PTPRε Acts as a Metastatic Promoter in Hepatocellular Carcinoma by Facilitating Recruitment of SMAD3 to TGF-ß Receptor 1.

BACKGROUND AND AIMS: Transforming growth factor beta (TGF-ß) suppresses early stages of tumorigenesis, but contributes to the migration and metastasis of cancer cells. However, the role of TGF-ß signaling in invasive prometastatic hepatocellular carcinoma (HCC) is poorly understood. In this study, we investigated the roles of canonical TGF-ß/mothers against decapentaplegic homolog 3 (SMAD3) signaling and identified downstream effectors on HCC migration and metastasis. APPROACH AND RESULTS: By using in vitro trans-well migration and invasion assays and in vivo metastasis models, we demonstrated that SMAD3 and protein tyrosine phosphatase receptor epsilon (PTPRε) promote migration, invasion, and metastasis of HCC cells in vitro and in vivo. Further mechanistic studies revealed that, following TGF-ß stimulation, SMAD3 binds directly to PTPRε promoters to activate its expression. PTPRε interacts with TGFBR1/SMAD3 and facilitates recruitment of SMAD3 to TGFBR1, resulting in a sustained SMAD3 activation status. The tyrosine phosphatase activity of PTPRε is important for binding with TGFBR1, recruitment and activation of SMAD3, and its prometastatic role in vitro. A positive correlation between pSMAD3/SMAD3 and PTPRε expression was determined in HCC samples, and high expression of SMAD3 or PTPRε was associated with poor prognosis of patients with HCC. CONCLUSIONS: PTPRε positive feedback regulates TGF-ß/SMAD3 signaling to promote HCC metastasis.

The crosstalk network of XIST/miR-424-5p/OGT mediates RAF1 glycosylation and participates in the progression of liver cancer.

BACKGROUND: Liver cancer is a major public health concern, but the mechanistic actions of biomarkers contributing to liver cancer remain to be determined. In this study, we aimed to investigate the regulatory cascade of microRNA-424-5p (miR-424-5p), X-inactive-specific transcript (XIST) and O-GlcNAc transferase (OGT) in liver cancer. METHODS: Differentially expressed miRNAs and target genes related to liver cancer were predicted by bioinformatics analyses, and their expression was determined in liver tissues of patients with liver cancer and liver cancer cells. The RNA immunoprecipitation (RIP), RNA pull-down and dual luciferase reporter assay were used to examine the binding affinity among XIST and miR-424-5p and OGT. Then, gain- and loss-of-function assays were conducted to evaluate the effects of the XIST/miR-424-5p/OGT axis on malignant phenotypes. A nude mouse model of liver cancer was further established for in vivo substantiation. RESULTS: XIST and OGT were up-regulated in liver cancer tissues and cells, responsible for poor prognosis in patients with liver cancer, while miR-424-5p was down-regulated. XIST competitively bound to miR-424-5p to increase OGT expression. XIST silencing inhibited malignant phenotypes of liver cancer cells, while miR-424-5p down-regulation negated its effect. miR-424-5p suppressed RAF1 glycosylation by negatively regulating OGT expression and promoted its ubiquitination/degradation. Furthermore, XIST knockdown inhibited tumour growth and metastasis in nude mice, while ectopic OGT reversed its effect. CONCLUSION: These results reveal a novel mechanism by which the interaction of XIST/miR-424-5p/OGT participates in the malignancy and metastasis of liver cancer.

CRISPR/Cas9-mediated knockout of HBsAg inhibits proliferation and tumorigenicity of HBV-positive hepatocellular carcinoma cells.

Chronic hepatitis B virus (HBV) infection remains the most common risk factor for hepatocellular carcinoma (HCC). High HBV surface antigen (HBsAg) levels are highly correlated with hepatocarcinogenesis and HBV-associated HCC development. However, the role and detailed mechanisms associated with HBsAg in HCC development remain elusive. In this study, we designed specific single guide RNAs (sgRNAs) targeting the open reading frames, preS1/preS2/S, of the HBV genome and established HBsAg knockout HCC cell lines using the CRISPR/Cas9 system. We showed that knockout of HBsAg in HCC cell lines decreased HBsAg expression and significantly attenuated HCC proliferation in vitro, as well as tumorigenicity in vivo. We also found that overexpression of HBsAg, including the large (LHBs), middle (MHBs), and small (SHBs) surface proteins promoted proliferation and tumor formation in HCC cells. Moreover, we demonstrated that knockout of HBsAg in HCC cells decreased interleukin (IL)-6 production and inhibited signal transducer and activator of transcription 3 (STAT3) signaling, while overexpression of HBsAg induced a substantial accumulation of pY-STAT3. Collectively, these results highlighted the tumorigenic role of HBsAg and implied that the IL-6-STAT3 pathway may be implicated in the HBsAg-mediated malignant potential of HBV-associated HCC.

Suberoylanilide hydroxamic acid enhances chemosensitivity to 5-fluorouracil in hepatocellular carcinoma via inhibition of thymidylate synthase.

Hepatocellular carcinoma (HCC) is associated with a high rate of mortality worldwide. Here, we investigated the effect of combination treatment with suberoylanilide hydroxamic acid (SAHA) and 5-fluorouracil (5-FU) on HCC cells. HepG2, SMMC7721, and BEL7402 cells were treated with SAHA and/or 5-FU and subjected to cell viability, colony formation, and soft agarose assays; cell cycle, apoptosis, and reverse transcription-PCR analyses; western blotting; immunohistochemistry; and xenograft tumorigenicity assay. SAHA and 5-FU inhibited the proliferation of the three cell lines, and combination treatment with SAHA and 5-FU resulted in a combination index <1 and a dose-reduction index value >1, indicating a synergistic effect. Co-treatment with SAHA and 5-FU caused G0/G1 phase arrest and induced caspase-dependent apoptosis, inhibiting tumorigenicity in vitro and in vivo. 5-FU upregulated thymidylate synthase, whereas SAHA downregulated its expression. Our results indicate that SAHA and 5-FU act synergistically to inhibit cell growth and tumorigenicity in HCC via the induction of cell-cycle arrest and apoptosis through a mechanism involving the inhibition of thymidylate synthase, suggesting that combination treatment with 5-FU and SAHA may be beneficial for the treatment of inoperable HCC.

USP11 potentiates HGF/AKT signaling and drives metastasis in hepatocellular carcinoma.

USP11 is a member of the ubiquitin-specific protease family and plays a crucial role in tumor progression in various cancers. However, the precise mechanism by which USP11 promotes EMT and metastasis in hepatocellular carcinoma (HCC) is not fully understood. In this study, we demonstrated that the USP11 expression was dramatically upregulated in HCC tissues and cell lines. Increased USP11 expression was closely associated with tumor number, vascular invasion, and poor prognosis. Functional experiments demonstrated that USP11 markedly promoted metastasis and EMT in HCC via induction of the transcription factor Snail. Mechanistically, USP11 interacted with and deubiquitinated eEF1A1 on Lys439, thereby inhibiting its ubiquitin-mediated degradation. Subsequently, the elevated expression of eEF1A1 resulted in its binding to SP1, which in turn drove the binding of SP1 to its target HGF gene promoter to increase its transcription. This led to an enhanced expression of HGF and the activation of the downstream PI3K/AKT signaling pathway. We demonstrated that USP11 promotes EMT and metastasis in HCC via eEF1A1/SP1/HGF dependent-EMT. Our findings suggest that the USP11/ eEF1A1/SP1/HGF axis contributes to metastasis in HCC, and therefore, could be considered as a potential therapeutic target for the treatment of HCC.

Inhibition of PTPRE suppresses tumor progression and improves sorafenib response in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) has a poor prognosis, and the efficacy of current therapeutic strategies is extremely limited in advanced diseases. Our previous study reported that protein tyrosine phosphatase receptor epsilon (PTPRE) is a promoting factor in HCC progression. In this study, our objective was to evaluate the treatment effect of PTPRE inhibitors in different HCC preclinical models. Our results indicated that the PTPRE inhibitory compound 63 (Cpd-63) inhibited tumor cell proliferation, migration, and HCC organoid growth. Mechanism research revealed that Cpd-63 could inhibit the expression of MYC and MYC targets by inhibiting the activation of SRC. Additionally, we found that Cpd-63 could improve the response of sorafenib in HCC cells. In conclusion, we demonstrated that the PTPRE inhibitors represented a potential therapeutic agent for HCC management.

The Role of Nuclear Receptors in the Pathogenesis and Treatment of Non-alcoholic Fatty Liver Disease.

Non-alcoholic fatty liver disease (NAFLD) is a global health burden closely linked to insulin resistance, obesity, and type 2 diabetes. The complex pathophysiology of NAFLD involves multiple cellular pathways and molecular factors. Nuclear receptors (NRs) have emerged as crucial regulators of lipid metabolism and inflammation in NAFLD, offering potential therapeutic targets for NAFLD. Targeting PPARs and FXRs has shown promise in ameliorating NAFLD symptoms and halting disease progression. However, further investigation is needed to address side effects and personalize therapy approaches. This review summarizes the current understanding of the involvement of NRs in the pathogenesis of NAFLD and explores their therapeutic potential. We discuss the role of several NRs in modulating lipid homeostasis in the liver, including peroxisome proliferator-activated receptors (PPARs), liver X receptors (LXRs), farnesoid X receptors (FXRs), REV-ERB, hepatocyte nuclear factor 4α (HNF4α), constitutive androstane receptor (CAR) and pregnane X receptor (PXR).The expanding knowledge of NRs in NAFLD offers new avenues for targeted therapies, necessitating exploration of novel treatment strategies and optimization of existing approaches to combat this increasingly prevalent disease.

Disrupting CCDC137-mediated LZTS2 and ß-TrCP interaction in the nucleus inhibits hepatocellular carcinoma development via ß-catenin and AKT.

Hepatocellular carcinoma (HCC) is a highly heterogeneous solid tumor, with its biological characteristics intricately linked to the activation of oncogenes. This research specifically explored CCDC137, a molecule within the CCDC family exhibiting the closest association with HCC. Our investigation aimed to unravel the role, underlying mechanisms, and potential therapeutic implications of CCDC137 in the context of HCC. We observed a close correlation between elevated CCDC137 expression and poor prognosis in HCC patients, along with a promotive effect on HCC progression in vitro and in vivo. Mechanistically, we identified LZTS2, a negative regulator of ß-catenin, as the binding protein of CCDC137. CCDC137 facilitated K48-linked poly-ubiquitination of LZTS2 at lysine 467 via recruiting E3 ubiquitin ligase ß-TrCP in the nucleus, triggering AKT phosphorylation and activation of ß-catenin pathway. Moreover, the 1-75 domain of CCDC137 was responsible for the formation of the CCDC137-LZTS2-ß-TrCP complex. Subsequently, designed peptides targeting the 1-75 domain of CCDC137 to disrupt CCDC137-LZTS2 interaction demonstrated efficacy in inhibiting HCC progression. This promising outcome was further supported by HCC organoids and patient-derived xenograft (PDX) models, underscoring the potential clinical utility of the peptides. This study elucidated the mechanism of the CCDC137-LZTS2-ß-TrCP protein complex in HCC and offered clinically significant therapeutic strategies targeting this complex.

CREB3 suppresses hepatocellular carcinoma progression by depressing AKT signaling through competitively binding with insulin receptor and transcriptionally activating RNA-binding motif protein 38.

cAMP responsive element binding protein 3 (CREB3), belonging to bZIP family, was reported to play multiple roles in various cancers, but its role in hepatocellular carcinoma (HCC) is still unclear. cAMP responsive element binding protein 3 like 3 (CREB3L3), another member of bZIP family, was thought to be transcription factor (TF) to regulate hepatic metabolism. Nevertheless, except for being TFs, other function of bZIP family were poorly understood. In this study, we found CREB3 inhibited growth and metastasis of HCC in vitro and in vivo. RNA sequencing indicated CREB3 regulated AKT signaling to influence HCC progression. Mass spectrometry analysis revealed CREB3 interacted with insulin receptor (INSR). Mechanistically, CREB3 suppressed AKT phosphorylation by inhibiting the interaction of INSR with insulin receptor substrate 1 (IRS1). In our study, CREB3 was firstly proved to affect activation of substrates by interacting with tyrosine kinase receptor. Besides, CREB3 could act as a TF to transactivate RNA-binding motif protein 38 (RBM38) expression, leading to suppressed AKT phosphorylation. Rescue experiments further confirmed the independence between the two functional manners. In conclusion, CREB3 acted as a tumor suppressor in HCC, which inhibited AKT phosphorylation through independently interfering interaction of INSR with IRS1, and transcriptionally activating RBM38.

YTHDF2 Is a Therapeutic Target for HCC by Suppressing Immune Evasion and Angiogenesis Through ETV5/PD-L1/VEGFA Axis.

N6-methyladenosine (m6A) modification orchestrates cancer formation and progression by affecting the tumor microenvironment (TME). For hepatocellular carcinoma (HCC), immune evasion and angiogenesis are characteristic features of its TME. The role of YTH N6-methyladenosine RNA binding protein 2 (YTHDF2), as an m6A reader, in regulating HCC TME are not fully understood. Herein, it is discovered that trimethylated histone H3 lysine 4 and H3 lysine 27 acetylation modification in the promoter region of YTHDF2 enhanced its expression in HCC, and upregulated YTHDF2 in HCC predicted a worse prognosis. Animal experiments demonstrated that Ythdf2 depletion inhibited spontaneous HCC formation, while its overexpression promoted xenografted HCC progression. Mechanistically, YTHDF2 recognized the m6A modification in the 5'-untranslational region of ETS variant transcription factor 5 (ETV5) mRNA and recruited eukaryotic translation initiation factor 3 subunit B to facilitate its translation. Elevated ETV5 expression induced the transcription of programmed death ligand-1 and vascular endothelial growth factor A, thereby promoting HCC immune evasion and angiogenesis. Targeting YTHDF2 via small interference RNA-containing aptamer/liposomes successfully both inhibited HCC immune evasion and angiogenesis. Together, this findings reveal the potential application of YTHDF2 in HCC prognosis and targeted treatment.

Alkaline phosphatase combined with γ-glutamyl transferase is an independent predictor of prognosis of hepatocellular carcinoma patients receiving programmed death-1 inhibitors.

Background: Immunotherapy plays an increasingly critical role in the systemic treatment of HCC. This current study aimed to establish a novel prognostic predictor of Programmed death 1 (PD-1) inhibitor therapy in hepatocellular carcinoma (HCC) independent of Child-Pugh grade. Methods: Our study screened patients with HCC who received PD-1 inhibitors at Tongji Hospital Affiliated to Tongji Medical College of Huazhong University of Science and Technology from January 2018 to December 2020. ALG grade was determined by the patient's serum ALP and GGT levels before the initiation of PD-1 inhibitors. The endpoints of our study were overall survival (OS) and progression free survival (PFS). Follow-up ended at May 31, 2022. Results: Eighty- five patients (77 with Child-Pugh grade A, 8 with Child-Pugh grade B at baseline) were enrolled according to the inclusion criteria. Patients with Child-Pugh grade A achieved longer PFS and OS than those with Child-Pugh grade B. Patients with ALG grade 3 at baseline showed worse tumor response and poorer survival, and ALG grade could stratify patients with Child-Pugh grade A into subgroups with significantly different prognosis. Conclusions: ALG grade, combining ALP and GGT, is a novel and readily available prognostic marker and the predictive effect of ALG grade on patient prognosis is independent of Child-Pugh grade.

Bone Lesion-Derived Extracellular Vesicles Fuel Prometastatic Cascades in Hepatocellular Carcinoma by Transferring ALKBH5-Targeting miR-3190-5p.

Bone is the second leading metastatic site for hepatocellular carcinoma (HCC). Patients with HCC and bone metastasis suffer poor quality of life and reduced survival time. Extracellular vesicles (EVs) are widely involved in HCC formation and metastasis. However, the communication between primary HCC and bone lesions mediated by EVs remains unclear and the possible effect of bone metastasis on the progression of HCC remains largely unknown. Here, bone-metastasized HCC-derived EVs (BM-EVs) are found to localize to orthotropic HCC cells and promote HCC progression. Mechanistically, miR-3190-5p (miR-3190) is upregulated in intracellular HCC cells isolated from bone lesions as well as in their derived EVs. miR-3190 in BM-EVs is transferred into orthotopic tumor cells and enhances their metastatic capacity by downregulating AlkB homolog 5 (ALKBH5) expression. Decreased level of ALKBH5 exacerbates the prometastatic characteristics of HCC by modulating gene expression in N6-methyladenosine-dependent and -independent ways. Finally, antagomir-miR-3190-loaded liposomes with HCC affinity successfully suppress HCC progression in mice treated with BM-EVs. These findings reveal that BM-EVs initiate prometastatic cascades in orthotopic HCC by transferring ALKBH5-targeting miR-3190 and miR-3190 is serving as a promising therapeutic target for inhibiting the progression of HCC in patients with bone metastasis.

Strategies for indica rice adapted to high-temperature stress in the middle and lower reaches of the Yangtze River.

High temperatures caused by climate warming severely affect the grain yield and quality of rice. In this study, the rice cultivars Longliangyou Huazhan (LLYHZ) and Quanliangyou 2118 (QLY2118) were selected as the experimental materials for investigation of an optimal cultivation system under high-temperature treatment. In addition, the heat-resistant cultivar Huanghuazhan (HHZ) and heat-sensitive cultivar Huiliangyou 858 (HLY858) were chosen as the experimental materials to study the effects of exogenous plant growth regulators on heat stress responses under high-temperature treatment. The results showed that mechanical transplanting of carpet seedlings and delayed sowing effectively increased the leaf area index and reduced the canopy temperature of LLYHZ and QLY2118. Furthermore, carpet seedling mechanical transplantation and delayed sowing improved grain yield and quality. Spray application of five plant growth regulators revealed that brassinolide and salicylic acid had the strongest effects on significantly improving antioxidant enzyme activities in the panicle, which would reduce the damage caused by the accumulation of reactive oxygen species and enhance plant tolerance of high-temperature stress. In addition, brassinolide and salicylic acid enhanced the percentage of anther dehiscence and percentage seed set. In this study, a set of simplified eco-friendly cultivation techniques for single-season indica rice adaptation to high-temperature stress was established. These results will be of great importance in alleviating the effects of high-temperature stress on rice production.

DDR1 promotes hepatocellular carcinoma metastasis through recruiting PSD4 to ARF6.

Discoidin domain receptor 1 (DDR1) is a member of the receptor tyrosine kinase family, and its ligand is collagen. Previous studies demonstrated that DDR1 is highly expressed in many tumors. However, its role in hepatocellular carcinoma (HCC) remains obscure. In this study, we found that DDR1 was upregulated in HCC tissues, and the expression of DDR1 in TNM stage II-IV was higher than that in TNM stage I in HCC tissues, and high DDR1 expression was associated with poor prognosis. Gene expression analysis showed that DDR1 target genes were functionally involved in HCC metastasis. DDR1 positively regulated the migration and invasion of HCC cells and promoted lung metastasis. Human Phospho-Kinase Array showed that DDR1 activated ERK/MAPK signaling pathway. Mechanically, DDR1 interacted with ARF6 and activated ARF6 through recruiting PSD4. The kinase activity of DDR1 was required for ARF6 activation and its role in metastasis. High expression of PSD4 was associated with poor prognosis in HCC. In summary, our findings indicate that DDR1 promotes HCC metastasis through collagen induced DDR1 signaling mediated PSD4/ARF6 signaling, suggesting that DDR1 and ARF6 may serve as novel prognostic biomarkers and therapeutic targets for metastatic HCC.

18[Formula: see text]-Glycyrrhetinic Acid Inhibits TGF-[Formula: see text]-Induced Epithelial-to-Mesenchymal Transition and Metastasis of Hepatocellular Carcinoma by Targeting STAT3.

18[Formula: see text]-glycyrrhetinic acid (GA) is the active ingredient of the traditional Chinese medicinal herb Glycyrrhizae radix et rhizoma. We previously demonstrated that GA inhibited tumor growth in hepatocellular carcinoma (HCC). However, the effect of GA on transforming growth factor-[Formula: see text] (TGF-[Formula: see text]-induced epithelial-mesenchymal transition (EMT) and metastasis were still unclear. In this study, in vitro transwell assays and immunofluorescence (IF) demonstrated that GA inhibited TGF-[Formula: see text]-induced migration, invasion and EMT of HCC cells. However, it had little effect on the inhibition of proliferation by TGF-[Formula: see text]. Moreover, we confirmed that GA suppressed the metastasis of HCC cells in vivousing an ectopic lung metastasis model. Furthermore, we found that GA inhibited TGF-[Formula: see text]-induced EMT mainly by reducing the phosphorylation of signal transducer and activator of transcription 3 (STAT3), which played an essential role in TGF-[Formula: see text]-induced EMT and cell mobility. Mechanistically, GA inhibited the phosphorylation of STAT3 by increasing the expression of Src homology 2 domain-containing protein tyrosine phosphatases 1 and 2 (SHP1 and SHP2). Therefore, we concluded that GA inhibited TGF-[Formula: see text]-induced EMT and metastasis via the SHP1&SHP2/STAT3/Snail pathway. Our data provide an attractive therapeutic target for future multimodal management of HCC.

Discoidin domain receptor 1 promotes hepatocellular carcinoma progression through modulation of SLC1A5 and the mTORC1 signaling pathway.

PURPOSE: Hepatocellular carcinoma (HCC) is one of the most common cancers in the world with a high mortality rate. Receptor tyrosine kinases play important roles in the occurrence and development of various cancers. Discoid protein domain receptor 1 (DDR1) is a special type of transmembrane receptor tyrosine kinase. Here, we show that the expression of DDR1 is significantly increased in HCC and is related to a poor clinical prognosis. METHODS: The expression of DDR1 in HCC cell lines and primary HCC specimens was evaluated using Western blotting and immunohistochemistry. A correlation between DDR1 and SLC1A5 expression was also investigated in primary HCC specimens. Cell proliferation was evaluated using in vitro CCK8 and colony formation assays. Gene knock-down and overexpression assays, CHX, NH4CL and Mg132 interference tests and immunoprecipitation, as well as nude mouse xenograft models were used to assess the mechanism by which DDR1 promotes tumorigenesis in vitro and in vivo. RESULTS: We found that DDR1 promotes the proliferation of HCC cells and accelerates the growth of HCC tumor xenografts, while DDR1 downregulation had the opposite effect. We also found that loss or gain of DDR1 expression affected HCC cell cycle progression. Mechanistically, we found that DDR1 interacts with SLC1A5, which belongs to the solute carrier (SLC) family of transporters, and regulates its stability, thereby affecting the mTORC1 signaling pathway. In addition, we found that SLC1A5 regulation by DDR1 can be restored by lysosome inhibitors. We also found that DDR1 is highly expressed in HCC tissues and that increased DDR1 expression predicts a shorter overall survival (OS) time. We additionally found that the expression of SLC1A5 was positively correlated with that of DDR1. Together, our data indicate that DDR1 acts as a tumor-promoting factor that can control HCC cell proliferation and cell cycle progression by stabilizing SLC1A5 in a lysosome-dependent way. CONCLUSIONS: Our study reveals a new mechanism by which DDR1 plays a liver cancer-promoting role. We also found that DDR1 expression serves as an independent prognostic marker, and that DDR1 and SLC1A5 expression levels are positively correlated in clinical samples. Our findings provide a new perspective for understanding HCC development and offers new targets for the treatment and management of HCC.

TDO2 Was Downregulated in Hepatocellular Carcinoma and Inhibited Cell Proliferation by Upregulating the Expression of p21 and p27.

BACKGROUND: Tryptophan-2,3-dioxygenase (TDO2) converts tryptophan into kynurenine in the initial limiting step of the kynurenine pathway. During the past decade, the overexpression of TDO2 has been found in various human tumors. However, the role of TDO2 in hepatocellular carcinoma is controversial, and we sought to clarify it in this study. METHODS: Western blot analysis and immunochemistry were used to detect the expression of TDO2 in human tissue specimens. The effect of TDO2 on cell proliferation in vitro was assessed using CCK8 and colony formation assays, and a xenograft mouse model was used to detect the effect of TDO2 on tumor growth in vivo. Flow cytometry was used to assess the cell cycle status. RESULTS: Low TDO2 expression was found in HCC and was associated with poor prognosis and adverse clinical outcomes. Conversely, TDO2 could restrain the proliferation of HCC cells in vivo and in vitro. Furthermore, TDO2 upregulated the expression of p21 and p27, inducing cell-cycle arrest. CONCLUSIONS: The loss of TDO2 expression in HCC was correlated with a poor prognosis and adverse clinical outcomes. At the same time, TDO2 could restrain the growth of HCC in vivo and in vitro. The results indicate that TDO2 is a potential biomarker and therapeutic target for HCC.

The 14-3-3σ protein promotes HCC anoikis resistance by inhibiting EGFR degradation and thereby activating the EGFR-dependent ERK1/2 signaling pathway.

Resistance to anoikis, cell death due to matrix detachment, is acquired during tumor progression. The 14-3-3σ protein is implicated in the development of chemo- and radiation resistance, indicating a poor prognosis in multiple human cancers. However, its function in anoikis resistance and metastasis in hepatocellular carcinoma (HCC) is currently unknown. Methods: Protein expression levels of 14-3-3σ were measured in paired HCC and normal tissue samples using western blot and immunohistochemical (IHC) staining. Statistical analysis was performed to evaluate the clinical correlation between 14-3-3σ expression, clinicopathological features, and overall survival. Artificial modulation of 14-3-3σ (downregulation and overexpression) was performed to explore the role of 14-3-3σ in HCC anoikis resistance and tumor metastasis in vitro and in vivo. Association of 14-3-3σ with epidermal growth factor receptor (EGFR) was assayed by co-immunoprecipitation. Effects of ectopic 14-3-3σ expression or knockdown on EGFR signaling, ligand-induced EGFR degradation and ubiquitination were examined using immunoblotting and co-immunoprecipitation, immunofluorescence staining, and flow cytometry analysis. The levels of EGFR ubiquitination, the interaction between EGFR and 14-3-3σ, and the association of EGFR with c-Cbl after EGF stimulation, in 14-3-3σ overexpressing or knockdown cells were examined to elucidate the mechanism by which 14-3-3σ inhibits EGFR degradation. Using gain-of-function or loss-of-function strategies, we further investigated the role of the EGFR signaling pathway and its downstream target machinery in 14-3-3σ-mediated anoikis resistance of HCC cells. Results: We demonstrated that 14-3-3σ was upregulated in HCC tissues, whereby its overexpression was correlated with aggressive clinicopathological features and a poor prognosis. In vitro and in vivo experiments indicated that 14-3-3σ promoted anoikis resistance and metastasis of HCC cells. Mechanistically, we show that 14-3-3σ can interact with EGFR and significantly inhibit EGF-induced degradation of EGFR, stabilizing the activated receptor, and therefore prolong the activation of EGFR signaling. We demonstrated that 14-3-3σ downregulated ligand-induced EGFR degradation by inhibiting EGFR-c-Cbl association and subsequent c-Cbl-mediated EGFR ubiquitination. We further verified that activation of the ERK1/2 pathway was responsible for 14-3-3σ-mediated anoikis resistance of HCC cells. Moreover, EGFR inactivation could reverse the 14-3-3σ-mediated effects on ERK1/2 phosphorylation and anoikis resistance. Expression of 14-3-3σ and EGFR were found to be positively correlated in human HCC tissues. Conclusions: Our results indicate that 14-3-3σ plays a pivotal role in the anoikis resistance and metastasis of HCC cells, presumably by inhibiting EGFR degradation and regulating the activation of the EGFR-dependent ERK1/2 pathway. To our best knowledge, this is the first report of the role of 14-3-3σ in the anoikis resistance of HCC cells, offering new research directions for the treatment of metastatic cancer by targeting 14-3-3σ.

Type-2 11ß-hydroxysteroid dehydrogenase promotes the metastasis of colorectal cancer via the Fgfbp1-AKT pathway.

Type-2 11ß-hydroxysteroid dehydrogenase (HSD11B2) is a key enzyme which converts cortisol to inactive cortisone and is involved in tumor progression and metastasis. Several studies have shown that the promotion of tumor progression and metastasis by HSD11B2 resulted from its physiological function of inactivating glucocorticoids (GC). However, the underlying molecular mechanisms by which HSD11B2 drives metastasis, in addition to inactivating GC, are still unclear. In our study, a series of in vivo and in vitro assays were performed to determine the function of HSD11B2 and the possible mechanisms underlying its role in CRC metastasis. mRNA transcriptome array analysis was used to identify the possible downstream targets of HSD11B2. We found that the ectopic expression of HSD11B2 significantly promoted the migration, invasion and metastasis of colorectal cancer (CRC) cells both in vitro and in vivo, while it did not affect their proliferation in either case. Mechanically, HSD11B2 appeared to enhance cell migration and invasion by upregulating the expression of fibroblast growth factor binding protein 1 (Fgfbp1), and subsequently increasing the phosphorylation of AKT. Furthermore, AKT activation partially mediated the increased expression of Fgfbp1 induced by HSD11B2. HSD11B2 expression was positively correlated with Fgfbp1 and p-AKT expression in clinical samples of CRC. Additionally, knockdown of either Fgfbp1 or AKT impaired the migration and invasion capability of CRC cells with HSD11B2 overexpression, suggesting that HSD11B2 promoted the migration, invasion and metastasis of CRC cells via the Fgfbp1-AKT pathway. Therefore, targeting HSD11B2 or Fgfbp1 may be a novel treatment strategy for inhibiting the metastasis of CRC.

FBXO22 promotes the development of hepatocellular carcinoma by regulating the ubiquitination and degradation of p21.

BACKGROUND: Deregulation of ubiquitin ligases is related to the malignant progression of human cancers. F-box only protein 22 (FBXO22), an F-box E3 ligase, is a member of the F-box protein family. However, the biological function of FBXO22 in HCC and the underlying molecular mechanisms are still unclear. In this study, we explored the role of FBXO22 in HCC and its mechanism of promoting tumor development. METHODS: We examined the expression of FBXO22 in normal liver cell lines, HCC cell lines, HCC tissue microarrays and fresh specimens. The correlation between FBXO22 and clinical features was analyzed in a retrospective study of 110 pairs of HCC tissue microarrays. Univariate and multivariate survival analyses were used to explore the prognostic value of FBXO22 in HCC. At the same time, the correlation between the FBXO22 and p21 was also studied in HCC samples. Knock-down and overexpression experiments, CHX and Mg132 intervention experiments, ubiquitination experiments, rescue experiments and nude mouse xenograft models were used to determine the potential mechanism by which FBXO22 promotes tumorigenesis in vitro and in vivo. RESULTS: The expression of FBXO22 in HCC tissues was significantly higher than in normal liver tissues. The overall survival rate and disease-free survival time of patients with high expression of FBXO22 were significantly shorter than those of patients with low expression of FBXO22. The high expression of FBXO22 in HCC tissues were significantly correlated with serum AFP (p = 0. 003, Pearson's chi-squared test), tumor size (p = 0. 019, Pearson's chi-squared test) and vascular invasion (p = 0. 031, Pearson's chi-squared test). Especially, Multivariate analysis showed that tumor size and the expression of FBXO22 were independent prognostic indicator of OS (95% CI: 1.077-5.157, P<0.05). Correlation analysis also showed that FBXO22 was negatively correlated with p21 in tissue microarrays (r = - 0.3788, P<0.001, Pearson correlation) and fresh specimens (r = - 0.4037, P<0.01, Pearson correlation). Moreover, both in vitro and in vivo experiments showed that knocking down FBXO22 expression could inhibit cell proliferation, while overexpression of FBXO22 promoted tumor formation. Furthermore, we identified that FBXO22 interacts with p21 by regulating protein stability and by influencing the ubiquitination process. A knockdown of FBXO22 decreased the ubiquitylation of p21, while overexpression enhanced it. CONCLUSIONS: This study uncovered a new mechanism by which FBXO22 functions as an oncogene in HCC pathogenesis and progression by mediating the ubiquitination and degradation of p21. It was also found that tumor size and the expression of FBXO22 were independent prognostic indicator of OS and the expression of FBXO22 and p21 was negatively correlated in clinical samples. Our findings present a new perspective for understanding the development of HCC, which may provide a new target for the treatment and management of this challenging cancer.