. The present study performed integrated bioinformatics analysis and explored the potential action of heme oxygenase 1 (HMOX1) in sorafenib-resistant HCC cells. Differentially expressed genes (DEGs) of the sorafenib-resistant group as compared to the sorafenib-sensitive group from GSE140202 and GSE143233 were extracted. Fifty common DEGs between GSE140202 and GSE143233 were extracted. Ten hub genes were identified from the protein-protein interaction network based on common DEGs. Experimental results revealed the upregulation of HMOX1 in sorafenib-resistant HCC cells. HMOX1 silence promoted the sensitivity to sorafenib in sorafenib-resistant HCC cells; overexpression of HMOX1 attenuated the sensitivity. In addition, HMOX1 silence downregulated the mRNA expression of ABC transporters in sorafenib-resistant HCC cells, while HMOX1 overexpression upregulated mRNA expression of ABC transporter expression in HCC cells. Further analysis also revealed that high expression of HMOX1 was associated with shorter OS and DSS in HCC patients. In conclusion, our analysis identified ten hub genes associated with sorafenib resistance in HCC. Further validation studies demonstrated that HMOX1 promoted sorafenib resistance of HCC cells via modulating ABC transporter expression.

MiR-15a and miR-20b sensitize hepatocellular carcinoma cells to sorafenib through repressing CDC37L1 and consequent PPIA downregulation

Sorafenib is a classical targeted drug for the treatment of advanced hepatocellular carcinoma (HCC), but intrinsic resistance severely limited its therapeutic effects. In the present study, we aimed to identify crucial genes in HCC cells that affect sorafenib resistance by a CRISPR/Cas9 genome-scale screening. The results indicated that the deficiency of miR-15a and miR-20b contributed to sorafenib resistance, whereas exogenous expression of miR-15a and miR-20b enhanced sorafenib sensitivity of HCC cells by cell viability, colony formation, and flow cytometry analyses. Further analyses revealed that cell division cycle 37 like 1 (CDC37L1) as a common target of miR-15a and 20b, was negatively regulated by the two miRNAs and could enhance sorafenib resistance of HCC cells in vitro and in vivo. Mechanistically, CDC37L1, as a cochaperone, effectively increased the expression of peptidylprolyl isomerase A (PPIA) through strengthening the binding between heat shock protein 90 (HSP90) and PPIA. The results from immunohistochemical staining of a HCC tissue microarray revealed a positive association between CDC37L1 and PPIA expression, and high expression of CDC37L1 and PPIA predicted worse prognosis of HCC patients after sorafenib therapy. Taken together, our findings reveal crucial roles of miR-15a, miR-20b, CDC37L1, and PPIA in sorafenib response of HCC cells. These factors may serve as therapeutic targets and predict prognosis for HCC treated with sorafenib.

DJ-1/FGFR-1 Signaling Pathway Contributes to Sorafenib Resistance in Hepatocellular Carcinoma.

Sorafenib is the first-line therapeutic regimen targeting against advanced or metastatic stage of hepatocellular carcinoma (HCC). However, HCC patients at these stages will eventually fail sorafenib treatment due to the drug resistance. At present, molecular mechanisms underlying sorafenib resistance are not completely understood. Our past studies have shown that DJ-1 is upregulated in HCC, while DJ-1 knockdown inhibits HCC xenograft-induced tumor growth and regeneration, implying that DJ-1 may be a potential target in for HCC treatment. However, whether DJ-1 plays a regulatory role between tumor cells and vascular endothelial cells and whether DJ-1 contributes to sorafenib resistance in HCC cells are largely unclear. To address these questions, we have performed a series of experiments in the current study, and we found that (1) DJ-1, one of the molecules secreted from HCC cells, promoted angiogenesis and migration of vascular endothelial cells (i.e., ECDHCC-1), by inducing phosphorylation of fibroblast growth factor receptor-1 (FGFR-1), phosphorylation of mTOR, phosphorylation of ERK, and phosphorylation of STAT3; (2) downregulation of FGFR1 inhibited tube formation and migration of ECDHCC-1 cells stimulated by DJ-1; (3) FGFR1 knockdown attenuated the phosphorylation of FGFR1 and impaired the activity of Akt, ERK, and STAT3 signals induced by DJ-1 in ECDHCC-1 cells; (4) knocking down FGFR1 led to the elevated expression of proapoptotic molecules but deceased level of antiapoptotic molecules in sorafenib-resistant HCC cells; and (5) Downregulation of FGFR1 suppressed tumor growth and angiogenesis of sorafenib-resistant HCC cells in vivo. Altogether, our results hinted that DJ-1 plays vital roles in tumor microenvironment in HCC development, and DJ-1/FGFR1 signaling pathway may be a therapeutic target for overcoming sorafenib resistance in treating HCC patients at the late stage.

Abstract 5347: miR-15a & miR-20b enhance sorafenib sensitivity of hepatocellular carcinoma through repressing CDC37L1 and consequent PPIA downregulation

Abstract Sorafenib is a classical targeted drug for the treatment of advanced hepatocellular carcinoma (HCC), but intrinsic resistance severely limits its therapeutic effects. In the present study, we aimed to identify crucial genes in HCC cells that affect sorafenib resistance by a CRISPR/Cas9 genome-scale screening. Among the candidates, two miRNAs, miR-15a and miR-20b, were finally identified as the essential sorafenib-sensitizing genes of HCC cells. In parallel, Cell Counting Kit-8 (CCK-8) assay,colony formation assays and flow cytometric analysis were carried out to assess cell proliferation and survival, whereas tumor growth in vivo was assessed using xenograft models. The results indicated that the deficiency of miR-15a and miR-20b contributed to sorafenib resistance of HCC cells. By combination of various bioinformatics tools, Cell division cycle 37 like 1 (CDC37L1) was identified as a common target gene of miR-15a and miR-20b. Further cellular and molecular biology experiments demonstrated CDC37L1 was negatively regulated by the two miRNAs, and CDC37L1 could enhance sorafenib resistance of HCC in vitro and in vivo. Mechanistically, we carried out SDS-PAGE and silver staining assays followed by mass spectrometry to analyze the immunoprecipitated proteins of CDC37L1. Our findings indicated that CDC37L1, as a cochaperone, effectively increased the expression of PPIA through strengthen the binding between HSP90 and PPIA. Additionally, CDC37L1 and PPIA expression were examined in a tissue microarray containing 80 pairs of HCC and normal tissues derived from patients who received sorafenib treatment via immunohistochemistry (IHC) staining, the results revealed a positive association between CDC37L1 and PPIA expression, and high expression of CDC37L1 and PPIA predicted worse prognosis of HCC patients after sorafenib therapy. Taken together, our findings suggest that both miR-15a and miR-20b play sorafenib-sensitizing roles in HCC by facilitating the complex of CDC37L1/HSP90/PPIA. Citation Format: Li Li, Shijun Yu, Jingde Chen, Ming Quan, Yong Gao, Yandong Li. miR-15a & miR-20b enhance sorafenib sensitivity of hepatocellular carcinoma through repressing CDC37L1 and consequent PPIA downregulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5347.

Orlistat Resensitizes Sorafenib-Resistance in Hepatocellular Carcinoma Cells through Modulating Metabolism.

Sorafenib is one of the options for advanced hepatocellular carcinoma treatment and has been shown to extend median overall survival. However, sorafenib resistance often develops a few months after treatment. Hence, developing various strategies to overcome sorafenib resistance and understand the possible mechanisms is urgently needed. We first established sorafenib-resistant hepatocellular carcinoma (HCC) cells. Then, we found that sorafenib-resistant Huh7 cells (Huh7/SR) exhibit higher glucose uptakes and express elevated fatty acid synthesis and glucose metabolism-related proteins than their parental counterparts (Huh7). The current study investigated whether sorafenib resistance could be reversed by suppressing fatty acid synthesis, using a fatty acid synthase (FASN) inhibitor, orlistat, in HCC cells. FASN inhibition-caused changes in protein expressions and cell cycle distribution were analyzed by Western blot and flow cytometry, and changes in glucose uptakes were also evaluated by 18F-FDG uptake. Orlistat remarkably enhanced the cytotoxicity of sorafenib in both Huh7 and Huh7/SR cells, and flow cytometry showed that combination treatment significantly increased the sub-G1 population in both cell lines. Western blot revealed that the combination treatment effectively increased the ratio of Bax/Bcl-2 and decreased expressions of pERK; additionally, the combination treatment also strongly suppressed fatty acid synthesis-related proteins (e.g., FASN and SCD) in both cell lines. Lastly, the 18F-FDG uptake was repressed by the combination treatment in both cell lines. Our results indicated that orlistat-mediated FASN inhibition could overcome sorafenib resistance and enhance cell killing in HCC by changing cell metabolism.