Hepatocellular Carcinoma Xenograft Tumors Research Articles

Circ_0084615 promotes epithelial‐mesenchymal transition‐mediated tumor progression in hepatocellular carcinoma

AbstractAimCircRNAs have been identified as crucial regulators in tumorigenesis and progression. This study aimed to explore the biological role and underlying mechanism of circ_0084615 in hepatocellular carcinoma (HCC).MethodsThe expression of RNAs was detected by quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR). The effects of circ_0084615 silencing on malignant behaviors of HCC cells were assessed by CCK‐8, colony formation, wound healing, and Transwell assays in vitro and tumor transplantation experiment in vivo. The expression of proteins was detected by Western blotting. Dual‐luciferase reporter assay and RNA‐binding protein immunoprecipitation were performed to explore the mechanism of circ_0084615.ResultsA significant upregulation of circ_0084615 was observed in HCC tissues, and positively correlated with the TNM staging. Silencing of circ_0084615 impeded HCC cell viability, colony formation, migration, invasion, epithelial‐mesenchymal transition, and xenograft tumor growth. Mechanistically, circ_0084615 could bind to miR‐1200 and eliminate its ability to destroy actin‐like 6A (ACTL6A) mRNA, thereby increasing ACTL6A expression and facilitating the malignant behaviors of HCC cells.ConclusionsThis study clarified the oncogenic activity and mechanism of circ_0084615, thereby providing potential diagnostic biomarker and therapeutic target for inhibiting HCC progression.

CircSNX6 promotes proliferation, metastasis, and angiogenesis in hepatocellular carcinoma via miR-383-5p/VEGFA signaling pathway

The role of circular RNA (circRNAs) in hepatocellular carcinoma (HCC) has been extensively studied. Previous research has highlighted the regulatory role of circSNX6 in HCC cells and tissues. However, the precise mechanism underlying HCC progression still requires comprehensive investigation. The study initially utilized quantitative reverse transcription-polymerase chain reaction (qRT-PCR) to assess circSNX6 expression levels in HCC cell lines and tissues. Subsequently, the stability of circRNA was evaluated through Ribonuclease R and actinomycin D treatment assays. The impact of circSNX6 knockdown on proliferation, migration, invasion, and angiogenesis abilities was determined using various assays including colony formation, Transwell culture system, tube formation assay, and cell counting kit (CCK)-8 assays. Additionally, RNA immunoprecipitation chip and dual-luciferase reporter assays were employed to investigate the interactions between circSNX6 and miR-383-5p. Finally, an HCC xenograft tumor model in mice was established to assess the in vivo expression of circSNX6 and its functional role in HCC. Our findings revealed an elevated circSNX6 expression in HCC tissues, which was correlated with poor patient prognosis. Knockdown of circSNX6 suppressed HCC cell growth, invasion, metastasis, and angiogenesis. The downregulation of miR-383-5p, a target of circSNX6, significantly attenuated the tumor-suppressive effects induced by circSNX6 knockdown. Moreover, circSNX6 was found to modulate VEGFA expression by targeting miR-383-5p. The inhibition of HCC cell proliferation by miR-383-5p could be partially reversed by overexpressing VEGFA. Silencing circSNX6 also suppressed tumor formation and the metastasis of HCC cells in a mouse model. In summary, our findings suggest that circSNX6 promotes cell proliferation, metastasis, and angiogenesis in HCC by regulating the miR-383-5p/VEGFA pathway.

Abstract 3172: Significance of PELP1 signaling in liver cancer progression

Abstract Background: Hepatocellular carcinoma (HCC) is the fastest-rising cause of cancer-related mortality in the United States. Furthermore, advanced HCC has a very poor prognosis, with a median survival time of only about ten months. Effective new targeted therapies are urgently needed for the treatment of HCC. Proline, glutamic acid-, and leucine-rich protein 1 (PELP1) is a protooncogene. PELP1 signaling has been implicated in the development of several cancers, however, its role in the progression of HCC remains unclear. The goal of this project is to investigate the significance and therapeutic potential of targeting PELP1 in HCC. Methods: In this investigation, we have used six HCC cell lines (HUH7, HEP3B, SNU398, SNU475, SNU423, and SNU449). Using immunohistochemistry (IHC), the expression of PELP1 in HCC tissue microarrays was examined. PELP1shRNA lentiviral particles were used to create PELP1 knockdown (KD) cells. Using well-established in vitro tests for cell proliferation, colony formation, and apoptosis, the effects of PELP1 KD or PELP1 inhibitor (SMIP34) were investigated. RNA-seq, RT-qPCR, Western blotting, IHC, reporter gene assays, and signaling pathway analysis were used in mechanistic investigations. Preclinical evaluations were conducted using mice xenograft models. Results: TNM plot analysis showed that HCC samples had increased PELP1 expression relative to normal tissue. Immunohistochemistry analysis of tissue microarray confirmed that HCC specimens overexpressed PELP1 in comparison to normal specimens. PELP1 knockdown with shRNA dramatically decreased HCC cell invasion, clonogenicity, and proliferation. Similarly, PELP1 inhibitor SMIP34 treatment significantly reduced cell viability, invasiveness, colony forming capacity, and induced apoptosis of HCC cells. RNA-seq analyses using PELP1 KD cells confirmed that PELP1 modulates the ribosome and the eukaryotic translation elongation pathways. Mechanistic studies confirmed that SMIP34 treatment contributed to the disruption of the Rix complex, which is crucial for ribosomal biogenesis. Puromycin labelling studies confirmed that PELP1 KD or SMIP34 treatment contributes to the reduction of ribosomal biogenesis and new protein synthesis. Furthermore, PELP1 KD or treatment with SMIP34 dramatically slowed the progression of HCC tumors in xenograft models. IHC analysis revealed reduced levels of the proliferation marker Ki67 in PELP1 KD/SMIP34 treated HCC xenograft tumors compared to controls. Conclusions: Overall, the results of our study point to PELP1 as a potential therapeutic target for HCC intervention. Citation Format: Uday Pratap, Khaled Mohamed Nassar, Xue Yang, Adriana Baker, Rahul Gopalam, William C. Arnold, Timilehin Adeniran, Marian Helena Hernandez Fernandez, Gangadhara R. Sareddy, Suryavathi Viswanadhapalli, Luzhe Sun, Ratna K. Vadlamudi. Significance of PELP1 signaling in liver cancer progression [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 3172.

Ellagic acid inhibits tumor growth and potentiates the therapeutic efficacy of sorafenib in hepatocellular carcinoma

BackgroundSorafenib is a classic molecular targeted drug approved for hepatocellular carcinoma (HCC) therapy. However, a poor response rate and increasing resistance to sorafenib make its therapeutic efficacy suboptimal. Combination treatment with an agent capable of potentiating sorafenib sensitivity may be a promising solution. AimThe aim of this study was to determine the synergistic effect of ellagic acid (EA), a natural polyphenol, and sorafenib on HCC. MethodsCCK-8, EdU incorporation and colony formation assays were used to study the effect of EA on HCC cell proliferation. Apoptosis was detected by flow cytometry in HCC cells and TUNEL assay in xenograft tumors. Transcriptome analysis was utilized to investigate alterations in signaling pathways with EA treatment. A xenograft mouse model was used to confirm the synergistic effect of sorafenib and EA on HCC tumors in vivo. ResultsWe found that EA inhibited growth and induced apoptosis in both HCC cells and xenograft tumors. Mechanistically, EA treatment reduced the activation of the MAPK and Akt/mTOR signaling pathways in HCC cells. Furthermore, combined EA and sorafenib treatment further inhibited the MAPK and Akt/mTOR signaling pathways compared to EA or sorafenib alone. EA synergistically potentiated the anticancer activity of sorafenib against HCC both in vitro and in vivo. ConclusionEA inhibits HCC growth by inducing apoptosis through attenuation of the MAPK and Akt/mTOR signaling pathways. EA potentiates the response of HCC tumors to sorafenib both in vitro and in vivo, an effect that may be attributed to further inhibition of the MAPK and Akt/mTOR signaling pathways. These results suggest that EA is an effective adjuvant option for sorafenib therapy.

MiR-4270 suppresses hepatocellular carcinoma progression by inhibiting DNMT3A-mediated methylation of HGFAC promoter

Background miR-4270 is a regulatory factor has been linked with the progression of various cancers, such as nasopharyngeal carcinoma, hepatocellular carcinoma (HCC), and gastric cancer. However, the underlying mechanisms through which miR-4270 modulates HCC development are not fully understood. Methods miR-4270 expression levels were analyzed in various HCC cell lines and tissue samples. An online bioinformatics tool was then utilized to predict the miR-4270 target gene. The binding relationship between miR-4270 and its target gene DNMT3A was verified using dual-luciferase reporter and Ago2–RIP assays. Then, co-immunoprecipitation (Co-IP) and chromatin immunoprecipitation (ChIP) assays were conducted to investigate the association between DNMT3A and the hepatocyte growth factor activator (HGFAC) promoter region. To assess the methylation level of the HGFAC promoter, methylation-specific PCR (MSP) was employed. Furthermore, rescue analyses were carried out to evaluate the functional relevance of miR-4270 and HGFAC in the modulation of the malignant properties of HCC cells. Finally, HepG2 cells overexpressing miR-4270 were subcutaneously injected into nude mice to estimate the impact of miR-4270 on the xenograft tumor growth of HCC. Results A substantial miR-4270 downregulation was revealed in HCC patient samples and cell lines. miR-4270 upregulation suppressed both cell proliferation and invasion while promoting apoptosis. At the molecular level, miR-4270 was found to bind to the 3′untranslated region (3′UTR) of DNMT3A, thereby inhibiting DNMT3A-mediated methylation of the HGFAC promoter. Functional assays indicated that inhibition of miR-4270 stimulated HCC cell growth, an effect counteracted by overexpression of HGFAC. In vivo assays further verified that miR-4270 effectively suppressed the progression of HCC xenograft tumors. Conclusions miR-4270 was found to mitigate the malignant characteristics of HCC by inhibiting DNMT3A-mediated methylation of the HGFAC promoter, suggesting a potential therapeutic avenue for the management of HCC.

Targeting the TCA cycle through cuproptosis confers synthetic lethality on ARID1A-deficient hepatocellular carcinoma

ARID1A is among the most commonly mutated tumor suppressor genes in hepatocellular carcinoma (HCC). In this study, we conduct a CRISPR-Cas9 synthetic lethality screen using ARID1A-deficient HCC cells to identify approaches to treat HCC patients harboring ARID1A deficiency. This strategy reveals that the survival of these ARID1A-deficient HCC cells is highly dependent on genes related to the tricarboxylic acid (TCA) cycle. Mechanistically, ARID1A loss represses expression of key glycolysis-related gene PKM, shifting cellular glucose metabolism from aerobic glycolysis to dependence on the TCA cycle and oxidative phosphorylation. Cuproptosis is a recently defined form of copper-induced cell death reported to directly target the TCA cycle. Here, we find that ARID1A-deficient HCC cells and xenograft tumors are highly sensitive to copper treatment. Together, these results offer evidence of the synthetic lethality between ARID1A deficiency and mitochondrial respiration impairment, suggesting that copper treatment constitutes a promising therapeutic strategy for selectively targeting ARID1A-deficient HCC.

Curcumin Induces Apoptosis by Suppressing XRCC4 Expression in Hepatocellular Carcinoma

Curcumin is a chemical with various pharmacological activities used for cancer treatment. It inhibits hepatocellular carcinoma (HCC) by inducing apoptosis. Here, the mechanism underlying the effect of curcumin on the apoptosis of HCC cells was studied. Cell counting kit-8 and plate cloning assays were used to assess the proliferation of HCC cells, and acridine orange/ethidium bromide and Annexin V/PI staining were used to analyze their apoptosis. HCC xenograft tumor models were established to validate anti-cancer effects of curcumin. Expression levels of XRCC4 protein in tumor tissues were assessed by immunohistochemistry. Correlation between XRCC4 expression and the prognosis of patients with HCC was analyzed by integrating publicly available gene expression data. Curcumin inhibited HCC cells proliferation in a dose-dependent manner. Compared with the control group, curcumin significantly promoted the apoptosis of HCC cells in vitro and in vivo. Immunohistochemical analysis revealed that curcumin downregulated XRCC4 expression levels in HCC tissues. Prognosis of HCC patients with high XRCC4 expression was poorer than that of patients with low XRCC4 expression. Therefore, curcumin exerts anti-cancer effects by inhibiting cell proliferation and promoting cell apoptosis in HCC. This may be due to curcumin interference in the repair process of the nonhomologous DNA terminal link of HCC cells by downregulating XRCC4 expression.

ETV4 facilitates angiogenesis in hepatocellular carcinoma by upregulating MMP14 expression

Abnormal vascularization plays a crucial role in cell proliferation, tumor invasion and metastasis of hepatocellular carcinoma (HCC). It has been reported that ETV4 functions as an oncogenic gene in driving the carcinogenesis and progression, and promoting invasion and metastasis of HCC. However, the function of ETV4 on angiogenesis in HCC remains unclear. In the current study, immunohistochemistry showed that knockdown of ETV4 reduced angiogenesis in HCC xenograft tumor tissues. In vitro, tube formation assay verified that ETV4 expression promoted angiogenesis through simulating the angiogenic environment in HCC cells. Transcriptome sequencing indicated that MMP14 was one of the differentially expressed genes enriched in angiogenesis process. Subsequently, it was confirmed that MMP14 was regulated by ETV4 at the transcription level in HCC cells, clinical tissue samples and online databases. Further, we demonstrated that MMP14 induced angiogenesis in ETV4-mediated HCC microenvironment. Collectively, this research further reveals the biological mechanism of ETV4 in promoting the migration and invasion of HCC, and provides novel mechanistic insights and strategic guidance for anti-angiogenic therapy in HCC.

GPC3-IL7-CCL19-CAR-T primes immune microenvironment reconstitution for hepatocellular carcinoma therapy.

Chimeric antigen receptor (CAR)-T-cell therapy is a revolutionary treatment that has become a mainstay of advanced cancer treatment. Conventional glypican-3 (GPC3)-CAR-T cells have not produced ideal clinical outcomes in advanced hepatocellular carcinoma (HCC), and the mechanism is unclear. This study aims to investigate the clinical utility of novel GPC3-7-19-CAR-T cells constructed by our team and to explore the mechanisms underlying their antitumor effects. We engineered a novel GPC3-targeting CAR including an anti-GPC3 scFv, CD3ζ, CD28 and 4-1BB that induces co-expression of IL-7 at a moderate level (500pg/mL) and CCL19 at a high level (15000pg /mL) and transduced it into human T cells. In vitro, cell killing efficacy was validated by the xCELLigence RTCA system, LDH nonradioactive cytotoxicity assay and was confirmed in primary HCC organoid models employing a 3D microfluid chip. In vivo, the antitumor capacity was assessed in a humanized NSG mouse xenograft model. Finally, we initiated a phase I clinical trial to evaluate the safety and effect of GPC3-7-19-CAR-T cells in the clinic. GPC3-7-19-CAR-T cells had 1.5-2 times higher killing efficiency than GPC3-CAR-T cells. The tumor formation rates in GPC3-7-19-CAR-T cells treated model were reduced (3/5vs.5/5), and the average tumor volumes were 0.74 cm3 ± 1.17 vs. 0.34 cm3 ± 0.25. Of note, increased proportion of CD4+ TEM and CD8+ TCM cells was infiltrated in GPC3-7-19-CAR-T cells group. GPC3-7-19-CAR-T cells obviously reversed the immunosuppressive tumor microenvironment (TME) by reducing polymorphonuclear (PMN)-myeloid-derived suppressor cells (MDSCs) and regulatory T (Treg) cells infiltration and recruiting more dendritic cells (DCs) to HCC xenograft tumor tissues. In one patient with advanced HCC, GPC3-7-19-CAR-T-cell treatment resulted in tumor reduction 56days after intravenous infusion. In conclusion, GPC3-7-19-CAR-T cells achieved antitumor effects superior to those of conventional GPC3-CAR-T cells by reconstructing the TME induced by the dominant CD4+ TEM and CD8+ TCM cell subsets. Most importantly, GPC3-7-19-CAR-T cells exhibited good safety and antitumor efficacy in HCC patients in the clinic. ► Novel GPC3-7-19-CAR-T cells designed with mediate level of IL-7 secretion and high level of CCL19 secretion, which could recruit more mature DCs to assist killing on GPC3+HCCs. ►DC cells recruited by CCL19 could interact with CD4+ T cells and promote the differentiation of CD4+TEFF cells into CD4+TEM and CD8+TCM subsets, leading a better anti-tumor effect on GPC3+HCCs. ►Compared with conventional GPC3-CAR-T, GPC3-7-CCL19-CAR-T cells could reverse tumor immunosuppressive microenvironment by reducing PMN-MDSC and Treg cell infiltration.

Engineered small extracellular vesicles loaded with miR-654-5p promote ferroptosis by targeting HSPB1 to alleviate sorafenib resistance in hepatocellular carcinoma

Sorafenib (sora) is the initial therapy for patients with progressive hepatocellular carcinoma (HCC), but the emergence of drug resistance has seriously impacted its therapeutic efficacy. However, the mechanism of sora resistance remains unclear, and effective strategies to overcome drug resistance are still lacking. By establishing a sora-resistant hepatocellular carcinoma cell line, we found that Heat Shock Protein Family B (small) Member 1 (HSPB1) was markedly upregulated in sora-resistant HCC cells. Further research revealed that the ferroptosis resistance induced by HSPB1 upregulation plays a crucial role in sora resistance. In addition, we confirmed that miR-654-5p enhances sora-induced ferroptosis by binding to HSPB1 and reducing its protein levels. To enhance miRNA stability and delivery efficiency in vivo, we used small extracellular vesicles (sEV) derived from human adipose mesenchymal stem cells as miR-654-5p carriers, creating engineered sEV (m654-sEV). The research demonstrated that m654-sEV effectively delivers miR-654-5p to HCC cells, targeting HSPB1 and enhancing sora-induced ferroptosis. This improves therapeutic effects on sora-resistant HCC cells and xenograft tumors, restoring their sensitivity to sora. In summary, m654-sEV, which targets HSPB1 via miR-654-5p delivery, represents a promising strategy for addressing sora-resistant issue. The combined use of m654-sEV and sora has the potential to significantly enhance therapeutic efficacy for patients with sora-resistant HCC.

The Role of miR-1183: A Potential Suppressor in Hepatocellular Carcinoma via Regulating Splicing Factor SRSF1.

Hepatocellular carcinoma (HCC) is a severe global health problem, causing many deaths of patients all over the world. Serine and arginine-rich splicing factor 1 (SRSF1) functions as an important oncogenic role in tumorigenesis and progression in HCC. Therefore, therapies targeting SRSF1 may provide promising therapeutic approaches. MiRNAs are virtually involved at the post-transcriptional level and bind to 3' untranslated region (3'-UTR) of their target messenger RNA (mRNA) to suppress expression. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to detect the expression of SRSF1 and miR-1183 in HCC cell lines. CCK8 assay, colony formation assay and wound healing assay were used to detect the function of miR-1183 in HCC cell lines in vitro. Luciferase reporter assay and Western blot were applied to detect the regulation of particular molecules. Xenograft tumor assay was used to detect the function of miR-1183 in HCC cell lines in vivo. Immunohistochemistry (IHC) was used to detect the expression of SRSF1 in HCC tissues and Xenograft tumors. In this study, we identified that miR-1183 was downregulated in HCC cell lines. Functional assays indicated that miR-1183-upregulation cells show weakened proliferation ability and migration ability in vitro and inhibit subcutaneous tumor formation in vivo. With respect to the underlying mechanism, we found that miR-1183 function as a tumor suppressor by specifically binding to SRSF1. This study is the first to demonstrate that miR-1183 function as an important tumor-suppressing role by binding to the 3'-UTR of SRSF1 mRNA and suppressing its protein level in HCC cells in vitro and in vivo. Further, miR-1183 may be a potential target in the prognosis and treatment of HCC.

Curcumin inhibits proliferation of hepatocellular carcinoma cells by blocking PTPN1 and PTPN11 expression.

The antitumor mechanism of curcumin is unclear, especially in hepatocellular carcinoma (HCC) cells. To clarify the mechanism of action of curcumin in the effective treatment of HCC, the targets of curcumin were screened and validated. Candidate genes of curcumin for HCC were screened using the traditional Chinese medicine systems pharmacology (TCMSP) database and validated using The Cancer Genome Atlas (TCGA) database. The correlation of mRNA expression levels between key candidate genes was identified in the TCGA liver hepatocellular carcinoma (LIHC) dataset. The effects on prognosis were analyzed to identify the target gene of curcumin, which inhibits HCC cell proliferation. Based on the subcutaneous xenograft model of human HCC in nude mice, the expression levels of target proteins were observed using immunohistochemistry. The analysis results of the present study identified the target genes of curcumin, which were obtained by screening the TCSMP database. The protein tyrosine phosphatase non-receptor type 1 (PTPN1) was obtained from TCGA database analysis of the targeted genes. The expression levels of PTPN1 and its homologous sequence genes in TCGA LIHC project was analyzed to identify the potential target gene of curcumin, for use in HCC treatment. Next, xenograft experiments were performed to investigate the therapeutic effects of curcumin in an animal model. Curcumin was demonstrated to inhibit the growth of HCC xenograft tumors in mice. Immunohistochemistry results demonstrated that the protein expression levels of PTPN1 and PTPN11 in the curcumin group were significantly lower compared with those in the control group. In conclusion, these results demonstrated that curcumin inhibits the proliferation of HCC cells by inhibiting the expression of PTPN1 and PTPN11.

TCR T cells overexpressing c-Jun have better functionality with improved tumor infiltration and persistence in hepatocellular carcinoma.

The overall 5-year survival rate of hepatocellular carcinoma (HCC), a major form of liver cancer, is merely 20%, underscoring the need for more effective therapies. We recently identified T cell receptors (TCR) specific for the HLA-A2/alpha fetoprotein amino acids 158-166 (AFP158) and showed that these TCR engineered T cells could control HCC xenografts in NSG mice. However, their efficacy was limited by poor expansion, loss of function, and short persistence of the TCR T cells. Here, we studied whether overexpression of c-Jun, a transcription factor required for T cell activation, in the TCR T cells could enhance their expansion, function, and persistence in HCC tumor models. Recombinant lentiviral vectors (lv), expressing either the HLA-A2/AFP158-specific TCR or both the TCR and c-Jun (TCR-JUN), were constructed and used to transduce primary human T cells to generate the TCR or TCR-JUN T cells, respectively. We compared the expansion, effector function, and exhaustion status of the TCR and TCR-JUN T cells in vitro after HCC tumor stimulation. Additionally, we studied the persistence and antitumor effects of the TCR and TCR-JUN T cells using the HCC xenografts in NSG mice. We could effectively transduce primary human T cells to express both TCR and c-Jun. Compared to the HLA-A2/AFP158 TCR T cells, the TCR-JUN T cells have better expansion potential in culture, with enhanced functional capacity against HCC tumor cells. In addition, the TCR-JUN T cells were less apoptotic and more resistant to exhaustion after HepG2 tumor stimulation. In the HCC xenograft tumor model, c-Jun overexpression enhanced the TCR T cell expansion and increased the overall survival rate of the treated mice. Importantly, the TCR-JUN T cells were less exhausted in the tumor lesions and demonstrated enhanced tumor infiltration, functionality, and persistence. c-Jun overexpression can enhance the expansion, function, and persistence of the A2/AFP158 TCR engineered T cells. The c-Jun gene co-delivery has the potential to enhance the antitumor efficacy of AFP specific TCR T cells when treating patients with HCC.

Gamabufotalin Induces Apoptosis and Cytoprotective Autophagy through the mTOR Signaling Pathway in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is a malignant tumor with a high rate of recurrence and a poor prognosis. Here, we investigated the effect and the potential antitumor mechanism of Gamabufotalin (CS-6) against HCC. Our results show that CS-6 strikingly reduced cell viability, inhibited colony formation, and promoted apoptosis in Hep3B and Huh7 cells. In vivo, CS-6 inhibited HCC xenograft tumor growth with no toxicity to normal tissues. Mechanistically, we found that CS-6 could induce cytoprotective autophagy through the mTOR-ULK1 signaling pathway through downregulation of p62 and upregulation of LC3 II/LC3 I. Meanwhile, CS-6 activated caspase-3 and PARP mediated apoptosis, and the caspase inhibitor Z-VAD-FMK blocked the CS-6-induced cell death in HCC cells. Moreover, autophagy and apoptosis were found to have antagonistic effects in Hep3B and Huh7 cells. Both the autophagy inhibitor chloroquine (CQ) and the mTOR activator MHY1485 blocked autophagy and further enhanced CS-6-induced apoptosis. Taken together, we demonstrated for the first time that CS-6 promotes apoptosis and cytoprotective autophagy through the mTOR signaling pathway in HCC, which proposes a novel strategy for HCC therapy.

A Water-Soluble Hydrogen Sulfide Donor Suppresses the Growth of Hepatocellular Carcinoma via Inhibiting the AKT/GSK-3β/β-Catenin and TGF-β/Smad2/3 Signaling Pathways.

Hepatocellular carcinoma (HCC) is a disease with high morbidity, high mortality, and low cure rate. Hyaluronic acid (HA) is widely adopted in tissue engineering and drug delivery. 5-(4-Hydroxyphenyl)-3H-1, 2-dithiol-3-thione (ADT-OH) is one of commonly used H2S donors. In our previous study, HA-ADT was designed and synthesized via coupling of HA and ADT-OH. In this study, compared with sodium hydrosulfide (NaHS, a fast H2S-releasing donor) and morpholin-4-ium (4-methoxyphenyl)-morpholin-4-ylsulfanylidenesulfido-λ5-phosphane (GYY4137, a slow H2S-releasing donor), HA-ADT showed stronger inhibitory effect on the proliferation, migration, invasion, and cell cycle of human HCC cells. HA-ADT promoted apoptosis by suppressing the expressions of phospho (p)-protein kinase B (PKB/AKT), p-glycogen synthase kinase-3β (GSK-3β), p-β-catenin, and also inhibited autophagy via the downregulation of the protein levels of p-Smad2, p-Smad3, and transforming growth factor-β (TGF-β) in human HCC cells. Moreover, HA-ADT inhibited HCC xenograft tumor growth more effectively than both NaHS and GYY4137. Therefore, HA-ADT can suppress the growth of HCC cells by blocking the AKT/GSK-3β/β-catenin and TGF-β/Smad2/3 signaling pathways. HA-ADT and its derivatives may be developed as promising antitumor drugs.

Interfering with lipid metabolism through targeting CES1 sensitizes hepatocellular carcinoma for chemotherapy.

Hepatocellular carcinoma (HCC) is the most common lethal form of liver cancer. Apart from surgical removal and transplantation, other treatments have not yet been well established for patients with HCC. Herein, we found that carboxylesterase 1 (CES1) was expressed at various levels in HCC. We further revealed that blockage of CES1 by pharmacological and genetical approaches leads to altered lipid profiles that are directly linked to impaired mitochondrial function. Mechanistically, LC-MS/MS and lipidomic analyses revealed that lipid signaling molecules, including polyunsaturated fatty acids (PUFAs), which activate PPARα/γ, were dramatically reduced upon CES1 inhibition. As a result, SCD, a PPARα/γ target gene involved in tumor progression and chemoresistance, was significantly downregulated. Consistently, clinical analysis demonstrated a strong correlation between the protein levels of CES1 and SCD in HCC. Interference with lipid signaling by targeting the "CES1-PPARα/γ-SCD" axis sensitized HCC cells to cisplatin treatment. As a demonstration, the growth of HCC xenograft tumors in NU/J mice was potently limited by co-administration of cisplatin and CES1 inhibition. Our results suggest that CES1 is a promising therapeutic target for HCC treatment.

Trichosanthin Promotes Anti-Tumor Immunity through Mediating Chemokines and Granzyme B Secretion in Hepatocellular Carcinoma.

Trichosanthin (TCS) is a type I ribosome-inactivating protein extracted from the tuberous root of the plant Trichosanthes. TCS shows promising potential in clinical drug abortion, anti-tumor and immunological regulation. However, the molecular mechanisms of its anti-tumor and immune regulation properties are still not well discovered. In the present study, we investigated the anti-tumor activity of TCS in hepatocellular carcinoma (HCC), both in vitro and in vivo. Both HCC cell lines and xenograft tumor tissues showed considerable growth inhibition after they were treated with TCS. TCS provoked caspase-mediated apoptosis in HCC cells and xenograft tumor tissues. The recruitment of CD8+ T cells to HCC tissues and the expression of chemokines, CCL2 and CCL22, were promoted upon TCS treatment. In addition, TCS induced an upregulation of Granzyme B (GrzB), TNF-α and IFN-γ in HCC tissues, which are the major cytotoxic mediators produced by T cells. Furthermore, TCS also resulted in an increase of mannose-6-phosphate receptor (M6PR), the major receptor of GrzB, in HCC tissues. In summary, these results suggest that TCS perhaps increases T-cell immunity via promoting the secretion of chemokines and accelerating the entry of GrzB to HCC cells, which highlights the potential role of TCS in anti-tumor immunotherapy.

Protein phosphatase 2A-B55β mediated mitochondrial p-GPX4 dephosphorylation promoted sorafenib-induced ferroptosis in hepatocellular carcinoma via regulating p53 retrograde signaling.

Rationale: As a key endogenous negative regulator of ferroptosis, glutathione peroxidase 4 (GPX4) can regulate its antioxidant function through multiple post-translational modification pathways. However, the effects of the phosphorylation/dephosphorylation status of GPX4 on the regulation of inducible ferroptosis in hepatocellular carcinoma (HCC) remain unclear. Methods: To investigate the effects and molecular mechanism of GPX4 phosphorylation/dephosphorylation modification on ferroptosis in HCC cells. Sorafenib (Sora) was used to establish the ferroptosis model in HCC cells in vitro. Using the site-directed mutagenesis method, we generated the mimic GPX4 phosphorylation or dephosphorylation HCC cell lines at specific serine sites of GPX4. The effects of GPX4 phosphorylation/dephosphorylation modification on ferroptosis in HCC cells were examined. The interrelationships among GPX4, p53, and protein phosphatase 2A-B55β subunit (PP2A-B55β) were also explored. To explore the synergistic anti-tumor effects of PP2A activation on Sora-administered HCC, we established PP2A-B55β overexpression xenograft tumors in a nude mice model in vivo. Results: In the Sora-induced ferroptosis model of HCC in vitro, decreased levels of cytoplasmic and mitochondrial GPX4, mitochondrial dysfunction, and enhanced p53 retrograde signaling occurred under Sora treatment. Further, we found that mitochondrial p53 retrograded remarkably into the nucleus and aggravated Sora-induced ferroptosis. The phosphorylation status of GPX4 at the serine 2 site (GPX4Ser2) revealed that mitochondrial p-GPX4Ser2 dephosphorylation was positively associated with ferroptosis, and the mechanism might be related to mitochondrial p53 retrograding into the nucleus. In HCC cells overexpressing PP2A-B55β, it was found that PP2A-B55β directly interacted with mitochondrial GPX4 and promoted Sora-induced ferroptosis in HCC. Further, PP2A-B55β reduced the interaction between mitochondrial GPX4 and p53, leading to mitochondrial p53 retrograding into the nucleus. Moreover, it was confirmed that PP2A-B55β enhanced the ferroptosis-mediated tumor growth inhibition and mitochondrial p53 retrograde signaling in the Sora-treated HCC xenograft tumors. Conclusion: Our data uncovered that the PP2A-B55β/p-GPX4Ser2/p53 axis was a novel regulatory pathway of Sora-induced ferroptosis. Mitochondrial p-GPX4Ser2 dephosphorylation triggered ferroptosis via inducing mitochondrial p53 retrograding into the nucleus, and PP2A-B55β was an upstream signal modulator responsible for mitochondrial p-GPX4Ser2 dephosphorylation. Our findings might serve as a potential theranostic strategy to enhance the efficacy of Sora in HCC treatment through the targeted intervention of p-GPX4 dephosphorylation via PP2A-B55β activation.

Exosome-transmitted circ_002136 promotes hepatocellular carcinoma progression by miR-19a-3p/RAB1A pathway

BackgroundCircular RNAs (circRNAs) are enriched in exosomes and are extremely stable. Exosome-mediated intercellular transfer of specific biologically active circRNA molecules can drive the transformation of the tumor microenvironment and accelerate or inhibit the local spread and multifocal growth of hepatocellular carcinoma (HCC). In this study, we explored in depth about the biological roles of HCC cell-derived exosomes and exosome-transported circRNAs on HCC in vivo and in vitro.MethodsExosomes extracted from HCC cells (Huh7 and HA22T) were characterized using transmission electron microscopy, nanoparticle size tracer analysis, and western blotting. Exosomes were observed for endocytosis using fluorescent labeling. The effects of HCC cell-derived exosomes and the circ_002136 they carried on cell growth, metastasis and apoptosis were determined by CCK-8 assay, transwell assay, flow cytometry analysis and TUNEL staining, respectively. The expressions of circ_002136, miR-19a-3p and RAB1A were detected by quantitative RT-PCR (qRT-PCR). Targeted binding between miR-19a-3p and circ_002136 or RAB1A was predicted and verified by bioinformatics analysis, dual-luciferase reporter and RNA pull-down experiments. The in vivo effect of circ_002136 was determined by constructing a xenograft tumor model.ResultsThe findings revealed that Huh7 and HA22T exosomes conferred enhanced viability as well as invasive ability to recipient HCC cells. Circ_002136 was shown for the first time to be differentially upregulated in HCC tissues and cells and transferred by HCC cell-derived exosomes. More importantly, selective silencing of circ_002136 depleted the malignant biological behaviors of HCC exosome-activated Huh7 and HA22T cells. Depletion of circ_002136 in vivo effectively retarded the growth of HCC xenograft tumors. Furthermore, a well-established circ_002136 ceRNA regulatory network was constructed, namely circ_002136 blocked miR-19a-3p expression, elevated RAB1A expression activity and stimulated HCC development. Finally, high levels of circ_002136 or RAB1A, as well as low levels of miR-19a-3p, negatively affected HCC patient survival.ConclusionThe study on circ_002136 provides good data to support our insight into the mechanism of to-be-silenced circRNA as a therapeutic agent in the progression of HCC.

PI3K/AKT1 Signaling Pathway Mediates Sinomenine-Induced Hepatocellular Carcinoma Cells Apoptosis: An in Vitro and in Vivo Study.

Hepatocellular carcinoma (HCC) is one of the most frequent cancers. Sinomenine (SIN) is a compound derived from Sinomenium acutum. Our previous investigations have found that SIN inhibited protein kinase B (AKT) signaling to induce autophagic death of tumor cells. However, whether inhibition of this pathway by SIN could impact the proliferation of HCC cells is unknown. Thus, we applied SIN to SK-Hep-1 cells and used cell counting kit 8 (CCK8), lactate dehydrogenase (LDH), colony formation and 5-ethynyl-20-deoxyuridine (EdU) incorporation experiments to detect cell viability. Then, staining with annexin V/propidium iodide (PI) coupled with terminal deoxynucleotidyl transferase-mediated biotinylated uridine 5'-triphosphate (UTP) nick end labeling (TUNEL) staining were utilized to monitor apoptosis. Changes in cell mitochondrial membrane capacity were explored via 5,5',6,6'-Tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) staining, whilst Western blot or immunohistochemistry was applied to evaluate the expression levels of key proteins, consisting of Cleaved Caspase 3, AKT1, B-cell leukemia/lymphoma 2 (BCL-2), phosphatidylinositol 3-kinase (PI3K) p85α, and Cleaved Caspase 9 etc. The Balb/c nude mice were utilized to establish HCC xenograft tumor model, administered by SIN. After treatments, the tumor volume along with weight were measured. The results illustrated that SIN suppressed SK-Hep-1 HCC cells' proliferation, enhanced the collapse of potential of the mitochondrial membrane, triggered cell apoptosis, down-regulated PI3K p85α, AKT1, BCL-2, Pro-Caspase 9, Pro-Caspase 3 expressions, and up-regulated Cleaved Caspase 9 and Cleaved Caspase 3 expressions in vitro and in vivo. Meanwhile, SIN reduced the tumor volume along with weight of mice. In addition, insulin-like growth factor-1 (IGF-1), a powerful activator of the PI3K/AKT pathway, could reverse the high apoptosis of SK-Hep-1 HCC cells induced by SIN. Overall, inhibition of PI3K/AKT1 signaling cascade by SIN induced HCC cells apoptosis.