Reactive oxygen species-responsive nanocarrier ameliorates murine colitis by intervening colonic innate and adaptive immune responses.

Ulcerative colitis (UC) is a chronic or relapsing inflammatory disease with limited therapeutic outcomes. Pterostilbene (PSB) is a polyphenol-based anti-oxidant that has received extensive interest for its intrinsic anti-inflammatory and anti-oxidative activities. This work aims to develop a reactive oxygen species (ROS)-responsive, folic acid (FA)-functionalized nanoparticle (NP) for efficient PSB delivery to treat UC. The resulting PSB@NP-FA had a nano-scaled diameter of 231 nm and a spherical shape. With ROS-responsive release and ROS-scavenging properties, PSB@NP could effectively scavenge H2O2, thereby protecting cells from H2O2-induced oxidative damage. After FA modification, the resulting PSB@NP-FA could be internalized by RAW 264.7 and Colon-26 cells efficiently and preferentially localized to the inflamed colon. In dextran sulfate sodium (DSS)-induced colitis models, PSB@NP-FA showed a prominent ROS-scavenging capacity and anti-inflammatory activity, therefore relieving murine colitis effectively. Mechanism results suggested that PSB@NP-FA ameliorated colitis by regulating dendritic cells (DCs), promoting macrophage polarization, and regulating T cell infiltration. Both innate and adaptive immunity were involved. More importantly, the combination of the PSB and dexamethasone (DEX) enhanced the therapeutic efficacy of colitis. This ROS-responsive and ROS-scavenging nanocarrier represents an alternative therapeutic approach to UC. It can also be used as an enhancer for classic anti-inflammatory drugs.

HIF-1α-activated TMEM237 promotes hepatocellular carcinoma progression via the NPHP1/Pyk2/ERK pathway.

BACKGROUND: Hypoxia-inducible factors (HIFs) are the most essential endogenous transcription factors in the hypoxic microenvironment and regulate multiple genes involved in the proliferation, migration, invasion, and EMT of hepatocellular carcinoma (HCC) cells. However, the regulatory mechanism of HIFs in driving HCC progression remains poorly understood. METHODS: Gain- and loss-of-function experiments were carried out to investigate the role of TMEM237 in vitro and in vivo. The molecular mechanisms involved in HIF-1α-induced TMEM237 expression and TMEM237-mediated enhancement of HCC progression were confirmed by luciferase reporter, ChIP, IP-MS and Co-IP assays. RESULTS: TMEM237 was identified as a novel hypoxia-responsive gene in HCC. HIF-1α directly bound to the promoter of TMEM237 to transactivate its expression. The overexpression of TMEM237 was frequently detected in HCC and associated with poor clinical outcomes in patients. TMEM237 facilitated the proliferation, migration, invasion, and EMT of HCC cells and promoted tumor growth and metastasis in mice. TMEM237 interacted with NPHP1 and strengthened the interaction between NPHP1 and Pyk2 to trigger the phosphorylation of Pyk2 and ERK1/2, thereby contributing to HCC progression. The TMEM237/NPHP1 axis mediates hypoxia-induced activation of the Pyk2/ERK1/2 pathway in HCC cells. CONCLUSIONS: Our study demonstrated that HIF-1α-activated TMEM237 interacted with NPHP1 to activate the Pyk2/ERK pathway, thereby promoting HCC progression.

The regulation of amino acid metabolism in tumor cell death: from the perspective of physiological functions.

Amino acids (AAs) are crucial molecules for the synthesis of mammalian proteins as well as a source of energy and redox equilibrium maintenance. The development of tumors also requires AAs as nutrients. Increased AAs metabolism is frequently seen in tumor cells to produce enough biomass, energy, and reduction agents. However, increased AA demand may result in auxotrophy in some cancer cells, highlighting the vulnerabilities of cancers and exposing the AA metabolism as a potential target for cancer therapy. The dynamic balance of cell survival and death is required for cellular homeostasis, growth, and development. Malignant cells manage to avoid cell death through a range of mechanisms, such as developing an addiction to amino acids through metabolic adaptation. In order to offer some guidance for AA-targeted cancer therapy, we have outlined the function of AA metabolism in tumor progression, the modalities of cell death, and the regulation of AA metabolism on tumor cell death in this review.

Orally administration of cerium oxide nanozyme for computed tomography imaging and anti-inflammatory/anti-fibrotic therapy of inflammatory bowel disease.

BACKGROUND: Inflammatory bowel disease (IBD) is a chronic nonspecific disease with unknown etiology. Currently, the anti-inflammatory therapeutic approaches have achieved a certain extent of effects in terms of inflammation alleviation. Still, the final pathological outcome of intestinal fibrosis has not been effectively improved yet. RESULTS: In this study, dextran-coated cerium oxide (D-CeO2) nanozyme with superoxide dismutase (SOD) and catalase (CAT) activities was synthesized by chemical precipitation. Our results showed that D-CeO2 could efficiently scavenge reactive oxide species (ROS) as well as downregulate the pro-inflammatory cytokines (IL-1ß, IL-6, TNF-α, and iNOS) to protect cells from H2O2-induced oxidative damage. Moreover, D-CeO2 could suppress the expression of fibrosis-related gene levels, such as α-SMA, and Collagen 1/3, demonstrating the anti-fibrotic effect. In both TBNS- and DSS-induced colitis models, oral administration of D-CeO2 in chitosan/alginate hydrogel alleviated intestinal inflammation, reduced colonic damage by scavenging ROS, and decreased inflammatory factor levels. Notably, our findings also suggested that D-CeO2 reduced fibrosis-related cytokine levels, predicting a contribution to alleviating colonic fibrosis. Meanwhile, D-CeO2 could also be employed as a CT contrast agent for noninvasive gastrointestinal tract (GIT) imaging. CONCLUSION: We introduced cerium oxide nanozyme as a novel therapeutic approach with computed tomography (CT)-guided anti-inflammatory and anti-fibrotic therapy for the management of IBD. Collectively, without appreciable systemic toxicity, D-CeO2 held the promise of integrated applications for diagnosis and therapy, pioneering the exploration of nanozymes with ROS scavenging capacity in the anti-fibrotic treatment of IBD.

Carbon dot nanozymes as free radicals scavengers for the management of hepatic ischemia-reperfusion injury by regulating the liver inflammatory network and inhibiting apoptosis.

BACKGROUND: Hepatic ischemia-reperfusion injury (HIRI) is a pathophysiological process during liver transplantation, characterized by insufficient oxygen supply and subsequent restoration of blood flow leading to an overproduction of reactive oxygen species (ROS), which in turn activates the inflammatory response and leads to cellular damage. Therefore, reducing excess ROS production in the hepatic microenvironment would provide an effective way to mitigate oxidative stress injury and apoptosis during HIRI. Nanozymes with outstanding free radical scavenging activities have aroused great interest and enthusiasm in oxidative stress treatment. RESULTS: We previously demonstrated that carbon-dots (C-dots) nanozymes with SOD-like activity could serve as free radicals scavengers. Herein, we proposed that C-dots could protect the liver from ROS-mediated inflammatory responses and apoptosis in HIRI, thereby improving the therapeutic effect. We demonstrated that C-dots with anti-oxidative stress and anti-inflammatory properties improved the survival of L-02 cells under H2O2 and LPS-treated conditions. In the animal model, Our results showed that the impregnation of C-dots could effectively scavenge ROS and reduce the expression of inflammatory cytokines, such as IL-1ß, IL-6, IL-12, and TNF-α, resulting in a profound therapeutic effect in the HIRI. To reveal the potential therapeutic mechanism, transcriptome sequencing was performed and the relevant genes were validated, showing that the C-dots exert hepatoprotective effects by modulating the hepatic inflammatory network and inhibiting apoptosis. CONCLUSIONS: With negligible systemic toxicity, our findings substantiate the potential of C-dots as a therapeutic approach for HIRI, thereby offering a promising intervention strategy for clinical implementation.

All-In-One Biomimetic Nanoplatform Based on Hollow Polydopamine Nanoparticles for Synergistically Enhanced Radiotherapy of Colon Cancer.

Even though radiotherapy is the most important therapeutic strategy for colon cancer treatment, there is an enormous demand to improve radiosensitivity in solid tumor destruction. For this purpose, a biomimetic nanoplatform based on hollow polydopamine nanoparticles (HP) with homologous targeting and pH-responsive drug release properties is designed. In this work, HP is constructed by using a chelation competition-induced polymerization strategy and then modified with the cancer cell membrane. Hollow polydopamine integrated with Pt nanoparticles (Pt@HP) has a catalase-like activity, which can be used to trigger endogenous H2 O2 into O2 , relieving hypoxia of the tumor microenvironment (TME). With mesoporous shells and large cavities, Pt@HP shows efficient apoptin100-109 (AP) and verteporfin (VP) loading to form AVPt@HP@M. Under X-ray irradiation, AVPt@HP@M exerts a radiosensitization effect via multiple strategies, including relieving hypoxia (Pt NPs), enhancing tumor apoptosis (AP), and X-ray-induced photodynamic therapy (X-PDT) (VP). Further metabonomics analysis shows that the specific mechanism of the AVPt@HP@M is through influencing purine metabolism. Without appreciable systemic toxicity, this nanoplatform highlights a new strategy for effective radiosensitization and provides a reference for treating malignant tumors.

Oral administration of turmeric-derived exosome-like nanovesicles with anti-inflammatory and pro-resolving bioactions for murine colitis therapy.

BACKGROUND: Ulcerative colitis (UC) is an inflammatory bowel disease (IBD) characterized by diffuse inflammation of the colonic mucosa and a relapsing and remitting course. The current therapeutics are only modestly effective and carry risks for unacceptable adverse events, and thus more effective approaches to treat UC is clinically needed. RESULTS: For this purpose, turmeric-derived nanoparticles with a specific population (TDNPs 2) were characterized, and their targeting ability and therapeutic effects against colitis were investigated systematically. The hydrodynamic size of TDNPs 2 was around 178 nm, and the zeta potential was negative (- 21.7 mV). Mass spectrometry identified TDNPs 2 containing high levels of lipids and proteins. Notably, curcumin, the bioactive constituent of turmeric, was evidenced in TDNPs 2. In lipopolysaccharide (LPS)-induced acute inflammation, TDNPs 2 showed excellent anti-inflammatory and antioxidant properties. In mice colitis models, we demonstrated that orally administrated of TDNPs 2 could ameliorate mice colitis and accelerate colitis resolution via regulating the expression of the pro-inflammatory cytokines, including TNF-α, IL-6, and IL-1ß, and antioxidant gene, HO-1. Results obtained from transgenic mice with NF-κB-RE-Luc indicated that TDNPs 2-mediated inactivation of the NF-κB pathway might partially contribute to the protective effect of these particles against colitis. CONCLUSION: Our results suggest that TDNPs 2 from edible turmeric represent a novel, natural colon-targeting therapeutics that may prevent colitis and promote wound repair in colitis while outperforming artificial nanoparticles in terms of low toxicity and ease of large-scale production.

All-in-one theranostic nano-platform based on polymer nanoparticles for BRET/FRET-initiated bioluminescence imaging and synergistically anti-inflammatory therapy for ulcerative colitis.

BACKGROUND: Ulcerative colitis (UC), a subtype of inflammatory bowel disease (IBD), has evolved into a global burden given its high incidence. There is a clinical need to create better diagnostic and therapeutic approaches to UC. RESULTS: We fabricated P-selectin binding peptide-decorated poly lactic-co-glycolic acid (PBP-PLGA-NP) doped with two lipophilic dyes, DiL and DiD. Meanwhile, two low-toxic anti-inflammatory natural products (betulinic acid [BA] and resveratrol [Res]) were co-loaded in the PBP-PLGA-NP system. The BA/Res-loaded NPs had an average size of around 164.18 nm with a negative zeta potential (- 25.46 mV). Entrapment efficiencies of BA and Res were 74.54% and 52.33%, respectively, and presented a sustained drug release profile. Further, the resulting PBP-PLGA-NP could be internalized by RAW 264.7 cells and Colon-26 cells efficiently in vitro and preferentially localized to the inflamed colon. When intravenously injected with luminol, MPO-dependent bioluminescence imaging to visualize tissue inflammation was activated by the bioluminescence and fluorescence resonance energy transfer (BRET-FRET) effect. Importantly, injected NPs could remarkably alleviate UC symptoms yet maintain intestinal microbiota homeostasis without inducing organ injuries in the mice models of colitis. CONCLUSIONS: This theranostic nano-platform not only serves as a therapeutic system for UC but also as a non-invasive and highly-sensitive approach for accurately visualizing inflammation.

Identification and validation of redox-immune based prognostic signature for hepatocellular carcinoma.

The intimate interaction between redox signaling and immunity has been widely revealed. However, the clinical application of relevant therapeutic is unavailable due to the absence of validated markers that stratify patients. Here, we identified novel biomarkers for prognosis prediction in hepatocellular carcinoma (HCC). Prognostic redox-immune-related genes for predicting overall survival (OS) of HCC were identified using datasets from TCGA, LIRI-JP, and GSE14520. LASSO Cox regression was employed to construct the signature model and generate a risk score in the TCGA cohort. The signature contained CDO1, G6PD, LDHA, GPD1L, PPARG, FABP4, CCL20, SPP1, RORC, HDAC1, STC2, HDGF, EPO, and IL18RAP. Patients in the high-risk group had a poor prognosis compared to the low-risk group. Univariate and multivariate Cox regressions identified this signature as an independent factor for predicting OS. Nomogram constructed by multiple clinical parameters showed good performance for predicting OS indicated by the c-index, the calibration curve, and AUC. GSEA showed that oxidoreductase activity and peroxisome-related metabolic pathways were enriched in the low-risk group, while glycolysis activity and hypoxia were higher in the high-risk group. Furthermore, immune profiles analysis showed that the immune score and stromal score were significantly decreased in the high-risk group in the TCGA cohort. There was a considerably lower infiltration of anti-tumor immune cells while a higher proportion of pro-tumor immune cells in silico. Immune markers were distinctly expressed between the subgroups, and redox-sensitive immunoregulatory biomarkers were at higher levels in the high-risk group. Altogether, we identified a redox-immune prognostic signature. A more severe redox perturbation-driven immunosuppressive environment in the high-risk group stratified by the signature may account for poor survival. This may provide a clue to the combined therapy targeting redox and immune in HCC.

Long noncoding RNA PICSAR/miR-588/EIF6 axis regulates tumorigenesis of hepatocellular carcinoma by activating PI3K/AKT/mTOR signaling pathway.

Accumulating evidence has identified long noncoding RNAs (lncRNAs) as regulators in tumor progression and development. Here, we elucidated the function and possible molecular mechanisms of the effect of lncRNA-PICSAR (p38 inhibited cutaneous squamous cell carcinoma associated lincRNA) on the biological behaviors of HCC. In the present study, we found that PICSAR was upregulated in HCC tissues and cells and correlated with progression and poor prognosis in HCC patients. Gain- and loss-of-function experiments indicated that PICSAR enhanced cell proliferation, colony formation, and cell cycle progression and inhibited apoptosis of HCC cells. PICSAR could function as a competing endogenous RNA by sponging microRNA (miR)-588 in HCC cells. Mechanically, miR-588 inhibited HCC progression and alternation of miR-588 reversed the promotive effects of PICSAR on HCC cells. In addition, we confirmed that eukaryotic initiation factor 6 (EIF6) was a direct target of miR-588 in HCC and mediated the biological effects of miR-588 and PICSAR in HCC, resulting in PI3K/AKT/mTOR pathway activation. Our data identified PICSAR as a novel oncogenic lncRNA associated with malignant clinical outcomes in HCC patients. PICSAR played an oncogenic role by targeting miR-588 and subsequently promoted EIF6 expression and PI3K/AKT/mTOR activation in HCC. Our results revealed that PICSAR could be a potential prognostic biomarker and therapeutic target for HCC.

p300 Acetyltransferase Is a Cytoplasm-to-Nucleus Shuttle for SMAD2/3 and TAZ Nuclear Transport in Transforming Growth Factor ß-Stimulated Hepatic Stellate Cells.

Nuclear translocation of mothers against decapentaplegic homolog 2/3 (SMAD2/3), core transcription factors of transforming growth factor ß (TGF-ß) signaling, is critical for hepatic stellate cell (HSC) differentiation into metastasis-promoting myofibroblasts. SMAD2/3 have multiple coactivators, including WW domain-containing transcription regulator protein 1 (WWTR1 or TAZ) and p300 acetyltransferase. In the nucleus, TAZ binds to SMAD2/3 to prevent SMAD2/3 nuclear export. However, how TAZ and SMAD2/3 enter the nucleus remains poorly understood because neither contains a nuclear localization signal (NLS), an amino acid sequence tagging proteins for nuclear transport. p300 is an NLS-containing large scaffold protein, so we hypothesized that SMAD2/3 and TAZ may undergo nuclear import through complexing with p300. Coimmunoprecipitation, immunofluorescence, and nuclear fractionation assays revealed that TGF-ß1 promoted binding of SMAD2/3 and TAZ to p300 and that p300 inactivation disrupted TGF-ß1-mediated SMAD2/3 and TAZ nuclear accumulation. Deleting the p300 NLS blocked TGF-ß1-induced SMAD2/3 and TAZ nuclear transport. Consistently, p300 inactivation suppressed TGF-ß1-mediated HSC activation and transcription of genes encoding tumor-promoting factors, such as connective tissue growth factor, Tenascin C, Periostin, platelet-derived growth factor C, and fibroblast growth factor 2, as revealed by microarray analysis. Chromatin immunoprecipitation-real-time quantitative PCR showed that canonical p300-mediated acetylation of histones also facilitated transcription in response to TGF-ß1 stimulation. Interestingly, although both TGF-ß1-mediated and stiffness-mediated HSC activation require p300, comparison of gene expression data sets revealed that transcriptional targets of TGF-ß1 were distinct from those of stiffness-p300 mechanosignaling. Lastly, in tumor/HSC coinjection and intrasplenic tumor injection models, targeting p300 of activated-HSC/myofibroblasts by C646, short hairpin RNA, or cre-mediated gene disruption reduced tumor and liver metastatic growth in mice. Conclusion: p300 facilitates TGF-ß1-stimulated HSC activation by both noncanonical (cytoplasm-to-nucleus shuttle for SMAD2/3 and TAZ) and canonical (histone acetylation) mechanisms. p300 is an attractive target for inhibiting HSC activation and the prometastatic liver microenvironment.

A novel lncRNA MCM3AP-AS1 promotes the growth of hepatocellular carcinoma by targeting miR-194-5p/FOXA1 axis.

BACKGROUND: Hepatocellular carcinoma (HCC) is the most common malignant liver tumor with poor clinical outcomes. Increasing amount of long non-coding RNAs (lncRNAs) have been revealed to be implicated in the carcinogenesis and progression of HCC. However, the expressions, clinical significances, and roles of most lncRNAs in HCC are still unknown. METHODS: The expression of lncRNA MCM3AP antisense RNA 1 (MCM3AP-AS1) in HCC tissues and cell lines was detected by qRT-PCR and fluorescence in situ hybridization. Immunoblotting, CCK-8, EdU, colony formation and flow cytometry were performed to investigate the role of MCM3AP-AS1 in HCC cell proliferation, cell cycle and apoptosis in vitro. A subcutaneous tumor mouse model was constructed to analyze in vivo growth of HCC cells after MCM3AP-AS1 knockdown. The interactions among MCM3AP-AS1, miR-194-5p and FOXA1 were measured by RNA pull-down, RNA immunoprecipitation and luciferase reporter assay. RESULTS: We revealed a novel oncogenic lncRNA MCM3AP-AS1, which is overexpressed in HCC and positively correlated with large tumor size, high tumor grade, advanced tumor stage and poor prognosis of HCC patients. MCM3AP-AS1 knockdown suppressed HCC cell proliferation, colony formation and cell cycle progression, and induced apoptosis in vitro, and depletion of MCM3AP-AS1 inhibited tumor growth of HCC in vivo. Mechanistically, MCM3AP-AS1 directly bound to miR-194-5p and acted as competing endogenous RNA (ceRNA), and subsequently facilitated miR-194-5p's target gene forkhead box A1 (FOXA1) expression in HCC cells. Interestingly, FOXA1 restoration rescued MCM3AP-AS1 knockdown induced proliferation inhibition, G1 arrest and apoptosis of HCC cells. CONCLUSIONS: Our results recognized MCM3AP-AS1 as a novel oncogenic lncRNA, which indicated poor clinical outcomes in patients with HCC. MCM3AP-AS1 exerted an oncogenic role in HCC via targeting miR-194-5p and subsequently promoted FOXA1 expression. Our findings suggested that MCM3AP-AS1 could be a potential prognostic biomarker and therapeutic target for HCC.

P300 Acetyltransferase Mediates Stiffness-Induced Activation of Hepatic Stellate Cells Into Tumor-Promoting Myofibroblasts.

BACKGROUND & AIMS: Hepatic stellate cells (HSCs) contribute to desmoplasia and stiffness of liver metastases by differentiating into matrix-producing myofibroblasts. We investigated whether stiffness due to the presence of tumors increases activation of HSCs into myofibroblasts and their tumor-promoting effects, as well as the role of E1A binding protein p300, a histone acetyltransferase that regulates transcription, in these processes. METHODS: HSCs were isolated from liver tissues of patients, mice in which the p300 gene was flanked by 2 loxP sites (p300F/F mice), and p300+/+ mice (controls). The HSCs were placed on polyacrylamide gels with precisely defined stiffness, and their activation (differentiation into myofibroblasts) was assessed by immunofluorescence and immunoblot analyses for alpha-smooth muscle actin. In HSCs from mice, the p300 gene was disrupted by cre recombinase. In human HSCs, levels of p300 were knocked down with small hairpin RNAs or a mutant form of p300 that is not phosphorylated by AKT (p300S1834A) was overexpressed. Human HSCs were also cultured with inhibitors of p300 (C646), PI3K signaling to AKT (LY294002), or RHOA (C3 transferase) and effects on stiffness-induced activation were measured. RNA sequencing and chromatin immunoprecipitation-quantitative polymerase chain reaction were used to identify HSC genes that changed expression levels in response to stiffness. We measured effects of HSC-conditioned media on proliferation of HT29 colon cancer cells and growth of tumors following subcutaneous injection of these cells into mice. MC38 colon cancer cells were injected into portal veins of p300F/Fcre and control mice, and liver metastases were measured. p300F/Fcre and control mice were given intraperitoneal injections of CCl4 to induce liver fibrosis. Liver tissues were collected and analyzed by immunofluorescence, immunoblot, and histology. RESULTS: Substrate stiffness was sufficient to activate HSCs, leading to nuclear accumulation of p300. Disrupting p300 level or activity blocked stiffness-induced activation of HSCs. In HSCs, substrate stiffness activated AKT signaling via RHOA to induce phosphorylation of p300 at serine 1834; this caused p300 to translocate to the nucleus, where it up-regulated transcription of genes that increase activation of HSCs and metastasis, including CXCL12. MC38 cells, injected into portal veins, formed fewer metastases in livers of p300F/Fcre mice than control mice. Expression of p300 was increased in livers of mice following injection of CCl4; HSC activation and collagen deposition were reduced in livers of p300F/Fcre mice compared with control mice. CONCLUSIONS: In studies of mice, we found liver stiffness to activate HSC differentiation into myofibroblasts, which required nuclear accumulation of p300. p300 increases HSC expression of genes that promote metastasis.

MicroRNA-1296 inhibits metastasis and epithelial-mesenchymal transition of hepatocellular carcinoma by targeting SRPK1-mediated PI3K/AKT pathway.

BACKGROUND: Increasing evidences demonstrate that miRNAs contribute to development and progression of hepatocellular carcinoma (HCC). Underexpression of miR-1296 is recently reported to promote growth and metastasis of human cancers. However, the expression and role of miR-1296 in HCC remain unknown. METHODS: The levels of miR-1296 in HCC tissues and cells were detected by qRT-PCR. Immunoblotting and immunofluorescence were used for detection of epithelial-to-mesenchymal transition (EMT) progression in HCC cells. Transwell assays were performed to determine migration and invasion of HCC cells. A lung metastasis mouse model was used to evaluated metastasis of HCC in vivo. The putative targets of miR-1296 were disclosed by public databases and a dual-luciferase reporter assay. RESULTS: We found that the expression of miR-1296 was reduced in HCC tissues and cell lines, and it was associated with metastasis and recurrence of HCC. Notably, miR-1296 overexpression inhibited migration, invasion and EMT progress of HCCLM3 cells, while miR-1296 loss facilitated these biological behaviors of Hep3B cells in vitro and in vivo. In addition, miR-1296 inversely regulated SRPK1 abundance by directly binding to its 3'-UTR, which subsequently resulted in suppression of p-AKT. Either SRPK1 re-expression or PI3K/AKT pathway activation, at least partially, abolished the effects of miR-1296 on migration, invasion and EMT progress of HCC cells. Furthermore, miR-1296 and SRPK1 expression were markedly correlated with adverse clinical features and poor prognosis of HCC patients. We showed that hypoxia was responsible for the underexpression of miR-1296 in HCC. And the promoting effects of hypoxia on metastasis and EMT of HCC cells were reversed by miR-1296. CONCLUSIONS: Underexpression of miR-1296 potentially serves as a prognostic biomarker in HCC. Hypoxia-induced miR-1296 loss promotes metastasis and EMT of HCC cells probably by targeting SRPK1/AKT pathway.

Long non-coding RNA CASC2 suppresses epithelial-mesenchymal transition of hepatocellular carcinoma cells through CASC2/miR-367/FBXW7 axis.

BACKGROUND: Recently, it has been reported that long non-coding RNA (lncRNA) cancer susceptibility candidate 2 (CASC2), a novel tumor suppressor, participates in regulating the carcinogenesis and suppresses tumor progression by sponging microRNAs (miRNAs). However, the expression and function of CASC2 in hepatocellular carcinoma (HCC) remain unclear. METHODS: The expression of CASC2 and miR-367 in HCC specimens and cell lines were detected by real-time PCR. Western blotting and immunohistochemistry were carried out for detection of epithelial-to-mesenchymal transition (EMT) markers in HCC. Transwell assays were used to determine migration and invasion of HCC cells. A mouse model for lung metastasis was established to evaluated HCC metastasis in vivo. The correlation among CASC2, miR-367 and F-box and WD repeat domain containing 7 (FBXW7) were disclosed by a dual-luciferase reporter assay, RIP assay and biotin pull-down assay. RESULTS: Here, CASC2 expression was significantly downregulated in HCC tissues, especially in aggressive and recurrent cases. In accordance, CASC2 underexpression was observed in HCC cell lines compared to LO2. In vitro and in vivo experiments revealed that CASC2 inhibited migration and invasion of HCC cells. Additionally, CASC2 repressed EMT process of HCC cells. Further studies demonstrated that CASC2 could function as a competing endogenous RNA (ceRNA) by sponging miR-367 in HCC cells. Functionally, gain- and loss-of-function studies showed that miR-367 promoted migration, invasion and EMT progression of HCC cells. Moreover, further investigations disclosed that FBXW7 was a downstream target of miR-367 and CASC2 prohibited EMT progression and subsequently exerted its anti-metastatic effects via CASC2/miR-367/FBXW7 axis in HCC cells. Clinically, CASC2 underexpression and miR-367 overexpression were closely correlated with the metastasis-associated clinicopathologic features. Notably, CASC2 low-expressing and miR-367 high-expressing HCC patients showed the poorest clinical outcome. CONCLUSIONS: Overall, we conclude that the CASC2/miR-367/FBXW7 axis may be a ponderable and promising therapeutic target for HCC.

HSP90 promotes cell glycolysis, proliferation and inhibits apoptosis by regulating PKM2 abundance via Thr-328 phosphorylation in hepatocellular carcinoma.

BACKGROUND: Heat shock protein 90 (HSP90) functions as a well-known onco-protein to regulate protein conformation, stability and degradation. Pyruvate kinase M2 (PKM2), a critical regulator of the metabolism, growth and metastasis of cancer cells, has been confirmed to be overexpressed in various human cancer including hepatocellular carcinoma (HCC). However, the molecular mechanisms underlying the oncogenic functions of HSP90 and PKM2 overexpression in HCC remain unknown. METHODS: The expression of HSP90 and PKM2 in HCC specimens and cells were detected by immunoblotting and immunostaining. The interaction between HSP90 and PKM2 was confirmed by tandem affinity purification, co-immunoprecipitation and Glutathione S transferase (GST)-pulldown assay. RESULTS: In this study, we found that HSP90 could bind to PKM2 and subsequently increased PKM2 abundance in HCC cells. Immunohistochemistry (IHC) staining showed that HSP90 level was positively correlated with PKM2 level in HCC tissues. Mechanistically, HSP90 was found to increase the phosphorylation of PKM2 at Thr-328. Protein kinase glycogen synthase kinase-3ß (GSK-3ß) formed a protein complex with HSP90 and PKM2, and directly mediated Thr-328 phosphorylation of PKM2 induced by HSP90. Thr-328 phosphorylation was critical for maintaining PKM2 stability and its biological functions in regulating glycolysis, mitochondria respiration, proliferation and apoptosis. Functionally, we found that HSP90 promoted the glycolysis and proliferation and inhibited apoptosis of HCC cells in a PKM2 dependent manner. In vivo experiments disclosed that PKM2 was required for the promoting effects of HSP90 on the growth of HCC cells in mice. Furthermore, we demonstrated that positive expression of HSP90 and PKM2 was correlated with poor clinicopathological features including high alpha fetoprotein (AFP) level, large tumor size, portal vein tumor thrombus (PVTT) and advanced tumor-node-metastasis (TNM) stage. Furthermore, we demonstrated that positive expression of HSP90 and PKM2, and a combination of these proteins could strongly predict the poor prognosis of HCC patients. CONCLUSIONS: We suggest that HSP90 potentiates the glycolysis and proliferation, reduces the apoptosis and thus enhances the growth of HCC cells through PKM2.

Hypoxia Accelerates Aggressiveness of Hepatocellular Carcinoma Cells Involving Oxidative Stress, Epithelial-Mesenchymal Transition and Non-Canonical Hedgehog Signaling.

BACKGROUND/AIMS: Hypoxic microenvironment, a common feature of hepatocellular carcinoma (HCC), can induce HIF-1α expression and promote the epithelial-mesenchymal transition (EMT) and invasion of cancer cells. However, the underlying molecular mechanisms have not fully elucidated. METHODS: HCC cells were cultured under controlled hypoxia conditions or normoxic conditions. Transwell assays were used to examine the migration and invasion capacity. HIF-1α siRNA, cyclopamine (a SMO antagonist) and GLI1 siRNA were used to inhibit HIF-1α transcription or Hh signaling activation. RESULTS: In present study, we first observed a strongly positive correlation between HIF-1α and GLI1 expression in HCC tissues. Then, we showed that hypoxia significantly promoted EMT process and invasion of HCC cells, associated with activating the non-canonical Hh pathway without affecting SHH and PTCH1 expression. HIF-1α knockdown mitigated hypoxia-induced SMO and GLI1 expression, EMT invasion of HCC cells. Moreover, the SMO inhibitor or GLI1 siRNA also reversed the hypoxia-driven EMT and invasion of HCC cells under hypoxia condition. Here, we show that non-canonical Hh signaling is required as an important role to switch on hypoxia-induced EMT and invasion in HCC cells. In addition, we found that hypoxia increased ROS production and that ROS inhibitors (NAC) blocked GLI1-dependent EMT process and invasion under hypoxic conditions. To determine a major route of ROS production, we tested whether nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 4 (NOX4) is involved in hypoxia-induced ROS production. NOX4 expression was found to be increased at both mRNA and protein levels in hypoxic HCC cells. Furthermore, siRNA-mediated knockdown of NOX4 expression abolished hypoxia induced ROS generation and GLI1-dependent activation and invasion of HCC cells. CONCLUSION: Our findings indicate that hypoxia triggers ROS-mediated GLI1-dependent EMT progress and invasion of HCC cells through induction of NOX4 expression. Thus, hypoxia-driven ROS mediated non-canonical Hh signaling may play an important role in the initiation of EMT and provides a potential marker for cancer prevention and treatment.